Edith E. Machowski

The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro

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.

VapC Toxins from Mycobacterium tuberculosis Are Ribonucleases that Differentially Inhibit Growth and Are Neutralized by Cognate VapB Antitoxins

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as ‘non-toxic’. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of ‘non-toxic’ VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617 – VapC proteins with similarity to Rv0549c and Rv3320c, respectively – these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.

Essential roles for imuA′- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2–type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA′) and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA′ and DnaE2, as well as with the β-clamp. Moreover, disruption of the ImuB-β clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA′, imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the α-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA′-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance.

Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the beta-clamp, consistent with its canonical C-terminal beta-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N(2)-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.

DNA alkylation damage as a sensor of nitrosative stress in Mycobacterium tuberculosis.

ABSTRACT One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N′-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is restricted.

In Vitro Analysis of Rates and Spectra of Mutations in a Polymorphic Region of the Rv0746 PE_PGRS Gene of Mycobacterium tuberculosis

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.

Comparative genomics for mycobacterial peptidoglycan remodelling enzymes reveals extensive genetic multiplicity

BackgroundMycobacteria comprise diverse species including non-pathogenic, environmental organisms, animal disease agents and human pathogens, notably Mycobacterium tuberculosis. Considering that the mycobacterial cell wall constitutes a significant barrier to drug penetration, the aim of this study was to conduct a comparative genomics analysis of the repertoire of enzymes involved in peptidoglycan (PG) remodelling to determine the potential of exploiting this area of bacterial metabolism for the discovery of new drug targets.ResultsWe conducted an in silico analysis of 19 mycobacterial species/clinical strains for the presence of genes encoding resuscitation promoting factors (Rpfs), penicillin binding proteins, endopeptidases, L,D-transpeptidases and N-acetylmuramoyl-L-alanine amidases. Our analysis reveals extensive genetic multiplicity, allowing for classification of mycobacterial species into three main categories, primarily based on their rpf gene complement. These include the M. tuberculosis Complex (MTBC), other pathogenic mycobacteria and environmental species. The complement of these genes within the MTBC and other mycobacterial pathogens is highly conserved. In contrast, environmental strains display significant genetic expansion in most of these gene families. Mycobacterium leprae retains more than one functional gene from each enzyme family, underscoring the importance of genetic multiplicity for PG remodelling. Notably, the highest degree of conservation is observed for N-acetylmuramoyl-L-alanine amidases suggesting that these enzymes are essential for growth and survival.ConclusionPG remodelling enzymes in a range of mycobacterial species are associated with extensive genetic multiplicity, suggesting functional diversification within these families of enzymes to allow organisms to adapt.

Cleavage of the moaX-encoded fused molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity

BackgroundMolybdopterin cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1, moaD2, moaE1, moaE2, the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis.ResultsUsing a heterologous Mycobacterium smegmatis host and the activity of the MoCo-dependent nitrate reductase, we confirmed that moaD2 and moaE2 from M. tuberculosis together encode a functional MPT synthase. In contrast, moaD1 displayed no functionality in this system, even in the presence of the MoeBR sulphurtransferase, which contains the rhodansese-like domain, predicted to activate MoaD subunits. We demonstrated that cleavage of MoaX into its constituent MoaD and MoaE subunits was required for MPT synthase activity and confirmed that cleavage occurs between the Gly82 and Ser83 residues in MoaX. Further analysis of the Gly81-Gly82 motif confirmed that both of these residues are necessary for catalysis and that the Gly81 was required for recognition/cleavage of MoaX by an as yet unidentified protease. In addition, the MoaE component of MoaX was able to function in conjunction with M. smegmatis MoaD2 suggesting that cleavage of MoaX renders functionally interchangeable subunits. Expression of MoaX in E. coli revealed that incorrect post-translational processing is responsible for the lack of activity of MoaX in this heterologous host.ConclusionsThere is a degree of functional interchangeability between the MPT synthase subunits of M. tuberculosis. In the case of MoaX, post-translational cleavage at the Gly82 residue is required for function.

Resuscitation promoting factors are required for biofilm formation in Mycobacterium smegmatis

The cell wall of pathogenic mycobacteria is composed of peptidoglycan, arabinogalactan, mycolic acids, and an outer capsule. This inherent complexity renders it resistant to many antibiotics. Consequently, its biosynthesis and remodeling during growth directly impact viability. Resuscitation-promoting factors (Rpfs), enzymes with lytic transglycosylase activity, have been associated with the revival of dormant cells and subsequent resumption of vegetative growth. Mycobacterium smegmatis, a soil saprophyte and close relative of the human pathogen Mycobacterium tuberculosis, encodes four distinct Rpfs. Herein, we assessed the relationship between Rpfs and biofilm formation, which is used as a model to study drug tolerance and bacterial signaling in mycobacteria. We demonstrated that progressive deletion of rpf genes hampered the development of biofilms and reduced drug tolerance. These effects were accompanied by a reduction in muropeptide production and altered peptidoglycan cross-linking. Collectively, these observations point to an important role for Rpfs in mycobacterial communication and drug tolerance. ABSTRACT Resuscitation-promoting factors (Rpfs) have previously been shown to act as growth-stimulatory molecules via their lysozyme-like activity on peptidoglycan in the bacterial cell wall. In this study, we investigated the ability of Mycobacterium smegmatis strains lacking rpf genes to form biofilms and tested their susceptibilities to cell wall-targeting agents. M. smegmatis contains four distinct rpf homologues, namely, MSMEG_5700 (rpfA), MSMEG_5439 (rpfB), MSMEG_4640 (rpfE2), and MSMEG_4643 (rpfE). During axenic growth of the wild-type strain, all four mRNA transcripts were expressed to various degrees, but the expression of MSMEG_4643 was significantly greater during exponential growth. Similarly, all rpf mRNA transcripts could be detected in biofilms grown for 7, 14, and 28 days, with MSMEG_4643 expressed at the highest abundance after 7 days. In-frame unmarked deletion mutants (single and combinatorial) were generated and displayed altered colony morphologies and the inability to form typical biofilms. Moreover, any strain lacking rpfA and rpfB simultaneously exhibited increased susceptibility to rifampin, vancomycin, and SDS. Exogenous Rpf supplementation in the form of culture filtrate failed to restore biofilm formation. Liquid chromatography-mass spectrometry (LC-MS) analysis of peptidoglycan (PG) suggested a reduction in 4-3 cross-linked PG in the ΔrpfABEE2 mutant strain. In addition, the level of PG-repeat units terminating in 1,6-anhydroMurNAc appeared to be significantly reduced in the quadruple rpf mutant. Collectively, our data have shown that Rpfs play an important role in biofilm formation, possibly through alterations in PG cross-linking and the production of signaling molecules. IMPORTANCE The cell wall of pathogenic mycobacteria is composed of peptidoglycan, arabinogalactan, mycolic acids, and an outer capsule. This inherent complexity renders it resistant to many antibiotics. Consequently, its biosynthesis and remodeling during growth directly impact viability. Resuscitation-promoting factors (Rpfs), enzymes with lytic transglycosylase activity, have been associated with the revival of dormant cells and subsequent resumption of vegetative growth. Mycobacterium smegmatis, a soil saprophyte and close relative of the human pathogen Mycobacterium tuberculosis, encodes four distinct Rpfs. Herein, we assessed the relationship between Rpfs and biofilm formation, which is used as a model to study drug tolerance and bacterial signaling in mycobacteria. We demonstrated that progressive deletion of rpf genes hampered the development of biofilms and reduced drug tolerance. These effects were accompanied by a reduction in muropeptide production and altered peptidoglycan cross-linking. Collectively, these observations point to an important role for Rpfs in mycobacterial communication and drug tolerance.

Genetic Mimetics of Mycobacterium tuberculosis and MethicillinResistant Staphylococcus aureus as Verification Standards for Molecular Diagnostics

ABSTRACT Molecular diagnostics have revolutionized the management of health care through enhanced detection of disease or infection and effective enrollment into treatment. In recognition of this, the World Health Organization approved the rollout of nucleic acid amplification technologies for identification of Mycobacterium tuberculosis using platforms such as GeneXpert MTB/RIF, the GenoType MTBDRplus line probe assay, and, more recently, GeneXpert MTB/RIF Ultra. These assays can simultaneously detect tuberculosis infection and assess rifampin resistance. However, their widespread use in health systems requires verification and quality assurance programs. To enable development of these, we report the construction of genetically modified strains of Mycobacterium smegmatis that mimic the profile of Mycobacterium tuberculosis on both the GeneXpert MTB/RIF and the MTBDRplus line probe diagnostic tests. Using site-specific gene editing, we also created derivatives that faithfully mimic the diagnostic result of rifampin-resistant M. tuberculosis, with mutations at positions 513, 516, 526, 531, and 533 in the rifampin resistance-determining region of the rpoB gene. Next, we extended this approach to other diseases and demonstrated that a Staphylococcus aureus gene sequence can be introduced into M. smegmatis to generate a positive response for the SCCmec probe in the GeneXpert SA Nasal Complete molecular diagnostic cartridge, designed for identification of methicillin-resistant S. aureus. These biomimetic strains are cost-effective, have low biohazard content, accurately mimic drug resistance, and can be produced with relative ease, thus illustrating their potential for widespread use as verification standards for diagnosis of a variety of diseases.