Lung microenvironments harbor Mycobacterium tuberculosis phenotypes with distinct treatment responses.

Tuberculosis lung lesions are complex and harbor heterogeneous microenvironments that influence antibiotic effectiveness. Major strides have been made recently in understanding drug pharmacokinetics in pulmonary lesions, but the bacterial phenotypes that arise under these conditions and their contribution to drug tolerance are poorly understood. A pharmacodynamic marker called the RS ratio® quantifies ongoing rRNA synthesis based on the abundance of newly synthesized precursor rRNA relative to mature structural rRNA. Application of the RS ratio in the C3HeB/FeJ mouse model demonstrated that Mycobacterium tuberculosis populations residing in different tissue microenvironments are phenotypically distinct and respond differently to drug treatment with rifampin, isoniazid, or bedaquiline. This work provides a foundational basis required to address how anatomic and pathologic microenvironmental niches may contribute to long treatment duration and drug tolerance during the treatment of human tuberculosis.

Drug distribution and efficacy of the DprE1 inhibitor BTZ-043 in the C3HeB/FeJ mouse tuberculosis model.

BTZ-043, a suicide inhibitor of the Mycobacterium tuberculosis cell wall synthesis decaprenylphosphoryl-beta-D-ribose 2' epimerase, is under clinical development as a potential new anti-tuberculosis agent. BTZ-043 is potent and bactericidal in vitro but has limited activity against non-growing bacilli in rabbit caseum. To better understand its behavior in vivo, BTZ-043 was evaluated for efficacy and spatial drug distribution as a single agent in the C3HeB/FeJ mouse model presenting with caseous necrotic pulmonary lesions upon Mycobacterium tuberculosis infection. BTZ-043 promoted significant reductions in lung and spleen bacterial burdens in the C3HeB/FeJ mouse model after 2 months of therapy. BTZ-043 penetrates cellular and necrotic lesions and was retained at levels above the serum-shifted minimal inhibitory concentration in caseum. The calculated rate of kill was found to be highest and dose-dependent during the second month of treatment. BTZ-043 treatment was associated with improved histology scores of pulmonary lesions, especially compared to control mice, which experienced advanced fulminant neutrophilic alveolitis in the absence of treatment. These positive treatment responses to BTZ-043 monotherapy in a mouse model of advanced pulmonary disease can be attributed to favorable distribution in tissues and lesions, retention in the caseum, and its high potency and bactericidal nature at drug concentrations achieved in necrotic lesions.

Combination of Mycobacterium tuberculosis RS Ratio and CFU Improves the Ability of Murine Efficacy Experiments to Distinguish between Drug Treatments.

Murine tuberculosis drug efficacy studies have historically monitored bacterial burden based on CFU of Mycobacterium tuberculosis in lung homogenate. In an alternative approach, a recently described molecular pharmacodynamic marker called the RS ratio quantifies drug effect on a fundamental cellular process, ongoing rRNA synthesis. Here, we evaluated the ability of different pharmacodynamic markers to distinguish between treatments in three BALB/c mouse experiments at two institutions. We confirmed that different pharmacodynamic markers measure distinct biological responses. We found that a combination of pharmacodynamic markers distinguishes between treatments better than any single marker. The combination of the RS ratio with CFU showed the greatest ability to recapitulate the rank order of regimen treatment-shortening activity, providing proof of concept that simultaneous assessment of pharmacodynamic markers measuring different properties will enhance insight gained from animal models and accelerate development of new combination regimens. These results suggest potential for a new era in which antimicrobial therapies are evaluated not only on culture-based measures of bacterial burden but also on molecular assays that indicate how drugs impact the physiological state of the pathogen.

Spectinamide MBX-4888A exhibits favorable lesion and tissue distribution and promotes treatment shortening in advanced murine models of tuberculosis.

The spectinamides are novel, narrow-spectrum semisynthetic analogs of spectinomycin, modified to avoid intrinsic efflux by Mycobacterium tuberculosis . Spectinamides, including lead MBX-4888A (Lee-1810), exhibit promising therapeutic profiles in mice, as single drugs and as partner agents with other anti-tuberculosis antibiotics including rifampin and/or pyrazinamide. To demonstrate that this translates to more effective cure, we first confirmed the role of rifampin, with or without pyrazinamide, as essential to achieve effective bactericidal responses and sterilizing cure in the current standard of care regimen in chronically infected C3HeB/FeJ mice compared to BALB/c mice. Thus, demonstrating added value in testing clinically relevant regimens in murine models of increasing pathologic complexity. Next we show that MBX-4888A, given by injection with the front-line standard of care regimen, is treatment shortening in multiple murine tuberculosis infection models. The positive treatment responses to MBX-4888A combination therapy in multiple mouse models including mice exhibiting advanced pulmonary disease can be attributed to favorable distribution in tissues and lesions, retention in caseum, along with favorable effects with rifampin and pyrazinamide under conditions achieved in necrotic lesions. This study also provides an additional data point regarding the safety and tolerability of spectinamide MBX-4888A in long-term murine efficacy studies.

Transcriptional adaptation of drug-tolerant Mycobacterium tuberculosis in mice.

Transcriptome evaluation of Mycobacterium tuberculosis in the lungs of laboratory animals during long-term treatment has been limited by extremely low abundance of bacterial mRNA relative to eukaryotic RNA. Here we report a targeted amplification RNA sequencing method called SEARCH-TB. After confirming that SEARCH-TB recapitulates conventional RNA-seq in vitro, we applied SEARCH-TB to Mycobacterium tuberculosis-infected BALB/c mice treated for up to 28 days with the global standard isoniazid, rifampin, pyrazinamide, and ethambutol regimen. We compared results in mice with 8-day exposure to the same regimen in vitro. After treatment of mice for 28 days, SEARCH-TB suggested broad suppression of genes associated with bacterial growth, transcription, translation, synthesis of rRNA proteins and immunogenic secretory peptides. Adaptation of drug-stressed Mycobacterium tuberculosis appeared to include a metabolic transition from ATP-maximizing respiration towards lower-efficiency pathways, modification and recycling of cell wall components, large-scale regulatory reprogramming, and reconfiguration of efflux pumps expression. Despite markedly different expression at pre-treatment baseline, murine and in vitro samples had broadly similar transcriptional change during treatment. The differences observed likely indicate the importance of immunity and pharmacokinetics in the mouse. By elucidating the long-term effect of tuberculosis treatment on bacterial cellular processes in vivo, SEARCH-TB represents a highly granular pharmacodynamic monitoring tool with potential to enhance evaluation of new regimens and thereby accelerate progress towards a new generation of more effective tuberculosis treatment.

Transcriptional adaptation of Mycobacterium tuberculosis that survives prolonged multi-drug treatment in mice.

To address the ongoing global tuberculosis crisis, there is a need for shorter, more effective treatments. A major reason why tuberculosis requires prolonged treatment is that, following a short initial phase of rapid killing, the residual Mycobacterium tuberculosis withstands drug killing. Because existing methods lack sensitivity to quantify low-abundance mycobacterial RNA in drug-treated animals, cellular adaptations of drug-exposed bacterial phenotypes in vivo remain poorly understood. Here, we used a novel RNA-seq method called SEARCH-TB to elucidate the Mycobacterium tuberculosis transcriptome in mice treated for up to 28 days with standard doses of isoniazid, rifampin, pyrazinamide, and ethambutol. We compared murine results with in vitro SEARCH-TB results during exposure to the same regimen. Treatment suppressed genes associated with growth, transcription, translation, synthesis of rRNA proteins, and immunogenic secretory peptides. Bacteria that survived prolonged treatment appeared to transition from ATP-maximizing respiration toward lower-efficiency pathways and showed modification and recycling of cell wall components, large-scale regulatory reprogramming, and reconfiguration of efflux pump expression. Although the pre-treatment in vivo and in vitro transcriptomes differed profoundly, genes differentially expressed following treatment in vivo and in vitro were similar, with differences likely attributable to immunity and drug pharmacokinetics in mice. These results reveal cellular adaptations of Mycobacterium tuberculosis that withstand prolonged drug exposure in vivo, demonstrating proof of concept that SEARCH-TB is a highly granular pharmacodynamic readout. The surprising finding that differential expression is concordant in vivo and in vitro suggests that insights from transcriptional analyses in vitro may translate to the mouse. IMPORTANCE A major reason that curing tuberculosis requires prolonged treatment is that drug exposure changes bacterial phenotypes. The physiologic adaptations of Mycobacterium tuberculosis that survive drug exposure in vivo have been obscure due to low sensitivity of existing methods in drug-treated animals. Using the novel SEARCH-TB RNA-seq platform, we elucidated Mycobacterium tuberculosis phenotypes in mice treated for with the global standard 4-drug regimen and compared them with the effect of the same regimen in vitro. This first view of the transcriptome of the minority Mycobacterium tuberculosis population that withstands treatment in vivo reveals adaptation of a broad range of cellular processes, including a shift in metabolism and cell wall modification. Surprisingly, the change in gene expression induced by treatment in vivo and in vitro was largely similar. This apparent "portability" from in vitro to the mouse provides important new context for in vitro transcriptional analyses that may support early preclinical drug evaluation.

Mycobacterium tuberculosis precursor rRNA as a measure of treatment-shortening activity of drugs and regimens.

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.

Methionine Antagonizes para-Aminosalicylic Acid Activity via Affecting Folate Precursor Biosynthesis in Mycobacterium tuberculosis.

para-Aminosalicylic acid (PAS) is a second-line anti-tubercular drug that is used for the treatment of drug-resistant tuberculosis (TB). PAS efficacy in the treatment of TB is limited by its lower potency against Mycobacterium tuberculosis relative to many other drugs in the TB treatment arsenal. It is known that intrinsic metabolites, such as, para-aminobenzoic acid (PABA) and methionine, antagonize PAS and structurally related anti-folate drugs. While the basis for PABA-mediated antagonism of anti-folates is understood, the mechanism for methionine-based antagonism remains undefined. In the present study, we used both targeted and untargeted approaches to identify factors associated with methionine-mediated antagonism of PAS activity. We found that synthesis of folate precursors as well as a putative amino acid transporter, designated MetM, play crucial roles in this process. Disruption of metM by transposon insertion resulted in a ≥30-fold decrease in uptake of methionine in M. bovis BCG, indicating that metM is the major facilitator of methionine transport. We also discovered that intracellular biotin confers intrinsic PAS resistance in a methionine-independent manner. Collectively, our results demonstrate that methionine-mediated antagonism of anti-folate drugs occurs through sustained production of folate precursors.