EUKARYOTIC SNARE VAMP3 DYNAMICALLY INTERACTS WITH MULTIPLE CHLAMYDIAL INCLUSION MEMBRANE PROTEINS

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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The trans-Golgi SNARE syntaxin 6 is recruited to the chlamydial inclusion membrane

Microbiology ◽

10.1099/mic.0.045856-0 ◽

2011 ◽

Vol 157 (3) ◽

pp. 830-838 ◽

Cited By ~ 35

Author(s):

Elizabeth R. Moore ◽

David J. Mead ◽

Cheryl A. Dooley ◽

Janet Sager ◽

Ted Hackstadt

Keyword(s):

Protein Synthesis ◽

Cell Model ◽

Parasitophorous Vacuole ◽

Endocytic Pathway ◽

Snare Proteins ◽

Inclusion Membrane ◽

Protein Receptor ◽

Infected Cells ◽

Obligate Intracellular Pathogen ◽

Chlamydial Inclusion

Chlamydia trachomatis is an obligate intracellular pathogen that replicates within a parasitophorous vacuole termed an inclusion. The chlamydial inclusion is isolated from the endocytic pathway but fusogenic with Golgi-derived exocytic vesicles containing sphingomyelin and cholesterol. Sphingolipids are incorporated into the chlamydial cell wall and are considered essential for chlamydial development and viability. The mechanisms by which chlamydiae obtain eukaryotic lipids are poorly understood but require chlamydial protein synthesis and presumably modification of the inclusion membrane to initiate this interaction. A polarized cell model of chlamydial infection has demonstrated that chlamydiae preferentially intercept basolaterally directed, sphingomyelin-containing exocytic vesicles. Here we examine the localization and potential function of trans-Golgi and/or basolaterally associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in chlamydia-infected cells. The trans-Golgi SNARE protein syntaxin 6 is recruited to the chlamydial inclusion in a manner that requires chlamydial protein synthesis and is conserved among all chlamydial species examined. The localization of syntaxin 6 to the chlamydial inclusion requires a tyrosine motif or plasma membrane retrieval signal (YGRL). Thus in addition to expression of at least two inclusion membrane proteins that contain SNARE-like motifs, chlamydiae also actively recruit eukaryotic SNARE-family proteins.

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

Infection and Immunity ◽

10.1128/iai.00159-11 ◽

2011 ◽

Vol 79 (8) ◽

pp. 3036-3045 ◽

Cited By ~ 14

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.

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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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Role for the Src Family Kinase Fyn in Sphingolipid Acquisition by Chlamydiae

Infection and Immunity ◽

10.1128/iai.05692-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4559-4568 ◽

Cited By ~ 13

Author(s):

Jeffrey Mital ◽

Ted Hackstadt

Keyword(s):

Host Proteins ◽

Content Type ◽

Obligate Intracellular ◽

Screening Protocol ◽

Infected Cells ◽

Protective Environment ◽

Membrane Bound ◽

Obligate Intracellular Pathogen ◽

Interfering Rna ◽

Chlamydial Inclusion

ABSTRACTThe bacterial obligate intracellular pathogenChlamydia trachomatisreplicates within a membrane-bound vacuole termed the inclusion. From within this protective environment, chlamydiae usurp numerous functions of the host cell to promote chlamydial survival and replication. Here we utilized a small interfering RNA (siRNA)-based screening protocol designed to identify host proteins involved in the trafficking of sphingomyelin to the chlamydial inclusion. Twenty-six host proteins whose deficiency significantly decreased sphingomyelin trafficking to the inclusion and 16 proteins whose deficiency significantly increased sphingomyelin trafficking to the inclusion were identified. The reduced sphingomyelin trafficking caused by downregulation of the Src family tyrosine kinase Fyn was confirmed in more-detailed analyses. Fyn silencing did not alter sphingomyelin synthesis or trafficking in the absence of chlamydial infection but reduced the amount of sphingomyelin trafficked to the inclusion in infected cells, as determined by two independent quantitative assays. Additionally, inhibition of Src family kinases resulted in increased cellular retention of sphingomyelin and significantly decreased incorporation into elementary bodies of bothC. trachomatisandChlamydophila caviae.

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Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

Infection and Immunity ◽

10.1128/iai.00584-13 ◽

2013 ◽

Vol 81 (9) ◽

pp. 3326-3337 ◽

Cited By ~ 13

Author(s):

Emily J. Kabeiseman ◽

Kyle Cichos ◽

Ted Hackstadt ◽

Andrea Lucas ◽

Elizabeth R. Moore

Keyword(s):

Membrane Protein ◽

De Novo ◽

Snare Proteins ◽

Dependent Manner ◽

Binding Partner ◽

And Function ◽

Sirna Knockdown ◽

Interfering Rna ◽

Chlamydial Inclusion

ABSTRACTThe predominant players in membrane fusion events are thesolubleN-ethylmaleimide-sensitive factorattachment proteinreceptor (SNARE) family of proteins. We hypothesize that SNARE proteins mediate fusion events at the chlamydial inclusion and are important for chlamydial lipid acquisition. We have previously demonstrated thattrans-Golgi SNARE syntaxin 6 localizes to the chlamydial inclusion. To investigate the role of syntaxin 6 at the chlamydial inclusion, we examined the localization and function of anothertrans-Golgi SNARE and syntaxin 6-binding partner, vesicle-associated membrane protein 4 (VAMP4), at the chlamydial inclusion. In this study, we demonstrate that syntaxin 6 and VAMP4 colocalize to the chlamydial inclusion and interact at the chlamydial inclusion. Furthermore, in the absence of VAMP4, syntaxin 6 is not retained at the chlamydial inclusion. Small interfering RNA (siRNA) knockdown of VAMP4 inhibited chlamydial sphingomyelin acquisition, correlating with a log decrease in infectious progeny. VAMP4 retention at the inclusion was shown to be dependent onde novochlamydial protein synthesis, but unlike syntaxin 6, VAMP4 recruitment is observed in a species-dependent manner. Notably, VAMP4 knockdown inhibits sphingomyelin trafficking only to inclusions in which it localizes. These data support the hypothesis that VAMP proteins play a central role in mediating eukaryotic vesicular interactions at the chlamydial inclusion and, thus, support chlamydial lipid acquisition and chlamydial development.

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Pmp-Like Proteins Pls1 and Pls2 Are Secreted into the Lumen of the Chlamydia trachomatis Inclusion

Infection and Immunity ◽

10.1128/iai.00632-08 ◽

2008 ◽

Vol 76 (9) ◽

pp. 3940-3950 ◽

Cited By ~ 28

Author(s):

Ine Jorgensen ◽

Raphael H. Valdivia

Keyword(s):

Chlamydia Trachomatis ◽

Gradient Centrifugation ◽

Effector Proteins ◽

Cellular Functions ◽

Passenger Domain ◽

Inclusion Membrane ◽

Associated Proteins ◽

Infected Cells ◽

Low Levels ◽

Obligate Intracellular Pathogen

ABSTRACT The obligate intracellular pathogen Chlamydia trachomatis secretes effector proteins across the membrane of the pathogen-containing vacuole (inclusion) to modulate host cellular functions. In an immunological screen for secreted chlamydial proteins, we identified CT049 and CT050 as potential inclusion membrane-associated proteins. These acidic, nonglobular proteins are paralogously related to the passenger domain of the polymorphic membrane protein PmpC and, like other Pmp proteins, are highly polymorphic among C. trachomatis ocular and urogenital strains. We generated antibodies to these Pmp-like secreted (Pls) proteins and determined by immunofluorescence microscopy that Pls1 (CT049) and Pls2 (CT050) localized to globular structures within the inclusion lumen and at the inclusion membrane. Fractionation of membranes and cytoplasmic components from infected cells by differential and density gradient centrifugation further indicated that Pls1 and Pls2 associated with membranes distinct from the bulk of bacterial and inclusion membranes. The accumulation of Pls1 and, to a lesser extent, Pls2 in the inclusion lumen was insensitive to the type III secretion inhibitor C1, suggesting that this translocation system is not essential for Pls protein secretion. In contrast, Pls secretion and stability were sensitive to low levels of β-lactam antibiotics, suggesting that a functional cell wall is required for Pls secretion from the bacterial cell. Finally, we tested the requirement for these proteins in Chlamydia infection by microinjecting anti-Pls1 and anti-Pls2 antibodies into infected cells. Coinjection of anti-Pls1 and -Pls2 antibodies partially inhibited expansion of the inclusion. Because Pls proteins lack classical sec-dependent secretion signals, we propose that Pls proteins are secreted into the inclusion lumen by a novel mechanism to regulate events important for chlamydial replication and inclusion expansion.

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Faculty Opinions recommendation of Chlamydia psittaci inclusion membrane protein IncB associates with host protein Snapin.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718359715.793538725 ◽

2017 ◽

Author(s):

Fabienne Paumet ◽

Jordan Wesolowski

Keyword(s):

Membrane Protein ◽

Chlamydia Psittaci ◽

Host Protein ◽

Inclusion Membrane

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Structural difference between the 5' termini of viral and cellular mRNA in poliovirus-infected cells: possible basis for the inhibition of host protein synthesis.

Journal of Virology ◽

10.1128/jvi.18.2.719-726.1976 ◽

1976 ◽

Vol 18 (2) ◽

pp. 719-726 ◽

Cited By ~ 82

Author(s):

R Fernandez-Munoz ◽

J E Darnell

Keyword(s):

Protein Synthesis ◽

Structural Difference ◽

Host Protein ◽

Infected Cells ◽

Host Protein Synthesis

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Hepatitis C Virus Regulates its Replication by Maturing miR-122 Through Akt-Dependent Phosphorylation of KSRP

10.1101/654392 ◽

2019 ◽

Author(s):

Camille Baudesson ◽

Céline Amadori ◽

Hassan Danso ◽

Flora Donati ◽

Quentin Nevers ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Direct Interaction ◽

Micro Rna ◽

Host Protein ◽

Hcv Rna ◽

Serine Residue ◽

Reciprocal Regulation ◽

Mrna Targets ◽

Infected Cells

AbstractThe liver-specific micro-RNA-122 (miR-122) is required for the replication of hepatitis C virus (HCV). The direct interaction between miR-122 and the 5’ untranslated region of the HCV genome promotes viral replication and protects HCV RNA from degradation. Because HCV RNA is its own substrate for replication, infected cells are submitted to the sequestration of increasing levels of miR-122 and to global de-repression of host miR-122 mRNA targets. Whether and how HCV regulates miR-122 maturation to create an environment favorable to its replication remains unexplored. We discovered that Akt-dependent phosphorylation of KSRP host protein at Serine residue 193 is essential for miR-122 maturation in hepatocytes. Moreover, we showed the existence of a reciprocal regulation loop where HCV replication can modulate the proviral effect mediated by KSRP-dependent maturation of miR-122. These data support a mechanism by which HCV regulates the expression of miR-122 by hijacking KSRP, thereby fueling its own replication.

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