Expression and Localization of Predicted Inclusion Membrane Proteins in Chlamydia trachomatis

Abstract

Chlamydia trachomatis is 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 for C. 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. Using Yersinia as 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. Chlamydiae are Gram-negative obligate intracellular pathogens that are of human and veterinary importance. Chlamydia trachomatis is a major cause of bacterial sexually transmitted infections and is the causative agent of trachoma, a leading cause of infectious blindness. The species includes at least 15 serovars that differ in disease state and severity, including trachoma (serovars A to C), a sexually transmitted infection (serovars D to K), and a systemic disease referred to as lymphogranuloma venereum (LGV) (serovars L1 to L3) (1). C. pneumoniae is a respiratory pathogen that is a common cause of pneumonia and bronchitis and is associated with chronic diseases, such as atherosclerosis (2). C. psittaci is a zoonotic pathogen associated with a rare but serious pneumonia (3). Other Chlamydia species, including C. muridarum, C. caviae, and C. felis, are associated with other animals, i.e., mice, guinea pigs, and cats, respectively (4-6).All chlamydiae exhibit a biphasic developmental cycle in which the bacteria alternate between an infectious, metabolically dormant elementary body (EB) and the replicative form, referred to as the reticulate body (RB) (7). Upon infection, the EB is internalized by endocytosis into a membrane-bound compartment that is rapidly modified by the pathogen to establish a replicative niche termed the inclusion (8). The inclusion avoids f...

Quantitative Protein Profiling of Chlamydia trachomatis Growth Forms Reveals Defense Strategies Against Tryptophan Starvation

“…5 A , supplemental Table S3). In addition, the group of uncategorized proteins included the inclusion membrane proteins (Inc proteins), as described (24, 25, 28). We found enrichment of this group represented by IncA, IncG, Ct147, Ct223, Ct228, Ct229, Ct249, and Ct618 previously described as encoded by “early genes” (35), and confirmed the presence of these proteins predominantly in the RB form.…”

“…The identified proteins were annotated in functional categories based on the information available at http://stdgen.northwestern.edu, and supplemented according to results from the literature (24–28), KEGG (http://www.genome.jp/kegg/), Uniprot (http://www.uniprot.org/) and NCBI http://www.ncbi.nlm.nih.gov/protein/). Unknown, uncategorized, hypothetical, and “other category” proteins were grouped together as uncategorized proteins.…”

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

“…C. trachomatis serovar L2 was transformed with pACT-incA as previously described (14,15,28). Plasmid DNA was transformed into C. trachomatis serovar L2 (LGV 434/Bu) density gradient-purified EBs using CaCl 2 buffer (10 mM Tris [pH 7.5], 50 mM CaCl 2 ).…”

“…The presence of this domain has been used to predict up to 59 Incs in C. trachomatis (8)(9)(10)(11); the number of Incs varies in other chlamydial species. Generation of specific antibodies (8,9,12,13) and expression of epitope-tagged recombinant Incs in C. trachomatis (14,15) have validated at least 37 of these as bona fide Incs; however, the function of most of these proteins is unknown.…”

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

“…Fluorophore-encoding genes can be substituted with a gene of interest for expression under the control of the incD promoter. (B) pBOMB4 vectors (21) are derived from an intact thase kinase), and TEM-1 ␤-lactamase, can be used to monitor effector secretion and translocation (20,21,29,(149)(150)(151)(152).…”