Identification ofChlamydia trachomatisOuter Membrane Complex Proteins by Differential Proteomics

Abstract

The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra-and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses of Chlamydia trachomatis serovar L2 434/Bu EB, COMC, and Sarkosylsoluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.Bacteria in the phylum Chlamydiae are characterized by their complex intracellular developmental cycles. Chlamydiae must assume at least two functionally distinct morphotypes, the intracellular, replicative reticulate body (RB) and the extracellular, infectious elementary body (EB), to replicate and be transmitted to new hosts (50). The divergence of distinct RB and EB forms may have been driven by the different pressures these pathogens face inside host cells during replication and outside host cells during transmission. For example, the outer membrane of EB contains a poorly immunogenic truncated lipopolysaccharide (LPS) (14, 30) and immunodominant epitopes of the major outer membrane protein (MOMP) vary substantially among closely related chlamydial strains (13). EB also lack detectable peptidoglycan (2, 20, 60), although functional murein biosynthetic enzymes (2,5,16,21,32,43,45,46) are expressed in RB during productive and persistent infection (44). To compensate for the loss of murein, EB are enveloped by a protein P-layer, which lends osmotic stability to the infectious particle (29).Attempts to identify components of the P-layer and outer membrane proteins of Chlamydia were advanced by the observation that these layers can be separated from many soluble EB proteins using the detergent N-lauroyl sarcosine (Sarkosyl). Caldwell et al. dubbed the Sarkosyl-insoluble fraction the chlamydial outer membrane complex (COMC) and noted that purified COMC maintained the shape of intact EB and contained a complete outer membrane, and they reported that a single outer membrane protein, MOMP, accounted for more than 60% of total COMC protein content (15). Other studies revealed that the COMC is ...

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

“…Proteomic studies (40)(41)(42) have also identified aminoacyl-tRNA synthetases in EBs. Since growth of HS1 was rescued when this mutant was shifted from 40°C to 37°C as late as 18 hpi (Fig.…”

Lipooligosaccharide is required for the generation of infectious elementary bodies in Chlamydia trachomatis

“…By analogy, we hypothesize that the osmotically fragile cell membrane of RBs must reorganize during development to form the rigid, highly disulfide crosslinked surface of EBs (35,36). OmcB, OmcC, MOMP, and a large family of polymorphic membrane proteins, constitute the bulk of the Chlamydia outer membrane complex, a proteinaceous shell that provides structural integrity to the outer envelope of EBs (37,38). OmcB and OmcC are developmentally regulated with maximum transcription and translation occurring late in the infectious cycle as RBs begin to transition to EBs (39,40).…”

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

“…The intermolecular network at the outer membrane is called chlamydial outer membrane complex (COMC) and is thought to substitute for the very limited amount of chlamydial cell wall peptidoglycan, a component that provides rigidity and structural strength against osmotic pressure to the wall of Gram‐negative bacteria 22. The major constituents of COMC are MOMP and two cysteine rich proteins OmcA and OmcB 25, 26. MOMP is part of the outer shell of two distinct chlamydial life‐cycle manifestations: (1) elementary bodies (EB), which are non‐replicating but infectious and (2) reticulate bodies (RB) which are the non‐infectious, metabolically active, replicating form of the bacteria.…”