scholarly journals Expression and Localization of Predicted Inclusion Membrane Proteins in Chlamydia trachomatis

2015 ◽  
Vol 83 (12) ◽  
pp. 4710-4718 ◽  
Author(s):  
Mary M. Weber ◽  
Laura D. Bauler ◽  
Jennifer Lam ◽  
Ted Hackstadt
Keyword(s):  
Chlamydia Trachomatis ◽  
Membrane Proteins ◽  
Membrane Microdomains ◽  
Membrane Expression ◽  
Hydrophobic Domain ◽  
Inclusion Membrane ◽  
Content Type ◽  
Genetic Tools ◽  
Type Iii ◽  
Inclusion Membrane Proteins

Chlamydia trachomatisis an obligate intracellular pathogen that replicates in a membrane-bound vacuole termed the inclusion. Early in the infection cycle, the pathogen extensively modifies the inclusion membrane through incorporation of numerous type III secreted effector proteins, called inclusion membrane proteins (Incs). These proteins are characterized by a bilobed hydrophobic domain of 40 amino acids. The presence of this domain has been used to predict up to 59 putative Incs forC. trachomatis; however, localization to the inclusion membrane with specific antibodies has been demonstrated for only about half of them. Here, we employed recently developed genetic tools to verify the localization of predicted Incs that had not been previously localized to the inclusion membrane. Expression of epitope-tagged putative Incs identified 10 that were previously unverified as inclusion membrane localized and thus authentic Incs. One novel Inc and 3 previously described Incs were localized to inclusion membrane microdomains, as evidenced by colocalization with phosphorylated Src (p-Src). Several predicted Incs did not localize to the inclusion membrane but instead remained associated with the bacteria. UsingYersiniaas a surrogate host, we demonstrated that many of these are not secreted via type III secretion, further suggesting they may not be true Incs. Collectively, our results highlight the utility of genetic tools for demonstrating secretion from chlamydia. Further mechanistic studies aimed at elucidating effector function will advance our understanding of how the pathogen maintains its unique intracellular niche and mediates interactions with the host.

scholarly journals Absence of Specific Chlamydia trachomatis Inclusion Membrane Proteins Triggers Premature Inclusion Membrane Lysis and Host Cell Death

Cell Reports ◽  
2017 ◽  
Vol 19 (7) ◽  
pp. 1406-1417 ◽  
Author(s):  
Mary M. Weber ◽  
Jennifer L. Lam ◽  
Cheryl A. Dooley ◽  
Nicholas F. Noriea ◽  
Bryan T. Hansen ◽  
...  
Keyword(s):  
Cell Death ◽  
Chlamydia Trachomatis ◽  
Membrane Proteins ◽  
Host Cell ◽  
Inclusion Membrane ◽  
Host Cell Death ◽  
Membrane Lysis ◽  
Inclusion Membrane Proteins

scholarly journals Characterization of Fifty Putative Inclusion Membrane Proteins Encoded in the Chlamydia trachomatis Genome

2008 ◽  
Vol 76 (6) ◽  
pp. 2746-2757 ◽  
Author(s):  
Zhongyu Li ◽  
Chaoqun Chen ◽  
Ding Chen ◽  
Yimou Wu ◽  
Youmin Zhong ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Membrane Proteins ◽  
Chlamydial Infection ◽  
Distribution Patterns ◽  
Western Blot Assay ◽  
Inclusion Membrane ◽  
Urogenital Infection ◽  
Infected Cells ◽  
Inclusion Membrane Proteins

ABSTRACT Although the Chlamydia trachomatis genome is predicted to encode 50 inclusion membrane proteins, only 18 have been experimentally localized in the inclusion membrane of C. trachomatis-infected cells. Using fusion proteins and anti-fusion protein antibodies, we have systematically evaluated all 50 putative inclusion membrane proteins for their localization in the infected cells, distribution patterns, and effects on subsequent chlamydial infection when expressed ectopically, as well as their immunogenicity during chlamydial infection in humans. Twenty-two of the 50 proteins were localized in the inclusion membrane, and 7 were detected inside the inclusions, while the location of the remaining 21 was not defined. Four (CT225, CT228, CT358, and CT440) of the 22 inclusion membrane-localized proteins were visualized in the inclusion membrane of Chlamydia-infected cells for the first time in the current study. The seven intra-inclusion-localized proteins were confirmed to be chlamydial organism proteins in a Western blot assay. Further characterization of the 50 proteins revealed that neither colocalization with host cell endoplasmic reticulum nor inhibition of subsequent chlamydial infection by ectopically expressed proteins correlated with the inclusion membrane localization. Interestingly, antibodies from women with C. trachomatis urogenital infection preferentially recognized proteins localized in the inclusion membrane, and the immunodominant regions were further mapped to the region predicted to be on the cytoplasmic side of the inclusion membrane. These observations suggest that most of the inclusion membrane-localized proteins are both expressed and immunogenic during C. trachomatis infection in humans and that the cytoplasmic exposure may enhance the immunogenicity.

scholarly journals A Functional Core of IncA Is Required for Chlamydia trachomatis Inclusion Fusion

2016 ◽  
Vol 198 (8) ◽  
pp. 1347-1355 ◽  
Author(s):  
Mary M. Weber ◽  
Nicholas F. Noriea ◽  
Laura D. Bauler ◽  
Jennifer L. Lam ◽  
Janet Sager ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Membrane Fusion ◽  
Parasitophorous Vacuole ◽  
Endocytic Pathway ◽  
Inclusion Membrane ◽  
Content Type ◽  
Genetic Tools ◽  
Insertional Inactivation ◽  
Sensitive Factor ◽  
Infected Cells

ABSTRACTChlamydia trachomatisis an obligate intracellular pathogen that is the etiological agent of a variety of human diseases, including blinding trachoma and sexually transmitted infections. Chlamydiae replicate within a membrane-bound compartment, termed an inclusion, which they extensively modify by the insertion of type III secreted proteins called Inc proteins. IncA is an inclusion membrane protein that encodes two coiled-coil domains that are homologous to eukaryotic SNARE (solubleN-ethylmaleimide-sensitive factor attachment receptor) motifs. Recent biochemical evidence suggests that a functional core, composed of SNARE-like domain 1 (SLD-1) and part of SNARE-like domain 2 (SLD-2), is required for the characteristic homotypic fusion ofC. trachomatisinclusions in multiply infected cells. To verify the importance of IncA in homotypic fusion inChlamydia, we generated anincA::blamutant. Insertional inactivation ofincAresulted in the formation of nonfusogenic inclusions, a phenotype that was completely rescued by complementation with full-length IncA. Rescue of homotypic inclusion fusion was dependent on the presence of the functional core consisting of SLD-1 and part of SLD-2. Collectively, these results confirmin vitromembrane fusion assays identifying functional domains of IncA and expand the genetic tools available for identification of chlamydia with a method for complementation of site-specific mutants.IMPORTANCEChlamydia trachomatisreplicates within a parasitophorous vacuole termed an inclusion. The chlamydial inclusions are nonfusogenic with vesicles in the endocytic pathway but, in multiply infected cells, fuse with each other to form a single large inclusion. This homotypic fusion is dependent upon the presence of a chlamydial inclusion membrane-localized protein, IncA. Specificity of membrane fusion in eukaryotic cells is regulated by SNARE (solubleN-ethylmaleimide sensitive factor attachment receptor) proteins on the cytosolic face of vesicles and target membranes. IncA contains two SNARE-like domains. Newly developed genetic tools for the complementation of targeted mutants inC. trachomatisare used to confirm the minimal requirement of SNARE-like motifs necessary to promote the homotypic fusion of inclusions.

Identification and characterization of a Chlamydia trachomatis early operon encoding four novel inclusion membrane proteins

1999 ◽  
Vol 33 (4) ◽  
pp. 753-765 ◽  
Author(s):  
Marci A. Scidmore-Carlson ◽  
Edward I. Shaw ◽  
Cheryl A. Dooley ◽  
Elizabeth R. Fischer ◽  
Ted Hackstadt
Keyword(s):  
Chlamydia Trachomatis ◽  
Membrane Proteins ◽  
Inclusion Membrane ◽  
Inclusion Membrane Proteins ◽  
Identification And Characterization

scholarly journals Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions

2011 ◽  
Vol 79 (8) ◽  
pp. 3036-3045 ◽  
Author(s):  
B. Chellas-Géry ◽  
K. Wolf ◽  
J. Tisoncik ◽  
T. Hackstadt ◽  
K. A. Fields
Keyword(s):  
Type Iii Secretion ◽  
De Novo ◽  
Ectopic Expression ◽  
Effector Proteins ◽  
Developmental Cycle ◽  
Inclusion Membrane ◽  
Content Type ◽  
Type Iii ◽  
Intracellular Parasites ◽  
The Many

ABSTRACTChlamydiaspp. are among the many pathogenic Gram-negative bacteria that employ a type III secretion system (T3SS) to overcome host defenses and exploit available resources. Significant progress has been made in elucidating contributions of T3S to the pathogenesis of these medically important, obligate intracellular parasites, yet important questions remain. Chief among these is how secreted effector proteins traverse eukaryotic membranes to gain access to the host cytosol. Due to a complex developmental cycle, it is possible that chlamydiae utilize a different complement of proteins to accomplish translocation at different stages of development. We investigated this possibility by extending the characterization ofC. trachomatisCopB and CopB2. CopB is detected early during infection but is depleted and not detected again until about 20 h postinfection. In contrast, CopB2 was detectible throughout development. CopB is associated with the inclusion membrane. Biochemical and ectopic expression analyses were consistent with peripheral association of CopB2 with inclusion membranes. This interaction correlated with development and required both chlamydialde novoprotein synthesis and T3SS activity. Collectively, our data indicate that it is unlikely that CopB serves as the sole chlamydial translocation pore and that CopB2 is capable of association with the inclusion membrane.

Inclusion membrane proteins of Chlamydiaceae

2008 ◽  
Vol 2 (2) ◽  
pp. 148-159 ◽  
Author(s):  
E. S. Kostryukova ◽  
V. N. Lazarev ◽  
V. M. Govorum
Keyword(s):  
Membrane Proteins ◽  
Inclusion Membrane ◽  
Inclusion Membrane Proteins
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