The chlamydial inclusion membrane as an engine of survival

ABSTRACT Chlamydia psittaci produces a collection of proteins, termed IncA, IncB, and IncC, that are localized to the chlamydial inclusion membrane. In this report we demonstrate that IncA is also produced by Chlamydia trachomatis. C. trachomatis IncA is structurally similar to C. psittaci IncA and is also localized to the inclusion membrane. Immunoblot analysis demonstrated that sera from C. trachomatis-infected patients and from experimentally infected monkeys both recognized C. trachomatis IncA.

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Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole inChlamydia trachomatisInfected Human Cells

10.1101/616896 ◽

2019 ◽

Author(s):

Macy G. Olson ◽

Ray E. Widner ◽

Lisa M. Jorgenson ◽

Alyssa Lawrence ◽

Dragana Lagundzin ◽

...

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions ◽

Host Pathogen Interactions ◽

Inclusion Membrane ◽

Eukaryotic Proteins ◽

Host Pathogen ◽

Membrane Bound ◽

Labeling System ◽

Chlamydial Inclusion

AbstractAs an obligate intracellular pathogenic bacterium,C. trachomatisdevelops within a membrane-bound vacuole, termed the inclusion. The inclusion membrane is modified by chlamydial inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. Anin vivounderstanding of Inc-Inc and Inc-eukaryotic protein interactions and how these contribute to overall host-chlamydial interactions at this unique membrane is lacking. Previous bacterial two-hybrid studies established that certain Incs have the propensity to bind other Incs while others have limited Inc-Inc interactions. We hypothesize some Incs organize the inclusion membrane whereas other Incs bind eukaryotic proteins to promote chlamydial-host interactions. To test this hypothesis, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotinin vivo, and chose to analyze Inc proteins with varying Inc-binding propensities. We inducibly expressed these Incs fused to APEX2 inChlamydia trachomatisL2, verified their localization and labeling activities by transmission electron microscopy, and used affinity purification-mass spectrometry to identify biotinylated proteins. To analyze our mass spectrometry results for statistical significance, we used Significance Analysis of INTeractome (SAINT), which demonstrated that our Inc-APEX2 constructs labeled Inc proteins as well as known and previously unreported eukaryotic proteins that localize to the inclusion. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. One eukaryotic protein, LRRFIP1 (LRRF1) was found in all of our Inc-APEX2 datasets, which is consistent with previously published AP-MS datasets. For the first time, we demonstrate by confocal and super-resolution microscopy that endogenous LRRF1 localizes to the chlamydial inclusion. We also used bacterial two-hybrid studies and pulldown assays to determine if LRRF1 was identified as a true interacting protein or was proximal to our Inc-APEX2 constructs. Combined, our data highlight the utility of APEX2 to capture the complexin vivoprotein-protein interactions at the chlamydial inclusion.Author summaryMany intracellular bacteria, including the obligate intracellular pathogenChlamydia trachomatis, grow within a membrane-bound “bacteria containing vacuole” (BCV) that, in most cases, prevents association with the lysosome. Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. Here, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotinin vivo, to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. The inclusion membrane is modified by chlamydial type III secreted inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. Our data highlight the utility of APEX2 to capture the complex protein-protein interactions at a membrane sitein vivoin the context of infection.

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Host Complement Regulatory Protein CD59 Is Transported to the Chlamydial Inclusion by a Golgi Apparatus-Independent Pathway

Infection and Immunity ◽

10.1128/iai.01062-08 ◽

2009 ◽

Vol 77 (4) ◽

pp. 1285-1292 ◽

Cited By ~ 8

Author(s):

Ayako Hasegawa ◽

L. Farah Sogo ◽

Ming Tan ◽

Christine Sütterlin

Keyword(s):

Golgi Apparatus ◽

Cell Surface ◽

Regulatory Protein ◽

Cytoplasmic Inclusion ◽

Surface Proteins ◽

Host Protein ◽

Membrane Microdomains ◽

Gpi Anchor ◽

Inclusion Membrane ◽

Chlamydial Inclusion

ABSTRACT Chlamydia is an obligate intracellular bacterium that grows and replicates inside a cytoplasmic inclusion. We report that a host protein, CD59, which regulates complement function at the surfaces of uninfected cells, can be detected at the membrane of the chlamydial inclusion. This localization to the inclusion membrane was specific for CD59 and not a general feature of other glycosylphosphatidylinositol (GPI)-anchored proteins or representative cell surface proteins. Using differential permeabilization studies, we showed that CD59 is localized to the luminal but not the cytoplasmic face of the inclusion membrane, consistent with membrane association via its GPI anchor. Furthermore, CD59 was present at the inclusion even when we prevented it from associating with membrane microdomains via the GPI anchor or when we inhibited general protein transport to the cell surface, indicating that a conventional Golgi apparatus-dependent trafficking mechanism was not involved. Based on these findings, we propose that selected host proteins are trafficked to the inclusion by a Golgi apparatus-independent pathway during a Chlamydia infection.

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Modulation of cytokines and transcription factors (T-Bet and GATA3) in CD4 enriched cervical cells of Chlamydia trachomatis infected fertile and infertile women upon stimulation with chlamydial inclusion membrane proteins B and C

Reproductive Biology and Endocrinology ◽

10.1186/1477-7827-7-84 ◽

2009 ◽

Vol 7 (1) ◽

pp. 84 ◽

Cited By ~ 15

Author(s):

Rishein Gupta ◽

Harsh Vardhan ◽

Pragya Srivastava ◽

Sudha Salhan ◽

Aruna Mittal

Keyword(s):

Transcription Factors ◽

Chlamydia Trachomatis ◽

Membrane Proteins ◽

Inclusion Membrane ◽

Infertile Women ◽

Inclusion Membrane Proteins ◽

Cervical Cells ◽

Chlamydial Inclusion

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Proximity Labeling To Map Host-Pathogen Interactions at the Membrane of a Bacterium-Containing Vacuole in Chlamydia trachomatis-Infected Human Cells

Infection and Immunity ◽

10.1128/iai.00537-19 ◽

2019 ◽

Vol 87 (11) ◽

Cited By ~ 5

Author(s):

Macy G. Olson ◽

Ray E. Widner ◽

Lisa M. Jorgenson ◽

Alyssa Lawrence ◽

Dragana Lagundzin ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Inclusion Membrane ◽

Content Type ◽

Host Interactions ◽

Eukaryotic Proteins ◽

Host Pathogen ◽

Chlamydial Inclusion

ABSTRACT Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound bacterium-containing vacuole (BCV). Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. We used the ascorbate peroxidase (APEX2) proximity labeling system, which labels proximal proteins with biotin in vivo, to study the protein-protein interactions that occur at the chlamydial vacuolar, or inclusion, membrane. An in vivo understanding of the secreted chlamydial inclusion membrane protein (Inc) interactions (e.g., Inc-Inc and Inc-eukaryotic protein) and how these contribute to overall host-chlamydia interactions at this unique membrane is lacking. We hypothesize some Incs organize the inclusion membrane, whereas other Incs bind eukaryotic proteins to promote chlamydia-host interactions. To study this, Incs fused to APEX2 were expressed in C. trachomatis L2. Affinity purification-mass spectrometry (AP-MS) identified biotinylated proteins, which were analyzed for statistical significance using significance analysis of the interactome (SAINT). Broadly supporting both Inc-Inc and Inc-host interactions, our Inc-APEX2 constructs labeled Incs as well as known and previously unreported eukaryotic proteins localizing to the inclusion. We demonstrate, using bacterial two-hybrid and coimmunoprecipitation assays, that endogenous LRRFIP1 (LRRF1) is recruited to the inclusion by the Inc CT226. We further demonstrate interactions between CT226 and the Incs used in our study to reveal a model for inclusion membrane organization. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.

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EUKARYOTIC SNARE VAMP3 DYNAMICALLY INTERACTS WITH MULTIPLE CHLAMYDIAL INCLUSION MEMBRANE PROTEINS

Infection and Immunity ◽

10.1128/iai.00409-20 ◽

2020 ◽

Author(s):

Duc-Cuong Bui ◽

Lisa M. Jorgenson ◽

Scot P. Ouellette ◽

Elizabeth A. Rucks

Keyword(s):

De Novo ◽

Host Protein ◽

Snare Proteins ◽

Developmental Cycle ◽

Inclusion Membrane ◽

Transient Nature ◽

Binding Partners ◽

Infected Cells ◽

Obligate Intracellular Pathogen ◽

Chlamydial Inclusion

Chlamydia trachomatis, an obligate intracellular pathogen, undergoes a biphasic developmental cycle within a membrane-bound vacuole called the chlamydial inclusion. To facilitate interactions with the host cell, Chlamydia modifies the inclusion membrane with type III secreted proteins, called Incs. As with all chlamydial proteins, Incs are temporally expressed, modifying the chlamydial inclusion during the early- and mid-developmental cycle. VAMP3 and VAMP4 are eukaryotic SNARE proteins that mediate membrane fusion and are recruited to the inclusion to facilitate inclusion expansion. Their recruitment requires de novo chlamydial protein synthesis during the mid-developmental cycle. Thus, we hypothesize that VAMPs 3 and 4 are recruited by Incs. In chlamydial infected cells, identifying Inc binding partners for SNARE proteins specifically has been elusive. To date, most studies examining chlamydial Inc-eukaryotic proteins have benefitted from stable interacting partners or a robust interaction at a specific time point post-infection. While these types of interactions are the predominant class that have been identified, they are likely the ‘exception’ to chlamydial-host interactions. Therefore, we applied two separate but complementary experimental systems to identify candidate chlamydial Inc binding partners for VAMPs. Based on these results, we created transformed strains of C. trachomatis serovar L2 to inducibly express a candidate Inc-FLAG protein. In chlamydial infected cells, we found that five Incs temporally and transiently interact with VAMP3. Further, loss of incA or ct813 expression altered VAMP3 localization to the inclusion. For the first time, our studies demonstrate the transient nature of certain host protein-Inc interactions that contribute to the chlamydial developmental cycle.

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