Sulfite reduction in mycobacteria

Mycobacterium tuberculosis places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in Mycobacterium smegmatis was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from M. tuberculosis were determined under strict anaerobic conditions [kcat = 1.0 (±0.1) electron consumed per second, and Km(SO3−2) = 27 (±1) μM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (sirA, originally misannotated nirA) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed che1) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe2+ into siroheme. Deletion of che1 causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery

Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria

Successful treatment of human tuberculosis requires 6–9 months' therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably as a result of reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters are not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms – organized communities of surface-attached cells – but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, which contain an extracellular matrix rich in free mycolic acids, and harbour an important drug-tolerant population that persist despite exposure to high levels of antibiotics

Mycobacterium tuberculosis nuoG Is a Virulence Gene That Inhibits Apoptosis of Infected Host Cells

The survival and persistence of Mycobacterium tuberculosis depends on its capacity to manipulate multiple host defense pathways, including the ability to actively inhibit the death by apoptosis of infected host cells. The genetic basis for this anti-apoptotic activity and its implication for mycobacterial virulence have not been demonstrated or elucidated. Using a novel gain-of-function genetic screen, we demonstrated that inhibition of infection-induced apoptosis of macrophages is controlled by multiple genetic loci in M. tuberculosis. Characterization of one of these loci in detail revealed that the anti-apoptosis activity was attributable to the type I NADH-dehydrogenase of M. tuberculosis, and was mainly due to the subunit of this multicomponent complex encoded by the nuoG gene. Expression of M. tuberculosis nuoG in nonpathogenic mycobacteria endowed them with the ability to inhibit apoptosis of infected human or mouse macrophages, and increased their virulence in a SCID mouse model. Conversely, deletion of nuoG in M. tuberculosis ablated its ability to inhibit macrophage apoptosis and significantly reduced its virulence in mice. These results identify a key component of the genetic basis for an important virulence trait of M. tuberculosis and support a direct causal relationship between virulence of pathogenic mycobacteria and their ability to inhibit macrophage apoptosis

ESX1-dependent fractalkine mediates chemotaxis and Mycobacterium tuberculosis infection in humans

SummaryMycobacterium tuberculosis-induced cellular aggregation is essential for granuloma formation and may assist establishment and early spread of M. tuberculosis infection. The M. tuberculosis ESX1 mutant, which has a non-functional type VII secretion system, induced significantly less production of the host macrophage-derived chemokine fractalkine (CX3CL1). Upon infection of human macrophages ESX1-dependent fractalkine production mediated selective recruitment of CD11b+ monocytic cells and increased infection of neighbouring cells consistent with early local spread of infection. Fractalkine levels were raised in vivo at tuberculous disease sites in humans and were significantly associated with increased CD11b+ monocytic cellular recruitment and extent of granulomatous disease. These findings suggest a novel fractalkine-dependent ESX1-mediated mechanism in early tuberculous disease pathogenesis in humans. Modulation of M. tuberculosis-mediated fractalkine induction may represent a potential treatment option in the future, perhaps allowing us to switch off a key mechanism required by the pathogen to spread between cells

Sequence and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae causes swine enzootic pneumonia, an important disease in the swine industry. Adherence of M. hyopneumoniae to the cilia of the tracheal epithelial cells is required to establish infection. Previous studies have identified a 97-kDa protein (P97) as the putative ciliary adhesin. To further characterize the P97 protein, the gene has been cloned, its DNA sequence analyzed, and the function of the P97 protein expressed in Escherichia coli studied. These results demonstrated that recombinant P97 has swine ciliary adherence activity. Further analysis revealed that the P97 gene was translated as a 124.9-kilodalton (kDa) protein and subjected to proteolytic cleavage at amino acid 195, resulting in a protein with a predicted molecular weight of 102 kDa. The translated P97 protein showed a high degree of hydrophilicity and contained no cysteine residues or acylation sites, confirming previous findings that P97 was not an integral membrane protein. Two repeat sequences, R1 and R2, were identified at the carboxy end of P97. The R1 sequence contained 15 repeats of AAKP(V/E) and was shown to function as the P97 ciliary binding motif. The adherence-blocking monoclonal antibody F1B6 antigenic epitope was also mapped to the 5' end of the R1 sequence. Analysis of different M. hyopneumoniae isolates displaying variation in cilia binding activity, showed that the variation was not due to expression of P97, the number of R1 copies, or changes in the AAKP(V/E) repeat sequence. Thus, another mechanism must be functioning to control cilium binding activity in M. hyopneumoniae. The R2 sequence contained four repeats of NQGKK(S/A)EG(A/T)P and showed a high degree of homology to ribosome binding proteins. Analysis of the P97 contig region showed that the P97 gene was the first of a two gene operon, designated the P97 operon. The second open reading frame coded for a possible membrane protein with a predicted molecular weight of 102 kDa. It had no sequence homology with any known sequence. Hybridization studies showed that the P97 operon sequence existed as multiple copies in the M. hyopneumoniae chromosome. These findings support the hypothesis that P97 is involved in ciliary adherence of M. hyopneumoniae.</p

Sulfite Reduction in Mycobacteria▿

Mycobacterium tuberculosis places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in Mycobacterium smegmatis was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from M. tuberculosis were determined under strict anaerobic conditions [kcat = 1.0 (±0.1) electron consumed per second, and Km(SO3−2) = 27 (±1) μM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (sirA, originally misannotated nirA) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed che1) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe2+ into siroheme. Deletion of che1 causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery

Overexpression of EPH Receptor B2 in Malignant Mesothelioma Correlates with Oncogenic Behavior

Introduction:Malignant pleural mesothelioma (MM) is an aggressive asbestos-associated malignancy with limited therapeutic options. This study describes the overexpression of Ephrin B2 receptor (EPHB2) in MM cell lines and tumors, and the effect of its manipulation on proliferative and invasive qualities of the disease.Methods:Using expression arrays, we investigated EPHB2 in MM tumors compared with normal mesothelium. EPHB2 and downstream target expression were evaluated using reverse-transcriptase polymerase chain reaction and immunoblotting methods. The biological significance of EPHB2 in MM was evaluated using in vitro functional assays with and without targeting by EPHB2-short hairpin RNA or blocking peptide in two mesothelioma cell lines, HP-1 and H2595.Results:EPHB2 is overexpressed in all MM cell lines, but not in benign mesothelial cells, and is significantly elevated in MM tumor tissue compared with matched normal peritoneum. Targeted knockdown of EPHB2 in HP-1 and H2595 cell lines reduced its expression and that of EPHB2 downstream targets such as matrix metalloproteinase (MMP-2) and vascular endothelial growth factor, whereas caspase 2 and caspase 8 had increased expression. Inhibition of EPHB2 resulted in a significant decrease in scratch closure (1.25-fold–1.8-fold), proliferation (1.5-fold), and invasion (1.7-fold–1.8-fold) compared with the controls. Most notably, however, EPHB2 silencing resulted in a significant increase in apoptotic proteins and activity.Conclusion:EPHB2 seems to play an important role in MM pathogenesis and these findings indicate that EPHB2 could serve as a potential novel therapeutic target for treatment of the disease