Iron is one of the crucial elements required for the growth of Mycobacterium tuberculosis. However, excess free iron becomes toxic for the cells because it catalyzes the production of reactive oxygen radicals, leading to oxidative damage. Hence, it is essential for the pathogen to have the ability to store intracellular iron in an iron-rich environment and utilize it under iron depletion. M. tuberculosis has two iron storage proteins, namely BfrA (Rv1876; a bacterioferritin) and BfrB (Rv3841; a ferritin-like protein). However, the demonstration of biological significance requires the disruption of relevant genes and the evaluation of the resulting mutant for its ability to survive in the host and cause disease. In this study, we have disrupted bfrA and bfrB of M. tuberculosis and demonstrated that these genes are crucial for the storage and supply of iron for the growth of bacteria and to withstand oxidative stress in vitro. In addition, the bfrA bfrB double mutant (H37Rv ⌬bfrA ⌬bfrB) exhibited a marked reduction in its ability to survive inside human macrophages. Guinea pigs infected with H37Rv ⌬bfrA ⌬bfrB exhibited a marked diminution in the dissemination of the bacilli to spleen compared to that of the parental strain. Moreover, guinea pigs infected with H37Rv ⌬bfrA ⌬bfrB exhibited significantly reduced pathological damage in spleen and lungs compared to that of animals infected with the parental strain. Our study clearly demonstrates the importance of these iron storage proteins in the survival and pathogenesis of M. tuberculosis in the host and establishes them as attractive targets for the development of new inhibitors against mycobacterial infections.

This study highlights the importance of mycobactins in the growth and virulence of M. tuberculosis and establishes the enzymes of mycobactin biosynthesis as novel targets for the development of therapeutic interventions against tuberculosis.

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.

BackgroundThe failure of Mycobacterium bovis Bacille Calmette-Guérin to impart satisfactory protection against adult pulmonary tuberculosis has necessitated the development of more effective TB vaccines. The assumption that the vaccine strain should be antigenically as similar as possible to the disease causing pathogen has led to the evaluation of M.tuberculosis mutants as candidate tuberculosis vaccines.Methods/Principal FindingsIn this study, we have generated a mutant of M.tuberculosis (Mtb∆mms) by disrupting 3 virulence genes encoding a mycobacterial secretory acid phosphatase (sapM) and two phosphotyrosine protein phosphatases (mptpA and mptpB) and have evaluated its protective efficacy in guinea pigs. We observed that Mtb∆mms was highly attenuated in THP-1 macrophages. Moreover, no bacilli were recovered from the lungs and spleens of guinea pigs after 10 weeks of Mtb∆mms inoculation, although, initially, the mutant exhibited some growth in the spleens. Subsequently, when Mtb∆mms was evaluated for its protective efficacy, we observed that similar to BCG vaccination, Mtb∆mms exhibited a significantly reduced CFU in the lungs of guinea pigs when compared with the unvaccinated animals at 4 weeks after challenge. In addition, our observations at 12 weeks post challenge demonstrated that Mtb∆mms exhibited a more sustainable and superior protection in lungs as compared to BCG. However, the mutant failed to control the hematogenous spread as the splenic bacillary load between Mtb∆mms vaccinated and sham immunized animals was not significantly different. The gross pathological observations and histopathological observations corroborated the bacterial findings. Inspite of disruption of phosphatase genes in MtbΔmms, the lipid profiles of M.tuberculosis and MtbΔmms were identical indicating thereby that the phenotype of the mutant was ascribed to the loss of phosphatase genes and the influence was not related to any alteration in the lipid composition.Conclusions/SignificanceThis study highlights the importance of M.tuberculosis mutants in imparting protection against pulmonary TB.

BackgroundSevere acute respiratory infections in children can be fatal, rapid identification of the causative agent and timely treatment can be life saving. Multiplex real time RT-PCR helps in simultaneous detection of multiple viruses saving cost, time and labour. Commercially available multiplex real time RT-PCR kits are very expensive. Therefore the aim of the present study was to develop a cost effective multiplex real time RT-PCR for the detection of 18 respiratory viruses and compare it with an in-vitro diagnostics approved Fast Track Diagnostic Respiratory Pathogens 21 Kit (FTD).MethodsNasopharyngeal aspirates and throat swabs were collected and processed for extraction of nucleic acid using an automated extraction system and multiplex real time RT-PCR was performed using the FTD kit and a custom assay on 356 samples.ResultsCustom and FTD assays detected one or more respiratory viruses in 268 (75.29 %) and 262 (73.60 %) samples respectively. The concordance between the custom assay and the FTD assay was 100 % for HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, HPeV, Flu A, and Influenza A(H1N1)pdm09 and 94.66 – 99.71 % for the remaining viruses; Flu B (99.71 %), HRV (99.71 %), HPIV-3 (98.87 %), HPIV-4 (99.43 %), HCoV NL63 (99.71 %), HMPV A/B (99.71 %), RSV A/B (94.66 %), EV (98.31 %), HCoV HKU1 (99.71 %), HAdV (99.71 %). Major discrepancy was observed for RSV A/B, which was over detected in 18 samples by the custom assay as compared to the FTD assay. The custom assay was much cheaper than the FTD assay and the time taken was only 29 min more.ConclusionThe custom primer and probe mix was found to be comparable to the FTD assay with good concordance but was much cheaper and the time taken for reporting was only 29 min more. The low cost custom multiplex RT-PCR can be a useful alternative to the costly FTD kit for rapid identification of viral aetiology in resource limited settings.

Background & objectives:Severe acute respiratory infection (SARI) is one of the leading causes of death among children worldwide. As different respiratory viruses exhibit similar symptoms, simultaneous detection of these viruses in a single reaction mixture can save time and cost. The present study was done in a tertiary care children's hospital for rapid identification of viruses causing SARI among children less than or equal to five years of age using multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) kit.Methods:A total of 155 throat swabs were collected from equal number of children suspected to have SARI and processed for extraction of nucleic acids using automated extraction system. Multiplex real-time RT-PCR was done to identify the viruses in the samples.Results:The overall positivity for viruses in the study was found to be 72.9 per cent with a co-infection rate of 19.5 per cent. Human metapneumovirus (HMPV) was the predominant virus detected in 25.7 per cent children followed by influenza A (H1N1)pdm09, human rhinovirus (HRV) and human adenovirus (HAdV) in 19.9, 11.0 and 8.8 per cent children, respectively. The HMPV was at its peak in February 2013, HAdV showed two peaks in March-April, 2012 and November 2012-March 2013 while HRV was detected throughout the year.Interpretation & conclusions:Multiplex real-time PCR helped in rapid identification of viruses. Seventeen viruses were detected in SARI cases with overall positivity of 72.9 per cent. HMPV was the most predominant virus. However, for better clinico-virological correlation, studies are required with complete work up of all the aetiological agents, clinical profile of patients and treatment outcome.

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kefB is annotated as a potassium/proton antiporter in M. tuberculosis. There have been divergent reports on the involvement of KefB in phagosomal maturation in M. bovis BCG and no investigation has been carried out on its role in M. tuberculosis, the pathogenic species responsible for causing tuberculosis. This study was taken up to ascertain the involvement of KefB in the growth of M. tuberculosis and its role in phagosomal maturation and survival of the pathogen in guinea pigs. Our findings show that kefB mutant of M. tuberculosis (MtbΔkefB) was impaired i) for growth in high concentrations of potassium and ii) in arresting phagosomal acidification. However, the disruption of kefB had no adverse effect on the survival of M. tuberculosis in macrophages as well as in guinea pigs suggesting that the role of KefB in phagosomal acidification is unrelated to the survival of the pathogen in the host.