Yann Bordat

Is adipose tissue a place for Mycobacterium tuberculosis persistence

Background Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), has the ability to persist in its human host for exceptionally long periods of time. However, little is known about the location of the bacilli in latently infected individuals. Long-term mycobacterial persistence in the lungs has been reported, but this may not sufficiently account for strictly extra-pulmonary TB, which represents 10–15% of the reactivation cases. Methodology/Principal Findings We applied in situ and conventional PCR to sections of adipose tissue samples of various anatomical origins from 19 individuals from Mexico and 20 from France who had died from causes other than TB. M. tuberculosis DNA could be detected by either or both techniques in fat tissue surrounding the kidneys, the stomach, the lymph nodes, the heart and the skin in 9/57 Mexican samples (6/19 individuals), and in 8/26 French samples (6/20 individuals). In addition, mycobacteria could be immuno-detected in perinodal adipose tissue of 1 out of 3 biopsy samples from individuals with active TB. In vitro, using a combination of adipose cell models, including the widely used murine adipose cell line 3T3-L1, as well as primary human adipocytes, we show that after binding to scavenger receptors, M. tuberculosis can enter within adipocytes, where it accumulates intracytoplasmic lipid inclusions and survives in a non-replicating state that is insensitive to the major anti-mycobacterial drug isoniazid. Conclusions/Significance Given the abundance and the wide distribution of the adipose tissue throughout the body, our results suggest that this tissue, among others, might constitute a vast reservoir where the tubercle bacillus could persist for long periods of time, and avoid both killing by antimicrobials and recognition by the host immune system. In addition, M. tuberculosis-infected adipocytes might provide a new model to investigate dormancy and to evaluate new drugs for the treatment of persistent infection.

Phospholipases C are involved in the virulence of Mycobacterium tuberculosis

Phospholipases C play a role in the pathogenesis of several bacteria. Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses four genes encoding putative phospholipases C, plcA, plcB, plcC and plcD. However, the contribution of these genes to virulence is unknown. We constructed four single mutants of M. tuberculosis each inactivated in one of the plc genes, a triple plcABC mutant and a quadruple plcABCD mutant. The mutants all exhibited a lower phospholipase C activity than the wild‐type parent strain, demonstrating that the four plc genes encode a functional phospholipase C in M. tuberculosis. Functional complementation of the ΔplcABC triple mutant with the individual plcA, plcB and plcC genes restored in each case about 20% of the total Plc activity detected in the parental strain, suggesting that the three enzymes contribute equally to the overall Plc activity of M. tuberculosis. RT‐PCR analysis of the plc genes transcripts showed that the expression of these genes is strongly upregulated during the first 24 h of macrophage infection. Moreover, the growth kinetics of the triple and quadruple mutants in a mouse model of infection revealed that both mutants are attenuated in the late phase of the infection emphasizing the importance of phospholipases C in the virulence of the tubercle bacillus.

Production of phthiocerol dimycocerosates protects Mycobacterium tuberculosis from the cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the early immune response to infection

The growth of Mycobacterium tuberculosis mutants unable to synthesize phthiocerol dimycocerosates (DIMs) was recently shown to be impaired in mouse lungs. However, the precise role of these molecules in the course of infection remained to be determined. Here, we provide evidence that the attenuation of a DIM‐deficient strain takes place during the acute phase of infection in both lungs and spleen of mice, and that this attenuation results in part from the increased sensitivity of the mutant to the cidal activity of reactive nitrogen intermediates released by activated macrophages. We also show that the DIM‐deficient mutant, the growth and survival of which were not impaired within resting macrophages and dendritic cells, induced these cells to secrete more tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 than the wild‐type strain. Although purified DIM molecules by themselves had no effect on the activation of macrophages and dendritic cells in vitro, we found that the proper localization of DIMs in the cell envelope of M. tuberculosis is critical to their biological effects. Thus, our findings suggest that DIM production contributes to the initial growth of M. tuberculosis by protecting it from the nitric oxide‐dependent killing of macrophages and modulating the early immune response to infection.

Capsular glucan and intracellular glycogen of Mycobacterium tuberculosis: biosynthesis and impact on the persistence in mice

Mycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen‐like α‐glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface‐exposed glucan in the physiology and virulence of these bacteria, orthologues of the glg genes involved in the biosynthesis of glycogen in Escherichia coli were identified in M. tuberculosis H37Rv and inactivated by allelic replacement. Biochemical analyses of the mutants and complemented strains indicated that the synthesis of glucan and glycogen involves the α‐1,4‐glucosyltransferases Rv3032 and GlgA (Rv1212c), the ADP‐glucose pyrophosphorylase GlgC (Rv1213) and the branching enzyme GlgB (Rv1326c). Disruption of glgC reduced by half the glucan and glycogen contents of M. tuberculosis, whereas the inactivation of glgA and Rv3032 affected the production of capsular glucan and glycogen, respectively. Attempts to disrupt Rv3032 in the glgA mutant were unsuccessful, suggesting that a functional copy of at least one of the two α‐1,4‐glucosyltransferases is required for growth. Importantly, the glgA mutant was impaired in its ability to persist in mice, suggesting a role for the capsular glucan in the persistence phase of infection. Unexpectedly, GlgB was found to be an essential enzyme.

LppX is a lipoprotein required for the translocation of phthiocerol dimycocerosates to the surface of Mycobacterium tuberculosis

Cell envelope lipids play an important role in the pathogenicity of mycobacteria, but the mechanisms by which they are transported to the outer membrane of these prokaryotes are largely unknown. Here, we provide evidence that LppX is a lipoprotein required for the translocation of complex lipids, the phthiocerol dimycocerosates (DIM), to the outer membrane of Mycobacterium tuberculosis. Abolition of DIM transport following disruption of the lppX gene is accompanied by an important attenuation of the virulence of the tubercle bacillus. The crystal structure of LppX unveils an U‐shaped β‐half‐barrel dominated by a large hydrophobic cavity suitable to accommodate a single DIM molecule. LppX shares a similar fold with the periplasmic molecular chaperone LolA and the outer membrane lipoprotein LolB, which are involved in the localization of lipoproteins to the outer membrane of Gram‐negative bacteria. Based on the structure and although an indirect participation of LppX in DIM transport cannot yet be ruled out, we propose LppX to be the first characterized member of a family of structurally related lipoproteins that carry lipophilic molecules across the mycobacterial cell envelope.

Sulfolipid Deficiency Does Not Affect the Virulence of Mycobacterium tuberculosis H37Rv in Mice and Guinea Pigs

ABSTRACT Lipids that are found only in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl-branched fatty acids, have long been thought to play a role in pathogenesis. Among these complex lipids, sulfolipids have been the most extensively studied over the last 50 years. The numerous biological effects exhibited by purified sulfolipids on phagocytic cells led to the idea that these molecules are probably important virulence factors facilitating the intracellular survival of Mycobacterium tuberculosis. However, definitive evidence to support this concept has been lacking. The recent construction of an isogenic sulfolipid-deficient mutant of M. tuberculosis H37Rv (Sirakova et al., J. Biol. Chem. 276:16833-16839, 2001) has for the first time provided the opportunity to directly assess the contribution of these complex lipids to pathogenesis. In the present study, we show that against all expectations, sulfolipid deficiency does not significantly affect the replication, persistence, and pathogenicity of M. tuberculosis H37Rv in mice and guinea pigs or in cultured macrophages.

Mycobacterium tuberculosis phoP mutant: lipoarabinomannan molecular structure

Mycobacterium tuberculosis encodes two-component signal systems. Recently, it was established that the viability of the M. tuberculosis phoP mutant is attenuated in the mouse, suggesting the requirement of the phoP gene for M. tuberculosis intracellular growth. It is now largely acknowledged that M. tuberculosis mannosylated lipoarabinomannans (ManLAM) play a key role in M. tuberculosis intramacrophagic survival by altering the macrophage functions. So ManLAM were extracted and purified from the M. tuberculosis MT103 wild-type strain and from the M. tuberculosis phoP mutant. Their two major functional domains (i) the mannooligosaccharide caps and (ii) the mannosyl phosphatidylinositol anchor were here investigated. Using capillary electrophoresis, it is demonstrated that both mutant and wild-type M. tuberculosis strains share the same capping motifs: mono-, di- and trimannosyl alpha(1-->2) units, with the same relative abundance. Using two-dimensional NMR spectroscopy, the same acyl forms were found to be shared by both strains. However, their relative abundance was quite different. Indeed, in the phoP mutant a decrease of the triacylated ManLAM and an increase of the monoacylated ManLAM were observed. The difference in the proportion of ManLAM acyl forms and the reduced virulence of the M. tuberculosis phoP mutant are discussed.

Deficiency in mycolipenate- and mycosanoate-derived acyltrehaloses enhances early interactions of Mycobacterium tuberculosis with host cells

Lipids that are uniquely found in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl‐branched long‐chain fatty acids, have long been thought to play a role in host–pathogen interactions. The recent construction by Dubey et al. (2002) Mol Microbiol 45: 1451–1459, of a Mycobacterium tuberculosis mutant that is deficient in the synthesis of the di‐ and tri‐methylbranched fatty acids, mycolipenates and mycosanoates, found in some forms of diacyltrehaloses (DAT) and polyacyltrehaloses (PAT) provided the opportunity to assess the contribution of these complex lipids to pathogenesis directly. We provide evidence that DAT/PAT deficiency affects the surface global composition of the mycobacterial cell envelope improving the efficiency with which M. tuberculosis binds to and enters phagocytic and non‐phagocytic host cells. Interestingly, this property did not affect the overall replication and persistence of the tubercle bacillus in the lungs, spleen and liver of mice infected via the respiratory or intravenous route.

Disruption of the Gene Homologous to Mammalian Nramp1 in Mycobacterium tuberculosis Does Not Affect Virulence in Mice

ABSTRACT Natural-resistance-associated macrophage protein 1 (Nramp1) is a divalent cation transporter belonging to a family of transporter proteins highly conserved in eukaryotes and prokaryotes. Mammalian and bacterial transporters may compete for essential metal ions during mycobacterial infections. The mycobacterial Nramp homolog may therefore be involved in Mycobacterium tuberculosis virulence. Here, we investigated this possibility by inactivating the M. tuberculosis Nramp1 gene (Mramp) by allelic exchange mutagenesis. Disruption of Mramp did not affect the extracellular growth of bacteria under standard conditions. However, the Mramp mutation was associated with growth impairment under conditions of limited iron availability. The Mramp mutant displayed no impairment of growth or survival in macrophages derived from mouse bone marrow or in Nramp1+/+ and Nramp1−/− congenic murine macrophage cell lines. Following intravenous challenge in BALB/c mice, counts of parental and Mramp mutant strains were similar in the lungs and spleens of the animals at all time points studied. These results indicate that Mramp does not contribute to the virulence of M. tuberculosis in mice.

The allele encoding the mycobacterial Erp protein affects lung disease in mice

Erp (exported repetitive protein), also known as P36, Pirg and Rv3810, is a member of a mycobacteria‐specific family of extracellular proteins. These proteins consist of three domains, the N‐ and C‐terminal domains are similar in all mycobacterial species, however, the central domain contains a repeated PGLTS module and differs considerably between species. The erp knockout mutant of Mycobacterium tuberculosis displays very low levels of multiplication both in macrophage cell lines and in vivo in a mouse model of infection. The high interspecies variability of the central repeated region of the Erp protein led us to investigate whether these orthologous proteins were functionally equivalent in a mouse model of tuberculosis. We expressed a gene fusion with the erp gene of Mycobacterium smegmatis, Mycobacterium leprae or M. tuberculosis in trans in an erp–M. tuberculosis mutant and found that these three alleles restored multiplication to similar levels in the spleen of infected mice. However, these alleles gave different levels of colonization in the lung, for the early time‐points. Quantitative histological analyses of the lungs of infected animals showed that the nature of the erp allele strongly affected the number and the size of lung lesions, demonstrating the importance of surface determinants for virulence and tissue damage.