Quantitative Proteomics of the Infectious and Replicative Forms of Chlamydia trachomatis

Abstract

The obligate intracellular developmental cycle of Chlamydia trachomatis presents significant challenges in defining its proteome. In this study we have applied quantitative proteomics to both the intracellular reticulate body (RB) and the extracellular elementary body (EB) from C. trachomatis. We used C. trachomatis L2 as a model chlamydial isolate for our study since it has a high infectivity:particle ratio and there is an excellent quality genome sequence. EBs and RBs (>99% pure) were quantified by chromosomal and plasmid copy number using PCR, from which the concentrations of chlamydial proteins per bacterial cell/genome were determined. RBs harvested at 15h post infection (PI) were purified by three successive rounds of gradient centrifugation. This is the earliest possible time to obtain purified RBs, free from host cell components in quantity, within the constraints of the technology. EBs were purified at 48h PI. We then used two-dimensional reverse phase UPLC to fractionate RB or EB peptides before mass spectroscopic analysis, providing absolute amount estimates of chlamydial proteins. The ability to express the data as molecules per cell gave ranking in both abundance and energy requirements for synthesis, allowing meaningful identification of rate-limiting components. The study assigned 562 proteins with high confidence and provided absolute estimates of protein concentration for 489 proteins. Interestingly, the data showed an increase in TTS capacity at 15h PI. Most of the enzymes involved in peptidoglycan biosynthesis were detected along with high levels of muramidase (in EBs) suggesting breakdown of peptidoglycan occurs in the non-dividing form of the microorganism. All the genome-encoded enzymes for glycolysis, pentose phosphate pathway and tricarboxylic acid cycle were identified and quantified; these data supported the observation that the EB is metabolically active. The availability of detailed, accurate quantitative proteomic data will be invaluable for investigations into gene regulation and function.

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

“…Our overall findings of increased T3SS capacity and carbohydrate metabolism in the EB form are in contrast to the recent study in the L2 strain (14), but support previous observations from the L2 strain (13). The reasons for the discrepancy between the two studies investigating the same invasive strain are not known.…”

“…S3 B ) as has been reported for studies where 2D PAGE is used as separation step (32). The initial SDS-PAGE separation step allowed a higher amount of starting material and less complex peptide mixtures to be analyzed in each run by the LC-MS/MS system, and led to identification of 91 novel C. trachomatis proteins in this study compared with previous proteome studies (12–14) (Fig. 2 A ).…”

“…A , Venn diagram of the number of proteins from the D/UW-3X/CX strain identified in this study by a minimum of two peptides compared with previous studies on the L2 strain (12–14). B , Venn diagram showing the overlap of the proteins identified and quantified in the RB, EB, and ARB forms, in total 635 proteins.…”

“…Recently, Skipp et al characterized the EB and RB proteomes of L2 as a model strain for C. trachomatis using 2D-RPLC-MS. The study identified 562 proteins of which 556 have orthologs in the D/UW-3/CX proteome (14). …”

Novel Detection Strategy To Rapidly Evaluate the Efficacy of Antichlamydial Agents

“…Approaches that use this reporter and other techniques have been utilized to revisit the transcriptional control of ompA encoding the major outer membrane protein (MOMP). Because it is a predominant surface-exposed protein in both EBs and RBs (17)(18)(19)(20), MOMP plays a key role in the maintenance of bacterial integration, mediates initial contact with the host cell (21), and is a promising candidate for vaccine development (22,23). Three antimicrobials used for treating infections caused by intracellular bacterial pathogens, azithromycin, rifampin, and chloramphenicol, were examined in this study.…”