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Multiple functions of YopN in the Yersinia pseudotuberculosis type III secretion system: from regulation to in vivo infection
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). (Åke Forsberg)
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The type 3 secretion systems (T3SSs) are virulence mechanisms used by various Gram-negative bacteria to overcome the host immunity. They are often target-cell contact induced and activated. Activation results in targeting of virulence effector substrates into host cells. One class of secreted substrates, translocators, are required for the intracellular targeting of the second class, the virulence effectors, into host target cells. T3SSs are mainly regulated at 2 levels; a shift from environmental to host temperature results in low level induction of the system whereas target cell contact further induces and activates the system. In the Yersinia T3SS, YopN, one of the secreted substrates, is involved in the latter level of activation. Under non-inducing conditions, YopN complexes with TyeA, SycN and YscB and this complex suppresses the T3SS via an unknown mechanism. When the system is induced, the complex is believed to dissociate and YopN is secreted resulting in the activation of the system. Earlier studies indicated that YopN is not only secreted but also translocated into target cells in a T3SS dependent manner. TyeA, SycN and YscB bind to the C-terminal and N-terminal YopN respectively but so far the central region (CR) of YopN has not been characterized. In this study we have focused on the function of the YopN central region.

We therefore generated in-frame deletion mutants within the CR of YopN. One of these deletion mutants, aa 76-181, showed decreased early translocation of both YopE and YopH into infected host cells and also failed to efficiently block phagocytosis by macrophages. However, the YopNΔ76-181 protein was expressed at lower levels compared to wt YopN and also showed a slightly deregulated phenotype when expressed from its native promoter and were as a consequence not possible to use in in vivo infection studies.

Therefore, we generated mutants that disrupted a putative coiled coil domain located at the very N-terminal of CR. Similar to YopNΔ76-181, these substitution mutants were affected in the early translocation of effector proteins. Importantly, they were as stable as wt YopN when expressed from the native promoter. One of these mutants was unable to cause systemic infection in mice indicating that YopN indeed also has a direct role in virulence and is required for establishment of systemic infection in vivo.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2019. , p. 88
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2007
Keywords [en]
Type III secretion system, Yersinia, YopN, virulence, phagocytosis, mouse infection, secretion kinetics
National Category
Biochemistry and Molecular Biology Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-155193ISBN: 978-91-7601-996-2 (print)OAI: oai:DiVA.org:umu-155193DiVA, id: diva2:1277200
Public defence
2019-02-01, Major Groove, Building 6L, NUS, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2019-01-11 Created: 2019-01-09 Last updated: 2019-01-11Bibliographically approved
List of papers
1. Kinetics of Type III secretion in Yersinia and sub-cellular localization of the Yops under non-inducing conditions
Open this publication in new window or tab >>Kinetics of Type III secretion in Yersinia and sub-cellular localization of the Yops under non-inducing conditions
(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-155204 (URN)
Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-01-10
2. YopN Is Required for Efficient Effector Translocation and Virulence in Yersinia pseudotuberculosis
Open this publication in new window or tab >>YopN Is Required for Efficient Effector Translocation and Virulence in Yersinia pseudotuberculosis
Show others...
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
3. Identification of specific sequence motif of YopN of Yersinia pseudotuberculosis required for systemic infection
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

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