Mycobacterium tuberculosis modulates its cell surface via an oligopeptide permease (Opp) transport system

Abstract

Bacterial species utilize a vast repertoire of surface structures to interact with their surroundings and employ a number of strategies to reconfigure the cellular envelope according to specific stimuli. Gram-positive bacteria, exemplified by Streptomyces and Bacillus species, control production of some exposed molecules by importing oligopeptide signals via permeases (Opp). Such oligopeptides modulate intracellular signaling pathways. In this work, we functionally characterized an Opp of the human pathogen Mycobacterium tuberculosis (Mtb) and propose its reannotation. Using genome-wide transcriptional profiling, we found that Opp was required to modulate (fold-change ranging from -3.5 to 2.0) the expression of several genes, most of them encoding surface-exposed molecules. These included the virulence-associated lipids mycolic acids and phthiocerol dimycocerosates (PDIMs) as well as PE-family proteins. By thin-layer chromatography and MALDI-TOF-MS we confirmed changes in the lipid profile, including an altered accumulation of triacylglycerides and an affected ratio of mycolic acids to PDIMs. An Opp loss of function mutant showed no in vitro growth defect, but had diminished burden during chronic infection and produced a slightly delayed time to death of animals when compared to WT Mtb infection.

Figure 1.

Functional characterization and reannotation of the M. tuberculosis oligopeptide (Opp) transport system. A) WT M. tuberculosis and the dpp, PDIM− (fadD26), and opp mutants, including the complemented (Comp) opp mutant, were cultured on solid medium in the absence (left plate, NO) or presence (right plate, YES) of bialaphos at a 5 μg/ml concentration, and incubated at 37°C for 3 wk. B) WT, opp mutant (opp KO), and its complemented strains were plated for single colonies on solid medium and incubated for 7 wk at 37°C. C) Growth curve (CFU/ml) of WT, opp KO, and complemented strains.

Figure 2.

Regulation of genes encoding proteins predicted to be involved in PDIMs, mycolic acids, PPE-family, ESAT-6/CFP-10, carbon, and intermediary metabolism. Yellow-blue display summarizes the regulation of selected Opp-modulated genes, comparing WT vs. mutant (opp KO) expression. cDNA ratios of statistically significantly affected genes were averaged, log₂ transformed, and displayed according to the color code at the top of the display. Black fields indicate genes with no significant change at a particular growth phase. Experimental conditions were as indicated in Materials and Methods. Genes were selected based on a demonstrated or presumed function of the encoded proteins and grouped in the following categories: virulence-associated lipids (A) and proteins (B) or involved in carbon (C) or intermediary metabolism (D).

Figure 3.

PDIM biosynthesis, Myc biosynthesis, and TAGs are modulated via Opp. A) TLC analyses of lipids extracted from the M. tuberculosis WT, opp mutant (opp KO), and complemented (comp) strains. Arrows indicate the positions of PDIM-A, PDIM-B, and triacylglycerol (TAG). B–D) MALDI-TOF mass spectra of PDIM and mycolic acids isolated from M. tuberculosis WT (B), opp mutant (opp KO) (C), and complemented strains (D), at different growth stages. OD, optical density 600 nm.

Figure 4.

Lack of Opp affects the chronic phase of tuberculosis infection in mice. A, B) Bacterial CFUs (y axis) present in mouse lungs (A) and spleens (B) were log₁₀ transformed and plotted against time postinfection (x axis). C) Survival kinetics of WT BALB/c mice infected via aerosol with 10³ CFUs of either M. tuberculosis WT (circles), opp KO (squares), or the complemented opp KO strain (triangles). Surviving mice (y axis) are plotted throughout the time postinfection (x axis). D, E) Representative photographs of gross pathology (D) and histopathology (E) of bacterial infected tissues. Scale bars = 0.5 mm.