jefA (Rv2459), a drug efflux gene in Mycobacterium tuberculosis confers resistance to isoniazid & ethambutol

Background & objectives: Drug efflux pumps have been contributing factor(s) in the development of multidrug resistance in various clinically relevant bacteria. During efflux pump gene expression studies on mycobacteria, we have found a previously uncharacterized open reading frame (ORF) Rv2459 to be overexpressed in drug stressed conditions. The objective of the present study was to investigate the role of this ORF as a drug efflux pump, which might add new information in our understanding about the alternative mechanisms of drug resistance in mycobacteria. Methods: The open reading frame Rv2459 of Mycobacterium tuberculosis encoding a probable drug efflux protein has been cloned using pSD5 E.coli-Mycobacterium shuttle vector and overexpressed in M. tuberculosis H37Rv. This ORF was named as jefA. Overexpression of this gene in clones has been verified by real-time reverse transcription PCR. Minimum inhibitory concentrations (MICs) of recombinant as well as non-recombinant clones were determined by resazurin microtitre assay plate method (REMA) with and without efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and verapamil. Results: In recombinant strains of M. tuberculosis, the overexpression of this gene led to an increase in MIC of anti-tubercular drugs isoniazid and ethambutol when tested by REMA. In the presence of CCCP and verapamil, the recombinant strains showed decrease in MIC for these drugs. Bioinformatic analysis has shown a close relation of JefA protein with drug efflux pumps of other clinically relevant bacteria. In homology derived structure prepared from nearest available model, it was observed that amino acids forming TMH 1, 8 and 11 participated in ethambutol specificity and those forming TMH 2, 7 and 10 participated in isoniazid specificity in JefA. Interpretation & conclusion: The increased transcription of jefA leads to increased resistance to ethambutol and isoniazid in M. tuberculosis via efflux pump like mechanism and contributes in the development of resistance to these drugs. JefA amino acid sequence is well conserved among clinically important bacterial genera, which further provides evidence of being a potent drug efflux pump. The involvement in drug resistance and very little homology with any of the human proteins makes JefA important to be included in the list of potential drug targets

Apurinic/apyrimidinic endonucleases of Mycobacterium tuberculosis protect against DNA damage but are dispensable for the growth of the pathogen in guinea pigs.

In host cells, Mycobacterium tuberculosis encounters an array of reactive molecules capable of damaging its genome. Non-bulky DNA lesions are the most common damages produced on the exposure of the pathogen to reactive species and base excision repair (BER) pathway is involved in the repair of such damage. During BER, apurinic/apyrimidinic (AP) endonuclease enzymes repair the abasic sites that are generated after spontaneous DNA base loss or by the action of DNA glycosylases, which if left unrepaired lead to inhibition of replication and transcription. However, the role of AP endonucleases in imparting protection against DNA damage and in the growth and pathogenesis of M.tuberculosis has not yet been elucidated. To demonstrate the biological significance of these enzymes in M.tuberculosis, it would be desirable to disrupt the relevant genes and evaluate the resulting mutants for their ability to grow in the host and cause disease. In this study, we have generated M.tuberculosis mutants of the base excision repair (BER) system, disrupted in either one (MtbΔend or MtbΔxthA) or both the AP endonucleases (MtbΔendΔxthA). We demonstrate that these genes are crucial for bacteria to withstand alkylation and oxidative stress in vitro. In addition, the mutant disrupted in both the AP endonucleases (MtbΔendΔxthA) exhibited a significant reduction in its ability to survive inside human macrophages. However, infection of guinea pigs with either MtbΔend or MtbΔxthA or MtbΔendΔxthA resulted in the similar bacillary load and pathological damage in the organs as observed in the case of infection with wild-type M.tuberculosis. The implications of these observations are discussed

Secreted Acid Phosphatase (SapM) of Mycobacterium tuberculosis Is Indispensable for Arresting Phagosomal Maturation and Growth of the Pathogen in Guinea Pig Tissues

10.1371/journal.pone.0070514PLoS ONE87e70514-e7051

Secreted acid phosphatase (SapM) of Mycobacterium tuberculosis is indispensable for arresting phagosomal maturation and growth of the pathogen in guinea pig tissues.

Tuberculosis (TB) is responsible for nearly 1.4 million deaths globally every year and continues to remain a serious threat to human health. The problem is further complicated by the growing incidence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), emphasizing the need for the development of new drugs against this disease. Phagosomal maturation arrest is an important strategy employed by Mycobacterium tuberculosis to evade the host immune system. Secretory acid phosphatase (SapM) of M.tuberculosis is known to dephosphorylate phosphotidylinositol 3-phosphate (PI3P) present on phagosomes. However, there have been divergent reports on the involvement of SapM in phagosomal maturation arrest in mycobacteria. This study was aimed at reascertaining the involvement of SapM in phagosomal maturation arrest in M.tuberculosis. Further, for the first time, we have also studied whether SapM is essential for the pathogenesis of M.tuberculosis. By deleting the sapM gene of M.tuberculosis, we demonstrate that MtbΔsapM is defective in the arrest of phagosomal maturation as well as for growth in human THP-1 macrophages. We further show that MtbΔsapM is severely attenuated for growth in the lungs and spleen of guinea pigs and has a significantly reduced ability to cause pathological damage in the host when compared with the parental strain. Also, the guinea pigs infected with MtbΔsapM exhibited a significantly enhanced survival when compared with M.tuberculosis infected animals. The importance of SapM in phagosomal maturation arrest as well as in the pathogenesis of M.tuberculosis establishes it as an attractive target for the development of new therapeutic molecules against tuberculosis

Apurinic/Apyrimidinic Endonucleases of Mycobacterium tuberculosis Protect against DNA Damage but Are Dispensable for the Growth of the Pathogen in Guinea Pigs

10.1371/journal.pone.0092035PLoS ONE95e92035-e9203

Iron Storage Proteins Are Essential for the Survival and Pathogenesis of Mycobacterium tuberculosis in THP-1 Macrophages and the Guinea Pig Model of Infection

10.1128/jb.05553-11Journal of Bacteriology1943567-57

Influence of disruption of AP endonuclease(s) on the susceptibility of <i>M.tuberculosis</i> to DNA damaging agents.

<p>The figure represents the fraction of surviving bacteria after a 24(A) methyl methane sulfonate (MMS), (B) hydrogen peroxide (H₂O₂) and (C) mitomycin C (MMC). The results indicate that while both the AP endonucleases are important in protecting the pathogen against alkylation damage, End plays a more important role than XthA in protecting this pathogen against oxidative assault. End and XthA do not contribute to the repair of intrastrand and interstrand cross-linking of DNA. The percentage of the bacteria surviving after addition of the stress agent in comparison to the bacteria without the addition of stress agent is represented as the mean (±SE) of two independent experiments carried out in duplicates. NS, not significant; *, <i>P</i><0.05; **, <i>P</i><0.001 and ***, <i>P</i><0.001 (One way ANOVA).</p

Disruption of both the AP endonucleases impairs the growth of <i>M.tuberculosis</i> in THP-1 macrophages.

<p>THP-1 cells were infected with wild-type <i>M.tuberculosis</i>, MtbΔ<i>end</i>, MtbΔ<i>xthA</i> or MtbΔ<i>end</i>Δ<i>xthA</i> at an MOI of 1∶5 (bacteria∶macrophage). The number of intracellular viable bacteria was determined on each alternative day for 6 days. A significant attenuation in the growth of MtbΔ<i>end</i>Δ<i>xthA</i> in comparison to wild-type <i>M.tuberculosis</i> was observed on the 4^th and 6^th day of infection. The growth of single mutants MtbΔ<i>end</i> and MtbΔ<i>xthA</i> was comparable with wild-type <i>M.tuberculosis</i>. The values are represented as the mean (±SE) of three independent infections and the experiment was repeated three times. ***, <i>P</i><0.001 (Two way ANOVA).</p

Gross pathology of guinea pig organs infected with various <i>M.tuberculosis</i> strains at 16 weeks.

<p>The figure depicts representative photographs of gross pathological lesions and graphical depiction of gross scores of lung, liver and spleen of guinea pigs (n = 5) infected with <i>M.tuberculosis</i>, MtbΔ<i>sapM</i> and MtbΔ<i>sapM</i>Comp euthanized at 16 weeks post-infection. Each data point represents the score of an individual animal, and the bars depict medians (±interquartile range) for each group. Organs of the MtbΔ<i>sapM</i> infected animals exhibited minimal involvement with the presence of only a few visible tubercles when compared with the organs of guinea pigs infected with either <i>M.tuberculosis</i> or the MtbΔ<i>sapM</i>Comp that exhibited a heavy involvement with numerous large sized tubercles and necrosis. *, <i>P</i><0.05; **, <i>P</i><0.01 (Mann-Whitney U test). Missing data points represent the animals that succumbed to disease before the time of euthanasia.</p

Oligonucleotides employed in this study.

<p>Oligonucleotides employed in this study.</p