Gilles Etienne

Extracellular enzyme activities potentially involved in the pathogenicity of Mycobacterium tuberculosis

To evaluate the potential contribution of extracellular enzymes to the pathogenicity of mycobacteria, the presence of selected enzyme activities was investigated in the culture filtrates of the obligate human pathogen Mycobacterium tuberculosis, M. bovis BCG, the opportunistic pathogens M. kansasii and M. fortuitum, and the non-pathogenic species M. phlei and M. smegmatis. For M. tuberculosis and M. bovis, 22 enzyme activities were detected in the culture filtrates and/or cell surfaces, of which eight were absent from the culture fluids of non-pathogens: alanine dehydrogenase, glutamine synthetase, nicotinamidase, isonicotinamidase, superoxide dismutase, catalase, peroxidase and alcohol dehydrogenase. These activities, which correspond to secreted enzymes, formed a significant part (up to 92%) of the total enzyme activities of the bacteria and were absent from the culture fluids and the cell surfaces of the non-pathogenic species M. smegmatis and M. phlei. The extracellular location of superoxide dismutase and glutamine synthetase seemed to be restricted to the obligate pathogens examined. The difference in the enzyme profiles was not attributable to the growth rates of the two groups of bacteria. The presence of the eight enzyme activities in the outermost compartments of obligate pathogens and their absence in those of non-pathogens provides further evidence that these enzymes may be involved in the pathogenicity of mycobacteria. In addition, the eight enzyme activities were demonstrated in the cell extract of M. smegmatis. Stepwise erosion of the cell surface of M. smegmatis to expose internal capsular constituents showed that the various enzyme activities, with the possible exception of superoxide dismutase, were located more deeply in the cell envelope of this bacterium. This suggests that the molecular architecture of the mycobacterial envelopes may play an important role in the pathogenicity of these organisms.

The impact of the absence of glycopeptidolipids on the ultrastructure, cell surface and cell wall properties, and phagocytosis of Mycobacterium smegmatis

Glycopeptidolipids (GPLs) are a class of species- or type-specific mycobacterial lipids and major constituents of the cell envelopes of many non-tuberculous mycobacteria. To determine the function of GPLs in the physiology of these bacteria, a mutant of Mycobacterium smegmatis in which the gene encoding a mycobacterial nonribosomal peptide synthetase has been inactivated by transposon mutagenesis was analysed. Labelling experiments indicated that half of the bacterial GPLs were located on the cell surface and represented 85% of the surface-exposed lipids of the parent strain whereas the mutant was defective in the production of the GPLs. Compared to the parent smooth morphotype strain, the GPL-deficient mutant strain exhibited a rough colony morphology, an increase of the cell hydrophobicity and formed huge aggregates. As a consequence, the mutant cells were no longer able to bind ruthenium red, as observed by transmission electron microscopy. The altered surface properties of the mutant cells also affected the phagocytosis of individual bacilli by human monocyte-derived macrophages since mutant cells were internalized more rapidly than cells from the parent strain. Nevertheless, no specific release of surface constituents into the culture broth of the mutant was observed, indicating that the cell surface is composed of substances other than GPLs and that these are essential for maintaining the architecture of the outermost layer of the cell envelope. Importantly, the absence of these major extractable lipids of M. smegmatis from the mutant strain has a profound effect on the uptake of the hydrophobic chenodeoxycholate by cells, indicating that GPLs are involved in the cell wall permeability barrier of M. smegmatis. Altogether, these data showed that, in addition to being distinctive markers of numerous mycobacterial species, GPLs play a role in the bacterial phenotype, surface properties and cell wall permeability.

Multidrug Resistance of a Porin Deletion Mutant of Mycobacterium smegmatis

ABSTRACT Mycobacteria contain an outer membrane of unusually low permeability which contributes to their intrinsic resistance to many agents. It is assumed that small and hydrophilic antibiotics cross the outer membrane via porins, whereas hydrophobic antibiotics may diffuse through the membrane directly. A mutant of Mycobacterium smegmatis lacking the major porin MspA was used to examine the role of the porin pathway in antibiotic sensitivity. Deletion of the mspA gene caused high-level resistance of M. smegmatis to 256 μg of ampicillin/ml by increasing the MIC 16-fold. The permeation of cephaloridine in the mspA mutant was reduced ninefold, and the resistance increased eightfold. This established a clear relationship between the activity and the outer membrane permeation of cephaloridine. Surprisingly, the MICs of the large and/or hydrophobic antibiotics vancomycin, erythromycin, and rifampin for the mspA mutant were increased 2- to 10-fold. This is in contrast to those for Escherichia coli, whose sensitivity to these agents was not affected by deletion of porin genes. Uptake of the very hydrophobic steroid chenodeoxycholate by the mspA mutant was retarded threefold, which supports the hypothesis that loss of MspA indirectly reduces the permeability by the lipid pathway. The multidrug resistance of the mspA mutant highlights the prominent role of outer membrane permeability for the sensitivity of M. smegmatis to antibiotics. An understanding of the pathways across the outer membrane is essential to the successful design of chemotherapeutic agents with activities against mycobacteria.

Gap, a mycobacterial specific integral membrane protein, is required for glycolipid transport to the cell surface

The cell envelope of mycobacteria is a complex multilaminar structure that protects the cell from stresses encountered in the environment, and plays an important role against the bactericidal activity of immune system cells. The outermost layer of the mycobacterial envelope typically contains species‐specific glycolipids. Depending on the mycobacterial species, the major glycolipid localized at the surface can be either a phenolglycolipid or a peptidoglycolipid (GPL). Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood. In this study, by using a transposon library of Mycobacterium smegmatis and a simple dye assay, six genes involved in GPLs synthesis have been characterized. All of these genes are clustered in a single genomic region of approximately 60 kb. We show by biochemical analyses that two non‐ribosomal peptide synthetases, a polyketide synthase, a methyltransferase and a member of the MmpL family are required for the biosynthesis of the GPLs backbone. Furthermore, we demonstrate that a small integral membrane protein of 272 amino acids named Gap (gap: GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologues across the mycobacterial genus, thus delineating a new protein family. This Gap family represents a new paradigm for the transport of small molecules across the mycobacterial envelope, a critical determinant of mycobacterial virulence.

pks5 -recombination-mediated surface remodelling in Mycobacterium tuberculosis emergence

Mycobacterium tuberculosis is a major, globally spread, aerosol-transmitted human pathogen, thought to have evolved by clonal expansion from a Mycobacterium canettii-like progenitor. In contrast, extant M. canettii strains are rare, genetically diverse, and geographically restricted mycobacteria of only marginal epidemiological importance. Here, we show that the contrasting evolutionary success of these two groups is linked to loss of lipooligosaccharide biosynthesis and subsequent morphotype changes. Spontaneous smooth-to-rough M. canettii variants were found to be mutated in the polyketide-synthase-encoding pks5 locus and deficient in lipooligosaccharide synthesis, a phenotype restored by complementation. Importantly, these rough variants showed an altered host–pathogen interaction and increased virulence in cellular- and animal-infection models. In one variant, lipooligosaccharide deficiency occurred via homologous recombination between two pks5 genes and removal of the intervening acyltransferase-encoding gene. The resulting single pks5 configuration is similar to that fixed in M. tuberculosis, which is known to lack lipooligosaccharides. Our results suggest that pks5-recombination-mediated bacterial surface remodelling increased virulence, driving evolution from putative generalist mycobacteria towards professional pathogens of mammalian hosts.

A Glycosyltransferase Involved in Biosynthesis of Triglycosylated Glycopeptidolipids in Mycobacterium smegmatis: Impact on Surface Properties

The cell envelope of mycobacteria is a complex structure that plays an important role in the interactions of the cell with its environment and in the protection against the antimicrobial activity of the immune system. Glycopeptidolipids (GPLs) are species- or type species-specific glycolipids that are present at the surface of a number of mycobacteria and that are characterized by a high variability in glycosylation patterns. These GPLs possess various biological activities that depend mostly on the sugars capping the core molecule. In Mycobacterium smegmatis, the GPL core can be substituted by either two or three deoxyhexoses. In this study, we show that Gtf3 is a glycosyltransferase responsible for the synthesis of the triglycosylated GPLs. Biochemical analysis of these molecules, with a combination of mass spectrometry and chemical degradation methods, has shown that they contain three deoxyhexose moieties. The presence of the triglycosylated GPLs is associated with cell surface modifications that lead to a decrease in sliding motility as well as a modification in cellular aggregation and colony appearance on Congo red. Phylogenetic analysis indicated that Gtf3 is a member of a yet-uncharacterized glycosyltransferase family conserved among the mycobacteria.

Spontaneous transposition of IS1096 or ISMsm3 leads to glycopeptidolipid overproduction and affects surface properties in Mycobacterium smegmatis.

Natural modification of the colony appearance is a phenomenon that has not been fully understood in mycobacteria. Here, we show that Mycobacterium smegmatis ATCC607 displays a low-frequency spontaneous morphological variation that correlates with the acquisition of a panel of new phenotypes, such as aggregation, biofilm formation and sliding motility. These variants produce larger amounts of glycopeptidolipid (GPL), a cell-surface component, than did the wild-type strain. This conversion results from the transposition of two types of insertion sequences, IS1096 and ISMsm3, into two loci. One locus is the promoter region of the mps operon, the GPL biosynthesis gene cluster, leading to the overexpression of these genes. The other locus is the lsr2 gene, which encodes a small basic histone-like protein that likely plays a regulatory role at the mps promoter and also controls pigment production. This study demonstrates that insertion sequence mobility play a crucial role in the acquisition of new phenotypes.

SigF Controls Carotenoid Pigment Production and Affects Transformation Efficiency and Hydrogen Peroxide Sensitivity in Mycobacterium smegmatis

Carotenoids are complex lipids that are known for acting against photodynamic injury and free radicals. We demonstrate here that sigma(F) is required for carotenoid pigment production in Mycobacterium smegmatis. We further show that a sigF mutant exhibits a transformation efficiency 10(4)-fold higher than that of the parental strain, suggesting that sigma(F) regulates the production of components affecting cell wall permeability. In addition, a sigF mutant showed an increased sensitivity to hydrogen peroxide. An in silico search of the M. smegmatis genome identified a number of SigF consensus sites, including sites upstream of the carotenoid synthesis locus, which explains its SigF regulation.

Mycobacterium abscessus Phospholipase C Expression Is Induced during Coculture within Amoebae and Enhances M. abscessus Virulence in Mice

ABSTRACT Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria.

Lack of Fusion of Azurophil Granules with Phagosomes during Phagocytosis of Mycobacterium smegmatis by Human Neutrophils Is Not Actively Controlled by the Bacterium

ABSTRACT Biogenesis of phagolysosomes is a very rapid event in neutrophils which takes place with nascent unclosed phagosomes, leading to the release of lysosomal enzymes such as β-glucuronidase in the extracellular medium. We have previously shown that, under nonopsonic conditions, both pathogenic and nonpathogenic mycobacteria uncouple phagocytosis from fusion of azurophil granules (specialized secretory lysosomes) with phagosomes. In the present study we questioned whether they actively act on neutrophils to block this process or use phagocytic receptors that negatively control the biogenesis of phagolysosomes. As for live unicellular Mycobacterium smegmatis, we observed that nonopsonic phagocytosis of heat-killed mycobacteria did not induce the release of β-glucuronidase, indicating that M. smegmatis does not actively act on the fusion process in neutrophils. In contrast, phagocytosis of unicellular M. smegmatis opsonized in immune serum or that of small nonopsonized mycobacterial aggregates restored the biogenesis of phagolysosomes. Aggregates were internalized in a CR3- and cholesterol-dependent manner as unicellular mycobacteria. However, aggregates but not unicellular bacteria triggered F-actin and Hck recruitment at the phagosomes, events that have been associated with lysosome fusion. Thus, we propose that M. smegmatis does not actively control the fusion of azurophil granules at early time points postinfection and that mycobacterial aggregates recruit large clusters of receptors at the neutrophil surface which could trap proteins implicated in the biogenesis of phagolysosomes.