Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms

Abstract

Summary Chlamydia trachomatis is an obligate intracellular pathogen responsible for ocular and genital infections of significant public health importance. C. trachomatis undergoes a biphasic developmental cycle alternating between two distinct forms: the infectious elementary body (EB), and the replicative but non-infectious reticulate body (RB). The molecular basis for these developmental transitions and the metabolic properties of the EB and RB forms are poorly understood as these bacteria have traditionally been difficult to manipulate through classical genetic approaches. Using two-dimensional liquid chromatography – tandem mass spectrometry (LC/LC-MS/MS) we performed a large-scale, label-free quantitative proteomic analysis of C. trachomatis LGV-L2 EB and RB forms. Additionally, we carried out LC-MS/MS to analyze the membranes of the pathogen-containing vacuole (“inclusion”). We developed a label-free quantification approaches to measure protein abundance in a mixed-proteome background which we applied for EB and RB quantitative analysis. In this manner, we catalogued the relative distribution of >54% of the predicted proteins in the C. trachomatis LGV-L2 proteome. Proteins required for central metabolism and glucose catabolism were predominant in the EB, whereas proteins associated with protein synthesis, ATP generation and nutrient transport were more abundant in the RB. These findings suggest that the EB is primed for a burst in metabolic activity upon entry, whereas the RB form is geared towards nutrient utilization, a rapid increase in cellular mass, and securing the resources for an impending transition back to the EB form. The most revealing difference between the two forms was the relative deficiency of cytoplasmic factors required for efficient Type III secretion (T3S) in the RB stage at 18 hpi, suggesting a reduced T3S capacity or a low frequency of active T3S apparatus assembled on a “per organism” basis. Our results show that EB and RB proteomes are streamlined to fulfill their predicted biological functions: maximum infectivity for EBs and replicative capacity for RBs.

Biochemical and Structural Insights into Microtubule Perturbation by CopN from Chlamydia pneumoniae

“…encompassing the C-terminal disordered region). Regarding the time of CopN expression, proteomic data show that tCopN accumulates in the infectious elementary bodies, whereas it is not detected in the replicative reticulate bodies (51). The transcription profiles of copN also strongly argue for an accumulation of the protein late in elementary bodies (52,53).…”

Metabolic-Stress-Induced Rearrangement of the 14-3-3ζ Interactome Promotes Autophagy via a ULK1- and AMPK-Regulated 14-3-3ζ Interaction with Phosphorylated Atg9

“…Three microliters of peptide sample digests was analyzed by using a nanoAcquity UPLC system with 2D Technology coupled to a Synapt G2 HDMS mass spectrometer (Waters Corp., Milford, MA), using a method that generated five fractions at pH 10 with approximately equal loading in each fraction, which was similar to methods described previously (20)(21)(22). The sample was first trapped at 2 l/ min at a 97/3 (vol/vol) water/methyl cyanide (MeCN) ratio in 20 mM ammonium formate (pH 10) on a 5-m XBridge BEH130 C 18 300-m by 50-mm column.…”

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.…”