Revisiting the expression signature of pks15/1 unveils regulatory patterns controlling phenolphtiocerol and phenolglycolipid production in pathogenic mycobacteria.

One of the most important and exclusive characteristics of mycobacteria is their cell wall. Amongst its constituent components are two related families of glycosylated lipids, diphthioceranates and phthiocerol dimycocerosate (PDIM) and its variant phenolic glycolipids (PGL). PGL have been associated with cell wall impermeability, phagocytosis, defence against nitrosative and oxidative stress and, intriguingly, biofilm formation. In bacteria from the Mycobacterium tuberculosis complex (MTBC), the biosynthetic pathway of the phenolphthiocerol moiety of PGL depends upon the expression of several genes encoding type I polyketide synthases (PKS), namely ppsA-E and pks15/1 which constitute the PDIM + PGL locus, and that are highly conserved in PDIM/PGL-producing strains. Consensus has not been achieved regarding the genetic organization of pks15/1 locus and knowledge is lacking on its transcriptional signature. Here we explore publicly available datasets of transcriptome data (RNA-seq) from more than 100 MTBC experiments in 40 growth conditions to outline the transcriptional structure and signature of pks15/1, using a differential expression approach to infer the regulatory patterns involving these and related genes. We show that pks1 expression is highly correlated with fadD22, Rv2949c, lppX, fadD29 and, also, pks6 and pks12, with the first three putatively integrating into a polycistronic structure. We evidence dynamic transcriptional heterogeneity within the genes involved in phenolphtiocerol and phenolic glycolipid production, most exhibiting up-regulation upon acidic pH and antibiotic exposure and down-regulation under hypoxia, dormancy, and low/high iron concentration. We finally propose a model based on transcriptome data in which σD positively regulates pks1, pks15 and fadD22, while σB and σE factors exert negative regulation at an upper level.

Small RNAs in mycobacteria: an unfolding story.

Mycobacteria represent a class of powerful pathogens, including those causing tuberculosis and leprosy, which continue to be worldwide health challenges. In the last 20 years, an abundance of non-coding, small RNAs (sRNAs) have been discovered in model bacteria and gained significant attention as regulators of cellular responses, including pathogenesis. Naturally, a search in mycobacteria followed, revealing over 200 sRNAs thus far. Characterization of these sRNAs is only beginning, but differential expression under environmental stresses suggests relevance to mycobacterial pathogenesis. This review provides a comprehensive overview of the current knowledge of sRNAs in mycobacteria, including historical perspective and techniques used for identification and characterization.

Targeting the global regulator Lsr2 as a novel approach for anti-tuberculosis drug development.

Leprosy serum reactive clone 2 (Lsr2; Rv3597c) is a recently identified nucleoid-associated protein that acts as a global transcriptional regulator of Mycobacterium tuberculosis. Strikingly, Lsr2 appears to play a critical role in controlling the expression of virulence-associated genes. Here the authors outline the current knowledge concerning this novel global regulator and its potential as a target for chemotherapeutic intervention. Compounds that induce high level expression of lsr2 may lead to abolishment of virulence traits and render the bacterium incapable of causing infection and/or disease. Alternatively, compounds that either silence lsr2 expression or block the protein's function could be lethal since it has been postulated that lsr2 is essential in M. tuberculosis.

Are phylogenetic position, virulence, drug susceptibility and in vivo response to treatment in mycobacteria interrelated?

Phylogenetic analyses on the basis of multiple house-keeping genes and whole genome sequences have offered new insights in the phylogeny of the genus Mycobacterium. This genus yields obligate pathogens, the M. tuberculosis complex and M. leprae, as well as opportunistic pathogens (e.g. M. avium, M. intracellulare, M. kansasii, M. marinum, M. malmoense) and saprophytes (e.g. M. phlei, M. sphagni, M. gordonae). The most virulent mycobacteria, the M. tuberculosis complex, M. leprae and the M. kansasii-M. szulgai-M. marinum-M. ulcerans group are phylogenetically related and infections by these organisms are better treatable than those caused by less virulent and phylogenetically more distantly related Mycobacterium species. The most virulent Mycobacterium species are also characterized by high levels of natural drug susceptibility. In this paper, we review studies of phylogeny, drug susceptibility, and clinical significance to support our hypothesis that drug susceptibility in mycobacteria is acquired and reflects the low level of competition in -and adaptation to- a closer-to-human (environmental) niche. In turn, mycobacteria that inhabit the most competitive environmental niches are the least adapted to humans, thus of low clinical significance, but most tolerant to antibiotics derived from microbes with which they share their habitat, lowering the chances of cure in case of infection.

[Novel type of antimicrobial mechanism in host macrophages against mycobacterial infections].

Macrophages (M(phi)s) play a central role as anti-microbial effector cells in the expression of host resistance to mycobacterial infections. With respect to antimicrobial effector molecules of host M(phi) against mycobacterial pathogens, recent studies suggest the possibility that the reactive nitrogen intermediates (RNI)--and reactive oxygen intermediates-independent antimycobacterial mechanism(s) may be crucial for the antimycobacterial function of host M(phi). In this context, we previously found that free fatty acids (FFAs) such as arachidonic acid (AA) and linolenic acid exhibited potent antimicrobial activity against mycobacterial organisms, including Mycobacterium tuberculosis (MTB) and Mycobacterium avium complex (MAC). In addition, FFAs in combination with RNI played critical roles in manifestation of the activity of M(phi) against mycobacterial organisms. Moreover, our recent studies have shown the following findings. First, anti-MTB activity of IFN-gamma-activated M(phi)s was specifically blocked by arachidonyl trifluoromethylketone (aTFMK), an inhibitor of cytosolic phospholipase A2 (cPLA2). Second, ATP potentiated the anti-MAC bactericidal activity of M(phi)s cultivated in the presence of clarithromycin and rifamycin. This effect of ATP was closely related to intracellular Ca2+ mobilization and was specifically blocked by aTFMK. Third, intramacrophage translocation of membranous AA molecules to MAC-containing phagosomes was also specifically blocked by aTFMK. In the confocal microscopic observation of MAC-infected M(phi)s, ATP enhanced the intracellular translocation of cPLA2 into MAC-containing phagosomes. These findings suggest that FFAs (especially AA) produced by the enzymatic action of cPLA2 play important roles as antimycobacterial effectors in the expression of M(phi) antimicrobial activity against mycobacterial pathogens.

Genome scale portrait of cAMP-receptor protein (CRP) regulons in mycobacteria points to their role in pathogenesis.

cAMP Receptor Protein (CRP)/Fumarate Nitrate Reductase Regulator (FNR) family proteins are ubiquitous regulators of cell stress in eubacteria. These proteins are commonly associated with maintenance of intracellular oxygen levels, redox-state, oxidative and nitrosative stresses, and extreme temperature conditions by regulating expression of target genes that contain regulatory cognate DNA elements. We describe the use of informatics enabled comparative genomics to identify novel genes under the control of CRP regulator in Mycobacterium tuberculosis (M.tb). An inventory of CRP regulated genes and their operon context in important mycobacterial species such as M. leprae, M. avium subsp. paratuberculosis and M. smegmatis and several common genes within this genus including the important cellular functions, mainly, cell-wall biogenesis, cAMP signaling and metabolism associated with such regulons were identified. Our results provide a possible theoretical framework for better understanding of the stress response in mycobacteria. The conservation of the CRP regulated genes in pathogenic mycobacteria, as opposed to non-pathogenic ones, highlights the importance of CRP-regulated genes in pathogenesis.

Inactivation of Rv2525c, a substrate of the twin arginine translocation (Tat) system of Mycobacterium tuberculosis, increases beta-lactam susceptibility and virulence.

The twin arginine translocation (Tat) system is used by many bacteria to export fully folded proteins containing cofactors. Here, we show genetically that this system is essential for Mycobacterium tuberculosis, as the tatAC operon and tatB genes could be inactivated only in partially diploid strains. Using comparative genomics, the rv2525c gene of M. tuberculosis was identified as encoding a histidine-rich protein, with a twin arginine signal peptide, and orthologous genes were shown to be present in several but not all actinobacterial species. Conservation of this gene by Mycobacterium leprae, which has undergone reductive evolution, suggested an important role for rv2525c. An rv2525c knockout mutant was constructed, and biochemical analysis indicated that the mature Rv2525c protein is secreted. Upon exposure to antituberculous drugs, rv2525c expression is significantly up-regulated together with those of other genes involved in cell wall biogenesis. Phenotypic comparison of the mutant with the parental strain revealed an increase in susceptibility to some beta-lactam antibiotics and, despite slower growth in vitro, enhanced virulence in both cellular and murine models of tuberculosis. The Tat system thus contributes in multiple ways to survival of the tubercle bacillus.

Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak.

Genealogy can illuminate the evolutionary path of important human pathogens. In some microbes, strict clonal reproduction predominates, as with the worldwide dissemination of Mycobacterium leprae, the cause of leprosy. In other pathogens, sexual reproduction yields clones with novel attributes, for example, enabling the efficient, oral transmission of the parasite Toxoplasma gondii. However, the roles of clonal or sexual propagation in the origins of many other microbial pathogen outbreaks remain unknown, like the recent fungal meningoencephalitis outbreak on Vancouver Island, Canada, caused by Cryptococcus gattii. Here we show that the C. gattii outbreak isolates comprise two distinct genotypes. The majority of isolates are hypervirulent and have an identical genotype that is unique to the Pacific Northwest. A minority of the isolates are significantly less virulent and share an identical genotype with fertile isolates from an Australian recombining population. Genotypic analysis reveals evidence of sexual reproduction, in which the majority genotype is the predicted offspring. However, instead of the classic a-alpha sexual cycle, the majority outbreak clone appears to have descended from two alpha mating-type parents. Analysis of nuclear content revealed a diploid environmental isolate homozygous for the major genotype, an intermediate produced during same-sex mating. These studies demonstrate how cryptic same-sex reproduction can enable expansion of a human pathogen to a new geographical niche and contribute to the ongoing production of infectious spores. This has implications for the emergence of other microbial pathogens and inbreeding in host range expansion in the fungal and other kingdoms.

Characterization of a haemolysin from Mycobacterium tuberculosis with homology to a virulence factor of Serpulina hyodysenteriae.

Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysin/cytotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenteriae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tuberculosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium avium and Mycobacterium bovis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His6-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bovis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in vitro.

Gene knockout reveals a novel gene cluster for the synthesis of a class of cell wall lipids unique to pathogenic mycobacteria.

Surface-exposed unusual lipids containing phthiocerol and phenolphthiocerol are found only in the cell wall of slow-growing pathogenic mycobacteria and are thought to play important roles in host-pathogen interaction. The enzymology and molecular genetics of biosynthesis of phthiocerol and phenolphthiocerol are unknown. We postulate the domain organization of a set of multifunctional enzymes and a cluster of genes (pps) that would encode these enzymes for the biosynthesis of phthiocerol and phenolphthiocerol. A cosmid containing the postulated pps gene cluster was identified by screening a genomic library of Mycobacterium bovis BCG with the postulated homologous domains from mycocerosic acid synthase and fatty acid synthase genes as probes. Homologous cosmids were also identified in the genomic libraries of Mycobacterium tuberculosis and Mycobacterium leprae. M. bovis BCG was transformed with a pps disruption construct, made from the BCG cosmid by introducing the hygromycin resistance gene as the positive-selectable marker and the sacB gene as the counter-selectable marker. Gene disruption by homologous recombination with double crossover was confirmed by polymerase chain reaction and Southern hybridization. Chromatographic analysis showed that the phenolphthiocerol derivative, mycoside B, and phthiocerol dimycocerosates were not produced by the gene knockout mutants. This result confirms the identity of the pps genes. With the identification of the pps gene clusters in both M. tuberculosis and M. leprae, it should be possible to test the postulated roles of these unique lipids in tuberculosis and leprosy.

Effects of overexpression of the alkyl hydroperoxide reductase AhpC on the virulence and isoniazid resistance of Mycobacterium tuberculosis.

Mutations to the regulatory region of the ahpC gene, resulting in overproduction of alkyl hydroperoxide reductase, were encountered frequently in a large collection of isoniazid (INH)-resistant clinical isolates of Mycobacterium tuberculosis but not in INH-susceptible strains. Overexpression of ahpC did not seem to be important for INH resistance, however, as most of these strains were already defective for catalase-peroxidase, KatG, the enzyme required for activation of INH. Transformation of the INH-susceptible reference strain, M. tuberculosis H37Rv, with plasmids bearing the ahpC genes of M. tuberculosis or M. leprae did not result in a significant increase in the MIC. Two highly INH-resistant mutants of H37Rv, BH3 and BH8, were isolated in vitro and shown to produce no or little KatG activity and, in the case of BH3, to overproduce alkyl hydroperoxide reductase as the result of an ahpC regulatory mutation that was also found in some clinical isolates. The virulence of H37Rv, BH3, and BH8 was studied intensively in three mouse models: fully immunocompetent BALB/c and Black 6 mice, BALB/c major histocompatibility complex class II-knockout mice with abnormally low levels of CD4 T cells and athymic mice producing no cellular immune response. The results indicated that M. tuberculosis strains producing catalase-peroxidase were considerably more virulent in immunocompetent mice than the isogenic KatG-deficient mutants but that loss of catalase-peroxidase was less important when immunodeficient mice, unable to produce activated macrophages, were infected. Restoration of virulence was not seen in an INH-resistant M. tuberculosis strain that overexpressed ahpC, and this finding was confirmed by experiments performed with appropriate M. bovis strains in guinea pigs. Thus, in contrast to catalase-peroxidase, alkyl hydroperoxide reductase does not appear to act as a virulence factor in rodent infections or to play a direct role in INH resistance, although it may be important in maintaining peroxide homeostasis of the organism when KatG activity is low or absent.