Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria

Rôle des acides mycoliques oxygénés dans la physiopathologie de la tuberculose et inhibition de la méthyltransférase Hma impliquée dans leur biosynthèse

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Distinct Spatiotemporal Dynamics of Peptidoglycan Synthesis between Mycobacterium smegmatis and Mycobacterium tuberculosis

Peptidoglycan (PG), a polymer cross-linked by d-amino acid-containing peptides, is an essential component of the bacterial cell wall. We found that a fluorescent d-alanine analog (FDAA) incorporates chiefly at one of the two poles in Mycobacterium smegmatis but that polar dominance varies as a function of the cell cycle in Mycobacterium tuberculosis: immediately after cytokinesis, FDAAs are incorporated chiefly at one of the two poles, but just before cytokinesis, FDAAs are incorporated comparably at both. These observations suggest that mycobacterial PG-synthesizing enzymes are localized in functional compartments at the poles and septum and that the capacity for PG synthesis matures at the new pole in M. tuberculosis. Deeper knowledge of the biology of mycobacterial PG synthesis may help in discovering drugs that disable previously unappreciated steps in the process

N,C-Capped Dipeptides with Selectivity for Mycobacterial Proteasome over Human Proteasomes: Role of S3 and S1 Binding Pockets

We identified N,C-capped dipeptides that are selective for the Mycobacterium tuberculosis proteasome over human constitutive and immunoproteasomes. Differences in the S3 and S1 binding pockets appeared to account for the species selectivity. The inhibitors can penetrate mycobacteria and kill nonreplicating M. tuberculosis under nitrosative stress

Disruption of an <i>M. tuberculosis</i> Membrane Protein Causes a Magnesium-dependent Cell Division Defect and Failure to Persist in Mice

<div><p>The identification of <i>Mycobacterium tuberculosis</i> genes necessary for persistence <i>in vivo</i> provides insight into bacterial biology as well as host defense strategies. We show that disruption of <i>M. tuberculosis</i> membrane protein PerM (Rv0955) resulted in an IFN-γ-dependent persistence defect in chronic mouse infection despite the mutant’s near normal growth during acute infection. The <i>perM</i> mutant required increased magnesium for replication and survival; incubation in low magnesium media resulted in cell elongation and lysis. Transcriptome analysis of the <i>perM</i> mutant grown in reduced magnesium revealed upregulation of cell division and cell wall biosynthesis genes, and live cell imaging showed PerM accumulation at the division septa in <i>M. smegmatis</i>. The mutant was acutely sensitive to β-lactam antibiotics, including specific inhibitors of cell division-associated peptidoglycan transpeptidase FtsI. Together, these data implicate PerM as a novel player in mycobacterial cell division and pathogenesis, and are consistent with the hypothesis that immune activation deprives <i>M. tuberculosis</i> of magnesium.</p></div

<i>PerM</i>::tn exhibits an Mg²⁺-dependent cell division defect.

<p>(<b>A</b>) mRNA levels for selected genes were compared by quantitative real time PCR. RNA was isolated from Mtb grown for 5 days in Sauton’s media containing 2000 or 250 μM Mg²⁺. mRNA levels were normalized to expression of the housekeeping gene <i>sigA</i>, then standardized to the wt sample grown in 2000 μM Mg²⁺ from the same experiment. Data are means ± SEM from five independent experiments. Data correlate with results obtained by microarray analysis. (<b>B</b>) Mtb was grown for 5 days at the indicated Mg²⁺ concentrations and imaged by scanning electron microscopy. Bar, 5 μm. (<b>C</b>) Lengths of bacteria from (B). Bar indicates median length. At least 80 bacteria were measured for each strain and concentration.</p

<i>PerM</i>::tn is highly sensitive to β-lactam antibiotics.

<p><i>PerM</i>::tn is highly sensitive to β-lactam antibiotics.</p

PerM accumulates at the septum during cell division.

<p>Representative image series of replicating <i>M</i>. <i>smegmatis</i> expressing PerM-GFP. Arrows point to septa. Inset corresponds to septum indicated by a dashed arrow. Numbers indicate time in hours. Scale bar, 4 μm.</p