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Forsberg, Åke
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Publications (10 of 58) Show all publications
Massai, F., Saleeb, M., Doruk, T., Elofsson, M. & Forsberg, Å. (2019). Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa. Frontiers in Cellular and Infection Microbiology, 9, Article ID 250.
Open this publication in new window or tab >>Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa
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2019 (English)In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 9, article id 250Article in journal (Refereed) Published
Abstract [en]

Antibiotics are becoming less effective in treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa. Antimicrobial therapies based on the inhibition of specific virulence-related traits, as opposed to growth inhibitors, constitute an innovative and appealing approach to tackle the threat of P. aeruginosa infections. The twin-arginine translocation (Tat) pathway plays an important role in the pathogenesis of P. aeruginosa, and constitutes a promising target for the development of anti-pseudomonal drugs. In this study we developed and optimized a whole-cell, one-well assay, based on native phospholipase C activity, to identify compounds active against the Tat system. Statistical robustness, sensitivity and consequently suitability for high-throughput screening (HTS) were confirmed by a dry run/pre-screening test scoring a Z' of 0.82 and a signal-to-noise ratio of 49. Using this assay, we evaluated ca. 40,000 molecules and identified 59 initial hits as possible Tat inhibitors. Since phospholipase C is exported into the periplasm by Tat, and subsequently translocated across the outer membrane by the type II secretion system (T2SS), our assay could also identify T2SS inhibitors. To validate our hits and discriminate between compounds that inhibited either Tat or T2SS, two separate counter assays were developed and optimized. Finally, three Tat inhibitors and one T2SS inhibitor were confirmed by means of dose-response analysis and additional counter and confirming assays. Although none of the identified inhibitors was suitable as a lead compound for drug development, this study validates our assay as a simple, efficient, and HTS compatible method for the identification of Tat and T2SS inhibitors.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019
Keywords
Pseudomonas aeruginosa, high-throughput screening, twin arginine translocase, type II secretion, virulence inhibitors, phospholipase C
National Category
Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-161822 (URN)10.3389/fcimb.2019.00250 (DOI)000474778200001 ()31355152 (PubMedID)
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-13Bibliographically approved
Bamyaci, S., Nordfelth, R. & Forsberg, Å. (2019). Identification of specific sequence motif of YopN of Yersinia pseudotuberculosis required for systemic infection. Virulence, 10(1), 10-25
Open this publication in new window or tab >>Identification of specific sequence motif of YopN of Yersinia pseudotuberculosis required for systemic infection
2019 (English)In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 10, no 1, p. 10-25Article in journal (Refereed) Published
Abstract [en]

Type III secretion systems (T3SSs) are tightly regulated key virulence mechanisms shared by many Gram-negative pathogens. YopN, one of the substrates, is also crucial in regulation of expression, secretion and activation of the T3SS of pathogenic Yersinia species. Interestingly, YopN itself is also targeted into host cells but so far no activity or direct role for YopN inside host cells has been described. Recently, we were able show that the central region of YopN is required for efficient translocation of YopH and YopE into host cells. This was also shown to impact the ability of Yersinia to block phagocytosis. One difficulty in studying YopN is to generate mutants that are not impaired in regulation of the T3SS. In this study we extended our previous work and were able to generate specific mutants within the central region of YopN. These mutants were predicted to be crucial for formation of a putative coiled-coil domain (CCD). Similar to the previously described deletion mutant of the central region, these mutants were all impaired in translocation of YopE and YopH. Interestingly, these YopN variants were not translocated into host cells. Importantly, when these mutants were introduced in cis on the virulence plasmid, they retained full regulatory function of T3SS expression and secretion. This allowed us to evaluate one of the mutants, yopNGAGA, in the systemic mouse infection model. Using in vivo imaging technology we could verify that the mutant was also attenuated in vivo and highly impaired to establish systemic infection.

Place, publisher, year, edition, pages
Taylor & Francis, 2019
Keywords
Yersinia, T3SS, YopN, mouse infection, virulence
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-154803 (URN)10.1080/21505594.2018.1551709 (DOI)000453038200001 ()30488778 (PubMedID)2-s2.0-85058611719 (Scopus ID)
Funder
The Kempe Foundations, 161121Swedish Research Council, 2011-3439
Available from: 2019-01-07 Created: 2019-01-07 Last updated: 2019-01-09Bibliographically approved
Saleeb, M., Sundin, C., Aglar, Ö., Pinto, A. F., Ebrahimi, M., Forsberg, Å., . . . Elofsson, M. (2018). Structure–activity relationships for inhibitors of Pseudomonas aeruginosa exoenzyme S ADP-ribosyltransferase activity. European Journal of Medicinal Chemistry, 143, 568-576
Open this publication in new window or tab >>Structure–activity relationships for inhibitors of Pseudomonas aeruginosa exoenzyme S ADP-ribosyltransferase activity
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2018 (English)In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 143, p. 568-576Article in journal (Refereed) Published
Abstract [en]

During infection, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa employs its type III secretion system to translocate the toxin exoenzyme S (ExoS) into the eukaryotic host cell cytoplasm. ExoS is an essential in vivo virulence factor that enables P. aeruginosa to avoid phagocytosis and eventually kill the host cell. ExoS elicits its pathogenicity mainly via ADP-ribosyltransferase (ADPRT) activity. We recently identified a new class of ExoS ADPRT inhibitors with in vitro IC50 of around 20 μM in an enzymatic assay using a recombinant ExoS ADPRT domain. Herein, we report structure-activity relationships of this compound class by comparing a total of 51 compounds based on a thieno [2,3-d]pyrimidin-4(3H)-one and 4-oxo-3,4-dihydroquinazoline scaffolds. Improved inhibitors with in vitro IC50 values of 6 μM were identified. Importantly, we demonstrated that the most potent inhibitors block ADPRT activity of native full-length ExoS secreted by viable P. aeruginosa with an IC50 value of 1.3 μM in an enzymatic assay. This compound class holds promise as starting point for development of novel antibacterial agents.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
2-Aminobenzamide, ADP-Ribosyltransferase, Bacterial exotoxins, ExoS, Pseudomonas aeruginosa, Quinazolines, Type III secretion
National Category
Organic Chemistry Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-143429 (URN)10.1016/j.ejmech.2017.11.036 (DOI)000428216700046 ()29207339 (PubMedID)
Available from: 2017-12-23 Created: 2017-12-23 Last updated: 2018-08-24Bibliographically approved
Bamyaci, S., Ekestubbe, S., Nordfelth, R., Erttmann, S. F., Edgren, T. & Forsberg, Å. (2018). YopN Is Required for Efficient Effector Translocation and Virulence in Yersinia pseudotuberculosis. Infection and Immunity, 86(8), Article ID e00957-17.
Open this publication in new window or tab >>YopN Is Required for Efficient Effector Translocation and Virulence in Yersinia pseudotuberculosis
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2018 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 86, no 8, article id e00957-17Article in journal (Refereed) Published
Abstract [en]

Type III secretion systems (T3SSs) are used by various Gram-negative pathogens to subvert the host defense by a host cell contact-dependent mechanism to secrete and translocate virulence effectors. While the effectors differ between pathogens and determine the pathogenic life style, the overall mechanism of secretion and translocation is conserved. T3SSs are regulated at multiple levels, and some secreted substrates have also been shown to function in regulation. In Yersinia, one of the substrates, YopN, has long been known to function in the host cell contact-dependent regulation of the T3SS. Prior to contact, through its interaction with TyeA, YopN blocks secretion. Upon cell contact, TyeA dissociates from YopN, which is secreted by the T3SS, resulting in the induction of the system. YopN has also been shown to be translocated into target cells by a T3SS-dependent mechanism. However, no intracellular function has yet been assigned to YopN. The regulatory role of YopN involves the N-terminal and C-terminal parts, while less is known about the role of the central region of YopN. Here, we constructed different in-frame deletion mutants within the central region. The deletion of amino acids 76 to 181 resulted in an unaltered regulation of Yop expression and secretion but triggered reduced YopE and YopH translocation within the first 30 min after infection. As a consequence, this deletion mutant lost its ability to block phagocytosis by macrophages. In conclusion, we were able to differentiate the function of YopN in translocation and virulence from its function in regulation.

Keywords
phagocytosis, type III secretion, Yersinia, YopN, virulence
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-150354 (URN)10.1128/IAI.00957-17 (DOI)000439474900006 ()29760214 (PubMedID)
Available from: 2018-08-13 Created: 2018-08-13 Last updated: 2019-01-09Bibliographically approved
Avican, U., Doruk, T., Östberg, Y., Fahlgren, A. & Forsberg, Å. (2017). The Tat substrate SufI is critical for the ability of Yersinia pseudotuberculosis to cause systemic infection. Infection and Immunity, 85(4), Article ID e00867-16.
Open this publication in new window or tab >>The Tat substrate SufI is critical for the ability of Yersinia pseudotuberculosis to cause systemic infection
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2017 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 85, no 4, article id e00867-16Article in journal (Refereed) Published
Abstract [en]

The twin arginine translocation (Tat) system targets folded proteins across the inner membrane and is crucial for virulence in many important humanpathogenic bacteria. Tat has been shown to be required for the virulence of Yersinia pseudotuberculosis, and we recently showed that the system is critical for different virulence-related stress responses as well as for iron uptake. In this study, we wanted to address the role of the Tat substrates in in vivo virulence. Therefore, 22 genes encoding potential Tat substrates were mutated, and each mutant was evaluated in a competitive oral infection of mice. Interestingly, a.sufI mutant was essentially as attenuated for virulence as the Tat-deficient strain. We also verified that SufI was Tat dependent for membrane/periplasmic localization in Y. pseudotuberculosis. In vivo bioluminescent imaging of orally infected mice revealed that both the.sufI and Delta tatC mutants were able to colonize the cecum and Peyer's patches (PPs) and could spread to the mesenteric lymph nodes (MLNs). Importantly, at this point, neither the Delta tatC mutant nor the Delta sufI mutant was able to spread systemically, and they were gradually cleared. Immunostaining of MLNs revealed that both the Delta tatC and Delta sufI mutants were unable to spread from the initial infection foci and appeared to be contained by neutrophils, while wild-type bacteria readily spread to establish multiple foci from day 3 postinfection. Our results show that SufI alone is required for the establishment of systemic infection and is the major cause of the attenuation of the Delta tatC mutant.

Keywords
Yersinia pseudotuberculosis, Tat pathway, virulence, SufI, mesenteric lymph nodes, neutrophils
National Category
Microbiology Immunology
Identifiers
urn:nbn:se:umu:diva-128087 (URN)10.1128/IAI.00867-16 (DOI)000397581800003 ()28115509 (PubMedID)
Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2018-06-09Bibliographically approved
Pinto, A. F., Ebrahimi, M., Saleeb, M., Forsberg, Å., Elofsson, M. & Schüler, H. (2016). Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity. Journal of Biomolecular Screening, 21(6), 590-595
Open this publication in new window or tab >>Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity
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2016 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 21, no 6, p. 590-595Article in journal (Refereed) Published
Abstract [en]

The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen associated with drug resistance complications and, as such, an important object for drug discovery efforts. One attractive target for development of therapeutics is the ADP-ribosyltransferase Exotoxin-S (ExoS), an early effector of the type III secretion system that is delivered into host cells to affect their transcription pattern and cytoskeletal dynamics. The purpose of this study was to formulate a real-time assay of purified recombinant ExoS activity for high-throughput application. We characterized the turnover kinetics of the fluorescent dinucleotide 1,N-6-etheno-NAD+ as co-substrate for ExoS. Further, we found that the toxin relied on any of five tested isoforms of human 14-3-3 to modify vH-Ras and the Rho-family GTPases Rac1, -2, and -3 and RhoC. We then used 14-3-3-stimulated ExoS modification of vH-Ras to screen a collection of low-molecular-weight compounds selected to target the poly-ADP ribose polymerase family and identified 3-(4-oxo-3,5,6,7-tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-2-y l)propanoic acid as an ExoS inhibitor with micromolar potency. Thus, we present an optimized method to screen for inhibitors of ExoS activity that is amenable to high-throughput format and an intermediate affinity inhibitor that can serve both as assay control and as a starting point for further development.

Keywords
ADP-ribosylation, bacterial toxins, drug discovery, enzyme inhibitors, Pseudomonas aeruginosa
National Category
Biochemistry and Molecular Biology Medicinal Chemistry
Identifiers
urn:nbn:se:umu:diva-124333 (URN)10.1177/1087057116629923 (DOI)000379694900007 ()26850638 (PubMedID)
Available from: 2016-09-22 Created: 2016-08-04 Last updated: 2018-08-24Bibliographically approved
Ekestubbe, S., Bröms, J. E., Edgren, T., Fällman, M., Francis, M. S. & Forsberg, Å. (2016). The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence. Frontiers in Cellular and Infection Microbiology, 6, Article ID 175.
Open this publication in new window or tab >>The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence
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2016 (English)In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 175Article in journal (Refereed) Published
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.

Keywords
LcrV, type III secretion system, YopH, translocation, pore formation, Yersinia pseudotuberculosis, virulence
National Category
Microbiology in the medical area Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-130149 (URN)10.3389/fcimb.2016.00175 (DOI)000389194200001 ()
Available from: 2017-01-12 Created: 2017-01-12 Last updated: 2018-06-09Bibliographically approved
Avican, U., Beckstette, M., Heroven, A. K., Lavander, M., Dersch, P. & Forsberg, Å. (2016). Transcriptomic and Phenotypic Analysis Reveals New Functions for the Tat Pathway in Yersinia pseudotuberculosis. Journal of Bacteriology, 198(20), 2876-2886
Open this publication in new window or tab >>Transcriptomic and Phenotypic Analysis Reveals New Functions for the Tat Pathway in Yersinia pseudotuberculosis
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2016 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, no 20, p. 2876-2886Article in journal (Refereed) Published
Abstract [en]

The twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and Delta tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26 degrees C and 37 degrees C. The most significant changes in the transcriptome of the Delta tatC mutant were seen at 26 degrees C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. IMPORTANCE In addition to its established role in mediating the secretion of housekeeping enzymes, the Tat system has been recognized as being involved in infection. In some clinically relevant bacteria, such as Pseudomonas spp., several key virulence determinants can readily be identified among the Tat substrates. In enteropathogens, such as Yersinia spp., there are no obvious virulence determinants among the Tat substrates. Tat mutants show no growth defect in vitro but are highly attenuated in in vivo. This makes Tat an attractive target for the development of novel antimicrobials. Therefore, it is important to establish the causes of the attenuation. Here, we show that the attenuation is likely due to synergistic effects of different Tat-dependent phenotypes that each contributes to lowered in vivo fitness.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-127950 (URN)10.1128/JB.00352-16 (DOI)000384347500014 ()27501981 (PubMedID)
Available from: 2016-12-16 Created: 2016-11-21 Last updated: 2018-06-09Bibliographically approved
Avican, U., Beckstette, M., Heroven, A. K., Lavander, M., Dersch, P. & Forsberg, Å. (2016). Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis. Journal of Bacteriology, 198(20), 2876-2886
Open this publication in new window or tab >>Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis
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2016 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, no 20, p. 2876-2886Article in journal (Refereed) Published
Abstract [en]

The Twin-arginine translocation (Tat) system mediates secretion of folded proteins that in bacteria, plants and archaea are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and ∆tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26oC and 37oC. The most significant changes in the transcriptome of the ∆tatC mutant were seen at 26oC during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper conditions as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.

Place, publisher, year, edition, pages
Washington: American Society for Microbiology, 2016
Keywords
Yersinia pseudotuberculosis, Tat pathway, virulence, stress response, transcriptome analysis
National Category
Microbiology Bioinformatics and Systems Biology Biochemistry and Molecular Biology
Research subject
Infectious Diseases; Microbiology; Molecular Biology
Identifiers
urn:nbn:se:umu:diva-128029 (URN)10.1128/JB.00352-16 (DOI)000384347500014 ()
Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2018-06-09Bibliographically approved
Amer, A., Gurung, J., Costa, T., Ruuth, K., Zavialov, A., Forsberg, Å. & Francis, M. S. (2016). YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis. Frontiers in Cellular and Infection Microbiology, 6, Article ID 66.
Open this publication in new window or tab >>YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis
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2016 (English)In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 66Article in journal (Refereed) Published
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.

Keywords
protein-protein interaction, molecular modelling, protein secretion, mutagenesis, bacterial pathogenesis, regulation
National Category
Microbiology in the medical area Biochemistry and Molecular Biology
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-122904 (URN)10.3389/fcimb.2016.00066 (DOI)000378543500001 ()
Funder
Swedish Research Council
Available from: 2016-06-23 Created: 2016-06-23 Last updated: 2018-06-07Bibliographically approved
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