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.

Effects of cytokines on mycobacterial phagosome maturation.

One of the major mechanisms permitting intracellular pathogens to parasitize macrophages is their ability to alter maturation of the phagosome or affect its physical integrity. These processes are opposed by the host innate and adaptive immune defenses, and in many instances mononuclear phagocytes can be stimulated with appropriate cytokines to restrict the growth of the microorganisms within the phagosomal compartment. Very little is known about the effects that cytokines have on phagosome maturation. Here we have used green fluorescent protein (GFP)-labeled mycobacteria and a fixable acidotropic probe, LysoTracker Red DND-99, to monitor maturation of the mycobacterial phagosome. The macrophage compartments that stained with the LysoTracker probe were examined first. This dye was found to colocalize preferentially with the late endosomal and lysosomal markers rab7 and Lamp1, and with a fluid phase marker chased into the late endosomal compartments. In contrast, LysoTracker showed only a minor overlap with the early endosomal marker rab5. Pathogenic mycobacteria are believed to reside in nonacidified vacuoles sequestered away from late endosomal compartments as a part of their intracellular survival strategy. We examined the status of mycobacterial phagosomes in macrophages from IL-10 knockout mice, in quiescent cells, and in mononuclear phagocytes stimulated with the macrophage-activating cytokine IFN-(gamma). When macrophages were derived from the bone marrow of transgenic IL-10 mice lacking this major deactivating cytokine, colocalization of GFP-fluorescing mycobacteria with the LysoTracker staining appeared enhanced, suggestive of increased acidification of the mycobacterial phagosome relative to macrophages from normal mice. When bone marrow-derived macrophages from normal mice or a J774 murine macrophage cell line were stimulated with IFN-(gamma) and LPS, this resulted in increased colocalization of mycobacteria and LysoTracker, but no statistically significant enhancement was observed in IL-10 transgenic animals. These studies are consistent with the interpretation that proinflammatory and anti-inflammatory cytokines affect maturation of mycobacterial phagosomes. Although multiple mechanisms are likely to be at work, we propose the existence of a direct link between cytokine effects on the host cell and phagosome maturation in the macrophage.

Loss of oxyR in Mycobacterium tuberculosis.

The loss of the putative regulator oxyR and the associated dysfunction of oxidative stress response in Mycobacterium tuberculosis may have coincided with, or directly participated in, the evolution of this microorganism into the potent contemporary human pathogen. These phenomena may have implications for host-pathogen interactions in tuberculosis and for M. tuberculosis sensitivity to the front-line antituberculosis agent isoniazid.

An active domain of the nuclear RNase P RNA.

The P10/11-P12 RNA domain of yeast nuclear RNase P RNA has been characterized using genetic and biochemical analysis. This RNA domain contains some of the most conserved nucleotides throughout yeast species and shares considerable homology with the P10-P11-P12 bacterial RNase P RNA domain. Viable yeast variants generated by sequence randomization of the conserved internal loop nucleotides have demonstrated magnesium-sensitive growth defects. Partial purification and characterization of the RNase P holoenzyme from these variants reveals that the mutations affect the catalytic rate of the enzyme and increased magnesium concentrations are required to achieve maximal activity compared to wild type enzyme. Biochemical structure probing has been employed to address the interaction of the RNA domain with magnesium. Several nucleotides within the loop portion of the domain show magnesium-induced changes in reagent accessibility. These include the highly conserved nucleotides shared between yeast and bacteria, which become less accessible in the presence of magnesium. Conversely, accessibility of other regions of the RNA increases. The genetic and biochemical data suggest that the P10/11-P12 RNA domain, and the conserved nucleotides in particular, interacts with magnesium in a manner that affects catalysis by RNase P.

A conserved RNA motif involved in divalent cation utilization by nuclear RNase P.

Catalytic RNAs are metalloenzymes that require precise coordination of divalent cation cofactors. In RNase P RNA, a conserved structural subdomain that has been implicated in magnesium coordination contains the consensus sequence acAGaRA. Randomization mutagenesis of the analogous sequence in the Saccharomyces cerevisiae nuclear RNase P RNA gene, RPR1, gave viable sequence variants that confer magnesium-correctable growth defects and are defective in magnesium cofactor utilization by the RNase P holoenzyme in vitro. Kinetic analysis of the defective holoenzymes suggests that the primary effects were on catalytic rate, rather than substrate recognition. The possible involvement of this RNA subdomain in catalysis is discussed.

The extreme sensitivity of Mycobacterium tuberculosis to the front-line antituberculosis drug isoniazid.

Mycobacterium tuberculosis is a natural mutant in oxyR, a close homolog of the central regulator of peroxide stress response in enteric bacteria. Inactivation of oxyR is specific for M. tuberculosis and other members of the M. tuberculosis complex. This phenomenon appears as a paradox due to the ability of this organism to parasitize host macrophages, in which the ingested organisms are likely to be exposed to reactive oxygen intermediates. However, the surprising finding that M. tuberculosis has multiple deletions, nonsense and frameshift mutations in oxyR may help explain the exceptionally high sensitivity of M. tuberculosis to the potent antituberculosis agent isoniazid. One of the genes affected by oxyR lesions, ahpC (encoding an alkylhydroperoxide reductase) may determine the intrinsic sensitivity of mycobacteria to isoniazid.

Mutational analysis of Saccharomyces cerevisiae nuclear RNase P: randomization of universally conserved positions in the RNA subunit.

Three regions in the Saccharomyces cerevisiae RNase P RNA have been identified, at positions Sce 87-94, Sce 309-316, and Sce 339-349, that contain nucleotides that are invariant in identity and position among all the known RNase P RNAs. To study the importance of these conserved RPR1 RNA regions in enzyme function, three independent mutational libraries were created in which the positions of invariant nucleotides were randomized simultaneously. Screening in vivo was used to identify viable RPR1 variants when reconstituted into holoenzyme in cells. Despite the universal evolutionary conservation, most of these positions tolerate certain sequence changes without severely affecting function. Most changes, however, produced subtle defects in cell growth and RNase P function, supporting the importance of these conserved regions. Isolation of conditional growth mutants allowed the characterization of the effects of mutations on cell growth, RPR1 RNA maturation, and activity of the holoenzyme in vitro. Kinetic analysis showed that viable variants were usually more defective in catalytic rate (Kcat) than in substrate recognition (Km).

Analysis of conserved positions in nuclear RNase P RNA.

Secondary structure models of eubacterial and eukaryotic nuclear RNase P RNA subunits show extensive structural similarities, allowing the identification of highly conserved nucleotide positions and molecular modeling of the enzyme-substrate complex in three dimensions. Based on this information, we present a preliminary tertiary structure model of the yeast nuclear RNase P RNA. In addition, the most conserved positions in the structure have been subjected to sequence randomization, with viable sequence variations identified by selection in vivo and characterized for phenotypic consequences.

Structure-function analysis in nuclear RNase P RNA.

Eukaryotic ribonuclease P (RNase P) enzymes require both RNA and protein subunits for activity in vivo and in vitro. We have undertaken an analysis of the complex RNA subunit of the nuclear holoenzyme in an effort to understand its structure and its similarities to and differences from the bacterial ribozymes. Phylogenetic analysis, structure-sensitive RNA footprinting, and directed mutagenesis reveal conserved secondary and tertiary structures with both strong similarities to the bacterial consensus and distinctive features. The effects of mutations in the most highly conserved positions are being used to dissect the functions of individual subdomains.

Replacement of the Saccharomyces cerevisiae RPR1 gene with heterologous RNase P RNA genes.

Phylogenetic studies of yeast nuclear RNase P RNA genes have shown a striking conservation of secondary structure for the Saccharomyces and Schizosaccharomyces RNase P RNAs, yet much of the primary sequence and many substructures vary among the RNAs examined. To investigate which sequences and structural features can be varied and still allow function in a heterologous organism, RNase P genes from several yeast species were tested for the ability to substitute for the Saccharomyces cerevisiae RNA. The RNase P genes from Saccharomyces carlsbergensis and Saccharomyces kluyveri could act as the sole source of RNase P RNA within S. cerevisiae cells, whereas the genes from Saccharomyces globosus and Schizosaccharomyces pombe could not. Although heterologous RNase P RNAs were synthesized by the cells in all cases, the RNAs that complemented tended to be processed from longer precursor transcripts into mature-sized RNase P RNA, while the RNAs that did not complement tended to accumulate as the longer precursor form. The results identified sequences and structures in the RNA that are not essential for interaction with species-specific proteins, processing or localization, and suggested other positions that may be candidates for such processes.