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The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). (Åke Forsberg)
Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
Vise andre og tillknytning
2016 (engelsk)Inngår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, artikkel-id 175Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2016. Vol. 6, artikkel-id 175
Emneord [en]
LcrV, type III secretion system, YopH, translocation, pore formation, Yersinia pseudotuberculosis, virulence
HSV kategori
Identifikatorer
URN: urn:nbn:se:umu:diva-130149DOI: 10.3389/fcimb.2016.00175ISI: 000389194200001OAI: oai:DiVA.org:umu-130149DiVA, id: diva2:1064556
Tilgjengelig fra: 2017-01-12 Laget: 2017-01-12 Sist oppdatert: 2018-06-09bibliografisk kontrollert
Inngår i avhandling
1. Timing and targeting of Type III secretion translocation of virulence effectors in Yersinia
Åpne denne publikasjonen i ny fane eller vindu >>Timing and targeting of Type III secretion translocation of virulence effectors in Yersinia
2017 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The Type III secretion system (T3SS) is an important virulence mechanism that allows pathogenic bacteria to translocate virulence effectors directly into the cytoplasm of eukaryotic host cells to manipulate the host cells in favor of the pathogen. Enteropathogenic Yersinia pseudotuberculosis use a T3SS to translocate effectors, Yops, that prevent phagocytosis by immune cells, and is largely dependent on it to establish and sustain an infection in the lymphoid tissues of a mammalian host. Translocation into a host cell requires specific translocator proteins, and is tightly controlled from both the bacterial and host cell cytoplasm. We aimed to investigate two of the regulatory elements, YopN and LcrV, to gain more insight into the translocation mechanism. Two separate regulatory complexes regulate expression and secretion of Yops, however, the processes are linked so that expression is induced when secretion is activated. A complex, including YopD, prevents expression of Yops, while YopN-TyeA and LcrG block secretion. LcrV is required to relieve the secretion block, by sequestering LcrG. We verified that LcrG binds to the C-terminal part of LcrV, which is consistent with what has been shown in Y. pestis. In addition to their regulatory roles, both LcrV and YopD are translocators and are assumed to interact at the bacterial surface, where LcrV promotes insertion of YopB and YopD into the host cell membrane. However, here we show that purified YopD failed to interact with LcrV, instead YopD solely interacted with a complex of LcrV-LcrG. This indicates that LcrV and YopD interact in the bacterial cytosol, which may be important for regulation of Yop expression and secretion. The established role of YopN is to block secretion prior to host cell contact. We found that deleting the central region (amino acids 76-181) had no effect on the regulatory role of YopN in expression and secretion of Yops. Interestingly, we found that, even though the YopN∆76-181 mutant secreted the translocators with similar kinetics as the wild type strain, translocation of the effector YopH, into HeLa cells, was significantly reduced. Consequently, the YopN∆76-181 mutant was unable to block phagocytosis, almost to the same level as the ∆lcrV mutant which is completely unable to translocate YopH. Our results indicate that YopN is involved in the translocation step in addition to its role in regulating secretion. Further, we show that the amino terminal of LcrV, in the context of translocation, is involved in the early intracellular targeting of YopH in order to block phagocytosis efficiently and sustain an in vivo infection. LcrV mutants that failed to efficiently target YopH intracellularly were severely attenuated also for in vivo virulence. All together, we show that LcrV and YopN are involved in more steps in the regulation of translocation, than what was known before. Our studies also highlight that early translocation is essential for Yersinia to block phagocytosis, which in the end is essential for in vivo virulence.

sted, utgiver, år, opplag, sider
Umeå: Umeå University, 2017. s. 77
Emneord
Type III secretion system, virulence, translocation, Yersinia pseudotuberculosis, LcrV, YopN, effector targeting, phagocytosis inhibition, YopH, in vivo infection
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-130155 (URN)978-91-7601-639-8 (ISBN)
Disputas
2017-01-27, N200, Naturvetarhuset, Umeå, 09:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2017-01-13 Laget: 2017-01-12 Sist oppdatert: 2018-06-09bibliografisk kontrollert

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