| 1. |
- Akopyan, Karen, et al.
(författare)
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Translocation of surface-localized effectors in type III secretion
- 2011
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:4, s. 1639-1644
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Tidskriftsartikel (refereegranskat)abstract
- Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells. Upon host cell contact, purified YopH coated on Y. pseudotuberculosis was specifically and rapidly translocated across the target-cell membrane, which led to a physiological response in the infected cell. In addition, translocation of externally added YopH required a functional T3SS and a specific translocation domain in the effector protein. Efficient, T3SS-dependent translocation of purified YopH added in vitro was also observed when using coated Salmonella typhimurium strains, which implies that T3SS-mediated translocation of extracellular effector proteins is conserved among T3SS-dependent pathogens. Our results demonstrate that polarized T3SS-dependent translocation of proteins can be achieved through an intermediate extracellular step that can be reconstituted in vitro. These results indicate that translocation can occur by a different mechanism from the assumed single-step conduit model.
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| 2. |
- Bamyaci, Sarp, et al.
(författare)
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YopN Is Required for Efficient Effector Translocation and Virulence in Yersinia pseudotuberculosis
- 2018
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Ingår i: Infection and Immunity. - : AMER SOC MICROBIOLOGY. - 0019-9567 .- 1098-5522. ; 86:8
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
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| 4. |
- Björnfot, Ann-Catrin, 1981-, et al.
(författare)
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Involvement of the heat shock proteins DnaK/DnaJ in Yersinia T3S
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Yersinia pseudotuberculosis uses a type III secretion system (T3SS) to secrete and deliver effectors called Yops into target cells. These processes are highly regulated and the pathogen senses cell contact and respond accordingly by inducing Yop-effector expression. A key component of the T3SS is the YscF needle present on the surface of the pathogen. It has been suggested that the bacterium can switch from needle export to Yop expression and secretion and that this substrate switch is important for proper regulation during infection. YscU is an essential protein regulating the substrate switch and autoproteolysis of YscU is essential for accurate T3SS regulation. To study regulation of Yop translocation in more detail, we generated mutants defective for expression of the heat shock proteins (HSPs) DnaJ and DnaK, since earlier studies had indicated a role of these proteins in regulation of effector translocation in Salmonella. The dnaJ mutant and the double dnaK/J mutant showed significant defects in Yop translocation, but surprisingly both mutants were able to secrete Yops in vitro much like the wild type. However, both mutants showed a changed export pattern of the YscF needle with a pronounced increased export of the YscF needle protein after incubation in calcium containing media. This phenotype was linked to defects in YscU autoproteolysis and in this respect the hsp-mutants were identical to earlier identified autoprocessing defective mutants in YscU (Single amino acid exchange mutants N263A and P264A). The hsp-mutants and the processing mutants accumulated full-length YscU, which surprisingly was associated with the outer membrane, while the processed form of YscU was found in the inner membrane fraction. The dnaJ and dnaK/J mutants were strongly affected in YscU autoproteolysis, which indicates a possible direct role for DnaJ in this process. Indeed a specific interaction between YscU and DnaJ could be found suggesting a direct role of the HSPs in regulation of the substrate switch in the T3SS.
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| 5. |
- Costa, Tiago, et al.
(författare)
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Measurement of Yersinia translocon pore formation in erythrocytes
- 2019
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Ingår i: Pathogenic Yersinia. - New York, NY, U.S.A. : Humana Press. - 9781493995400 ; , s. 211-229
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Bokkapitel (refereegranskat)abstract
- Many Gram-negative pathogens produce a type III secretion system capable of intoxicating eukaryotic cells with immune-modulating effector proteins. Fundamental to this injection process is the prior secretion of two translocator proteins destined for injectisome translocon pore assembly within the host cell plasma membrane. It is through this pore that effectors are believed to travel to gain access to the host cell interior. Yersinia species especially pathogenic to humans and animals assemble this translocon pore utilizing two hydrophobic translocator proteins-YopB and YopD. Although a full molecular understanding of the biogenesis, function and regulation of this translocon pore and subsequent effector delivery into host cells remains elusive, some of what we know about these processes can be attributed to studies of bacterial infections of erythrocytes. Herein we describe the methodology of erythrocyte infections by Yersinia, and how analysis of the resultant contact-dependent hemolysis can serve as a relative measurement of YopB- and YopD-dependent translocon pore formation.
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| 6. |
- Costa, Tiago R. D., et al.
(författare)
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Active type III translocon assemblies that attenuate Yersinia virulence
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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 a-helix motif at the C-terminus. Mutants YopDI262P and YopDK267P poorly localised 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 YopDA263P and YopDA270P 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. It is therefore probable that an active translocon makes a range of contributions during bacteria-host cell contact that extends beyond effector delivery per se.
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| 7. |
- Costa, Tiago, et al.
(författare)
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Type III secretion translocon assemblies that attenuate Yersinia virulence
- 2013
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Ingår i: Cellular Microbiology. - : Wiley-Blackwell. - 1462-5814 .- 1462-5822. ; 15:7, s. 1088-1110
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
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| 9. |
- Edgren, Tomas, 1971-
(författare)
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Electron transport to nitrogenase in Rhodospirillum rubrum
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biological nitrogen fixation is a key step in the global nitrogen cycle. In this process, dinitrogen in the air is converted to biologically accessible ammonia, which is further assimilated in to the biosphere. Nitrogenase, the enzyme system responsible for dinitrogen reduction, is only found in prokaryotic organisms and biological nitrogen fixation is an energy-demanding process, requiring both ATP and low potential reducing equivalents. In the free-living purple non-sulfur anoxygenic phototroph Rhodospirillum rubrum, efficient electron transfer to nitrogenase is dependent on active electron transport in the chromatophore membrane.I have shown that reducing equivalents for nitrogen fixation is generated through the action of the proteins encoded by the fixABCX genes. The membrane associated protein complex encoded by these genes reduces a soluble ferredoxin, which in turn acts as the direct electron donor to nitrogenase in this organism. The heterodimeric flavoprotein FixAB has NADH dehydrogenase activity indicating that the reducing equivalents for nitrogen fixation are derived from the general metabolism of the cell. The membrane associated FixC protein is believed to drive the energetically unfavorable reduction of ferredoxin N using energy derived from the electron transfer processes in the chromatophore membrane in some unknown manner. The membrane associated protein complex encoded by fixABCX most likely constitutes the unknown link between photosynthesis and nitrogen fixation in R. rubrum.
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| 10. |
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