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1.
MethodsX ; 11: 102344, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37711139

ABSTRACT

Bacterial populations in the in vitro laboratory cultures, environment, and patients contain metabolically different subpopulations that respond differently to stress agents, including antibiotics, and emerge as stress tolerant or resistant strains. To contain the emergence of such strains, it is important to study the features of the metabolic status and response of the subpopulations to stress agents. For this purpose, an efficient method is required for the fractionation and isolation of the subpopulations from the cultures. Here we describe in detail the manual setting up of a simple, easy-to-do, reproducibly robust Percoll discontinuous density gradient centrifugation for the fractionation of subpopulations of short-sized cells (SCs) and normal/long-sized cells (NCs) from Mycobacterium smegmatis and Mycobacterium tuberculosis cultures, which we had reported earlier. About 90-98% enrichment was obtained respectively for SCs and NCs for M. smegmatis and 69-67% enrichment was obtained respectively for the SCs and NCs for M. tuberculosis.•The Percoll discontinuous density gradient centrifugation helps the fractionation and isolation of mycobacterial subpopulations that differ in density.•The method offers a consistently reproducible high enrichment of the subpopulations of SCs and NCs from the in vitro cultures of M. smegmatis and M. tuberculosis.•Our earlier reports on the consistency in the differential response of the subpopulations, enriched using the method, to oxidative, nitrite, and antibiotic stress proves its validity.

2.
Sci Immunol ; 8(88): eadg7015, 2023 10 27.
Article in English | MEDLINE | ID: mdl-37191508

ABSTRACT

Omicron SARS-CoV-2 variants escape vaccine-induced neutralizing antibodies and cause nearly all current COVID-19 cases. Here, we compared the efficacy of three booster vaccines against Omicron BA.5 challenge in rhesus macaques: mRNA-1273, the Novavax ancestral spike protein vaccine (NVX-CoV2373), or Omicron BA.1 spike protein version (NVX-CoV2515). All three booster vaccines induced a strong BA.1 cross-reactive binding antibody and changed immunoglobulin G (Ig) dominance from IgG1 to IgG4 in the serum. All three booster vaccines also induced strong and comparable neutralizing antibody responses against multiple variants of concern, including BA.5 and BQ.1.1, along with long-lived plasma cells in the bone marrow. The ratio of BA.1 to WA-1 spike-specific antibody-secreting cells in the blood was higher in NVX-CoV2515 animals compared with NVX-CoV2373 animals, suggesting a better recall of BA.1-specific memory B cells by the BA.1 spike-specific vaccine compared with the ancestral spike-specific vaccine. Further, all three booster vaccines induced low levels of spike-specific CD4 but not CD8 T cell responses in the blood. After challenge with SARS-CoV-2 BA.5 variant, all three vaccines showed strong protection in the lungs and controlled virus replication in the nasopharynx. In addition, both Novavax vaccines blunted viral replication in nasopharynx at day 2. The protection against SARS-CoV-2 BA.5 infection in the upper respiratory airways correlated with binding, neutralizing, and ADNP activities of the serum antibody. These data have important implications for COVID-19 vaccine development, because vaccines that lower nasopharyngeal virus may help to reduce transmission.


Subject(s)
2019-nCoV Vaccine mRNA-1273 , COVID-19 , Animals , Humans , COVID-19 Vaccines , COVID-19/prevention & control , Macaca mulatta , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Immunoglobulin G
3.
Front Microbiol ; 13: 920117, 2022.
Article in English | MEDLINE | ID: mdl-36338044

ABSTRACT

Bacteria regulate FtsZ protein levels through transcriptional and translational mechanisms for proper cell division. A cis-antisense RNA, StfZ, produced from the ftsA-ftsZ intergenic region, was proposed to regulate FtsZ level in Escherichia coli. However, its structural identity remained unknown. In this study, we determined the complete sequence of StfZ and identified the isoforms and its promoters. We find that under native physiological conditions, StfZ is expressed at a 1:6 ratio of StfZ:ftsZ mRNA at all growth phases from three promoters as three isoforms of 366, 474, and 552 nt RNAs. Overexpression of StfZ reduces FtsZ protein level, increases cell length, and blocks cell division without affecting the ftsZ mRNA stability. We did not find differential expression of StfZ under the stress conditions of heat shock, cold shock, or oxidative stress, or at any growth phase. These data indicated that the cis-encoded StfZ antisense RNA to ftsZ mRNA may be involved in the fine tuning of ftsZ mRNA levels available for translation as per the growth-phase-specific requirement at all phases of growth and cell division.

4.
Int J Mycobacteriol ; 11(3): 273-286, 2022.
Article in English | MEDLINE | ID: mdl-36260446

ABSTRACT

Background: We recently reported the de novo emergence of unusually high numbers of antibiotic resisters from the in vitro cultures of Mycobacterium tuberculosis and Mycobacterium smegmatis surviving in the presence of minimum bactericidal concentration (MBC) of antituberculosis antibiotics. The resisters emerged due to multiple asymmetric divisions of elongated mother cells containing multiple nucleoids and multiple septae. We had earlier found a minor subpopulation of short-sized cells (SCs) and a major subpopulation of normal-sized cells (NCs) (10% and 90%, respectively, of the whole population), with significant difference in antibiotic susceptibility and resister generation frequency, in the in vitro cultures of M. tuberculosis, M. smegmatis, and Mycobacterium xenopi, as well as in pulmonary tuberculosis patients' sputum. However, the mechanisms of growth and division promoting the emergence of antibiotic resisters from these subpopulations remained unknown. Therefore, here, we took up the first-time study to find out the mechanism of growth and division by which antibiotic resisters emerge from the antibiotic-surviving population of the two subpopulations of M. smegmatis. Methods: M. smegmatis SCs and NCs were fractionated from mid-log phase cultures using Percoll gradient centrifugation; their purity was checked and exposed to 10×, 2×, and 0.4× MBC of rifampicin for 120 h. The colony-forming units (CFUs) were determined on rifampicin-free plates for the total population and on rifampicin-containing plates for scoring rifampicin resisters. The phenotype and the morphology of the cells at various stages of the exposure were determined using transmission electron microscopy. The dynamic growth and division mechanisms of the cells to emerge as rifampicin resisters were monitored using live-cell time-lapse imaging. The rifampicin resisters were sequenced for mutations in the rifampicin resistance determining region of rpoB gene. Statistical significance was calculated using two-tailed paired t-test, with *P ≤ 0.05 and **P ≤ 0.01. Results: Multinucleated and multiseptated elongated cells emerged from their respective antibiotic-surviving populations. They produced a large number of sibling-daughter cells through multiple asymmetric divisions in short durations, showing abnormally high spurts in CFUs of antibiotic resisters. The CFUs were several-fold higher than that expected from the mass-doubling time of the subpopulations. Despite this commonality, the subpopulations showed specific differences in their response to different multiples of their respective MBC of rifampicin. Conclusions: Mycobacterial subpopulations come out of rifampicin stress by undergoing multiple nucleoid replications, multiple septation for nucleoid segregation, and acquisition of antibiotic target-specific mutations, followed by multiple asymmetric divisions to generate unusually a large number of rifampicin resisters. Because we had earlier shown that SCs and NCs are present in the pulmonary tuberculosis patients' sputum, the present findings have clinical relevance on the mechanism of emergence of antibiotic-resistant strains from mycobacterial subpopulations.


Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Humans , Rifampin/pharmacology , Anti-Bacterial Agents/pharmacology , Mycobacterium tuberculosis/genetics , Mycobacterium smegmatis/genetics
5.
Curr Res Microb Sci ; 3: 100148, 2022.
Article in English | MEDLINE | ID: mdl-35909613

ABSTRACT

Exposure to antibiotics most often generates oxidative stress in bacteria. Oxidative stress survival mechanisms would facilitate the evolution of antibiotic resistance. As part of an effort to understand oxidative stress survival mechanisms in mycobacteria, here we show that the minor subpopulation (SCs; short-sized cells constituting 10% of the population) of Mycobacterium smegmatis significantly increased the survival of its major kin subpopulation (NCs; normal/long-sized cells constituting 90% of the population) in the mid-log-phase (MLP) cultures against the oxidative stress induced by rifampicin and exogenously added H2O2 (positive control). We had earlier shown that the SCs in the MLP cultures inherently and naturally release significantly high levels of H2O2 into the medium. Addition of the SCs' culture supernatant, unlike the supernatant of the dimethylthiourea (H2O2 scavenger) exposed SCs, enhanced the survival of NCs. It indicated that NCs' survival required the H2O2 present in the SCs' supernatant. This H2O2 transcriptionally induced high levels of catalase-peroxidase (KatG) in the NCs. The naturally high KatG levels in the NCs significantly neutralised the endogenous H2O2 formed upon exposure to rifampicin or H2O2, thereby enhancing the survival of NCs against oxidative stress. The absence of such enhanced survival in the furA-katG and katG knockout (KO) mutants of NCs in the presence of wild-type SCs, confirmed the requirement of the H2O2 present in the SCs' supernatant and NCs' KatG for enhanced oxidative stress survival. The presence of SCs:NCs at 1:9 in the pulmonary tuberculosis patients' sputum alludes to the clinical significance of the finding.

6.
Int J Mycobacteriol ; 11(2): 150-158, 2022.
Article in English | MEDLINE | ID: mdl-35775547

ABSTRACT

Background: The antibiotic-exposed bacteria often contain the reactive oxygen species (ROS), hydroxyl radical, which inflicts genome-wide mutations, causing the de novo formation of antibiotic-resistant strains. Hydroxyl radical is generated by Fenton reaction of Fe (II) with the ROS, H2O2, which, in turn, is formed by the dismutation of the ROS, superoxide. Therefore, for the emergence of bacterial strains genetically resistant to antibiotics, increased levels of superoxide, H2O2, hydroxyl radical, and Fe (II) should be present in the antibiotic-exposed bacteria. Here, we verified this premise by finding out whether the in vitro cultures of M. smegmatis, exposed to MBC of moxifloxacin for a prolonged duration, contain significantly high levels of superoxide, H2O2, hydroxyl radical, and Fe (II). Methods: Biological triplicate cultures of M. smegmatis, were exposed to MBC of moxifloxacin for 84 h. The colony-forming units (CFUs) of the cultures were determined on moxifloxacin-free and moxifloxacin-containing plates for the entire 84 h at a regular interval of 6 h. The cultures were analyzed at specific time points of killing phase (KP), antibiotic-surviving phase (ASP), and regrowth phase (RGP) for the presence of superoxide, H2O2, hydroxyl radical, and Fe (II) using the ROS- and Fe (II)-detecting fluorescence probes. The experimental cultures were grown in the presence of ROS and Fe (II) quenchers also and determined the levels of fluorescence corresponding to the ROS- and Fe (II)-specific probes. This was performed to establish the specificity of detection of ROS and Fe (II). Biological triplicate cultures, unexposed to moxifloxacin but cultured for 84 h, were used as the control for the measurement of ROS and Fe (II) levels. The CFUs of the cultures were determined on moxifloxacin-free and moxifloxacin-containing plates for the entire 84 h at regular intervals of 6 h. Flow cytometry analyses were performed for the detection and quantitation of the levels of fluorescence of the ROS-and Fe (II)-specific probes. The experimental cultures were grown in the presence of thiourea and bipyridyl as the ROS and Fe (II) quenchers, respectively, for the determination of the levels of fluorescence corresponding to the ROS- and Fe (II)-specific probes. Paired t-test was used to calculate statistical significance (n = 3). Results: The moxifloxacin-exposed cultures, but not the cultures unexposed to moxifloxacin, showed a triphasic response with a KP, ASP, and RGP. The cells in the late KP and ASP contained significantly elevated levels of superoxide, H2O2, hydroxyl radical, and Fe (II). Thus, high levels of the ROS and Fe (II) were found in the small population (in the ASP) of M. smegmatis cells that survived the moxifloxacin-mediated killing. From this moxifloxacin-surviving population (in the ASP), moxifloxacin-resistant genetic resisters emerged de novo at high frequency, regrew, divided, and populated the cultures. The levels of these ROS, Fe (II), and the high moxifloxacin resister generation frequency were quenched in the cultures grown in the presence of the respective ROS and Fe (II) quenchers. The cultures unexposed to moxifloxacin did not show any of these responses, indicating that the whole response was specific to antibiotic exposure. Conclusions: Significantly high levels of superoxide, H2O2, hydroxyl radical, and Fe (II) were generated in the M. smegmatis cultures exposed to moxifloxacin for a prolonged duration. It promoted the de novo emergence of genetic resisters to moxifloxacin at high frequency.


Subject(s)
Hydroxyl Radical , Superoxides , Anti-Bacterial Agents/pharmacology , Humans , Hydrogen Peroxide/pharmacology , Moxifloxacin/pharmacology , Mycobacterium smegmatis , Reactive Oxygen Species
7.
Antimicrob Agents Chemother ; 66(5): e0228521, 2022 05 17.
Article in English | MEDLINE | ID: mdl-35435709

ABSTRACT

We had earlier reported the de novo emergence of genetic resisters of Mycobacterium tuberculosis and Mycobacterium smegmatis to rifampicin and moxifloxacin from the antibiotic-surviving population containing elevated levels of the non-DNA-specific mutagenic reactive oxygen species (ROS) hydroxyl radical. Since hydroxyl radical is generated by Fenton reaction between Fe(II) and H2O2, which is produced by superoxide dismutation, we here report significantly elevated levels of these three ROS and Fe(II) in the M. smegmatis rifampicin-surviving population. Elevated levels of superoxide and the consequential formation of high levels of H2O2 and Fe(II) led to the generation of hydroxyl radical, facilitating de novo high frequency emergence of antibiotic resisters. The M. smegmatis cultures, exposed to nontoxic concentrations of the ROS scavenger, thiourea (TU), and the NADH oxidase (one of the superoxide producers) inhibitor, diphenyleneiodonium chloride (DPI), showed a reduction in the levels of the three ROS, Fe(II), and antibiotic resister generation frequency. The non-antibiotic-exposed cultures grown in the absence/presence of TU/DPI did not show increased ROS, Fe(II) levels, or antibiotic resister generation frequency. The antibiotic-surviving population showed significantly increased expression and activity of superoxide-producing genes and decreased expression of antioxidant and DNA repair genes, revealing an environment conducive for the acquisition and retention of mutations. Since we recently reported significant comparability between the antibiotic-survival gene expression profiles of the saprophyte-cum-opportunistic pathogens M. smegmatis and the M. tuberculosis in tuberculosis patients undergoing treatment, we discuss the clinical relevance of the findings on the mechanism of emergence of antibiotic-resistant mycobacterial strains.


Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Ferrous Compounds/pharmacology , Humans , Hydrogen Peroxide/metabolism , Hydroxyl Radical/metabolism , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Reactive Oxygen Species/metabolism , Rifampin/metabolism , Rifampin/pharmacology , Superoxides/metabolism
8.
FEMS Microbiol Lett ; 368(14)2021 07 20.
Article in English | MEDLINE | ID: mdl-34240144

ABSTRACT

The bacterial populations surviving in the presence of antibiotics contain cells that have gained genetic resistance, phenotypic resistance and tolerance to antibiotics. Isolation of live bacterial population, surviving against antibiotics, from the milieu of high proportions of dead/damaged cells will facilitate the study of the cellular/molecular processes used by them for survival. Here we present a Percoll gradient centrifugation based method for the isolation of enriched population of Mycobacterium smegmatis surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin. From the time of harvest, throughout the enrichment and isolation processes, and up to the lysis of the cells for total RNA preparation, we maintained the cells in the presence of the antibiotic to avoid changes in their metabolic status. The total RNA extracted from the enriched population of live antibiotic-surviving population showed structural integrity and purity. We analysed the transcriptome profile of the antibiotic-surviving population and compared it with the orthologue genes of Mycobacterium tuberculosis that conferred antibiotic tolerance on tubercle bacilli isolated from the tuberculosis patients under treatment with four antitubercular antibiotics. Statistically significant comparability between the gene expression profiles of the antibiotic tolerance associated genes of M. smegmatis and M. tuberculosis validated the reliability/utility of the method.


Subject(s)
Bacteriological Techniques/methods , Moxifloxacin/pharmacology , Mycobacterium smegmatis/isolation & purification , Mycobacterium smegmatis/physiology , Rifampin/pharmacology , Antitubercular Agents/pharmacology , Drug Tolerance/genetics , Gene Expression Profiling , Humans , Microbial Sensitivity Tests , Microbial Viability/drug effects , Microbial Viability/genetics , Mycobacterium Infections, Nontuberculous/microbiology , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/genetics , Reproducibility of Results
9.
Cells ; 10(5)2021 05 11.
Article in English | MEDLINE | ID: mdl-34064643

ABSTRACT

Twenty to thirty percent of the septating mycobacterial cells of the mid-log phase population showed highly deviated asymmetric constriction during division (ACD), while the remaining underwent symmetric constriction during division (SCD). The ACD produced short-sized cells (SCs) and normal/long-sized cells (NCs) as the sister-daughter cells, but with significant differential susceptibility to antibiotic/oxidative/nitrite stress. Here we report that, at 0.2% glycerol, formulated in the Middlebrook 7H9 medium, a significantly high proportion of the cells were divided by SCD. When the glycerol concentration decreased to 0.1% due to cell-growth/division, the ACD proportion gradually increased until the ACD:SCD ratio reached ~50:50. With further decrease in the glycerol levels, the SCD proportion increased with concomitant decrease in the ACD proportion. Maintenance of glycerol at 0.1%, through replenishment, held the ACD:SCD proportion at ~50:50. Transfer of the cells from one culture with a specific glycerol level to the supernatant from another culture, with a different glycerol level, made the cells change the ACD:SCD proportion to that of the culture from which the supernatant was taken. RT-qPCR data showed the possibility of diadenosine tetraphosphate phosphorylase (MSMEG_2932), phosphatidylinositol synthase (MSMEG_2933), and a Nudix family hydrolase (MSMEG_2936) involved in the ACD:SCD proportion-change in response to glycerol levels. We also discussed its physiological significance.


Subject(s)
Glycerol/metabolism , Mycobacterium bovis/metabolism , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/metabolism , Acid Anhydride Hydrolases/metabolism , Antioxidants/metabolism , CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase/metabolism , Cell Proliferation , Culture Media , DNA, Complementary/metabolism , Glycerol/chemistry , Humans , Mutation , Oxidative Stress , Pyrophosphatases/metabolism , RNA/metabolism , Tuberculosis , Tuberculosis, Multidrug-Resistant/microbiology , Nudix Hydrolases
10.
mSphere ; 5(6)2020 11 18.
Article in English | MEDLINE | ID: mdl-33208519

ABSTRACT

The emergence of antibiotic genetic resisters of pathogenic bacteria poses a major public health challenge. The mechanism by which bacterial antibiotic genetic resister clones formed de novo multiply and establish a resister population remained unknown. Here, we delineated the unique mode of cell division of the antibiotic genetic resisters of Mycobacterium smegmatis and Mycobacterium tuberculosis formed de novo from the population surviving in the presence of bactericidal concentrations of rifampicin or moxifloxacin. The cells in the rifampicin/moxifloxacin-surviving population generated elevated levels of hydroxyl radical-inflicting mutations. The genetic mutants selected against rifampicin/moxifloxacin became multinucleated and multiseptated and developed multiple constrictions. These cells stochastically divided multiple times, producing sister-daughter cells phenomenally higher in number than what could be expected from their generation time. This caused an abrupt, unexpectedly high increase in the rifampicin/moxifloxacin resister colonies. This unique cell division behavior was not shown by the rifampicin resisters formed naturally in the actively growing cultures. We could detect such abrupt increases in the antibiotic resisters in others' and our earlier data on the antibiotic-exposed laboratory/clinical M. tuberculosis strains, M. smegmatis and other bacteria in in vitro cultures, infected macrophages/animals, and tuberculosis patients. However, it went unnoticed/unreported in all those studies. This phenomenon occurring in diverse bacteria surviving against different antibiotics revealed the broad significance of the present study. We speculate that the antibiotic-resistant bacillary clones, which emerge in patients with diverse bacterial infections, might be using the same mechanism to establish an antibiotic resister population quickly in the continued presence of antibiotics.IMPORTANCE The bacterial pathogens that are tolerant to antibiotics and survive in the continued presence of antibiotics have the chance to acquire genetically resistant mutations against the antibiotics and emerge de novo as antibiotic resisters. Once the antibiotic resister clone has emerged, often with compromise on growth characteristics, for the protection of the species, it is important to establish an antibiotic-resistant population quickly in the continued presence of the antibiotic. In this regard, the present study has unraveled multinucleation and multiseptation followed by multiple constrictions as the cellular processes used by the bacteria for quick multiplication to establish antibiotic-resistant populations. The study also points out the same phenomenon occurring in other bacterial systems investigated in our laboratory and others' laboratories. Identification of these specific cellular events involved in quick multiplication offers additional cellular processes that can be targeted in combination with the existing antibiotics' targets to preempt the emergence of antibiotic-resistant bacterial strains.


Subject(s)
Antibiotics, Antitubercular/pharmacology , Cell Division/drug effects , Drug Resistance, Bacterial/genetics , Mycobacterium smegmatis/drug effects , Mycobacterium tuberculosis/drug effects , Bacterial Proteins/genetics , Drug Tolerance , Moxifloxacin/pharmacology , Mutation , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/genetics , Rifampin/pharmacology
11.
Front Microbiol ; 10: 2339, 2019.
Article in English | MEDLINE | ID: mdl-31681204

ABSTRACT

Bacteria undergo adaptive morphological changes to survive under stress conditions. The present work documents the morphological changes in Mycobacterium tuberculosis (Mtb) cells cultured under hypoxic condition using Wayne's in vitro hypoxia model involving non-replicating persistence stages 1 and 2 (NRP stage 1 and NRP stage 2) and reveals their physiological significance. Transmission electron microscopy of the NRP stage 2 cells showed uneven but thick outer layer (TOL), unlike the evenly thin outer layer of the actively growing mid-log phase (MLP) cells. On the contrary, the saprophytic Mycobacterium smegmatis NRP stage 2 cells lacked TOL. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) of the Mtb NRP stage 2 cells confirmed the rough uneven surface unlike the smooth surface of the MLP cells. Zeta potential measurements showed high negative charge on the surface of NRP stage 2 cells and polysaccharide specific calcofluor white (CFW) staining of the cells revealed high content of polysaccharide in the TOL. This observation was supported by the real-time PCR data showing high levels of expression of the genes involved in the synthesis of sugars, such as trehalose, mannose and others, which are implicated in polysaccharide synthesis. Experiments to understand the physiological significance of the TOL revealed restricted entry of the biologically low-active 5-carboxyfluorescein-rifampicin (5-FAM-RIF), at concentrations equivalent to microbicidal concentrations of the unconjugated biologically active rifampicin, into the NRP stage 2 cells, unlike in the MLP cells. Further, as expected, mechanical removal of the TOL by mild bead beating or release of the NRP stage 2 cells from hypoxia into normoxia in fresh growth medium also significantly increased 5-FAM-RIF permeability into the NRP stage 2 cells to an extent comparable to that into the MLP cells. Taken together, these observations revealed that Mtb cells under hypoxia develop TOL that helps in restricting rifampicin entry, thereby conferring rifampicin tolerance.

12.
Redox Biol ; 19: 116-133, 2018 10.
Article in English | MEDLINE | ID: mdl-30149290

ABSTRACT

Oxidative stress response in bacteria is mediated through coordination between the regulators of oxidant-remediation systems (e.g. OxyR, SoxR) and nucleoid condensation (e.g. Dps, Fis). However, these genetic factors are either absent or rendered non-functional in the human pathogen Mycobacterium tuberculosis (Mtb). Therefore, how Mtb organizes genome architecture and regulates gene expression to counterbalance oxidative imbalance is unknown. Here, we report that an intracellular redox-sensor, WhiB4, dynamically links genome condensation and oxidative stress response in Mtb. Disruption of WhiB4 affects the expression of genes involved in maintaining redox homeostasis, central metabolism, and respiration under oxidative stress. Notably, disulfide-linked oligomerization of WhiB4 in response to oxidative stress activates the protein's ability to condense DNA. Further, overexpression of WhiB4 led to hypercondensation of nucleoids, redox imbalance and increased susceptibility to oxidative stress, whereas WhiB4 disruption reversed this effect. In accordance with the findings in vitro, ChIP-Seq data demonstrated non-specific binding of WhiB4 to GC-rich regions of the Mtb genome. Lastly, data indicate that WhiB4 deletion affected the expression of ~ 30% of genes preferentially bound by the protein, suggesting both direct and indirect effects on gene expression. We propose that WhiB4 structurally couples Mtb's response to oxidative stress with genome organization and transcription.


Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Mycobacterium tuberculosis/genetics , Oxidative Stress , Repressor Proteins/genetics , Tuberculosis/microbiology , Animals , Bacterial Proteins/metabolism , Gene Deletion , Genome, Bacterial , Humans , Mice , Mycobacterium tuberculosis/metabolism , Oxidation-Reduction , RAW 264.7 Cells , Repressor Proteins/metabolism , Up-Regulation
13.
Front Microbiol ; 8: 463, 2017.
Article in English | MEDLINE | ID: mdl-28377757

ABSTRACT

The present study shows the existence of two specific sub-populations of Mycobacterium smegmatis and Mycobacterium tuberculosis cells differing in size and density, in the mid-log phase (MLP) cultures, with significant differential susceptibility to antibiotic, oxidative, and nitrite stress. One of these sub-populations (~10% of the total population), contained short-sized cells (SCs) generated through highly-deviated asymmetric cell division (ACD) of normal/long-sized mother cells and symmetric cell divisions (SCD) of short-sized mother cells. The other sub-population (~90% of the total population) contained normal/long-sized cells (NCs). The SCs were acid-fast stainable and heat-susceptible, and contained high density of membrane vesicles (MVs, known to be lipid-rich) on their surface, while the NCs possessed negligible density of MVs on the surface, as revealed by scanning and transmission electron microscopy. Percoll density gradient fractionation of MLP cultures showed the SCs-enriched fraction (SCF) at lower density (probably indicating lipid-richness) and the NCs-enriched fraction (NCF) at higher density of percoll fractions. While live cell imaging showed that the SCs and the NCs could grow and divide to form colony on agarose pads, the SCF, and NCF cells could independently regenerate MLP populations in liquid and solid media, indicating their full genomic content and population regeneration potential. CFU based assays showed the SCF cells to be significantly more susceptible than NCF cells to a range of concentrations of rifampicin and isoniazid (antibiotic stress), H2O2 (oxidative stress),and acidified NaNO2 (nitrite stress). Live cell imaging showed significantly higher susceptibility of the SCs of SC-NC sister daughter cell pairs, formed from highly-deviated ACD of normal/long-sized mother cells, to rifampicin and H2O2, as compared to the sister daughter NCs, irrespective of their comparable growth rates. The SC-SC sister daughter cell pairs, formed from the SCDs of short-sized mother cells and having comparable growth rates, always showed comparable stress-susceptibility. These observations and the presence of M. tuberculosis SCs and NCs in pulmonary tuberculosis patients' sputum earlier reported by us imply a physiological role for the SCs and the NCs under the stress conditions. The plausible reasons for the higher stress susceptibility of SCs and lower stress susceptibility of NCs are discussed.

14.
Article in English | MEDLINE | ID: mdl-27895008

ABSTRACT

Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.


Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Bacterial/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Bacterial Proteins/genetics , DNA Gyrase/genetics , DNA-Directed RNA Polymerases/genetics , Dose-Response Relationship, Drug , Drug Resistance, Bacterial/drug effects , Fluoroquinolones/pharmacology , Genome, Bacterial , Hydroxyl Radical/metabolism , Moxifloxacin , Mutation , Mycobacterium tuberculosis/growth & development , Oxidative Stress/drug effects , Rifampin/administration & dosage , Rifampin/pharmacology
15.
PLoS One ; 10(12): e0143677, 2015.
Article in English | MEDLINE | ID: mdl-26630542

ABSTRACT

INTRODUCTION: Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. METHODS: Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. RESULTS: NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK. CONCLUSION: Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.


Subject(s)
Bacterial Proteins/metabolism , Cytoskeletal Proteins/metabolism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/biosynthesis , Nucleoside-Diphosphate Kinase/metabolism , Adenosine Triphosphate/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Microscopy, Electron, Transmission , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Nucleoside-Diphosphate Kinase/genetics , Polymerization , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
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