Mridula Bose

Population Genetics Study of Isoniazid Resistance Mutations and Evolution of Multidrug-Resistant Mycobacterium tuberculosis

ABSTRACT The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.

Role of embB Codon 306 Mutations in Mycobacterium tuberculosis Revisited: a Novel Association with Broad Drug Resistance and IS6110 Clustering Rather than Ethambutol Resistance

ABSTRACT Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P = 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P = 0.008) and katG315 (odds ratio, 1.99; P = 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.

Pulmonary epithelial cells are a source of interferon-gamma in response to Mycobacterium tuberculosis infection.

Recent report from our laboratory showed that A549 cells representing alveolar epithelial cells produce chemokine interleukin‐8 and nitric oxide (NO) when challenged with Mycobacterium tuberculosis. Interferon‐γ (IFN‐γ) played a critical role in priming these cells to generate NO in vitro. In the present study, we report that M. tuberculosis‐infected A549 cells are capable of elaborating IFN‐γ as shown by enzyme‐linked immunosorbent assay and intracellular staining for IFN‐γ. Secretion profile indicated that M. tuberculosis‐infected A549 released significantly high concentration of IFN‐γ at 48 and 72 h post‐infection. Low level of IFN‐γ release was also seen to be induced by γ‐irradiated M. tuberculosis and subcellular components of M. tuberculosis. Cell surface receptor analysis showed that the M. tuberculosis‐infected A549 cells expressed enhanced levels of IFN‐γ receptors. This observation suggests that the endogenously produced IFN‐γ in response to M. tuberculosis infection plays a role in intracellular regulation of innate immunity against intracellular pathogen such as M. tuberculosis. This observation is further strengthened by the fact that infected A549 cells expressed signal transducer and activator of transcription 1 (STAT1), an important mediator for IFN‐γ signaling pathway, leading to expression of inducible NO synthase and subsequent release of NO in sufficient concentration to be mycobactericidal. Our results show that production of IFN‐γ and enhanced expression of IFN‐γ receptors by infected A549 cells is a local phenomenon occurring as de novo intracellular activity, in response to M. tuberculosis infection. To the best of our knowledge, this is the first report to show that A549 cells interact actively with M. tuberculosis to produce IFN‐γ that might play an important role in innate immunity against tuberculosis.

Induction of nitric oxide release from the human alveolar epithelial cell line A549: an in vitro correlate of innate immune response to Mycobacterium tuberculosis

In view of the presence of a large number of epithelial cells in the alveoli of the lung and their ability to produce various cytokines and chemokines, the possible role of alveolar epithelial cells in the innate immune response to tuberculosis was examined. The human alveolar epithelial cell line A549 was used as a model. The ability of A549 cells to induce nitric oxide (NO) in response to Mycobacterium tuberculosis infection was taken as an in vitro correlate of innate immunity. M. tuberculosis infection induced A549 cells to produce significant levels of NO and to express inducible nitric oxide synthase mRNA at 48 hr of infection. However, the amount of NO released at this point was not mycobactericidal. Cytokine stimulation (interferon‐γ, tumour necrosis factor‐α, interleukin‐1β, alone or in combination) of the infected A549 cells induced a higher concentration of NO. The study of colony‐forming units (CFU) as a measure of the mycobactericidal capacity of A549 cells revealed a reduction in CFU of M. tuberculosis by 39·29% (from 10·62 ± 0·48 – 6·392 ± 0·54) following cytokine stimulation of the infected cells. Interestingly γ‐irradiated M. tuberculosis H37Rv could also induce higher than basal level of NO. Therefore we examined mycobacterial antigenic components for their possible role in NO production. We observed that A549 cells produced significantly higher amounts of NO at 48 hr when treated with mycobacterial whole cell lysates, cell wall or cell membrane preparations. The release of NO and the resultant mycobactericidal activity could be further enhanced by simultaneously conditioning the M. tuberculosis infected A549 cells with cytokine and mycobacterial components. These results suggest that alveolar epithelial cells respond to their microenvironment, which is constituted of various cytokines and macrophage‐processed antigens and may contribute to the innate immune response to tuberculosis.

Analysis of Expression Profile of Mammalian Cell Entry (mce) Operons of Mycobacterium tuberculosis

ABSTRACT The sequencing of the complete genome of M. tuberculosis H37Rv has resulted in the recognition of four mce operons in its genome by in silico analysis. In an attempt to understand the significance of the redundancy of mce operons, we analyzed the expression profile of mce operons after different periods of growth in culture as well as during in vivo infection. Our results strongly suggest that mce1 is expressed as a polycistronic message. In culture from day 8 to day 12, expression of only mce1 was observed, but as the cultures progress towards stationary phase the expression profile of mce operons was altered; the transcripts of the mce1 operon were barely detected while those of the mce4 operon were prominent. In an analysis of the expression of mce operons in tubercle material collected from infected animal tissues, we detected the expression of mce1, -3 and -4. Our results imply that mce operons other than mce1 are also expressed during infection and that it is necessary to examine their role in pathogenesis.

Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis Isolates from India and Mexico by a Molecular Beacon Assay

ABSTRACT We assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-resistant Mycobacterium tuberculosis by using clinical isolates from north India and Mexico, regions with a high incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short region of the M. tuberculosis rpoB gene contains mutations that predict rifampin resistance in most isolates. Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin susceptibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%) and specificity (99%) for use in countries with a high prevalence of tuberculosis.

Mycobacterium tuberculosis induces high production of nitric oxide in coordination with production of tumour necrosis factor-α in patients with fresh active tuberculosis but not in MDR tuberculosis

Mycobacterium tuberculosis is an intracellular pathogen that readily survives and replicates in human macrophages. Host cells have developed various mycobactericidal and immunoregulatory mechanisms, such as the production of nitric oxide and inflammatory cytokines to control intracellular replication of M. tuberculosis. Inducible nitric oxide synthase (iNOS) is transcriptionally under the control of IFN‐γ and TNF‐α. IL‐12 provides a crucial link between activated mononuclear phagocytes and T cells by regulating the production of IFN‐γ. In this study, we investigated the production of nitric oxide (NO), TNF‐α and IL‐12 by the peripheral blood monocytes (PB Mn) of patients suffering from multidrug‐resistant tuberculosis (MDR‐TB). The cells were infected with M. tuberculosis and stimulated with IFN‐γ or activated with mycobacterial subcellular components. The results were compared with those from cases of newly diagnosed TB and healthy controls. Nitric oxide production was significantly depressed in PB Mn from MDR‐TB patients. Infected monocytes from newly diagnosed TB patients produced significantly higher levels of NO as compared to those from MDR‐TB patients or normal controls. The subcellular fraction of M. tuberculosis‐like whole cell lysate (WCL), culture filtrate protein (CFP) and lipoarabinomannan (LAM) induced higher concentrations of NO release in PB Mn from newly diagnosed TB patients as compared to those from MDR‐TB patients. Cell culture supernatant from PB Mn assayed at 48 h after infection or stimulation demonstrated significantly depressed release of TNF‐α and IL‐12 from MDR‐TB cases as compared to the fresh cases. We observed a definite correlation between nitric oxide release and TNF‐α production, irrespective of low or high production in MDR‐TB or fresh cases, respectively. The present data suggest that peripheral blood monocytes of MDR‐TB patients typically show signs of immunosuppression. Whether such immunodepression is the cause or the effect of MDR‐TB merits further investigation.

Characterization of Mce4A protein of Mycobacterium tuberculosis: role in invasion and survival

BackgroundThe mce4 operon is one of the four homologues of mammalian cell entry (mce) operons of Mycobacterium tuberculosis. The mce4A (Rv3499c) gene within this operon is homologous to mce1A (Rv0169), that has a role in host cell invasion by M. tuberculosis. Our earlier reports show that mce4 operon is expressed during the stationary phase of growth of the bacillus in culture and during the course of infection in mammalian hosts. M. tuberculosis carrying mutation in mce4 operon shows growth defect and reduced survival in infected mice. However, the intracellular localization of Mce4A protein and its direct role in cell entry or survival of the bacillus has not been demonstrated so far.ResultsBy transmission electron microscopy we have demonstrated that recombinant Mce4A protein facilitates the invasion of non-pathogenic strain of E. coli into non-phagocytic HeLa cells. We observe that mce4A gene has a role comparable to mce1A in the survival of recombinant E. coli in human macrophages. Using antibodies raised against Mce4A protein, we show that the protein is localized in the cell wall fraction of M. tuberculosis H37Rv stationary phase culture only.ConclusionMce4A protein is expressed during the stationary phase of broth culture and localizes in the cell wall fraction of M. tuberculosis. Mce4A protein expressed in non-pathogenic E. coli enables it to enter and survive within HeLa cells and the macrophages. As Mce4A protein is expressed during later phase of mycobacterial growth, our results raise the possibility of it playing a role in maintenance of persistent tubercular infection.

Proinflammatory cytokines can significantly induce human mononuclear phagocytes to produce nitric oxide by a cell maturation-dependent process

The capacity of three proinflammatory cytokines, interferon-gamma (rhifn-gamma), tumour-necrosis factor-alpha (rHTNF-alpha) and interleukin-1 (rHIL-1), to induce release of nitric oxide (NO) from human mononuclear phagocytes were investigated. Peripheral blood monocytes were either used immediately or after culturing in vitro to develop into monocyte-derived macrophages (macrophages). Lipopolysaccharide (LPS) was used as second signal in all experiments. The three cytokines tested had significantly high enhancing influence on the production of nitric oxide by monocytes as well as by macrophages. However production was significantly higher by the monocytes matured in vitro to macrophages (P < 0.01). In our experimental system LPS had only marginal synergistic influence on production of NO2, and IFN-gamma demonstrated to be the most efficient of the three cytokines tested. Addition of L-arginine in the monocytes/macrophages culture further amplified production of NO2, whereas addition of NG-monomethylarginine abrogated this amplification. We conclude that human mononuclear phagocytes are capable of using inducible nitric oxide synthase pathway to produce nitric oxide.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.