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  • 1.
    Aili, Margareta
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Isaksson, Elin L
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Carlsson, Sara E
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Regulation of Yersinia Yop-effector delivery by translocated YopE2008In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 298, no 3-4, p. 183-192Article in journal (Refereed)
    Abstract [en]

    The bacterial pathogen Yersinia pseudotuberculosis uses a type III secretion (T3S) system to translocate Yop effectors into eukaryotic cells. Effectors are thought to gain access to the cytosol via pores formed in the host cell plasma membrane. Translocated YopE can modulate this pore formation through its GTPase-activating protein (GAP) activity. In this study, we analysed the role of translocated YopE and all the other known Yop effectors in the regulation of effector translocation. Elevated levels of Yop effector translocation into HeLa cells occurred by YopE-defective strains, but not those defective for other Yop effectors. Only Yersinia devoid of YopK exhibits a similar hyper-translocation phenotype. Since both yopK and yopE mutants also failed to down-regulate Yop synthesis in the presence of eukaryotic cells, these data imply that translocated YopE specifically regulates subsequent effector translocation by Yersinia through at least one mechanism that involves YopK. We suggest that the GAP activity of YopE might be working as an intra-cellular probe measuring the amount of protein translocated by Yersinia during infection. This may be a general feature of T3S-associated GAP proteins, since two homologues from Pseudomonas aeruginosa, exoenzyme S (ExoS) and exoenzyme T (ExoT), can complement the hyper-translocation phenotypes of the yopE GAP mutant.

  • 2.
    Amer, Ayad
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Costa, Tiago
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Farag, Salah
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Avican, Ummehan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 10, article id e77767Article in journal (Refereed)
    Abstract [en]

    Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.

  • 3.
    Amer, Ayad
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Costa, Tiago
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Gurung,, Jyoti
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Avican, Ummehan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Functional consequences of site-directed mutagenesis in theC-terminus of YopN, a Yersinia pseudotuberculosis regulator ofYop secretionManuscript (preprint) (Other academic)
    Abstract [en]

    Pathogenic Yersinia spp. utilizes the Ysc-Yop type III secretion system to targetYop effector proteins into the cytosol of host immune cells. Internalizedeffectors alter specific signaling pathways to neutralize immune cell-dependentphagocytosis, killing and pro-inflammatory responsiveness. This enablesextracellular bacterial multiplication and survival in immune tissue. Central tothe temporal control of Yop type III secretion is the regulator YopN. Incomplex with TyeA, YopN acts to plug the inner face of the type III secretionchannel, denying entry to other Yop substrates until after YopN has beensecreted. A +1 frameshift event in the 3-prime end of yopN results in thesynthesis of a singular secreted YopN-TyeA polypeptide chimera that retainssome regulatory function. As the C-terminal coding sequence of YopN in thishybrid product differs greatly from native sequence, we used site-directedmutagenesis to determine the functional significance of this segment. YopNtruncated at residue 287 or containing a shuffled sequence covering 288 to 293retains full function both in vitro and in vivo. Thus, the extreme C-terminus isapparently superfluous to YopN function. In contrast, a YopN varianttruncated after residue 278 was completely unstable, and these bacteria hadlost all control of T3S activity, and failed to defend against immune cell killing.Interestingly, inclusion of a shuffled sequence from residues 279 to 287recovered some T3S control over function. Hence, the YopN segmentencompassing 279 to 287 is essential for full function, although the exact aminoacid sequence is less important.

  • 4.
    Amer, Ayad
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Gurung, Jyoti
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Costa, Tiago
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Ruuth, Kristina
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Zavialov, Anton
    Joint Biotechnology Laboratory, Department of Chemistry, University of Turku, Turku, Finland.
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis2016In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 66Article in journal (Refereed)
    Abstract [en]

    Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopNW279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.

  • 5.
    Amer, Ayad
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Gurung, Jyoti
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Yersinia pseudotuberculosis type III secretion is reliant upon anauthentic N‐terminal YscX secretor domainManuscript (preprint) (Other academic)
    Abstract [en]

    Certain Gram‐negative bacteria use type III secretion systems to deliver effectorproteins into eukaryotic cells, serving either parasitic or mutualistic roles inside the hostcell. About 25 structural proteins are needed to assemble and deliver effector proteins.Collections of these proteins are quite well characterized, although the function ofsome continues to remain obscure. This is true for the Yersinia Ysc‐Yop systemcomponents YscX, a secreted substrate and YscY, its cognate non‐secreted chaperone.Despite recent evidence suggesting that they might coordinate Yop substrate secretion,YscX and YscY remain poorly characterized. To further investigate the function of theseproteins in the enteropathogen Y. pseudotuberculosis, we explored correlationsbetween the YscX N‐terminal segment, YscX secretion, as well as the secretion of otherYops. Analysis of a series of chimeric substrates in which the extreme YscX N‐terminushad been exchanged with equivalent functional secretion signals of other Ysc‐Yopsubstrates revealed that this segment contains non‐redundant information needed forYscX function, which includes permitting surface polymerization of the YscF needle andYops secretion. Further, in cis deletion of the YscX N‐terminus and ectopic expression ofepitope tagged YscX variants again correlated stable YscX production but not secretionto the type III secretion of Yops. Despite this, the first 5 codons were determined toconstitute a minimal signal capable of promoting secretion of the signalless ‐lactamasereporter. Hence, YscX does contain a fully equipped N‐terminal secretor domain topromote secretion of self. Nevertheless, the primary role of this N‐terminal segmentmust be to assemble an operational secretion system, and this occurs independently ofYscX secretion.

  • 6.
    Amer, Ayad
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Åhlund, Monika
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bröms, Jeanette
    Department of Medical Countermeasures, Swedish Defense Research Agency, Division of NBC12 Defense, Umeå, Sweden.
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Impact of the N-terminal secretor domain on YopD translocator function in Yersinia pseudotuberculosis type III secretion2011In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, no 23, p. 6683-6700Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems (T3SSs) secrete needle components, pore-forming translocators, and the translocated effectors. In part, effector recognition by a T3SS involves their N-terminal amino acids and their 5′ mRNA. To investigate whether similar molecular constraints influence translocator secretion, we scrutinized this region within YopD from Yersinia pseudotuberculosis. Mutations in the 5′ end of yopD that resulted in specific disruption of the mRNA sequence did not affect YopD secretion. On the other hand, a few mutations affecting the protein sequence reduced secretion. Translational reporter fusions identified the first five codons as a minimal N-terminal secretion signal and also indicated that the YopD N terminus might be important for yopD translation control. Hybrid proteins in which the N terminus of YopD was exchanged with the equivalent region of the YopE effector or the YopB translocator were also constructed. While the in vitro secretion profile was unaltered, these modified bacteria were all compromised with respect to T3SS activity in the presence of immune cells. Thus, the YopD N terminus does harbor a secretion signal that may also incorporate mechanisms of yopD translation control. This signal tolerates a high degree of variation while still maintaining secretion competence suggestive of inherent structural peculiarities that make it distinct from secretion signals of other T3SS substrates.

  • 7.
    Bröms, Jeanette E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Edqvist, Petra J
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Carlsson, Katrin E
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mapping of a YscY binding domain within the LcrH chaperone that is required for regulation of Yersinia type III secretion2005In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 187, no 22, p. 7738-7752Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of Yersinia pseudotuberculosis. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native Pseudomonas aeruginosa or when produced in Yersinia. In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the Yersinia type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of P. aeruginosa. YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of Yersinia and Pcr3 of P. aeruginosa. Still, Pcr4 was unable to complement a DeltayscY null mutant defective for type III secretion and yop-regulatory control in Yersinia, despite the ability of YscY to function in P. aeruginosa. Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to Yersinia type III secretion.

  • 8.
    Bröms, Jeanette E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Edqvist, Petra J
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Forsberg, Ake
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Tetratricopeptide repeats are essential for PcrH chaperone function in Pseudomonas aeruginosa type III secretion.2006In: FEMS Microbiol Lett, ISSN 0378-1097, Vol. 256, no 1, p. 57-66Article in journal (Refereed)
    Abstract [en]

    The type III secretion system (T3SS) is a specialized apparatus evolved by Gram-negative bacteria to deliver effector proteins into host cells, thus facilitating the establishment of an infection. Effector translocation across the target cell plasma membrane is believed to occur via pores formed by at least two secreted translocator proteins, the functions of which are dependent upon customized class II T3SS chaperones. Recently, three internal tetratricopeptide repeats (TPRs) were identified in this class of chaperones. Here, defined mutagenesis of the class II chaperone PcrH of Pseudomonas aeruginosa revealed these TPRs to be essential for chaperone activity towards the translocator proteins PopB and PopD and subsequently for the translocation of exoenzymes into host cells.

  • 9.
    Bröms, Jeanette E
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Ake
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Diminished LcrV secretion attenuates Yersinia pseudotuberculosis virulence.2007In: J Bacteriol, ISSN 0021-9193Article in journal (Refereed)
    Abstract [en]

    Many Gram negative bacterial pathogenicity factors that function beyond the outer membrane are secreted via a contact-dependent type III secretion system. Two types of substrates are predestined for this mode of secretion; anti-host effectors that are translocated directly into target cells and the translocators required for targeting of the effectors across the host cell membrane. N-terminal secretion signals are important for recognition of the protein cargo by the type III secretion machinery. Even though such signals are known for several effectors, a consensus signal sequence is not obvious. One of the tranclocators, LcrV, has been attributed other functions in addition to its role in translocation. These functions include regulation, presumably via interaction with LcrG inside the bacteria and immunomodulation via interaction with TLR2. Here we wanted to address the significance of the specific targeting of LcrV to the exterior for its function in regulation, effector targeting and virulence. The results, highlighting key N-terminal amino acids important for LcrV secretion, allowed us to dissect the role of LcrV in regulation from that in effector targeting/virulence. While only low levels of exported LcrV were required for in vitro effector translocation as deduced by a cell infection assay, fully functional export of LcrV was found to be a prerequisite for its role in virulence in the systemic murine infection model.

  • 10.
    Bröms, Jeanette
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    Forslund, Anna-Lena
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis.2003In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Microbiology, Vol. 149, no 9, p. 2615-2626Article in journal (Refereed)
    Abstract [en]

    The homologous pcrGVHpopBD and lcrGVHyopBD translocase operons of Pseudomonas aeruginosa and pathogenic Yersinia spp., respectively, are responsible for the translocation of anti-host effectors into the cytosol of infected eukaryotic cells. In Yersinia, this operon is also required for yop-regulatory control. To probe for key molecular interactions during the infection process, the functional interchangeability of popB/yopB and popD/yopD was investigated. Secretion of PopB produced in trans in a yopB null mutant of Yersinia was only observed when co-produced with its native chaperone PcrH, but this was sufficient to complement the yopB translocation defect. The Yersinia yopD null mutant synthesized and secreted PopD even in the absence of native PcrH, yet this did not restore YopD-dependent yop-regulatory control or effector translocation. Thus, this suggests that key residues in YopD, which are not conserved in PopD, are essential for functional Yersinia type III secretion.

  • 11.
    Bröms, Jeanette
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    Forslund, Anna-Lena
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Molecular Biology.
    Francis, Matthew
    Umeå University, Faculty of Medicine, Molecular Biology.
    PcrH of Pseudomonas aeruginosa is essential for secretion and assembly of the type III translocon2003In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 188, no 12, p. 1909-1921Article in journal (Refereed)
    Abstract [en]

    Pseudomonas aeruginosa harbors a type III secretion system that translocates antihost effectors into an infected eukaryotic cell. PcrH is a key component of type III secretion in this essential virulence strategy. In the absence of PcrH, P. aeruginosa is translocation deficient because of a specific reduction in presecretory stability and subsequent secretion of PopB and PopD, 2 proteins essential for the translocation process. PcrH exerts this chaperone function by binding directly to PopB and PopD. Consistent with the genetic relatedness of PcrH with LcrH of pathogenic Yersinia species, these proteins are functionally interchangeable with respect to their ability to complement the translocation defect associated with either a lcrH or pcrH null mutant, respectively. Thus, the translocator class of chaperones performs a critical function in ensuring the assembly of a translocation competent type III secreton. Finally, unlike the regulatory roles of other translocator-class chaperones (e.g., LcrH, SicA of Salmonella enterica, and IpgC of Shigella species), in vitro regulation of P. aeruginosa type III secretion does not involve PcrH.

  • 12.
    Bröms, Jeanette
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    Sundin, Charlotta
    Umeå University, Faculty of Medicine, Molecular Biology.
    Francis, Matthew
    Umeå University, Faculty of Medicine, Molecular Biology.
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Molecular Biology.
    Comparative analysis of type III effector translocation by Yersinia pseudotuberculosis expressing native LcrV or PcrV from Pseudomonas aeruginosa.2003In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 188, no 2, p. 239-249Article in journal (Refereed)
    Abstract [en]

    The homologues LcrV of Yersinia species and PcrV of Pseudomonas aeruginosa are pore-forming components. When expressed in a Yersinia lcrV background, PcrV formed smaller pores in infected erythrocyte membranes, correlating to a lowered translocation of Yersinia effectors. To understand this phenomenon, cytotoxins exoenzyme S of P. aeruginosa and YopE of Yersinia were introduced into a Yersinia background without Yop effectors but expressing LcrV or PcrV. Comparable translocation of each substrate indicated that substrate recognition by LcrV/PcrV is not a regulator of translocation. Yersinia harboring pcrV coexpressed with its native operon efficiently translocated effectors into HeLa cell monolayers and formed large LcrV-like pores in erythrocyte membranes. Thus, a PcrV complex with native P. aeruginosa translocon components is required to form fully functional pores for complete complementation of effector translocation in Yersinia.

  • 13.
    Carlsson, Katrin E
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Liu, Junfa
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Edqvist, Petra J
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Extracytoplasmic-stress-responsive pathways modulate type III secretion in Yersinia pseudotuberculosis.2007In: Infect Immun, ISSN 0019-9567, Vol. 75, no 8, p. 3913-24Article in journal (Refereed)
    Abstract [en]

    Three signal transduction pathways, the two-component systems CpxRA and BaeSR and the alternative sigma factor sigma(E), respond to extracytoplasmic stress that facilitates bacterial adaptation to changing environments. At least the CpxRA and sigma(E) pathways control the production of protein-folding and degradation factors that counter the effects of protein misfolding in the periplasm. This function also influences the biogenesis of multicomponent extracellular appendages that span the bacterial envelope, such as various forms of pili. Herein, we investigated whether any of these regulatory pathways in the enteropathogen Yersinia pseudotuberculosis affect the functionality of the Ysc-Yop type III secretion system. This is a multicomponent molecular syringe spanning the bacterial envelope used to inject effector proteins directly into eukaryotic cells. Disruption of individual components revealed that the Cpx and sigma(E) pathways are important for Y. pseudotuberculosis type III secretion of Yops (Yersinia outer proteins). In particular, a loss of CpxA, a sensor kinase, reduced levels of structural Ysc (Yersinia secretion) components in bacterial membranes, suggesting that these mutant bacteria are less able to assemble a functional secretion apparatus. Moreover, these bacteria were no longer capable of localizing Yops into the eukaryotic cell interior. In addition, a cpxA lcrQ double mutant engineered to overproduce and secrete Yops was still impaired in intoxicating cells. Thus, the Cpx pathway might mediate multiple influences on bacterium-target cell contact that modulate Yersinia type III secretion-dependent host cell cytotoxicity.

  • 14.
    Carlsson, Katrin E
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Liu, Junfa
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Edqvist, Petra J
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Influence of the Cpx extracytoplasmic-stress-responsive pathway on Yersinia sp.-eukaryotic cell contact.2007In: Infect Immun, ISSN 0019-9567, Vol. 75, no 9, p. 4386-99Article in journal (Refereed)
    Abstract [en]

    The extracytoplasmic-stress-responsive CpxRA two-component signal transduction pathway allows bacteria to adapt to growth in extreme environments. It controls the production of periplasmic protein folding and degradation factors, which aids in the biogenesis of multicomponent virulence determinants that span the bacterial envelope. This is true of the Yersinia pseudotuberculosis Ysc-Yop type III secretion system. However, despite using a second-site suppressor mutation to restore Yop effector secretion by yersiniae defective in the CpxA sensor kinase, these bacteria poorly translocated Yops into target eukaryotic cells. Investigation of this phenotype herein revealed that the expression of genes which encode several surface-located adhesins is also influenced by the Cpx pathway. In particular, the expression and surface localization of invasin, an adhesin that engages beta1-integrins on the eukaryotic cell surface, are severely restricted by the removal of CpxA. This reduces bacterial association with eukaryotic cells, which could be suppressed by the ectopic production of CpxA, invasin, or RovA, a positive activator of inv expression. In turn, these infected eukaryotic cells then became susceptible to intoxication by translocated Yop effectors. In contrast, bacteria harboring an in-frame deletion of cpxR, which encodes the cognate response regulator, displayed an enhanced ability to interact with cell monolayers, as well as elevated inv and rovA transcription. This phenotype could be drastically suppressed by providing a wild-type copy of cpxR in trans. We propose a mechanism of inv regulation influenced by the direct negative effects of phosphorylated CpxR on inv and rovA transcription. In this fashion, sensing of extracytoplasmic stress by CpxAR contributes to productive Yersinia sp.-eukaryotic cell interactions.

  • 15.
    Chen, Shiyun
    et al.
    Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
    Thompson, Karl
    Department of Microbiology, College of Medicine, Howard University, Washington, DC, USA.
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Environmental Regulation of Yersinia Pathophysiology2016In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 25Article in journal (Refereed)
    Abstract [en]

    Hallmarks of Yersinia pathogenesis include the ability to form biofilms on surfaces, the ability to establish close contact with eukaryotic target cells and the ability to hijack eukaryotic cell signaling and take over control of strategic cellular processes. Many of these virulence traits are already well-described. However, of equal importance is knowledge of both confined and global regulatory networks that collaborate together to dictate spatial and temporal control of virulence gene expression. This review has the purpose to incorporate historical observations with new discoveries to provide molecular insight into how some of these regulatory mechanisms respond rapidly to environmental flux to govern tight control of virulence gene expression by pathogenic Yersinia.

  • 16.
    Costa, Tiago
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Amer, Ayad
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Farag, Salah
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Edgren, Tomas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Type III secretion translocon assemblies that attenuate Yersinia virulence2013In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, no 7, p. 1088-1110Article in journal (Refereed)
    Abstract [en]

    Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted α-helix motif at the C-terminus. Mutants YopD(I262P) and YopD(K267P) poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopD(A263P) and YopD(A270P) had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.

  • 17.
    Costa, Tiago R. D.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Amer, Ayad A. A.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Coiled-coils in the YopD translocator family: A predicted structure unique to the YopD N-terminus contributes to full virulence of Yersinia pseudotuberculosis2012In: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 12, no 8, p. 1729-1742Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia all harbor a virulence plasmid-encoded Ysc–Yop T3SS. In this system, translocator function is performed by the hydrophobic proteins YopB and YopD. With the goal to better understand how YopD orchestrates yop-regulatory control, translocon pore formation and Yop effector translocation, we performed an in silico prediction of coiled-coil motifs in YopD and YopD-like sequences from other bacteria. Of interest was a predicted N-terminal coiled-coil that occurred solely in Yersinia YopD sequences. To investigate if this unique feature was biologically relevant, two in cis point mutations were generated with a view to disrupting this putative structure. Both mutants maintained full T3SS function in vitro in terms of environmental control of Yops synthesis and secretion, effector toxin translocation and evasion of phagocytosis and killing by cultured immune cells. However, these same mutants were attenuated for virulence in a murine oral-infection model. The cause of this tardy disease progression is unclear. However, these data indicate that any structural flaw in this element unique to the N-terminus will subtly compromise an aspect of YopD biology. Sub-optimal T3SSs are then formed that are unable to fortify Yersinia against attack by the host innate and adaptive immune response.

  • 18.
    Costa, Tiago R D
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Edqvist, Petra J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bröms, Jeanette E
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Åhlund, Monika K
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 33, p. 25269-25284Article in journal (Refereed)
    Abstract [en]

    YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an alpha-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.

  • 19.
    Deleuil, Fabienne
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    Mogemark, Lena
    Umeå University, Faculty of Medicine, Molecular Biology.
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Interaction between the Yersinia protein tyrosine phosphatase YopH and eukaryotic Cas/Fyb is an important virulence mechanism2003In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 5, no 1, p. 53-64Article in journal (Refereed)
    Abstract [en]

    The tyrosine phosphatase YopH is an essential virulence factor produced by pathogenic Yersinia species. YopH is translocated into host cells via a type III secretion system and its dephosphorylating activity causes disruption of focal complex structures and blockage of the phagocytic process. Among the host cell targets of YopH are the focal adhesion proteins Crk-associated substrate (p130Cas) and focal adhesion kinase (FAK) in epithelial cells, and p130Cas and Fyn-binding protein (Fyb) in macrophages. Previous studies have shown that the N-terminal domain of YopH acts as a substrate-binding domain. In this study, the mechanism and biological importance of the targeting of YopH to focal complexes relative to its interaction with p130Cas/Fyb was elucidated. Mutants of YopH that were defective in p130Cas/Fyb binding but otherwise indistinguishable from wild type were constructed. Mutants unable to bind p130Cas did not localize to focal complex structures in infected cells, indicating that the association with p130Cas is critical for appropriate subcellular localization of YopH. These yopH mutants were also clearly attenuated in virulence, showing that binding to p130Cas and/or Fyb is biologically relevant in Yersinia infections.

  • 20.
    Edqvist, Petra J
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Aili, Margareta
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Liu, Junfa
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Minimal YopB and YopD translocator secretion by Yersinia is sufficient for Yop-effector delivery into target cells.2007In: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 9, no 2, p. 224-233Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia sp. utilise a common type III secretion system to translocate several anti-host Yop effectors into the cytosol of target eukaryotic cells. The secreted YopB and YopD translocator proteins are essential for this process, forming pores in biological membranes through which the effectors are thought to gain access to the cell interior. The non-secreted cognate chaperone, LcrH, also plays an important role by ensuring pre-secretory stabilisation and efficient secretion of YopB and YopD. This suggests that LcrH-regulated secretion of the translocators could be used by Yersinia to control effector translocation levels. We collected several LcrH mutants impaired in chaperone activity. These poorly bound, stabilised and/or secreted YopB and YopD in vitro. However, these mutants generally maintained stable substrates during a HeLa cell infection and these infected cells were intoxicated by translocated effectors. Surprisingly, this occurred in the absence of detectable YopB- and YopD-dependent pores in eukaryotic membranes. A functional type III translocon must therefore only require minuscule amounts of secreted translocator proteins. Based on these observations, LcrH dependent control of translocation via regulated YopB and YopD secretion would need to be exquisitely tight.

  • 21.
    Edqvist, Petra J
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Bröms, Jeanette E
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Betts, Helen J
    Forsberg, Ake
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Pallen, Mark J
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.2006In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 59, no 1, p. 31-44Article in journal (Refereed)
    Abstract [en]

    Non-flagellar type III secretion systems (T3SSs) transport proteins across the bacterial cell and into eukaryotic cells. Targeting of proteins into host cells requires a dedicated translocation apparatus. Efficient secretion of the translocator proteins that make up this apparatus depends on molecular chaperones. Chaperones of the translocators (also called class-II chaperones) are characterized by the possession of three tandem tetratricopeptide repeats (TPRs). We wished to dissect the relations between chaperone structure and function and to validate a structural model using site-directed mutagenesis. Drawing on a number of experimental approaches and focusing on LcrH, a class-II chaperone from the Yersinia Ysc-Yop T3SS, we examined the contributions of different residues, residue classes and regions of the protein to chaperone stability, chaperone-substrate binding, substrate stability and secretion and regulation of Yop protein synthesis. We confirmed the expected role of the conserved canonical residues from the TPRs to chaperone stability and function. Eleven mutations specifically abrogated YopB binding or secretion while three mutations led to a specific loss of YopD secretion. These are the first mutations described for any class-II chaperone that allow interactions with one translocator to be dissociated from interactions with the other. Strikingly, all mutations affecting the interaction with YopB mapped to residues with side chains projecting from the inner, concave surface of the modelled TPR structure, defining a YopB interaction site. Conversely, all mutations preventing YopD secretion affect residues that lie on the outer, convex surface of the triple-TPR cluster in our model, suggesting that this region of the molecule represents a distinct interaction site for YopD. Intriguingly, one of the LcrH double mutants, Y40A/F44A, was able to maintain stable substrates inside bacteria, but unable to secrete them, suggesting that these two residues might influence delivery of substrates to the secretion apparatus.

  • 22.
    Ekestubbe, Sofie
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bröms, Jeanette E.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Edgren, Tomas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence2016In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 175Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems (T3SS) are dedicated to targeting anti-host effector proteins into the cytosol of the host cell to promote bacterial infection. Delivery of the effectors requires three specific translocator proteins, of which the hydrophilic translocator, LcrV, is located at the tip of the T3SS needle and is believed to facilitate insertion of the two hydrophobic translocators into the host cell membrane. Here we used Yersinia as a model to study the role of LcrV in T3SS mediated intracellular effector targeting. Intriguingly, we identified N-terminal IcrV mutants that, similar to the wild-type protein, efficiently promoted expression, secretion and intracellular levels of Yop effectors, yet they were impaired in their ability to inhibit phagocytosis by J774 cells. In line with this, the YopH mediated dephosphorylation of Focal Adhesion Kinase early after infection was compromised when compared to the wild type strain. This suggests that the mutants are unable to promote efficient delivery of effectors to their molecular targets inside the host cell upon host cell contact. The significance of this was borne out by the fact that the mutants were highly attenuated for virulence in the systemic mouse infection model. Our study provides both novel and significant findings that establish a role for LcrV in early targeting of effectors in the host cell.

  • 23.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Secretion systems and metabolism in the pathogenic Yersiniae2011In: Stress response in pathogenic bacteria / [ed] Stephen P. Kidd, United Kingdom: CAB International , 2011, 1, p. 185-220Chapter in book (Refereed)
    Abstract [en]

    The genus Yersinia comprises 11 species, three of which have clear etiology for causing human disease (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica). The obligate pathogen Y. pestis is the most infamous of these being the causal agent of plague, a bivalent disease that when left untreated is invariably fatal. The lifecycle of Y. pestis is complex, being dependent on two diverse hosts – the invertebrate flea Xenopsylla cheopis and a mammalian host (usually wild rodents). Although capable of catastrophic consequences, plague in humans is accidental – a consequence of being infected with Y. pestis via the bite of an infected flea that has been forced from its normal rodent host. In brief, the initial stage of disease presents as swollen lymph nodes (buboes) and is termed bubonic plague, whereas the second stage is a more vigorous systemic infection that results in bacterial colonization of multiple tissue organs including the lung. This form of disease is termed pneumonic plague; a highly contagious disease that enables bacteria to rapidly and effectively spread to new hosts via aerosol droplets. In light of this, global health organizations routinely list Y. pestis as a category A biowarfare agent. On the other hand, Y. pseudotuberculosis and Y. enterocolitica are essentially environmental bacteria that are capable of causing spasmodic enteric disease (known as yersiniosis) outbreaks linked to the ingestion of contaminated food or fluids. While these diseases cause gastrointestinal discomfort, they are usually self-limiting and rarely associated with systemic disease. In certain susceptible individuals however, chronic reactive arthritic sequelae can be attributed to these bacteria.

    On account of their ability to cause human disease, a prolific amount of information is available that describes these three human pathogens with respect to their ecology, epidemiology and the pathogenesis of disease. In contrast, very little information is available concerning the additional Yersinia species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, Y. aleksiciae, Y. mexicana and Y. aldovae). However, they might still be clinically relevant given their propensity to harbour a moderate number of genes that encode for products known to be associated with pathogenicity by other non-Yersinia bacteria. It is at least well established that Y. ruckeri is the causative agent of yersiniosis in infected salmonid fish, although the pathogenic mechanisms are comparatively poorly understood.

    In the first part of this chapter, the presence of known and suggested protein secretion mechanisms in the Yersiniae are described. Where appropriate, these individual processes are briefly discussed in the context of their contribution to bacterial pathogenesis to help the reader gain an understanding of their physiological importance within the various unique environments of an infected host. As considerably more is known about the pathogenic mechanisms of human pathogenic Yersinia, these examples will dominate the discussion. Then, focus will turn to the consequences of adaptation of pathogenic Yersinia to their surrounding environment. Where possible, emphasis will be given to the crosstalk between metabolism and the temporal and spatial regulatory control of these important secretion systems. This connection ensures that Yersinia conserve their valuable energy reserves to maximize their survival in stressful environments and only synthesize energetically expensive virulence determinants, such as multi-component secretion systems, when they will have utmost benefit during host infections.

  • 24.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The pathogenic Yersiniae: advances in the understanding of physiology and virulence2013In: Frontiers in Cellular and Infection Microbiology, ISSN 2235-2988, Vol. 3, no 51, p. 2p. 1-2Article, review/survey (Refereed)
  • 25.
    Francis, Matthew
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Aili, Margareta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wiklund, Magda-Lena
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    A study of the YopD-LcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD2000In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 38, no 1, p. 85-102Article in journal (Refereed)
    Abstract [en]

    The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersiniaouter protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic α-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic α-helix abolished the YopD–LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.

  • 26.
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Type III secretion chaperones: a molecular toolkit for all occasions2010In: Handbook of molecular chaperones: roles, structures and mechanisms / [ed] Piero Durante and Leandro Colucci, New York: Nova Science Publishers , 2010, 1, p. 79-148Chapter in book (Refereed)
    Abstract [en]

    Common to many bacteria is the ability to establish a symbiotic relationship or to evade innate immune responses of an animal, plant, fish or insect host. Most often this capacity is mediated by a type III secretion system (T3SS). The function of these complex molecular machines is likened to a syringe-needle injection device that is dedicated to the translocation of effector proteins directly into target eukaryotic cells. Each translocated effector tends to possess a distinct enzymatic activity that aids in subverting host cell signaling for the benefit of the bacterium. Their translocation requires another class of secreted protein – the translocator – which form pores in the target eukaryotic cell plasma membrane through which the effectors may transit to gain entry into the cell interior. Most often, each secreted substrate requires a dedicated small, non-secreted cytoplasmic chaperone for their efficient secretion. Unlike traditional molecular chaperones, these specialized type III chaperones do not assist in protein folding and are not energized by ATP. Controversy still surrounds their primary role; as bodyguards to prevent premature aggregation or as pilots to direct substrate secretion through the correct T3SS. The later is supported by recent evidence that these chaperones can dock directly to the cytoplasmic face of the T3S machinery, possibly serving as a recognition motif for substrate secretion. Added to this functional complexity is their important contribution to system regulation, which can ultimately confer temporal order to substrate secretion. Moreover, some chaperones display a bewildering propensity to interact with several additional T3S-associated proteins – the relevance of which remains uncertain. Structural data has now appeared for several important type III chaperones, either alone or in complex with their cognate substrate. This is proving a fillip in our attempts to understand the mercurial ways in which these versatile proteins operate in nature. It is hoped that this article will provide information on type III chaperone function, as well as highlighting key recent advances in the field. May it also be a testament to the value of continued intense effort in unravelling the mysteries of type III chaperone biology.

  • 27.
    Francis, Matthew S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Amer, Ayad A A
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Milton, Debra
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Department of Biological and Environmental Sciences, Troy University, Troy, AL, USA.
    Costa, Tiago R D
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Institute of Structural and Molecular Biology, University College London and Birkbeck, London, UK.
    Site-directed mutagenesis and its application in studying the interactions of T3S components2017In: Type 3 secretion systems: methods and protocols / [ed] Matthew L. Nilles and Danielle L. Jessen Condry, Humana Press, 2017, p. 11-31Chapter in book (Refereed)
    Abstract [en]

    Type III secretion systems are a prolific virulence determinant among Gram-negative bacteria. They are used to paralyze the host cell, which enables bacterial pathogens to establish often fatal infections—unless an effective therapeutic intervention is available. However, as a result of a catastrophic rise in infectious bacteria resistant to conventional antibiotics, these bacteria are again a leading cause of worldwide mortality. Hence, this report describes a pDM4-based site-directed mutagenesis strategy that is assisting in our foremost objective to better understand the fundamental workings of the T3SS, using Yersinia as a model pathogenic bacterium. Examples are given that clearly document how pDM4-mediated site-directed mutagenesis has been used to establish clean point mutations and in-frame deletion mutations that have been instrumental in identifying and understanding the molecular interactions between components of the Yersinia type III secretion system.

  • 28.
    Francis, Matthew S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lloyd, Scott A
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The type III secretion chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis2001In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 42, no 4, p. 1075-1093Article in journal (Refereed)
    Abstract [en]

    The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens secrete and subsequently translocate antihost effector proteins into target eukaryotic cells by a common type III secretion system (TTSS). In this process, YopD (Yersinia outer protein D) is essential to establish regulatory control of Yop synthesis and the ensuing translocation process. YopD function depends upon the non-secreted TTSS chaperone LcrH (low-calcium response H), which is required for presecretory stabilization of YopD. However, as a new role for TTSS chaperones in virulence gene regulation has been proposed recently, we undertook a detailed analysis of LcrH. A lcrH null mutant constitutively produced Yops, even when this strain was engineered to produce wild-type levels of YopD. Furthermore, the YopD-LcrH interaction was necessary to regain the negative regulation of virulence associated genes yops). This finding was used to investigate the biological significance of several LcrH mutants with varied YopD binding potential. Mutated LcrH alleles were introduced in trans into a lcrH null mutant to assess their impact on yop regulation and the subsequent translocation of YopE, a Rho-GTPase activating protein, across the plasma membrane of eukaryotic cells. Two mutants, LcrHK20E, E30G, I31V, M99V, D136G and LcrHE30G lost all regulatory control, even though YopD binding and secretion and the subsequent translocation of YopE was indistinguishable from wild type. Moreover, these regulatory deficient mutants showed a reduced ability to bind YscY in the two-hybrid assay. Collectively, these findings confirm that LcrH plays an active role in yop regulation that might be mediated via an interaction with the Ysc secretion apparatus. This chaperone-substrate interaction presents an innovative means to establish a regulatory hierarchy in Yersinia infections. It also raises the question as to whether or not LcrH is a true chaperone involved in stabilization and secretion of YopD or a regulatory protein responsible for co-ordinating synthesis of Yersinia virulence determinants. We suggest that LcrH can exhibit both of these activities.

  • 29.
    Francis, Matthew S
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Schesser, Kurt
    Forsberg, Åke
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Type III secretion systems in animal- and plant-interacting bacteria2004In: Cellular Microbiology, ASM Press, Washington, D.C. , 2004, p. 362-392Chapter in book (Other (popular science, discussion, etc.))
  • 30.
    Francis, Matthew S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    YopD of Yersinia pseudotuberculosis is translocated into the cytosol of HeLa epithelial cells: evidence of a structural domain necessary for translocation.1998In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 29, no 3, p. 799-813Article in journal (Refereed)
    Abstract [en]

    Yersinia pseudotuberculosis YopB and YopD proteins are essential for translocation of Yop effector proteins into the target cell cytosol. YopB is suggested to mediate pore formation in the target cell plasma membrane, allowing translocation of Yop effector proteins, although the function of YopD is unclear. To investigate the role in translocation for YopD, a mutant strain in Y. pseudotuberculosis was constructed containing an in frame deletion of essentially the entire yopD gene. As shown recently for the Y. pestis YopD protein, we found that the in vitro low calcium response controlling virulence gene expression was negatively regulated by YopD. This yopD null mutant (YPIII/pIB621) was also non-cytotoxic towards HeLa cell monolayers, supporting the role for YopD in the translocation process. Although other constituents of the Yersinia translocase apparatus (YopB, YopK and YopN) are not translocated into the host cell cytosol, fractionation of infected HeLa cells allowed us to identify the cytosolic localization of YopD by the wild-type strain (YPIII/pIB102), but not by strains defective in either YopD or YopB. YopD was also identified by immunofluorescence in the cytoplasm of HeLa cell monolayers infected with a multiple yop mutant strain (YPIII/pIB29MEKA). These results demonstrate a dual function for YopD in negative regulation of Yop production and Yop effector translocation, including the YopD protein itself. To investigate whether an amphipathic domain near the C-terminus of YopD is involved in the translocation process, a mutant strain (YPIII/pIB155deltaD278-292) was constructed that is devoid of this region. Phenotypically, this small in frame deltayopD278-292 deletion mutant was indistinguishable from the yopD null mutant. The truncated YopD protein and Yop effectors were not translocated into the cytosol of HeLa cell monolayers infected with this mutant. The comparable regulatory and translocation phenotypes displayed by the small in frame deltayopD278-292 deletion and deltayopD null mutants suggest that regulation of Yop synthesis and Yop translocation are intimately coupled. We present an intriguing scenario to the Yersinia infection process that highlights the need for polarized translocation of YopD to specifically establish translocation of Yop effectors. These observations are contrary to previous suggestions that members of the translocase apparatus were not translocated into the host cell cytosol.

  • 31.
    Francis, Matthew S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Ake
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Regulation of type III secretion systems2002In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 5, no 2, p. 166-172Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems are utilised by numerous Gram-negative bacteria to efficiently interact with a host. Appropriate expression of type III genes is achieved through the integration of several regulatory pathways that ultimately co-ordinate the activity of a central transcriptional activator usually belonging to the AraC family. The complex regulatory cascades allow this virulence strategy to be utilised by different bacteria even if they occupy diverse niches that define a unique set of environmental cues. Simulating the appropriate combination of signals in vitro to allow a meaningful interpretation of the type III assembly and secretion regulatory cascade remains a common goal for researchers. Pieces of the puzzle slowly emerge to provide insightful views into the complex regulatory networks that allow bacteria to assemble and utilise type III secretion to efficiently colonise a host.

  • 32.
    Francis, Matthew
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Schesser, Kurt
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Type III secretion systems in animal- and plant-interacting bacteria2000In: Cellular Microbiology, Washington, DC: ASM Press , 2000, 1, p. 239-263Chapter in book (Refereed)
  • 33.
    Gurung, Jyoti M.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Amer, Ayad
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Monika
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Costa, Tiago
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chen, Shiyun
    Zavialov, Anton V.
    Francis, Matthew S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Heterologous complementation studies with the YscX and YscY protein families reveals a specificity for Yersinia pseudotuberculosis type III secretion2018In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 8, article id 80Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems harbored by several Gram-negative bacteria are often used to deliver host-modulating effectors into infected eukaryotic cells. About 20 core proteins are needed for assembly of a secretion apparatus. Several of these proteins are genetically and functionally conserved in type III secretion systems of bacteria associated with invertebrate or vertebrate hosts. In the Ysc family of type III secretion systems are two poorly characterized protein families, the YscX family and the YscY family. In the plasmid-encoded Ysc-Yop type III secretion system of human pathogenic Yersinia species, YscX is a secreted substrate while YscY is its non-secreted cognate chaperone. Critically, neither an yscX nor yscY null mutant of Yersinia is capable of type III secretion. In this study, we show that the genetic equivalents of these proteins produced as components of other type III secretion systems of Pseudomonas aeruginosa (PscX and PscY), Aeromonas species (AscX and AscY), Vibrio species (VscX and VscY), and Photorhabdus luminescens (SctX and SctY) all possess an ability to interact with its native cognate partner and also establish cross-reciprocal binding to non-cognate partners as judged by a yeast two-hybrid assay. Moreover, a yeast three-hybrid assay also revealed that these heterodimeric complexes could maintain an interaction with YscV family members, a core membrane component of all type III secretion systems. Despite maintaining these molecular interactions, only expression of the native yscX in the near full-length yscX deletion and native yscY in the near full-length yscY deletion were able to complement for their general substrate secretion defects. Hence, YscX and YscY must have co-evolved to confer an important function specifically critical for Yersinia type III secretion.

  • 34.
    Henriksson, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Peden, Alex
    Aili, Margareta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Stefansson, Kristina
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Palmer, Ruth
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Aitken, Alastair
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo2002In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 269, no 20, p. 4921-4929Article in journal (Refereed)
    Abstract [en]

    14-3-3 proteins play an important role in a multitude of signalling pathways. The interactions between 14-3-3 and other signalling proteins, such as Raf and KSR (kinase suppressor of Ras), occur in a phospho-specific manner. Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and several proteins, for example 5-phosphatase, p75NTR-associated cell death executor (NADE) and the bacterial toxin Exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. In this study we have identified the amino acid residues on ExoS, which are responsible for its specific interaction with 14-3-3. Furthermore, we show that a peptide derived from ExoS, containing the 14-3-3 interaction site, effectively competes out the interaction between ExoS and 14-3-3. In addition, competition with this peptide blocks ExoS modification of Ras in our Ras modification assay. We show that the ExoS protein interacts with all isoforms of the 14-3-3 family tested. Moreover, in vivo an ExoS protein lacking the 14-3-3 binding site has a reduced capacity to ADP ribosylate cytoplasmic proteins, e.g. Ras, and shows a reduced capacity to change the morphology of infected cells.

  • 35.
    Li, Yunlong
    et al.
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
    Hu, Yangbo
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
    Francis, Matthew
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Chen, Shiyun
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
    RcsB positively regulates the Yersinia Ysc-Yop type III secretion system by activating expression of the master transcriptional regulator LcrF2015In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 17, no 4, p. 1219-1233Article in journal (Refereed)
    Abstract [en]

    The Rcs phosphorelay is a complex signaling pathway used by the family Enterobacteriaceae to sense, respond and adapt to environmental changes during free-living or host-associated lifestyles. In this study, we show that the Rcs phosphorelay pathway positively regulates the virulence plasmid encoded Ysc-Yop type III secretion system (T3SS) in the enteropathogen Yesinia pseudotuberculosis. Both the overexpression of the wild-type Rcs regulator RcsB or the constitutive active RscB(D56E) variant triggered more abundant Ysc-Yop synthesis and secretion, whereas the non-phosphorylatable mutant RcsB(D56Q) negated this. Congruently, enhanced Yops expression and secretion occurred in an in cis rscB(D56E) mutant but not in an isogenic rscB(D56Q) mutant. Screening for regulatory targets of RcsB identified the virG-lcrF operon that encodes for LcrF, the Ysc-Yop T3SS master regulator. Protein-DNA binding assays confirmed that RcsB directly bound to this operon promoter, which subsequently caused stimulated lcrF transcription. Moreover, active RcsB enhanced the ability of bacteria to deliver Yop effectors into immune cells during cell contact, and this promoted an increase in bacterial viability. Taken together, our study demonstrates the role of the Rcs system in regulating the Ysc-Yop T3SS in Yersinia and reports on RcsB being the first transcriptional activator known to directly control lcrF transcription.

  • 36. Li, Yunlong
    et al.
    Hu, Yangbo
    Francis, Matthew S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Chen, Shiyun
    RcsB positively regulates the Yersinia Ysc-Yop type III secretion system by activating expression of the master transcriptional regulator LcrF2015In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 17, no 4, p. 1219-1233Article in journal (Refereed)
    Abstract [en]

    The Rcs phosphorelay is a complex signaling pathway used by the family Enterobacteriaceae to sense, respond and adapt to environmental changes during free-living or host-associated lifestyles. In this study, we show that the Rcs phosphorelay pathway positively regulates the virulence plasmid encoded Ysc-Yop type III secretion system (T3SS) in the enteropathogen Yesinia pseudotuberculosis. Both the overexpression of the wild-type Rcs regulator RcsB or the constitutive active RscB(D56E) variant triggered more abundant Ysc-Yop synthesis and secretion, whereas the non-phosphorylatable mutant RcsB(D56Q) negated this. Congruently, enhanced Yops expression and secretion occurred in an in cis rscB(D56E) mutant but not in an isogenic rscB(D56Q) mutant. Screening for regulatory targets of RcsB identified the virG-lcrF operon that encodes for LcrF, the Ysc-Yop T3SS master regulator. Protein-DNA binding assays confirmed that RcsB directly bound to this operon promoter, which subsequently caused stimulated lcrF transcription. Moreover, active RcsB enhanced the ability of bacteria to deliver Yop effectors into immune cells during cell contact, and this promoted an increase in bacterial viability. Taken together, our study demonstrates the role of the Rcs system in regulating the Ysc-Yop T3SS in Yersinia and reports on RcsB being the first transcriptional activator known to directly control lcrF transcription.

  • 37.
    Li, Yunlong
    et al.
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Li, Lamei
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Huang, Li
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Francis, Matthew
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hu, Yangbo
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Chen, Shiyun
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Yersinia Ysc-Yop type III secretion feedback inhibition is relieved through YscV-dependent recognition and secretion of LcrQ2014In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 91, no 3, p. 494-507Article in journal (Refereed)
    Abstract [en]

    Human pathogenic Yersinia species share a virulence plasmid encoding the Ysc-Yop type III secretion system (T3SS). A plasmid-encoded anti-activator, LcrQ, negatively regulates the expression of this secretion system. Under inducible conditions, LcrQ is secreted outside of bacterial cells and this activates the T3SS, but the mechanism of targeting LcrQ for type III secretion remains largely unknown. In this study, we characterized the regulatory role of the export apparatus component YscV. Depletion or overexpression of YscV compromised Yop synthesis and this primarily prevented secretion of LcrQ. It followed that a lcrQ deletion reversed the repressive effects of excessive YscV. Further characterization demonstrated that the YscV residues 493–511 located within the C-terminal soluble cytoplasmic domain directly bound with LcrQ. Critically, YscV-LcrQ complex formation was a requirement for LcrQ secretion, since YscVΔ493–511 failed to secrete LcrQ. This forced a cytoplasmic accumulation of LcrQ, which predictably caused the feedback inhibition of Yops synthesis. Based on these observations, we proposed a model for the YscV-dependent secretion of LcrQ and its role in regulating Yop synthesis in Yersinia.

  • 38.
    Liu, Junfa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Obi, Ikenna R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Thanikkal, Edvin J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Kieselbach, Thomas
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Phosphorylated CpxR Restricts Production of the RovA Global Regulator in Yersinia pseudotuberculosis2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 8, p. e23314-Article in journal (Refereed)
    Abstract [en]

    Background: RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression.

    Methodology/Principal Findings: In this study, we characterized CpxR phosphorylation (CpxR similar to P) in vitro, and determined that phosphorylation was necessary for CpxR to efficiently bind to the PCR-amplified upstream regulatory region of rovA. The precise CpxR similar to P binding site was mapped by a nuclease protection assay and directed mutagenesis confirmed that in vivo binding to the rovA promoter inhibits transcription. Reduced RovA production was most pronounced following CpxR, P accumulation in the Yersinia cytoplasm during chronic Cpx pathway activation and by the indiscriminate phosphodonor action of acetyl phosphate.

    Conclusions/Significance: Cpx pathway activation restricts levels of the RovA global regulator. The regulatory influence of CpxR similar to P must therefore extend well beyond periplasmic quality control in the Yersinia envelope, to include genes involved in environmental survival and pathogenicity.

  • 39.
    Liu, Junfa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Thanikkal, Edvin
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Obi, Ikenna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Elevated CpxR~P levels repress the Ysc-Yop type III secretion system of Yersinia pseudotuberculosis2012In: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 163, no 8, p. 518-530Article in journal (Refereed)
    Abstract [en]

    One way that Gram-negative bacteria respond to extracytoplasmic stress is through the CpxA-CpxR system. An activated CpxA sensor kinase phosphorylates the CpxR response regulator to instigate positive auto-amplification of Cpx pathway activation, as well as synthesis of various bacterial survival factors. In the absence of CpxA, human enteropathogenic Yersinia pseudotuberculosis accumulates high CpxR~P levels aided by the action of low molecular weight phosphodonors such as acetyl~P. Critically, these bacteria are also defective for plasmid encoded Ysc-Yop-dependent type III synthesis and secretion, an essential determinant of virulence. Herein, we investigated whether elevated CpxR~P levels account for lost Ysc-Yop function. Decisively, reducing CpxR~P in Yersinia defective for CpxA phosphatase activity - through incorporating second-site suppressor mutations in ackA-pta or cpxR - dramatically restored Ysc-Yop T3S function. Moreover, the repressive effect of accumulated CpxR~P is a direct consequence of binding to the promoter regions of the T3S genes. Thus, Cpx pathway activation has two consequences in Yersinia; one, to maintain quality control in the bacterial envelope, and the second, to restrict ysc-yop gene expression to those occasions where it will have maximal effect.

  • 40. Lloyd, Scott A
    et al.
    Forsberg, Åke
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Targeting exported substrates to the Yersinia TTSS: different functions for different signals?2001In: Trends in Microbiology, ISSN 0966-842X, E-ISSN 1878-4380, Vol. 9, no 8, p. 367-371Article in journal (Refereed)
    Abstract [en]

    Many Gram-negative pathogens utilize a type III secretion system (TTSS) to inject toxins into the cytosol of eukaryotic cells. Previous studies have indicated that exported substrates are targeted to the Yersinia TTSS via the coding regions of their 5' mRNA sequences, as well as by their cognate chaperones. However, recent results from our laboratory have challenged the role of mRNA targeting signals, as we have shown that the amino termini of exported substrates are crucial for type III secretion. Here, we discuss the nature of these amino-terminal secretion signals and propose a model for the secretion of exported substrates by amino-terminal and chaperone-mediated signals. In addition, we discuss the roles of chaperones as regulators of virulence gene expression and present models suggesting that such regulation can occur independently of the delivery of their substrates to the secretion apparatus.

  • 41.
    Lu, Pei
    et al.
    Chinese Academy of Sciences, Wuhan, China.
    Zhang, Yong
    Chinese Academy of Sciences, Wuhan, China.
    Hu, Yangbo
    Chinese Academy of Sciences, Wuhan, China.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chen, Shiyun
    Chinese Academy of Sciences, Wuhan, China.
    A cis-encoded sRNA controls the expression of fabH2 in Yersinia2014In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 588, no 10, p. 1961-1966Article in journal (Refereed)
    Abstract [en]

    YsrH is a novel cis-encoded sRNA located on the opposite strand to fabH2, which is essential for fatty acid biosynthesis in bacteria. In this study, YsrH-mediated regulation of fabH2 expression was investigated in Yersinia pseudotuberculosis. Constitutive and inducible over-expression of YsrH decreased the mRNA level of fabH2, while expression of downstream fabD and fabG remained unaffected. Polynucleotide phosphorylase (PNPase) also played an important role in this regulation process by mediating YsrH decay in the exponential phase. Thus, our data defines a cis-encoded sRNA that regulates fatty acid synthesis via a regulatory mechanism also involving PNPase.

  • 42.
    Obi, Ikenna
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Demarcating SurA activities required for outer membrane targeting of Yersinia pseudotuberculosis adhesins2013In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 81, no 7, p. 2296-2308Article in journal (Refereed)
    Abstract [en]

    SurA is a periplasmic protein folding factor involved in chaperoning and trafficking of outer membrane proteins across the Gram-negative bacterial periplasm. In addition, SurA also possesses peptidyl-prolyl cis/trans isomerase activity. In enteropathogenic Yersinia pseudotuberculosis, we have previously reported that SurA is needed for bacterial virulence and envelope integrity. In this study, we investigated the role of SurA in the assembly of important Yersinia adhesins. Using genetic mutation, biochemical characterization and an in vitro-based bacterial host cell association assay, we confirmed that surface localization of the invasin adhesin is dependent on SurA. As a surA deletion also has some impact on the levels of individual components of the BAM complex in the Yersinia outer membrane, abolished invasin surface assembly could reflect both a direct loss of SurA-dependent periplasmic targeting as well as a potentially compromised BAM complex assembly platform in the outer membrane. To varying degrees, the assembly of two other adhesins, Ail and the pH 6 antigen fibrillum PsaA also depend on SurA. Consequently, loss of SurA leads to a dramatic reduction in Yersinia attachment to eukaryotic host cells. Genetic complementation of surA deletion mutants indicated a prominent role for SurA chaperone function in outer membrane protein assembly. Significantly, the N-terminus of SurA contributed most of this SurA chaperone function. Despite a dominant chaperoning role, it was also evident that SurA isomerization activity did make a modest contribution to this assembly process.

  • 43.
    Obi, Ikenna
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Nordfelth, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Varying dependency of periplasmic peptidylprolyl cis-trans isomerases in promoting Yersinia pseudotuberculosis stress tolerance and pathogenicity2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 439, no 2, p. 321-332Article in journal (Refereed)
    Abstract [en]

    Periplasmic PPIases (peptidylprolyl cis-trans isomerases) catalyse the cis-trans isomerization of peptidyl-prolyl bonds, which is a rate-limiting step during protein folding. We demonstrate that the surA, ppiA, ppiD, fkpA and fklB alleles each encode a periplasmic PPIase in the bacterial pathogen Yersinia pseudotuberculosis. Of these, four were purified to homogeneity. Purified SurA, FkpA and FklB, but not PpiD, displayed detectable PPIase activity in vitro. Significantly, only Y. pseudotuberculosis lacking surA caused drastic alterations to the outer membrane protein profile and FA (fatty acid) composition. They also exhibited aberrant cellular morphology, leaking LPS (lipopolysaccharide) into the extracellular environment. The SurA PPIase is therefore most critical for maintaining Y. pseudotuberculosis envelope integrity during routine culturing. On the other hand, bacteria lacking either surA or all of the genes ppiA, ppiD, fkpA and fklB were sensitive to hydrogen peroxide and were attenuated in mice infections. Thus Y. pseudotuberculosis exhibits both SurA-dependent and -independent requirements for periplasmic PPIase activity to ensure in vivo survival and a full virulence effect in a mammalian host.

  • 44.
    Olsson, Jan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Edqvist, Petra J
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bröms, Jeanette E
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Ake
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The YopD translocator of Yersinia pseudotuberculosis is a multifunctional protein comprised of discrete domains.2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 13, p. 4110-4123Article in journal (Refereed)
    Abstract [en]

    To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III translocon to microinject several anti-host Yop effectors into the cytosol of target eukaryotic cells. YopD has been implicated in several key steps during Yop effector translocation, including maintenance of yop regulatory control and pore formation in the target cell membrane through which effectors traverse. These functions are mediated, in part, by an interaction with the cognate chaperone, LcrH. To gain insight into the complex molecular mechanisms of YopD function, we performed a systematic mutagenesis study to search for discrete functional domains. We highlighted amino acids beyond the first three N-terminal residues that are dispensable for YopD secretion and confirmed that an interaction between YopD and LcrH is essential for maintenance of yop regulatory control. In addition, discrete domains within YopD that are essential for both pore formation and translocation of Yop effectors were identified. Significantly, other domains were found to be important for effector microinjection but not for pore formation. Therefore, YopD is clearly essential for several discrete steps during efficient Yop effector translocation. Recognition of this modular YopD domain structure provides important insights into the function of YopD.

  • 45. Ottmann, Christian
    et al.
    Yasmin, Lubna
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Weyand, Michael
    Veesenmeyer, Jeffrey L
    Diaz, Maureen H
    Palmer, Ruth H
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hauser, Alan R
    Wittinghofer, Alfred
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Phosphorylation-independent interaction between 14-3-3 and exoenzyme S: from structure to pathogenesis.2007In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 26, no 3, p. 902-913Article in journal (Refereed)
    Abstract [en]

    14-3-3 proteins are phosphoserine/phosphothreonine-recognizing adapter proteins that regulate the activity of a vast array of targets. There are also examples of 14-3-3 proteins binding their targets via unphosphorylated motifs. Here we present a structural and biological investigation of the phosphorylation-independent interaction between 14-3-3 and exoenzyme S (ExoS), an ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. ExoS binds to 14-3-3 in a novel binding mode mostly relying on hydrophobic contacts. The 1.5 A crystal structure is supported by cytotoxicity analysis, which reveals that substitution of the corresponding hydrophobic residues significantly weakens the ability of ExoS to modify the endogenous targets RAS/RAP1 and to induce cell death. Furthermore, mutation of key residues within the ExoS binding site for 14-3-3 impairs virulence in a mouse pneumonia model. In conclusion, we show that ExoS binds 14-3-3 in a novel reversed orientation that is primarily dependent on hydrophobic residues. This interaction is phosphorylation independent and is required for the function of ExoS.

  • 46. Pallen, Mark J
    et al.
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fütterer, Klaus
    Tetratricopeptide-like repeats in type-III-secretion chaperones and regulators2003In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 223, no 1, p. 53-60Article in journal (Refereed)
    Abstract [en]

    Efficient type-III secretion depends on cytosolic molecular chaperones, which bind specifically to the translocators and effectors. In the past there has been a tendency to shoe-horn all type-III-secretion chaperones into a single structural and functional class. However, we have shown that the LcrH/SycD-like chaperones consist of three central tetratricopeptide-like repeats that are predicted to fold into an all-alpha-helical array that is quite distinct from the known structure of the SycE class of chaperones. Furthermore, we predict that this array creates a peptide-binding groove that is utterly different from the helix-binding groove in SycE. We present a homology model of LcrH/SycD that is consistent with existing mutagenesis data. We also report the existence of tetratricopeptide-like repeats in regulators of type-III secretion, such as HilA from Salmonella enterica and HrpB from Ralstonia solanacearum. The discovery of tetratricopeptide-like repeats in type-III-secretion regulators and chaperones provides a new conceptual framework for structural and mutagenesis studies and signals a potential unification of prokaryotic and eukaryotic chaperone biology.

  • 47.
    Tengel, Tobias
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sethson, Ingmar
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia2002In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 269, no 15, p. 3659-3668Article in journal (Refereed)
    Abstract [en]

    To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278−300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic α helix ranging from Phe280 to Tyr291, followed by a type I β turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278−292 and YopD271−292, lacked helical structure, implicating the β turn in helix stability. An interaction between YopD278−300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide–LcrH complex. These effects were not observed for the unstructured peptide, YopD278−292, which confirms that the α helical structure of the YopD amphipathic domain is a critical binding region of LcrH.

  • 48.
    Thanikkal, Edvin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Mangu, Jagadish
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Interactions of the CpxA sensor kinase and cognate CpxR response regulator from Yersinia pseudotuberculosis2012In: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 5, no 1, p. 536-Article in journal (Refereed)
    Abstract [en]

    Background

    The CpxA sensor kinase-CpxR response regulator two-component regulatory system is a sentinel of bacterial envelope integrity. Integrating diverse signals, it can alter the expression of a wide array of components that serve to shield the envelope from damage and to promote bacterial survival. In bacterial pathogens such as Yersinia pseudotuberculosis, this also extends to pathogenesis. CpxR is thought to dimerize upon phosphorylation by the sensor kinase CpxA. This phosphorylation enables CpxR binding to specific DNA sequences where it acts on gene transcription. As Cpx pathway activation is dependent on protein-protein interactions, we performed an interaction analysis of CpxR and CpxA from Y. pseudotuberculosis.

    Results

    CpxR full-length and truncated versions that either contained or lacked a putative internal linker were all assessed for their ability to homodimerize and interact with CpxA. Using an adenylate cyclase-based bacterial two hybrid approach, full-length CpxR readily engaged with CpxA. The CpxR N-terminus could also homodimerize with itself and with a full-length CpxR. A second homodimerization assay based upon the lamda cI repressor also demonstrated that the CpxR C-terminus could homodimerize. While the linker was not specifically required, it enhanced CpxR homodimerization. Mutagenesis of cpxR identified the aspartate at residue 51, putative N-terminal coiled-coil and C-terminal winged-helix-turn-helix domains as mediators of CpxR homodimerization. Scrutiny of CpxA full-length and truncated versions revealed that dimerization involved the N-terminus and an internal dimerization and histidine phosphotransfer domain.

    Conclusions

    This interaction analysis mapped regions of CpxR and CpxA that were responsible for interactions with self or with each other. When combined with other physiological and biochemical tests both hybrid-based assays can be useful in dissecting molecular contacts that may underpin Cpx pathway activation and repression.

  • 49.
    Thanikkal, Edvin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Liu, Junfa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gurung, Jyoti
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Dersch, Petra
    Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Yersinia pseudotuberculosis Cpx envelope stress system contributes to transcription activation of rovMManuscript (preprint) (Other academic)
  • 50.
    Thorslund, Sara E
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Edgren, Tomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Pettersson, Jonas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nordfelth, Roland
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sellin, Mikael E
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ivanova, Ekaterina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Isaksson, Elin L
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 2, p. e16784-Article in journal (Refereed)
    Abstract [en]

    Many Gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated beta1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.

12 1 - 50 of 53
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