Deletion of MSMEG_1350 in Mycobacterium smegmatis causes loss of epoxy-mycolic acids, fitness alteration at low temperature and resistance to a set of mycobacteriophages.

Mycobacterium smegmatis is intrinsically resistant to thiacetazone, an anti-tubercular thiourea; however we report here that it causes a mild inhibition in growth in liquid medium. Since mycolic acid biosynthesis was affected, we cloned and expressed Mycobacterium smegmatis mycolic acid methyltransferases, postulated as targets for thiacetazone in other mycobacterial species. During this analysis we identified MSMEG_1350 as the methyltransferase involved in epoxy mycolic acid synthesis since its deletion led to their total loss. Phenotypic characterization of the mutant strain showed colony morphology alterations at all temperatures, reduced growth and a slightly increased susceptibility to SDS, lipophilic and large hydrophilic drugs at 20 °C with little effect at 37 °C. No changes were detected between parental and mutant strains in biofilm formation, sliding motility or sedimentation rate. Intriguingly, we found that several mycobacteriophages severely decreased their ability to form plaques in the mutant strain. Taken together our results prove that, in spite of being a minor component of the mycolic acid pool, epoxy-mycolates are required for a proper assembly and functioning of the cell envelope. Further studies are warranted to decipher the role of epoxy-mycolates in the M. smegmatis cell envelope.

Mycelial Growth and Antimicrobial Activity of Pleurotus Species (Agaricomycetes).

The objective of this study was to analyze mycelial growth under different culture conditions and antimicrobial activity of Pleurotus ostreatus (Jacq.: Fr.) Kumm. (DPUA 1533) and P. ostreatus (Jacq.: Fr.) Kumm. cv. Florida (DPUA 1534) against fungi and bacteria of medical importance. The growth of Pleurotus species was evaluated in natural and complex media, with and without light, at 25°C and 28°C for 8, 15, and 30 days. Candida albicans (DPUA 1336), Cryptococcus laurentii (DPUA 1501), Aspergillus flavus (DPUA 1836), Escherichia coli (DAUPE 224), and Mycobacterium smegmatis (ATCC 607) were used to test antibiosis. Under all growth conditions in vitro, Pleurotus species evidenced growth and high density of mycelia on potato dextrose agar and Sabouraud agar with yeast extract; mycelial growth but lesser mycelial density was observed on rice bran extract agar. Organic mycelial extracts of Pleurotus species exhibited potential antibacterial and antifungal activity, and were selective for the tested microorganisms.

Impairment of D-alanine biosynthesis in Mycobacterium smegmatis determines decreased intracellular survival in human macrophages.

d-Alanine is a structural component of mycobacterial peptidoglycan. The primary route of d-alanine biosynthesis in eubacteria is the enantiomeric conversion from l-alanine, a reaction catalysed by d-alanine racemase (Alr). Mycobacterium smegmatis alr insertion mutants are not dependent on d-alanine for growth and display a metabolic pattern consistent with an alternative pathway for d-alanine biosynthesis. In this study, we demonstrate that the M. smegmatis alr insertion mutant TAM23 can synthesize d-alanine at lower levels than the parental strain. The insertional inactivation of the alr gene also decreases the intracellular survival of mutant strains within primary human monocyte-derived macrophages. By complementation studies, we confirmed that the impairment of alr gene function is responsible for this reduced survival. Inhibition of superoxide anion and nitric oxide formation in macrophages suppresses the differential survival. In contrast, for bacteria grown in broth, both strains had approximately the same susceptibility to hydrogen peroxide, acidified sodium nitrite, low pH and polymyxin B. In contrast, TAM23 exhibited increased resistance to lysozyme. d-Alanine supplementation considerably increased TAM23 viability in nutritionally deficient media and within macrophages. These results suggest that nutrient deprivation in phagocytic cells combined with killing mediated by reactive intermediates underlies the decreased survival of alr mutants. This knowledge may be valuable in the construction of mycobacterial auxotrophic vaccine candidates.

Internalization of a non-pathogenic mycobacteria by macropinocytosis in human alveolar epithelial A549 cells.

Mycobacterium smegmatis (MSM) a non-pathogenic mycobacterium is often employed as a tool to understand many aspects of the mycobacterial infections. However, its own biology and particularly its mechanism of entry into non-phagocytic cells are not well known. Previously, we demonstrated that Mycobacterium tuberculosis (MTB) invades epithelial cells by macropinocytosis. In the present study, we investigated whether MSM also invades human epithelial type II pneumocytes (A549) by macropinocytosis. Infection of A549 cells with MSM elicited actin filaments redistribution, lamellipodia formation and increased fluid phase uptake, suggesting macropinocytosis. Furthermore, macropinocytosis inhibitors like cytochalasin D and amiloride caused inhibition of fluid phase and bacterial uptake. We can conclude that MSM, like MTB, takes advantage of macropinocytosis for entry into epithelial cells, however, unlike MTB, internalized MSM are killed by host cells. These findings suggest that induction of macropinocytosis and cell invasion is not an exclusive feature of pathogenic organisms.

Mycobacterium bovis bacillus Calmette-Guérin induces TLR2-mediated formation of lipid bodies: intracellular domains for eicosanoid synthesis in vivo.

Differentiation of macrophages into foamy (lipid-laden) macrophages is a common pathological observation in tuberculous granulomas both in experimental settings as well as in clinical conditions; however, the mechanisms that regulate intracellular lipid accumulation in the course of mycobacterial infection and their significance to pathophysiology of tuberculosis are not well understood. In this study, we investigated the mechanisms of formation and function of lipid-laden macrophages in a murine model of tuberculosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG), but not Mycobacterium smegmatis, induced a dose- and time-dependent increase in lipid body-inducible nonmembrane-bound cytoplasmic lipid domain size and numbers. Lipid body formation was drastically inhibited in TLR2-, but not in TLR4-deficient mice, indicating a role for TLR2 in BCG recognition and signaling to form lipid bodies. Increase in lipid bodies during infection correlated with increased generation of PGE2 and localization of cyclooxygenase-2 within lipid bodies. Moreover, we demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid that lipid bodies were the predominant sites of PGE2 synthesis in activated macrophages. Our findings demonstrated that BCG-induced lipid body formation is TLR2 mediated and these structures function as signaling platforms in inflammatory mediator production, because compartmentalization of substrate and key enzymes within lipid bodies has impact on the capacity of activated leukocytes to generate increased amounts of eicosanoids during experimental infection by BCG.

Binding of alpha2-laminins by pathogenic and non-pathogenic mycobacteria and adherence to Schwann cells.

The ability of Mycobacterium leprae to specifically bind alpha2-laminins of Schwann cells has been described recently as being an important property of the leprosy bacillus, which could explain the neural tropism of M. leprae. Therefore, the extent of the expression of alpha2-laminin-binding properties among mycobacteria was investigated. In an ELISA-based assay, all three species of Mycobacterium tested (M. tuberculosis, M. chelonae and M. smegmatis) expressed laminin-binding capacity, suggesting that the ability to bind alpha2-laminins is conserved within the genus Mycobacterium. This report also demonstrated that not only M. leprae but all the mycobacterial species tested readily interacted with the ST88-14 cells, a human schwannoma cell line, and that the addition of soluble alpha2-laminins significantly increased their adherence to these cells. These results failed to demonstrate the presence in M. leprae of a unique system based on alpha2-laminins for adherence to Schwann cells.

[Sliding motility and biofilm formation in mycobacteria]. / Movilidad por deslizamiento y formación de biofilms en las micobacterias.

Using as a model Mycobacterium smegmatis, a non-motile microorganism, we have studied for the first time in mycobacteria the phenomenon known as sliding motility, as well as the process of biofilm formation. A screen of random transposon mutants was performed in order to identify the genes required for mycobacterial sliding over the surface of motility plates. The genetic analysis described here has been published recently. The genes required for sliding and for biofilm formation (mps and tmtpC) are involved in the biosynthesis of the glycopeptidolipids (GPLs) and their transport to the mycobacterial capsule. Based on our results, we suggest a model for the role of the GPLs in both phenomena.