Valerie Mizrahi

Mycobacterium tuberculosis is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd- and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C ₃ ) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in M. tuberculosis pathogenesis. In addition to the well-characterized methylcitrate cycle, the M. tuberculosis genome contains a complete methylmalonyl pathway, including a mutAB -encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B ₁₂ -derived cofactor for activity. Here, we demonstrate the ability of M. tuberculosis to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B ₁₂ is supplied exogenously. We show that this ability is dependent on mutAB and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Importantly, although the glyoxylate and methylcitrate cycles supported robust growth of M. tuberculosis on the C ₁₇ fatty acid heptadecanoate, growth on valerate (C ₅ ) was significantly enhanced through vitamin B ₁₂ supplementation. Moreover, both wild-type and methylcitrate cycle mutant strains grew on B ₁₂ -supplemented valerate in the presence of 3-nitropropionate, an inhibitor of the glyoxylate cycle enzyme isocitrate lyase, indicating an anaplerotic role for the methylmalonyl pathway. The demonstrated functionality of MCM reinforces the potential relevance of vitamin B ₁₂ to mycobacterial pathogenesis and suggests that vitamin B ₁₂ availability in vivo might resolve the paradoxical dispensability of the methylcitrate cycle for the growth and persistence of M. tuberculosis in mice.

Summary: Mycobacterium tuberculosis contains five resuscitation‐promoting factor (Rpf)‐like proteins, RpfA‐E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA‐E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a ‘non‐culturable’ state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild‐type organisms confirming that the phenotype was associated with rpf‐like gene loss and that the ‘non‐culturable’ cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late‐stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

An assay modeled on a known polymorphism in the PE_PGRS9 gene of Mycobacterium tuberculosis was designed to assess the mutability of a sequence containing interspersed PGRS repeats. Application of the assay in Mycobacterium smegmatis revealed sequence plasticity: in addition to recapitulating the mutation on which it was based, other mutations likely mediated by replication slippage between PGRS repeats were detected. However, the mutation rates argued against marked hypermutability of such sequences in mycobacteria.

SUMMARY: The global tuberculosis (TB) control effort is focused on interrupting transmission of the causative agent, Mycobacterium tuberculosis , through chemotherapeutic intervention in active infectious disease. The insufficiency of this approach is manifest in the inexorable annual increase in TB infection and mortality rates and the emergence of multidrug-resistant isolates. Critically, the limited efficacy of the current frontline anti-TB drug combination suggests that heterogeneity of host and bacillary physiologies might impair drug activity. This review explores the possibility that strategies enabling adaptation of M. tuberculosis to hostile in vivo conditions might contribute to the subversion of anti-TB chemotherapy. In particular, evidence that infecting bacilli are exposed to environmental and host immune-mediated DNA-damaging insults suggests a role for error-prone DNA repair synthesis in the generation of chromosomally encoded antibiotic resistance mutations. The failure of frontline anti-TB drugs to sterilize a population of susceptible bacilli is independent of genetic resistance, however, and instead implies the operation of alternative tolerance mechanisms. Specifically, it is proposed that the emergence of persister subpopulations might depend on the switch to an altered metabolic state mediated by the stringent response alarmone, (p)ppGpp, possibly involving some or all of the many toxin-antitoxin modules identified in the M. tuberculosis genome.

The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three possible terminal oxidase complexes. The structure and function of the electron transport pathway leading from the menaquinol-menaquinone pool to the cytochrome bc ₁ complex and terminating in the aa ₃ -type cytochrome c oxidase was characterized. M. smegmatis strains with mutations in the bc ₁ complex and in subunit II of cyctochome c oxidase were found to be profoundly growth impaired, confirming the importance of this respiratory pathway for mycobacterial growth under aerobic conditions. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB , which encodes the bioenergetically less efficient and microaerobically induced cytochrome bd -type menaquinol oxidase that is required for the growth of M. smegmatis under O ₂ -limiting conditions. Further insights into the adaptation of this organism to rerouting of the electron flux through the branch terminating in the bd -type oxidase were revealed by expression profiling of the bc ₁ -deficient mutant strain using a partial-genome microarray of M. smegmatis that is enriched in essential genes. Although the expression profile was indicative of an increase in the reduced state of the respiratory chain, blockage of the bc ₁ - aa ₃ pathway did not induce the sentinel genes of M. smegmatis that are induced by oxygen starvation and are regulated by the DosR two-component regulator.

Mycobacterium tuberculosis contains five genes, rpfA through rpfE , that bear significant homology to the resuscitation-promoting factor ( rpf ) gene of Micrococcus luteus , whose product is required to resuscitate the growth of dormant cultures of M. luteus and is essential for the growth of this organism. Previous studies have shown that deletion of any one of the five rpf -like genes did not affect the growth or survival of M. tuberculosis in vitro. In conjunction with the results of whole-genome expression profiling, this finding was indicative of their functional redundancy. In this study, we demonstrate that the single deletion mutants are phenotypically similar to wild-type M. tuberculosis H37Rv in vivo. The deletion of individual rpf -like genes had no discernible effect on the growth or long-term survival of M. tuberculosis in liquid culture, and the ability to resuscitate spontaneously from a nonculturable state in a most probable number assay was also unaffected for the three strains tested (the Δ rpfB , ΔrpfD , and Δ rpfE strains). In contrast, two multiple strains, KDT8 (Δ rpfA -mutation ΔrpfC ΔrpfB ) and KDT9 (Δ rpfA ΔrpfC ΔrpfD ), which lack three of the five rpf -like genes, were significantly yet differentially attenuated in a mouse infection model. These mutants were also unable to resuscitate spontaneously in vitro, demonstrating the importance of the Rpf-like proteins of M. tuberculosis in resuscitation from the nonculturable state. These results strongly suggest that the biological functions of the five rpf -like genes of M. tuberculosis are not wholly redundant and underscore the potential utility of these proteins as targets for therapeutic intervention.

Mycobacterium tuberculosis , the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ . In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis . We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a Δ nrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE , nrdF2 , nrdF1 , and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O ₂ -limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O ₂ restriction. However, survival of the Δ nrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F ₁ mice infected with the Δ nrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model.