Mycobacterium Lrp/AsnC family transcriptional factor modulates the arginase pathway as both a sensor and a transcriptional repressor.

L-Arginine is the precursor of nitric oxide (NO), a host immune effector against intracellular pathogens including Mycobacterium tuberculosis (M. tb). Pathogens including M. tb have evolved various strategies targeting arginine to block the production of NO for better survival and proliferation. However, L-arginine metabolism and regulation in Mycobacterium are poorly understood. Here, we report the identification of M. smegmatis MSMEG_1415 (homolog of M. tb Rv2324) as an arginine-responsive transcriptional factor regulating the arginase pathway. In the absence of L-arginine, MSMEG_1415 acts as a repressor to inhibit the transcription of the roc (for arginine, ornithine catabolism) gene cluster, thereby switching off the arginase pathway. Treatment with L-arginine relieves the transcriptional inhibition of MSMEG_1415 on the roc gene cluster to activate the arginase pathway. Moreover, the L-arginine-MSMEG_1415 complex activates the transcription of the roc gene cluster by recognizing and binding a 15-bp palindrome motif, thereby preventing the excess accumulation of L-arginine in M. smegmatis. Physiologically, MSMEG_1415 confers mycobacteria resistance to starvation and fluoroquinolones exposure, suggestive of its important role in M. smegmatis persistence. The results uncover a unique regulatory mechanism of arginine metabolism in mycobacteria and identify M. tb Rv2324 as an attractive candidate target for the design of drugs against tuberculosis.

Differential DNA methylomes of clinical MDR, XDR and XXDR Mycobacterium tuberculosis isolates revealed by using single-molecule real-time sequencing.

Post-replicative DNA methylation is essential for diverse biological processes in both eukaryotes and prokaryotes. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, remains one of the most formidable threats worldwide. Although DNA methylation of M. tuberculosis has been documented, little information is available for clinical drug-resistant M. tuberculosis. Single-molecule real-time (SMRT) sequencing was used to profile the core methylome of three clinical isolates, namely multidrug-resistant (MDR), extensively drug-resistant (XDR) and extremely drug-resistant (XXDR) strains. 3812, 6808 and 6041 DNA methylated sites were identified in MDR-MTB, XDR-MTB and XXDR-MTB genome, respectively. There are two types of methylated motifs, namely N6-methyladenine (m6A) and N4-methylcytosine (m4C). A novel widespread 6 mA methylation motif 5'-CACGCAG-3' was found in XDR-MTB and XXDR-MTB. The methylated genes are involved in multiple cellular processes, especially metabolic enzymes engaged in glucose metabolism, fatty acid and TCA cycle. Many methylated genes are involved in mycobacterial virulence, antibiotic resistance and tolerance. This provided a comprehensive list of methylated genes in drug-resistant clinical isolates and the basis for further functional elucidation.

Global quantitative phosphoproteome reveals phosphorylation network of bovine lung tissue altered by Mycobacterium bovis.

Bovine tuberculosis caused by Mycobacterium bovis remains a major cause of economic loss in cattle industries worldwide. However, the pathogenic mechanisms remain poorly understood. Post-translation modifications (PTM) such as phosphorylation play a crucial role in pathogenesis. While the change of transcriptome and proteome during the interaction between M. bovis and cattle were studied, there are no reports on the phosphoproteome change. We apply Tandem Mass Tag-based (TMT) quantitative proteomics coupled with immobilized metal-chelated affinity chromatography (IMAC) enrichment to obtain the quantified phosphorylation in vivo of M. bovis infected cattle lung tissue. The phosphorylated proteins are widespread in the nucleus, cytoplasm and plasma membrane. By using a change fold of 1.2, 165 phosphosites from 147 proteins were enriched, with 88 upregulated and 77 downregulated sites respectively. We further constructed the protein-protein interaction (PPI) networks of STAT3, SRRM2 and IRS-1 based on their number of differential phosphorylation sites and KEGG pathways. Similar patterns of gene expression dynamics of selected genes were observed in Mycobacterium tuberculosis infected human sample GEO dataset, implicating crucial roles of these genes in pathogenic Mycobacteria - host interaction. The first phosphorproteome reveals the relationship between bovine tuberculosis and glucose metabolism, and will help further refinement of target proteins for mechanistic study.

Mycobacterium tuberculosis PE31 (Rv3477) Attenuates Host Cell Apoptosis and Promotes Recombinant M. smegmatis Intracellular Survival via Up-regulating GTPase Guanylate Binding Protein-1.

The Mycobacterium (M.) tuberculosis comprising proline-glutamic acid (PE) subfamily proteins associate with virulence, pathogenesis, and host-immune modulations. While the functions of most of this family members are not yet explored. Here, we explore the functions of "PE only" subfamily member PE31 (Rv3477) in virulence and host-pathogen interactions. We have expressed the M. tuberculosis PE31 in non-pathogenic Mycobacterium smegmatis strain (Ms_PE31) and demonstrated that PE31 significantly altered the cell facet features including colony morphology and biofilm formation. PE31 expressing M. smegmatis showed more resistant to the low pH, diamide, H2O2 and surface stress. Moreover, Ms_PE31 showed higher intracellular survival in macrophage THP-1 cells. Ms_PE31 significantly down-regulated the production of IL-12p40 and IL-6, while up-regulates the production of IL-10 in macrophages. Ms_PE31 also induced the expression of guanylate-binding protein-1 (GBP-1) in macrophages. Further analysis demonstrates that Ms_PE31 inhibits the caspase-3 activation and reduces the macrophages apoptosis. Besides, the NF-κB signaling pathway involves the interplay between Ms_PE31 and macrophages. Collectively, our finding identified that PE31 act as a functionally relevant virulence factor of M. tuberculosis.

Transport mechanism of Mycobacterium tuberculosis MmpL/S family proteins and implications in pharmaceutical targeting.

Tuberculosis caused by Mycobacterium tuberculosis remains a serious threat to public health. The M. tuberculosis cell envelope is closely related to its virulence and drug resistance. Mycobacterial membrane large proteins (MmpL) are lipid-transporting proteins of the efflux pump resistance nodulation cell division (RND) superfamily with lipid substrate specificity and non-transport lipid function. Mycobacterial membrane small proteins (MmpS) are small regulatory proteins, and they are also responsible for some virulence-related effects as accessory proteins of MmpL. The MmpL transporters are the candidate targets for the development of anti-tuberculosis drugs. This article summarizes the structure, function, phylogenetics of M. tuberculosis MmpL/S proteins and their roles in host immune response, inhibitors and regulatory system.

Biology of MarR family transcription factors and implications for targets of antibiotics against tuberculosis.

The emergence of multidrug resistant (MDR) Mycobacterium tuberculosis strains and increased incidence of HIV coinfection fueled the difficulty in controlling tuberculosis (TB). MarR (multiple antibiotic resistance regulator) family transcription factors can regulate marRAB operon and are involved in resistance to multiple environmental stresses. We have summarized the structure, function, distribution, and regulation of the MarR family proteins, as well as their implications for novel targets for antibiotics, especially for tuberculosis.

Mce-associated protein Rv0177 alters the cell wall structure of Mycobacterium smegmatis and promotes macrophage apoptosis via regulating the cytokines.

The success of Mycobacterium tuberculosis as a pathogen largely contributes to its ability to infect, modify and persist within the host cells. M. tuberculosis Rv0177 is a gene of the mce1 operon (Mammalian cell entry), encoding a conserved hypothetical protein, essential for M. tuberculosis survival and up-regulated within murine macrophages. To explore its function, Rv0177 was heterologously expressed in M. smegmatis. The recombinant protein was located in the cell wall. M. smegmatis recombinant strain expressing Rv0177 altered sliding motility, its cell wall architecture and the permeability. Moreover, M. smegmatis expressing Rv0177 could up-regulate MCP-1 and downregulate the IL-6 expression in RAW264.7 macrophages in comparison to the control. MS_Rv0177 increased the expression of MCP-1 inducible protein (MCPIP) and a C/EBP homologous protein (CHOP) owing to MCP-1. In addition, the JNK signaling pathway was engaged in the interplay between MS_Rv0177 and macrophages. The macrophage caspase-3 activation and cell apoptosis were induced by the recombinant. This provided novel functional cues for the MCE-associated Rv0177.