Rv1255c, a dormancy-related transcriptional regulator of TetR family in Mycobacterium tuberculosis, enhances isoniazid tolerance in Mycobacterium smegmatis.

Mycobacterium tuberculosis is exposed to diverse stresses inside the host during dormancy. Meanwhile, many metabolic and transcriptional regulatory changes occur, resulting in physiological modifications that help M. tuberculosis to adapt to these stresses. The same physiological changes also cause antibiotic tolerance in dormant M. tuberculosis. However, the transcriptional regulatory mechanism of antibiotic tolerance during dormancy remains unclear. Here, we showed that the expression of Rv1255c, an uncharacterised member of the tetracycline repressor family of transcriptional regulators, is upregulated during different stresses and hypoxia-induced dormancy. Antibiotic tolerance and efflux activities of Mycobacterium smegmatis constitutively expressing Rv1255c were analysed, and interestingly, it showed increased isoniazid tolerance and efflux activity. The intrabacterial isoniazid concentrations were found to be low in M. smegmatis expressing Rv1255c. Moreover, orthologs of the M. tuberculosis katG, gene of the enzyme which activates the first-line prodrug isoniazid, are overexpressed in this strain. Structural analysis of isoforms of KatG enzymes in M. smegmatis identified major amino acid substitutions associated with isoniazid resistance. Thus, we showed that Rv1255c helps M. smegmatis tolerate isoniazid by orchestrating drug efflux machinery. In addition, we showed that Rv1255c also causes overexpression of katG isoform in M. smegmatis which has amino acid substitutions as found in isoniazid-resistant katG in M. tuberculosis.

Overexpression of a membrane transport system MSMEG_1381 and MSMEG_1382 confers multidrug resistance in Mycobacterium smegmatis.

Mycobacterium tuberculosis is a leading cause of human mortality worldwide, and the emergence of drug-resistant strains demands the discovery of new classes of antimycobacterial that can be employed in the therapeutic pipeline. Previously, a secondary metabolite, chrysomycin A, isolated from Streptomyces sp. OA161 displayed potent bactericidal activity against drug-resistant clinical isolates of M. tuberculosis and different species of mycobacteria. The antibiotic inhibits mycobacterial topoisomerase I and DNA gyrase, leading to bacterial death, but the mechanisms that could cause resistance to this antibiotic are currently unknown. To further understand the resistance mechanism, using M. smegmatis as a model, spontaneous resistance mutants were isolated and subjected to whole-genome sequencing. Mutation in a TetR family transcriptional regulator MSMEG_1380 was identified in the resistant isolates wherein the gene was adjacent to an operon encoding membrane proteins MSMEG_1381 and MSMEG_1382. Sequence analysis and modeling studies indicated that MSMEG_1381 and MSMEG_1382 are components of the Mmp family of efflux pumps and over-expression of either the operon or individual genes conferred resistance to chrysomycin A, isoniazid, and ethambutol. Our study highlights the role of membrane transporter proteins in conferring multiple drug resistance and the utility of recombinant strains overexpressing membrane transporters in the drug screening pipeline.

Prevalence and heterogeneity of antibiotic resistance genes in Orientia tsutsugamushi and other rickettsial genomes.

Despite a million infections every year and an estimated one billion people at risk, scrub typhus is regarded as a neglected tropical disease. The causative bacterium Orientia tsutsugamushi, a member of rickettsiae, seems to be intrinsically resistant to several classes of antibiotics. The emergence of antibiotic-resistant scrub typhus is likely to become a global public health concern. Yet, it is unknown as to how common antibiotic resistance genes are in O. tsutsugamushi, and how variable these loci are among the genomes of rickettsiae. By using the comprehensive antibiotic resistance database, we explored 79 complete genomes from 24 species of rickettsiae for antibiotic resistance loci. There were 244 unique antibiotic resistance genes in rickettsiae. Both the total and unique antibiotic resistance genes in O. tsutsugamushi were significantly less compared to other members of rickettsiae. However, antibiotic resistance genes in O. tsutsugamushi genomes were more unique and highly variable. Many genes such as resistant variants of evgS, and vanS A/G were present in numerous copies. These results will have important implications in the context of antibiotic-resistant scrub typhus.

The Error-Prone Polymerase DnaE2 Mediates the Evolution of Antibiotic Resistance in Persister Mycobacterial Cells.

Applying antibiotics to susceptible bacterial cultures generates a minor population of persisters that remain susceptible to antibiotics but can endure them for extended periods. Recent reports suggest that antibiotic persisters (APs) of mycobacteria experience oxidative stress and develop resistance upon treatment with lethal doses of ciprofloxacin or rifampicin. However, the mechanisms driving the de novo emergence of resistance remained unclear. Here, we show that mycobacterial APs activate the SOS response, resulting in the upregulation of the error-prone DNA polymerase DnaE2. The sustained expression of dnaE2 in APs led to mutagenesis across the genome and resulted in the rapid evolution of resistance to antibiotics. Inhibition of RecA by suramin, an anti-Trypanosoma drug, reduced the rate of conversion of persisters to resistors in a diverse group of bacteria. Our study highlights suramin's novel application as a broad-spectrum agent in combating the development of drug resistance.

Base excision repair pathways of bacteria: new promise for an old problem.

Infectious diseases continue to be a major cause of human mortality. With the emergence of drug resistance, diseases that were long thought to have been curable by antibiotics are resurging. There is an urgent clinical need for newer antibiotics that target novel cellular pathways to overcome resistance to currently used therapeutics. The base excision repair (BER) pathways of the pathogen restore altered bases and safeguard the genomic integrity of the pathogen from the host's immune response. Although the BER machinery is of paramount importance to the survival of the pathogens, its potential as a drug target is largely unexplored. In this review, we discuss the importance of BER in different pathogenic organisms and the potential of its inhibition with small molecules.

The Capacity of Mycobacterium tuberculosis To Survive Iron Starvation Might Enable It To Persist in Iron-Deprived Microenvironments of Human Granulomas.

This study was conducted to investigate the role of iron deprivation in the persistence of Mycobacterium tuberculosis We present evidence of iron restriction in human necrotic granulomas and demonstrate that under iron starvation M. tuberculosis persists, refractive to antibiotics and capable of restarting replication when iron is made available. Transcriptomics and metabolomic analyses indicated that the persistence of M. tuberculosis under iron starvation is dependent on strict control of endogenous Fe utilization and is associated with upregulation of pathogenicity and intrinsic antibiotic resistance determinants. M. tuberculosis mutants compromised in their ability to survive Fe starvation were identified. The findings of this study advance the understanding of the physiological settings that may underpin the chronicity of human tuberculosis (TB) and are relevant to the design of effective antitubercular therapies.IMPORTANCE One-third of the world population may harbor persistent M. tuberculosis, causing an asymptomatic infection that is refractory to treatment and can reactivate to become potentially lethal tuberculosis disease. However, little is known about the factors that trigger and maintain M. tuberculosis persistence in infected individuals. Iron is an essential nutrient for M. tuberculosis growth. In this study, we show, first, that in human granulomas the immune defense creates microenvironments in which M. tuberculosis likely experiences drastic Fe deprivation and, second, that Fe-starved M. tuberculosis is capable of long-term persistence without growth. Together, these observations suggest that Fe deprivation in the lung might trigger a state of persistence in M. tuberculosis and promote chronic TB. We also identified vulnerabilities of iron-restricted persistent M. tuberculosis, which can be exploited for the design of new antitubercular therapies.

miR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila.

microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, our study reveals a mutual repression between miR-34 expression and ecdysone signaling, and identifies miR-34 as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity.

The mycobacterial iron-dependent regulator IdeR induces ferritin (bfrB) by alleviating Lsr2 repression.

Emerging evidence indicates that precise regulation of iron (Fe) metabolism and maintenance of Fe homeostasis in Mycobacterium tuberculosis (Mtb) are essential for its survival and proliferation in the host. IdeR is a central transcriptional regulator of Mtb genes involved in Fe metabolism. While it is well understood how IdeR functions as a repressor, how it induces transcription of a subset of its targets is still unclear. We investigated the molecular mechanism of IdeR-mediated positive regulation of bfrB, the gene encoding the major Fe-storage protein of Mtb. We found that bfrB induction by Fe required direct interaction of IdeR with a DNA sequence containing four tandem IdeR-binding boxes located upstream of the bfrB promoter. Results of in vivo and in vitro transcription assays identified a direct repressor of bfrB, the histone-like protein Lsr2. IdeR counteracted Lsr2-mediated repression in vitro, suggesting that IdeR induces bfrB transcription by antagonizing the repressor activity of Lsr2. Together, these results elucidate the main mechanism of bfrB positive regulation by IdeR and identify Lsr2 as a new factor contributing to Fe homeostasis in mycobacteria.

SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function.

microRNAs (miRNAs) are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22-24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs) that contain Argonaute (AGO) family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. Select pre-mRNA splicing factors have been implicated in small RNA-mediated gene silencing pathways in fission yeast, worms, flies and mammals, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP) implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. Finally, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Our study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be evolutionarily widespread.

Hypersensitivity of hypoxia grown Mycobacterium smegmatis to DNA damaging agents: implications of the DNA repair deficiencies in attenuation of mycobacteria.

Mycobacteria are an important group of pathogenic bacteria. We generated a series of DNA repair deficient strains of Mycobacterium smegmatis, a model organism, to understand the importance of various DNA repair proteins (UvrB, Ung, UdgB, MutY and Fpg) in survival of the pathogenic strains. Here, we compared tolerance of the M. smegmatis strains to genotoxic stress (ROS and RNI) under aerobic, hypoxic and recovery conditions of growth by monitoring their survival. We show an increased susceptibility of mycobacteria to genotoxic stress under hypoxia. UvrB deficiency led to high susceptibility of M. smegmatis to the DNA damaging agents. Ung was second in importance in strains with single deficiencies. Interestingly, we observed that while deficiency of UdgB had only a minor impact on the strain's susceptibility, its combination with Ung deficiency resulted in severe consequences on the strain's survival under genotoxic stress suggesting a strong interdependence of different DNA repair pathways in safeguarding genomic integrity. Our observations reinforce the possibility of targeting DNA repair processes in mycobacteria for therapeutic intervention during active growth and latency phase of the pathogen. High susceptibility of the UvrB, or the Ung/UdgB deficient strains to genotoxic stress may be exploited in generation of attenuated strains of mycobacteria.

Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila.

RNAi is an evolutionarily conserved gene regulatory process that operates in a wide variety of organisms. During RNAi, long double-stranded RNA precursors are processed by Dicer proteins into ∼21-nt siRNAs. Subsequently, siRNAs are incorporated into the RNA-induced silencing complexes (RISCs) that contain Argonaute-family proteins and guide RISC to target RNAs via complementary base pairing, leading to posttranscriptional gene silencing. Select pre-mRNA splicing factors have been implicated in RNAi in fission yeast, worms, and flies, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle implicated in splicing, is required for RNAi and antiviral immunity in cultured cells and in vivo. SmD1 interacts with both Dicer-2 and dsRNA precursors and is indispensable for optimal siRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the small interfering RISC without significantly affecting the expression of major canonical siRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the small interfering RISC, including Argonaute 2, both in flies and in humans. Notably, RNAi defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the RNAi and splicing machineries are physically and functionally distinct entities. Our results suggest that Drosophila SmD1 plays a direct role in RNAi-mediated gene silencing independently of its pre-mRNA splicing activity and indicate that the dual roles of splicing factors in posttranscriptional gene regulation may be evolutionarily widespread.

Development of a new generation of vectors for gene expression, gene replacement, and protein-protein interaction studies in mycobacteria.

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.

Distinct mechanisms of DNA repair in mycobacteria and their implications in attenuation of the pathogen growth.

About a third of the human population is estimated to be infected with Mycobacterium tuberculosis. Emergence of drug resistant strains and the protracted treatment strategies have compelled the scientific community to identify newer drug targets, and to develop newer vaccines. In the host macrophages, the bacterium survives within an environment rich in reactive nitrogen and oxygen species capable of damaging its genome. Therefore, for its successful persistence in the host, the pathogen must need robust DNA repair mechanisms. Analysis of M. tuberculosis genome sequence revealed that it lacks mismatch repair pathway suggesting a greater role for other DNA repair pathways such as the nucleotide excision repair, and base excision repair pathways. In this article, we summarize the outcome of research involving these two repair pathways in mycobacteria focusing primarily on our own efforts. Our findings, using Mycobacterium smegmatis model, suggest that deficiency of various DNA repair functions in single or in combinations severely compromises their DNA repair capacity and attenuates their growth under conditions typically encountered in macrophages.

Base excision and nucleotide excision repair pathways in mycobacteria.

About a third of the human population is estimated to be infected with Mycobacterium tuberculosis. The bacterium displays an excellent adaptability to survive within the host macrophages. As the reactive environment of macrophages is capable of inducing DNA damage, the ability of the pathogen to safeguard its DNA against the damage is of paramount significance for its survival within the host. Analysis of the genome sequence has provided important insights into the DNA repair machinery of the pathogen, and the studies on DNA repair in mycobacteria have gained momentum in the past few years. The studies have revealed considerable differences in the mycobacterial DNA repair machinery when compared with those of the other bacteria. This review article focuses especially on the aspects of base excision, and nucleotide excision repair pathways in mycobacteria.

Detrimental effects of hypoxia-specific expression of uracil DNA glycosylase (Ung) in Mycobacterium smegmatis.

Mycobacterium tuberculosis is known to reside latently in a significant fraction of the human population. Although the bacterium possesses an aerobic mode of metabolism, it adapts to persistence under hypoxic conditions such as those encountered in granulomas. While in mammalian systems hypoxia is a recognized DNA-damaging stress, aspects of DNA repair in mycobacteria under such conditions have not been studied. We subjected Mycobacterium smegmatis, a model organism, to the Wayne's protocol of hypoxia. Analysis of the mRNA of a key DNA repair enzyme, uracil DNA glycosylase (Ung), by real-time reverse transcriptase PCR (RT-PCR) revealed its downregulation during hypoxia. However, within an hour of recovery of the culture under normal oxygen levels, the Ung mRNA was restored. Analysis of Ung by immunoblotting and enzyme assays supported the RNA analysis results. To understand its physiological significance, we misexpressed Ung in M. smegmatis by using a hypoxia-responsive promoter of narK2 from M. tuberculosis. Although the misexpression of Ung during hypoxia decreased C-to-T mutations, it compromised bacterial survival upon recovery at normal oxygen levels. RT-PCR analysis of other base excision repair gene transcripts (UdgB and Fpg) suggested that these DNA repair functions also share with Ung the phenomenon of downregulation during hypoxia and recovery with return to normal oxygen conditions. We discuss the potential utility of this phenomenon in developing attenuated strains of mycobacteria.

Novel insertion and deletion mutants of RpoB that render Mycobacterium smegmatis RNA polymerase resistant to rifampicin-mediated inhibition of transcription.

The startling increase in the occurrence of rifampicin (Rif) resistance in the clinical isolates of Mycobacterium tuberculosis worldwide is posing a serious concern to tuberculosis management. The majority of Rif resistance in bacteria arises from mutations in the RpoB subunit of the RNA polymerase. We isolated M. smegmatis strains harbouring either an insertion (6 aa) or a deletion (10 aa) in their RpoB proteins. Although these strains showed a compromised fitness for growth in 7H9 Middlebrook medium, their resistance to Rif was remarkably high. The attenuated growth of the strains correlated with decreased specific activities of the RNA polymerases from the mutants. While the RNA polymerases from the parent or a mutant strain (harbouring a frequently occurring mutation, H442Y, in RpoB) were susceptible to Rif-mediated inhibition of transcription from calf thymus DNA, those from the insertion and deletion mutants were essentially refractory to such inhibition. Three-dimensional structure modelling revealed that the RpoB amino acids that interact with Rif are either deleted or unable to interact with Rif due to their unsuitable spatial positioning in these mutants. We discuss possible uses of the RpoB mutants in studying transcriptional regulation in mycobacteria and as potential targets for drug design.

Synergistic effects of UdgB and Ung in mutation prevention and protection against commonly encountered DNA damaging agents in Mycobacterium smegmatis.

The incorporation of dUMP during replication or the deamination of cytosine in DNA results in the occurrence of uracils in genomes. To maintain genomic integrity, uracil DNA glycosylases (UDGs) excise uracil from DNA and initiate the base-excision repair pathway. Here, we cloned, purified and biochemically characterized a family 5 UDG, UdgB, from Mycobacterium smegmatis to allow us to use it as a model organism to investigate the physiological significance of the novel enzyme. Studies with knockout strains showed that compared with the wild-type parent, the mutation rate of the udgB( -) strain was approximately twofold higher, whereas the mutation rate of a strain deficient in the family 1 UDG (ung(- )) was found to be approximately 8.4-fold higher. Interestingly, the mutation rate of the double-knockout (ung(-)/ udgB(-)) strain was remarkably high, at approximately 19.6-fold. While CG to TA mutations predominated in the ung(-) and ung(-)/udgB(-) strains, AT to GC mutations were enhanced in the udgB(-) strain. The ung(-)/udgB(-) strain was notably more sensitive to acidified nitrite and hydrogen peroxide stresses compared with the single knockouts (ung(-) or udgB(-)). These observations reveal a synergistic effect of UdgB and Ung in DNA repair, and could have implications for the generation of attenuated strains of Mycobacterium tuberculosis.

A distinct physiological role of MutY in mutation prevention in mycobacteria.

Oxidative damage to DNA results in the occurrence of 7,8-dihydro-8-oxoguanine (8-oxoG) in the genome. In eubacteria, repair of such damage is initiated by two major base-excision repair enzymes, MutM and MutY. We generated a MutY-deficient strain of Mycobacterium smegmatis to investigate the role of this enzyme in DNA repair. The MutY deficiency in M. smegmatis did not result in either a noteworthy susceptibility to oxidative stress or an increase in the mutation rate. However, rifampicin-resistant isolates of the MutY-deficient strain showed distinct mutations in the rifampicin-resistance-determining region of rpoB. Besides the expected C to A (or G to T) mutations, an increase in A to C (or T to G) mutations was also observed. Biochemical characterization of mycobacterial MutY (M. smegmatis and M. tuberculosis) revealed an expected excision of A opposite 8-oxoG in DNA. Additionally, excision of G and T opposite 8-oxoG was detected. MutY formed complexes with DNA containing 8-oxoG : A, 8-oxoG : G or 8-oxoG : T but not 8-oxoG : C pairs. Primer extension reactions in cell-free extracts of M. smegmatis suggested error-prone incorporation of nucleotides into the DNA. Based on these observations, we discuss the physiological role of MutY in specific mutation prevention in mycobacteria.

Important role of the nucleotide excision repair pathway in Mycobacterium smegmatis in conferring protection against commonly encountered DNA-damaging agents.

Mycobacteria are an important group of human pathogens. Although the DNA repair mechanisms in mycobacteria are not well understood, these are vital for the pathogen's persistence in the host macrophages. In this study, we generated a null mutation in the uvrB gene of Mycobacterium smegmatis to allow us to compare the significance of the nucleotide excision repair (NER) pathway with two important base excision repair pathways, initiated by uracil DNA glycosylase (Ung) and formamidopyrimidine DNA glycosylase (Fpg or MutM), in an isogenic strain background. The strain deficient in NER was the most sensitive to commonly encountered DNA-damaging agents such as UV, low pH, reactive oxygen species, hypoxia, and was also sensitive to acidified nitrite. Taken together with previous observations on NER-deficient M. tuberculosis, these results suggest that NER is an important DNA repair pathway in mycobacteria.