Standardized RS Ratio Metrics To Assess Tuberculosis Antimicrobial Efficacy and Potency.

The sigmoid Emax model was used to describe the rRNA synthesis ratio (RS ratio) response of Mycobacterium tuberculosis to antimicrobial concentration. RS-Emax measures the maximal ability of a drug to inhibit the RS ratio and can be used to rank-order drugs based on their RS ratio effect. RS-EC90 is the concentration needed to achieve 90% of the RS-Emax, which may guide dose selection to achieve a maximal RS ratio effect in vivo.

Population heterogeneity in Mycobacterium smegmatis and Mycobacterium abscessus.

Bacteria use population heterogeneity, the presence of more than one phenotypic variant in a clonal population, to endure diverse environmental challenges - a 'bet-hedging' strategy. Phenotypic variants have been described in many bacteria, but the phenomenon is not well-understood in mycobacteria, including the environmental factors that influence heterogeneity. Here, we describe three reproducible morphological variants in M. smegmatis - smooth, rough, and an intermediate morphotype that predominated under typical laboratory conditions. M. abscessus has two recognized morphotypes, smooth and rough. Interestingly, M. tuberculosis exists in only a rough form. The shift from smooth to rough in both M. smegmatis and M. abscessus was observed over time in extended static culture, however the frequency of the rough morphotype was high in pellicle preparations compared to planktonic culture, suggesting a role for an aggregated microenvironment in the shift to the rough form. Differences in growth rate, biofilm formation, cell wall composition, and drug tolerance were noted among M. smegmatis and M. abscessus variants. Deletion of the global regulator lsr2 shifted the M. smegmatis intermediate morphotype to a smooth form but did not fully phenocopy the naturally generated smooth morphotype, indicating Lsr2 is likely downstream of the initiating regulatory cascade that controls these morphotypes. Rough forms typically correlate with higher invasiveness and worse outcomes during infection and our findings indicate the shift to this rough form is promoted by aggregation. Our findings suggest that mycobacterial population heterogeneity, reflected in colony morphotypes, is a reproducible, programmed phenomenon that plays a role in adaptation to unique environments and this heterogeneity may influence infection progression and response to treatment.

Anaerobic Mycobacterium tuberculosis Cell Death Stems from Intracellular Acidification Mitigated by the DosR Regulon.

Mycobacterium tuberculosis is a strict aerobe capable of prolonged survival in the absence of oxygen. We investigated the ability of anaerobic M. tuberculosis to counter challenges to internal pH homeostasis in the absence of aerobic respiration, the primary mechanism of proton efflux for aerobic bacilli. Anaerobic M. tuberculosis populations were markedly impaired for survival under a mildly acidic pH relative to standard culture conditions. An acidic environmental pH greatly increased the susceptibilities of anaerobic bacilli to the collapse of the proton motive force by protonophores, to antimicrobial compounds that target entry into the electron transport system, and to small organic acids with uncoupling activity. However, anaerobic bacilli exhibited high tolerance against these challenges at a near-neutral pH. At a slightly alkaline pH, which was near the optimum intracellular pH, the addition of protonophores even improved the long-term survival of bacilli. Although anaerobic M. tuberculosis bacilli under acidic conditions maintained 40% lower ATP levels than those of bacilli under standard culture conditions, ATP loss alone could not explain the drop in viability. Protonophores decreased ATP levels by more than 90% regardless of the extracellular pH but were bactericidal only under acidic conditions, indicating that anaerobic bacilli could survive an extreme ATP loss provided that the external pH was within viable intracellular parameters. Acidic conditions drastically decreased the anaerobic survival of a DosR mutant, while an alkaline environment improved the survival of the DosR mutant. Together, these findings indicate that intracellular acidification is a primary challenge for the survival of anaerobic M. tuberculosis and that the DosR regulon plays a critical role in sustaining internal pH homeostasis.IMPORTANCE During infection, M. tuberculosis bacilli are prevalent in environments largely devoid of oxygen, yet the factors that influence the survival of these severely growth-limited and metabolically limited bacilli remain poorly understood. We determined how anaerobic bacilli respond to fluctuations in environmental pH and observed that these bacilli were highly susceptible to stresses that promoted internal acidic stress, whereas conditions that promoted an alkaline internal pH promoted long-term survival even during severe ATP depletion. The DosR regulon, a major regulator of general hypoxic stress, played an important role in maintaining internal pH homeostasis under anaerobic conditions. Together, these findings indicate that in the absence of aerobic respiration, protection from internal acidification is crucial for long-term M. tuberculosis survival.

Limited activity of clofazimine as a single drug in a mouse model of tuberculosis exhibiting caseous necrotic granulomas.

New drugs and drugs with a novel mechanism of action are desperately needed to shorten the duration of tuberculosis treatment, to prevent the emergence of drug resistance, and to treat multiple-drug-resistant strains of Mycobacterium tuberculosis. Recently, there has been renewed interest in clofazimine (CFZ). In this study, we utilized the C3HeB/FeJ mouse model, possessing highly organized, hypoxic pulmonary granulomas with caseous necrosis, to evaluate CFZ monotherapy in comparison to results with BALB/c mice, which form only multifocal, coalescing cellular aggregates devoid of caseous necrosis. While CFZ treatment was highly effective in BALB/c mice, its activity was attenuated in the lungs of C3HeB/FeJ mice. This lack of efficacy was directly related to the pathological progression of disease in these mice, since administration of CFZ prior to the formation of hypoxic, necrotic granulomas reconstituted bactericidal activity in this mouse strain. These results support the continued use of mouse models of tuberculosis infection which exhibit a granulomatous response in the lungs that more closely resembles the pathology found in human disease.

Archaeal RNA polymerase and transcription regulation.

To elucidate the mechanism of transcription by cellular RNA polymerases (RNAPs), high-resolution X-ray crystal structures together with structure-guided biochemical, biophysical, and genetics studies are essential. The recently solved X-ray crystal structures of archaeal RNAP allow a structural comparison of the transcription machinery among all three domains of life. The archaea were once thought of closely related to bacteria, but they are now considered to be more closely related to the eukaryote at the molecular level than bacteria. According to these structures, the archaeal transcription apparatus, which includes RNAP and general transcription factors (GTFs), is similar to the eukaryotic transcription machinery. Yet, the transcription regulators, activators and repressors, encoded by archaeal genomes are closely related to bacterial factors. Therefore, archaeal transcription appears to possess an intriguing hybrid of eukaryotic-type transcription apparatus and bacterial-like regulatory mechanisms. Elucidating the transcription mechanism in archaea, which possesses a combination of bacterial and eukaryotic transcription mechanisms that are commonly regarded as separate and mutually exclusive, can provide data that will bring basic transcription mechanisms across all life forms.

Functional analysis of the three TATA binding protein homologs in Methanosarcina acetivorans.

The roles of three TATA binding protein (TBP) homologs (TBP1, TBP2, and TBP3) in the archaeon Methanosarcina acetivorans were investigated by using genetic and molecular approaches. Although tbp2 and tbp3 deletion mutants were readily obtained, a tbp1 mutant was not obtained, and the growth of a conditional tbp1 expression strain was tetracycline dependent, indicating that TBP1 is essential. Transcripts of tbp1 were 20-fold more abundant than transcripts of tbp2 and 100- to 200-fold more abundant than transcripts of tbp3, suggesting that TBP1 is the primary TBP utilized during growth. Accordingly, tbp1 is strictly conserved in the genomes of Methanosarcina species. Deltatbp3 and Deltatbp2 strains exhibited an extended lag phase compared with the wild type, although the lag phase for the Deltatbp2 strain was less pronounced when this strain was transitioning from growth on methylotrophic substrates to growth on acetate. Acetate-adapted Deltatbp3 cells exhibited growth rates, final growth yields, and lag times that were significantly reduced compared with those of the wild type when the organisms were cultured with growth-limiting concentrations of acetate, and the acetate-adapted Deltatbp2 strain exhibited a final growth yield that was reduced compared with that of the wild type when the organisms were cultured with growth-limiting acetate concentrations. DNA microarray analyses identified 92 and 77 genes with altered transcription in the Deltatbp2 and Deltatbp3 strains, respectively, which is consistent with a role for TBP2 and TBP3 in optimizing gene expression. Together, the results suggest that TBP2 and TBP3 are required for efficient growth under conditions similar to the conditions in the native environment of M. acetivorans.

Bedaquiline and Pyrazinamide Treatment Responses Are Affected by Pulmonary Lesion Heterogeneity in Mycobacterium tuberculosis Infected C3HeB/FeJ Mice.

BALB/c and Swiss mice are routinely used to validate the effectiveness of tuberculosis drug regimens, although these mouse strains fail to develop human-like pulmonary granulomas exhibiting caseous necrosis. Microenvironmental conditions within human granulomas may negatively impact drug efficacy, and this may not be reflected in non-necrotizing lesions found within conventional mouse models. The C3HeB/FeJ mouse model has been increasingly utilized as it develops hypoxic, caseous necrotic granulomas which may more closely mimic the pathophysiological conditions found within human pulmonary granulomas. Here, we examined the treatment response of BALB/c and C3HeB/FeJ mice to bedaquiline (BDQ) and pyrazinamide (PZA) administered singly and in combination. BALB/c mice consistently displayed a highly uniform treatment response to both drugs, while C3HeB/FeJ mice displayed a bimodal response composed of responsive and less-responsive mice. Plasma pharmacokinetic analysis of dissected lesions from BALB/c and C3HeB/FeJ mice revealed that PZA penetrated lesion types from both mouse strains with similar efficiency. However, the pH of the necrotic caseum of C3HeB/FeJ granulomas was determined to be 7.5, which is in the range where PZA is essentially ineffective under standard laboratory in vitro growth conditions. BDQ preferentially accumulated within the highly cellular regions in the lungs of both mouse strains, although it was present at reduced but still biologically relevant concentrations within the central caseum when dosed at 25 mg/kg. The differential treatment response which resulted from the heterogeneous pulmonary pathology in the C3HeB/FeJ mouse model revealed several factors which may impact treatment efficacy, and could be further evaluated in clinical trials.

MreA functions in the global regulation of methanogenic pathways in Methanosarcina acetivorans.

UNLABELLED: Results are presented supporting a regulatory role for the product of the MA3302 gene locus (designated MreA) previously annotated as a hypothetical protein in the methanogenic species Methanosarcina acetivorans of the domain Archaea. Sequence analysis of MreA revealed identity to the TrmB family of transcription factors, albeit the sequence is lacking the sensor domain analogous to TrmBL2, abundant in nonmethanogenic species of the domain Archaea. Transcription of mreA was highly upregulated during growth on acetate versus methylotrophic substrates, and an mreA deletion (ΔmreA) strain was impaired for growth with acetate in contrast to normal growth with methylotrophic substrates. Transcriptional profiling of acetate-grown cells identified 280 genes with altered expression in the ΔmreA strain versus the wild-type strain. Expression of genes unique to the acetate pathway decreased whereas expression of genes unique to methylotrophic metabolism increased in the ΔmreA strain relative to the wild type, results indicative of a dual role for MreA in either the direct or indirect activation of acetate-specific genes and repression of methylotrophic-specific genes. Gel shift experiments revealed specific binding of MreA to promoter regions of regulated genes. Homologs of MreA were identified in M. acetivorans and other Methanosarcina species for which expression patterns indicate roles in regulating methylotrophic pathways. IMPORTANCE: Species in the domain Archaea utilize basal transcription machinery resembling that of the domain Eukarya, raising questions addressing the role of numerous putative transcription factors identified in sequenced archaeal genomes. Species in the genus Methanosarcina are ideally suited for investigating principles of archaeal transcription through analysis of the capacity to utilize a diversity of substrates for growth and methanogenesis. Methanosarcina species switch pathways in response to the most energetically favorable substrate, metabolizing methylotrophic substrates in preference to acetate marked by substantial regulation of gene expression. Although conversion of the methyl group of acetate accounts for most of the methane produced in Earth's biosphere, no proteins involved in the regulation of genes in the acetate pathway have been reported. The results presented here establish that MreA participates in the global regulation of diverse methanogenic pathways in the genus Methanosarcina. Finally, the results contribute to a broader understanding of transcriptional regulation in the domain Archaea.