cAMP-regulated Protein Lysine Acetylases in Mycobacteria

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellular cAMP found in both pathogenic and non-pathogenic mycobacteria suggest that additional and important biological processes are regulated by cAMP in these organisms. We describe here the biochemical characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase.     

Cyclic Amp-Dependent Resuscitation of Dormant Mycobacteria by Exogenous Free Fatty Acids

One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant (“non-culturable”) cells of Mycobacterium smegmatis (mc²155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6–10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3–10 mM) or dibutyryl-cAMP (0.5–1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.     

Oxidative Stress Response and Characterization of the oxyR-ahpC and furA-katG Loci in Mycobacterium marinum

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H₂O₂, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.     

The Mycobacterial MsDps2 Protein Is a Nucleoid-Forming DNA Binding Protein Regulated by Sigma Factors σA and σB

The Dps (DNA-binding protein from starved cells) proteins from Mycobacterium smegmatis MsDps1 and MsDps2 are both DNA-binding proteins with some differences. While MsDps1 has two oligomeric states, with one of them responsible for DNA binding, MsDps2 has only one DNA-binding oligomeric state. Both the proteins however, show iron-binding activity. The MsDps1 protein has been shown previously to be induced under conditions of starvation and osmotic stress and is regulated by the extra cellular sigma factors σ^H and σ^F. We show here, that the second Dps homologue in M. smegmatis, namely MsDps2, is purified in a DNA-bound form and exhibits nucleoid-like structures under the atomic force microscope. It appears that the N-terminal sequence of Dps2 plays a role in nucleoid formation. MsDps2, unlike MsDps1, does not show elevated expression in nutritionally starved or stationary phase conditions; rather its promoter is recognized by RNA polymerase containing σ^A or σ^B, under in vitro conditions. We propose that due to the nucleoid-condensing ability, the expression of MsDps2 is tightly regulated inside the cells.     

Identification of a novel gene product that promotes survival of Mycobacterium smegmatis in macrophages

Background

Bacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs.

Methodology/Principal Findings

We have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate.

Conclusions/Significance

MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria.     

Glycopeptidolipid of Mycobacterium smegmatis J15cs Affects Morphology and Survival in Host Cells

Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc²155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc²155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc²155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.     

Protection against oxidative stress in Escherichia coli stationary phase by a phosphate concentration-dependent genes expression

Escherichia coli gradually decline the capacity to resist oxidative stress during stationary phase. Besides the aerobic electron transport chain components are down-regulated in response to growth arrest. However, we have previously reported that E. coli cells grown in media containing at least 37 mM phosphate maintained ndh expression in stationary phase, having high viability and low NADH/NAD⁺ ratio. Here we demonstrated that, in the former condition, other aerobic respiratory genes (nuoAB, sdhC, cydA, and ubiC) expression was maintained. In addition, reactive oxygen species production was minimal and consequently the levels of thiobarbituric acid-reactive substances and protein carbonylation were lower than the expected for stationary cells. Interestingly, defense genes (katG and ahpC) expression was also maintained during this phase. Our results indicate that cells grown in high phosphate media exhibit advantages to resist endogenous and exogenous oxidative stress in stationary phase.     

Initiation of Methylglucose Lipopolysaccharide Biosynthesis in Mycobacteria

Background

Mycobacteria produce two unique families of cytoplasmic polymethylated polysaccharides - the methylglucose lipopolysaccharides (MGLPs) and the methylmannose polysaccharides (MMPs) - the physiological functions of which are still poorly defined. Towards defining the roles of these polysaccharides in mycobacterial physiology, we generated knock-out mutations of genes in their putative biosynthetic pathways.

Methodology/Principal Findings

We report here on the characterization of the Rv1208 protein of Mycobacterium tuberculosis and its ortholog in Mycobacterium smegmatis (MSMEG_5084) as the enzymes responsible for the transfer of the first glucose residue of MGLPs. Disruption of MSMEG_5084 in M. smegmatis resulted in a dramatic decrease in MGLP synthesis directly attributable to the almost complete abolition of glucosyl-3-phosphoglycerate synthase activity in this strain. Synthesis of MGLPs in the mutant was restored upon complementation with wild-type copies of the Rv1208 gene from M. tuberculosis or MSMEG_5084 from M. smegmatis.

Conclusions/Significance

This is the first evidence linking Rv1208 to MGLP biosynthesis. Thus, the first step in the initiation of MGLP biosynthesis in mycobacteria has been defined, and subsequent steps can be inferred.     

Novel Role of Phosphorylation-Dependent Interaction between FtsZ and FipA in Mycobacterial Cell Division

The bacterial divisome is a multiprotein complex. Specific protein-protein interactions specify whether cell division occurs optimally, or whether division is arrested. Little is known about these protein-protein interactions and their regulation in mycobacteria. We have investigated the interrelationship between the products of the Mycobacterium tuberculosis gene cluster Rv0014c-Rv0019c, namely PknA (encoded by Rv0014c) and FtsZ-interacting protein A, FipA (encoded by Rv0019c) and the products of the division cell wall (dcw) cluster, namely FtsZ and FtsQ. M. smegmatis strains depleted in components of the two gene clusters have been complemented with orthologs of the respective genes of M. tuberculosis. Here we identify FipA as an interacting partner of FtsZ and FtsQ and establish that PknA-dependent phosphorylation of FipA on T77 and FtsZ on T343 is required for cell division under oxidative stress. A fipA knockout strain of M. smegmatis is less capable of withstanding oxidative stress than the wild type and showed elongation of cells due to a defect in septum formation. Localization of FtsQ, FtsZ and FipA at mid-cell was also compromised. Growth and survival defects under oxidative stress could be functionally complemented by fipA of M. tuberculosis but not its T77A mutant. Merodiploid strains of M. smegmatis expressing the FtsZ(T343A) showed inhibition of FtsZ-FipA interaction and Z ring formation under oxidative stress. Knockdown of FipA led to elongation of M. tuberculosis cells grown in macrophages and reduced intramacrophage growth. These data reveal a novel role of phosphorylation-dependent protein-protein interactions involving FipA, in the sustenance of mycobacterial cell division under oxidative stress.     

Molecular characterization of a new gene cluster for steroid degradation in Mycobacterium smegmatis

The C‐19 steroids 4‐androstene‐3,17‐dione (AD), 1,4‐androstadiene‐3,17‐dione (ADD) or 9α‐hydroxy‐4‐androstene‐3,17‐dione (9OH‐AD), which have been postulated as intermediates of the cholesterol catabolic pathway in Mycobacterium smegmatis, cannot be used as sole carbon and energy sources by this bacterium. Only the ΔkstR mutant which constitutively expresses the genes repressed by the KstR regulator can metabolize AD and ADD with severe difficulties but still cannot metabolize 9OH‐AD, suggesting that these compounds are not true intermediates but side products of the cholesterol pathway. However, we have found that some M. smegmatis spontaneous mutants mapped in the PadR‐like regulator (MSMEG_2868) can efficiently metabolize all C‐19 steroids. We have demonstrated that the PadR mutants allow the expression of a gene cluster named C‐19+ (MSMEG_2851 to MSMEG_2901) encoding steroid degrading enzymes, that are not expressed under standard culture conditions. The C‐19+ cluster has apparently evolved independently from the upper cholesterol kstR‐regulon, but both clusters converge on the lower cholesterol kstR2‐regulon responsible for the metabolism of C and D steroid rings. Homologous C‐19+ clusters have been found only in other actinobacteria that metabolize steroids, but remarkably it is absent in Mycobacterium tuberculosis.