Comparison of Gene Expression Profiles Between Pansensitive and Multidrug-Resistant Strains of Mycobacterium tuberculosis

Mycobacterium tuberculosis has developed resistance to anti-tuberculosis first-line drugs. Multidrug-resistant strains complicate the control of tuberculosis and have converted it into a worldwide public health problem. Mutational studies of target genes have tried to envisage the resistance in clinical isolates; however, detection of these mutations in some cases is not sufficient to identify drug resistance, suggesting that other mechanisms are involved. Therefore, the identification of new markers of susceptibility or resistance to first-line drugs could contribute (1) to specifically diagnose the type of M. tuberculosis strain and prescribe an appropriate therapy, and (2) to elucidate the mechanisms of resistance in multidrug-resistant strains. In order to identify specific genes related to resistance in M. tuberculosis, we compared the gene expression profiles between the pansensitive H37Rv strain and a clinical CIBIN:UMF:15:99 multidrug-resistant isolate using microarray analysis. Quantitative real-time PCR confirmed that in the clinical multidrug-resistant isolate, the esxG, esxH, rpsA, esxI, and rpmI genes were upregulated, while the lipF, groES, and narG genes were downregulated. The modified genes could be involved in the mechanisms of resistance to first-line drugs in M. tuberculosis and could contribute to increased efficiency in molecular diagnosis approaches of infections with drug-resistant strains.     

The Genetic Requirements for Fast and Slow Growth in Mycobacteria

Mycobacterium tuberculosis infects a third of the world''s population. Primary tuberculosis involving active fast bacterial replication is often followed by asymptomatic latent tuberculosis, which is characterised by slow or non-replicating bacteria. Reactivation of the latent infection involving a switch back to active bacterial replication can lead to post-primary transmissible tuberculosis. Mycobacterial mechanisms involved in slow growth or switching growth rate provide rational targets for the development of new drugs against persistent mycobacterial infection. Using chemostat culture to control growth rate, we screened a transposon mutant library by Transposon site hybridization (TraSH) selection to define the genetic requirements for slow and fast growth of Mycobacterium bovis (BCG) and for the requirements of switching growth rate. We identified 84 genes that are exclusively required for slow growth (69 hours doubling time) and 256 genes required for switching from slow to fast growth. To validate these findings we performed experiments using individual M. tuberculosis and M. bovis BCG knock out mutants. We have demonstrated that growth rate control is a carefully orchestrated process which requires a distinct set of genes encoding several virulence determinants, gene regulators, and metabolic enzymes. The mce1 locus appears to be a component of the switch to slow growth rate, which is consistent with the proposed role in virulence of M. tuberculosis. These results suggest novel perspectives for unravelling the mechanisms involved in the switch between acute and persistent TB infections and provide a means to study aspects of this important phenomenon in vitro.     

Mycobacterium tuberculosis induces an atypical cell death mode to escape from infected macrophages

Background

Macrophage cell death following infection with Mycobacterium tuberculosis plays a central role in tuberculosis disease pathogenesis. Certain attenuated strains induce extrinsic apoptosis of infected macrophages but virulent strains of M. tuberculosis suppress this host response. We previously reported that virulent M. tuberculosis induces cell death when bacillary load exceeds ∼20 per macrophage but the precise nature of this demise has not been defined.

Methodology/Principal Findings

We analyzed the characteristics of cell death in primary murine macrophages challenged with virulent or attenuated M. tuberculosis complex strains. We report that high intracellular bacillary burden causes rapid and primarily necrotic death via lysosomal permeabilization, releasing hydrolases that promote Bax/Bak-independent mitochondrial damage and necrosis. Cell death was independent of cathepsins B or L and notable for ultrastructural evidence of damage to lipid bilayers throughout host cells with depletion of several host phospholipid species. These events require viable bacteria that can respond to intracellular cues via the PhoPR sensor kinase system but are independent of the ESX1 system.

Conclusions/Significance

Cell death caused by virulent M. tuberculosis is distinct from classical apoptosis, pyroptosis or pyronecrosis. Mycobacterial genes essential for cytotoxicity are regulated by the PhoPR two-component system. This atypical death mode provides a mechanism for viable bacilli to exit host macrophages for spreading infection and the eventual transition to extracellular persistence that characterizes advanced pulmonary tuberculosis.     

The rpsL gene and streptomycin resistance in single and multiple drug-resistant strains of Mycobacterium tuberculosis

The recent emergence of indolent and rapidly fatal drug-resistant strains of Mycobacterium tuberculosis has renewed interest in defining the molecular mechanisms of drug resistance in the tubercle bacilli. In this report, we have examined the mechanism of resistance to streptomycin (Sm) in M. tuberculosis through the cloning and nucleotide sequence analysis of the gene encoding the ribosomal S^R protein (rpsL gene) from streptomycin-resistant strains and their streptomycin-sensitive parental strains. We have demonstrated that five singly Sm^RM. tuberculosis strains and an Sm^R isolate that has reduced sensitivity to multiple antibiotics have identical point mutations at codon 43 of the rpsL gene. Mutations at this same site confer Sm^R in Escherichia coli. In contrast, two other multiple drug-resistant M. tuberculosis strains that are resistant to Sm have rpsL genes that have the same nucleotide sequence as their drug-sensitive parent strains, suggesting that different resistance mechanisms are involved in these strains.     

Comprehensive Definition of the SigH Regulon of Mycobacterium tuberculosis Reveals Transcriptional Control of Diverse Stress Responses

Expression of SigH, one of 12 Mycobacterium tuberculosis alternative sigma factors, is induced by heat, oxidative and nitric oxide stresses. SigH activation has been shown to increase expression of several genes, including genes involved in maintaining redox equilibrium and in protein degradation. However, few of these are known to be directly regulated by SigH. The goal of this project is to comprehensively define the Mycobacterium tuberculosis genes and operons that are directly controlled by SigH in order to gain insight into the role of SigH in regulating M. tuberculosis physiology. We used ChIP-Seq to identify in vivo SigH binding sites throughout the M. tuberculosis genome, followed by quantification of SigH-dependent expression of genes linked to these sites and identification of SigH-regulated promoters. We identified 69 SigH binding sites, which are located both in intergenic regions and within annotated coding sequences in the annotated M. tuberculosis genome. 41 binding sites were linked to genes that showed greater expression following heat stress in a SigH-dependent manner. We identified several genes not previously known to be regulated by SigH, including genes involved in DNA repair, cysteine biosynthesis, translation, and genes of unknown function. Experimental and computational analysis of SigH-regulated promoter sequences within these binding sites identified strong consensus -35 and -10 promoter sequences, but with tolerance for non-consensus bases at specific positions. This comprehensive identification and validation of SigH-regulated genes demonstrates an extended SigH regulon that controls an unexpectedly broad range of stress response functions.     

Integrative analysis of transcriptome and genome indicates two potential genomic islands are associated with pathogenesis of Mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb) is a successful human pathogen and widely prevalent throughout the world. Genomic islands (GIs) are thought to be related to pathogenicity. In this study, we predicted two potential genomic islands in M.tb genome, respectively named as GI-1 and GI-2. It is indicated that the genes belong to PE_PGRS family in GI-1 and genes involved in sulfolipid-1 (SL-1) synthesis in GI-2 are strongly associated with M.tb pathogenesis. Sequence analysis revealed that the five PGRS genes are more polymorphic than other PGRS members in full virulence M.tb complex strains at significance level 0.01 but not in attenuated strains. Expression analysis of microarrays collected from literatures displayed that GI-1 genes, especially Rv3508 might be correlated with the response to the inhibition of aerobic respiration. Microarray analysis also showed that SL-1 cluster genes are drastically down-expressed in attenuated strains relative to full virulence strains. We speculated that the effect of SL-1 on M.tb pathogenicity could be associated with long-term survival and persistence establishment during infection. Additionally, the gene Rv3508 in GI-1 was under positive selection. Rv3508 may involve the response of M.tb to the inhibition of aerobic respiration by low oxygen or drug PA-824, and it may be a common feature of genes in GI-1. These findings may provide some novel insights into M.tb physiology and pathogenesis.     

Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis

The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen’s persistence.     

Characterization of a Mycobacterium tuberculosis Nanocompartment and Its Potential Cargo Proteins

Mycobacterium tuberculosis has evolved various mechanisms by which the bacterium can maintain homeostasis under numerous environmental assaults generated by the host immune response. M. tuberculosis harbors enzymes involved in the oxidative stress response that aid in survival during the production of reactive oxygen species in activated macrophages. Previous studies have shown that a dye-decolorizing peroxidase (DyP) is encapsulated by a bacterial nanocompartment, encapsulin (Enc), whereby packaged DyP interacts with Enc via a unique C-terminal extension. M. tuberculosis also harbors an encapsulin homolog (CFP-29, Mt-Enc), within an operon with M. tuberculosis DyP (Mt-DyP), which contains a C-terminal extension. Together these observations suggest that Mt-DyP interacts with Mt-Enc. Furthermore, it has been suggested that DyPs may function as either a heme-dependent peroxidase or a deferrochelatase. Like Mt-DyP, M. tuberculosis iron storage ferritin protein, Mt-BfrB, and an M. tuberculosis protein involved in folate biosynthesis, 7,8-dihydroneopterin aldolase (Mt-FolB), have C-terminal tails that could also interact with Mt-Enc. For the first time, we show by co-purification and electron microscopy that mycobacteria via Mt-Enc can encapsulate Mt-DyP, Mt-BfrB, and Mt-FolB. Functional studies of free or encapsulated proteins demonstrate that they retain their enzymatic activity within the Mt-Enc nanocompartment. Mt-DyP, Mt-FolB, and Mt-BfrB all have antioxidant properties, suggesting that if these proteins are encapsulated by Mt-Enc, then this nanocage may play a role in the M. tuberculosis oxidative stress response. This report provides initial structural and biochemical clues regarding the molecular mechanisms that utilize compartmentalization by which the mycobacterial cell may aid in detoxification of the local environment to ensure long term survival.     

Keratinocyte Growth Factor Administration Attenuates Murine Pulmonary Mycobacterium tuberculosis Infection through Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF)-dependent Macrophage Activation and Phagolysosome Fusion

Augmentation of innate immune defenses is an appealing adjunctive strategy for treatment of pulmonary Mycobacterium tuberculosis infections, especially those caused by drug-resistant strains. The effect of intranasal administration of keratinocyte growth factor (KGF), an epithelial mitogen and differentiation factor, on M. tuberculosis infection in mice was tested in prophylaxis, treatment, and rescue scenarios. Infection of C57BL6 mice with M. tuberculosis resulted in inoculum size-dependent weight loss and mortality. A single dose of KGF given 1 day prior to infection with 10⁵ M. tuberculosis bacilli prevented weight loss and enhanced pulmonary mycobacterial clearance (compared with saline-pretreated mice) for up to 28 days. Similar effects were seen when KGF was delivered intranasally every third day for 15 days, but weight loss and bacillary growth resumed when KGF was withdrawn. For mice with a well established M. tuberculosis infection, KGF given every 3 days beginning on day 15 postinoculation was associated with reversal of weight loss and an increase in M. tuberculosis clearance. In in vitro co-culture experiments, M. tuberculosis-infected macrophages exposed to conditioned medium from KGF-treated alveolar type II cell (MLE-15) monolayers exhibited enhanced GM-CSF-dependent killing through mechanisms that included promotion of phagolysosome fusion and induction of nitric oxide. Alveolar macrophages from KGF-treated mice also exhibited enhanced GM-CSF-dependent phagolysosomal fusion. These results provide evidence that administration of KGF promotes M. tuberculosis clearance through GM-CSF-dependent mechanisms and enhances host defense against M. tuberculosis infection.     

Mitogen-activated protein kinases mediate <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>–induced CD44 surface expression in monocytes

CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.     

The MprB Extracytoplasmic Domain Negatively Regulates Activation of the Mycobacterium tuberculosis MprAB Two-Component System

Mycobacterium tuberculosis is an acid-fast pathogen of humans and the etiological agent of tuberculosis (TB). It is estimated that one-third of the world''s population is latently (persistently) infected with M. tuberculosis. M. tuberculosis persistence is regulated, in part, by the MprAB two-component signal transduction system, which is activated by and mediates resistance to cell envelope stress. Here we identify MprAB as part of an evolutionarily conserved cell envelope stress response network and demonstrate that MprAB-mediated signal transduction is negatively regulated by the MprB extracytoplasmic domain (ECD). In particular, we report that deregulated production of the MprB sensor kinase, or of derivatives of this protein, negatively impacts M. tuberculosis growth. The observed growth attenuation is dependent on MprAB-mediated signal transduction and is exacerbated in strains of M. tuberculosis producing an MprB variant lacking its ECD. Interestingly, full-length MprB, and the ECD of MprB specifically, immunoprecipitates the Hsp70 chaperone DnaK in vivo, while overexpression of dnaK inhibits MprAB-mediated signal transduction in M. tuberculosis grown in the absence or presence of cell envelope stress. We propose that under nonstress conditions, or under conditions in which proteins present in the extracytoplasmic space are properly folded, signaling through the MprAB system is inhibited by the MprB ECD. Following exposure to cell envelope stress, proteins present in the extracytoplasmic space become unfolded or misfolded, leading to removal of the ECD-mediated negative regulation of MprB and subsequent activation of MprAB.     

Complexity of roles and regulation of the PMK1-MAPK pathway in mycelium development,conidiation and appressorium formation in Magnaporthe oryzae

MST50, MST11, MST7, PMK1 and GAS1/GAS2 genes are the important components in the PMK1-MAPK signal transduction pathway in fungi. Mutants with deletion of these five genes of Magnaporthe oryzae, a pathogen of the rice blast, were constructed. A cDNA array containing 4108 unique genes of M. oryzae was developed and used to analyze the gene expression profiles of these mutants against the wild type to dissect the gene expression regulation networks responsible for conidiation and appressorium formation. With this approach, differentially regulated genes by these five components were identified. The vast majority of the regulated genes were mutant-specific, while only a small proportion were in common for all of the mutants, suggesting that each of these genes has its own regulon. Functional groups and expression patterns of the regulated genes showed that (1) gene members in the PMK1-MAPK pathway are associated with multiple signaling pathways; (2) the regulation of PMK1-mediated signaling pathways is very complex and likely involved in other signaling networks; (3) glucose metabolism and signals are required in mycelium development; and (4) appressorium formation likely shares the mechanisms responsible for sexual conjugation and meiosis, which is affected by carbohydrate metabolism.     

Functional characterization of Mycobacterium tuberculosis Rv2969c membrane protein

Identifying Mycobacterium tuberculosis membrane proteins involved in binding to and invasion of host cells is important in designing subunit-based anti-tuberculosis vaccines. The Rv2969c gene sequence was identified by PCR in M. tuberculosis complex strains, being transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG. Rabbits immunized with synthetic peptides from highly specific conserved regions of this protein produced antibodies recognizing 27 and 29 kDa bands in M. tuberculosis lysate, which is consistent with the molecular weight of the Rv2969c gene product in M. tuberculosis H37Rv. Immunoelectron microscopy revealed the protein was localized on the bacillus surface. Four and three specific high activity binding peptides (HABPs) to the A549 alveolar epithelial and U937 monocyte cell lines were found, respectively. Two of the HABPs found inhibited M. tuberculosis invasion of A549 cells, suggesting that these peptides might be good candidates to be included in a multiepitopic, subunit-based anti-tuberculosis vaccine.     

Mutations in the Essential Arabinosyltransferase EmbC Lead to Alterations in Mycobacterium tuberculosis Lipoarabinomannan

The Mycobacterium tuberculosis cell wall is a complex structure essential for the viability of the organism and its interaction with the host. The glycolipid lipoarabinomannan (LAM) plays an important role in mediating host-bacteria interactions and is involved in modulation of the immune response. The arabinosyltransferase EmbC required for LAM biosynthesis is essential. We constructed recombinant strains of M. tuberculosis expressing a variety of alleles of EmbC. We demonstrated that EmbC has a functional signal peptide in M. tuberculosis. Over- or underexpression of EmbC resulted in reduced or increased sensitivity to ethambutol, respectively. The C-terminal domain of EmbC was essential for activity because truncated alleles were unable to mediate LAM production in Mycobacterium smegmatis and were unable to complement an embC deletion in M. tuberculosis. The C-terminal domain of the closely related arabinosyltransferase EmbB was unable to complement the function of the EmbC C-terminal domain. Two functional motifs were identified. The GT-C motif contains two aspartate residues essential for function in the DDX motif. The proline-rich region contains two highly conserved asparagines (Asn-638 and Asn-652). Mutation of these residues was tolerated, but loss of Asn-638 resulted in the synthesis of truncated LAM, which appeared to lack arabinose branching. All embC alleles that were incapable of complementing LAM production in M. smegmatis were not viable in M. tuberculosis, supporting the hypothesis that LAM itself is essential in M. tuberculosis.