Emergence of antibiotic-specific phenotypes during prolonged treatment of mice.

A major challenge in tuberculosis (TB) therapeutics is that antibiotic exposure leads to changes in the physiology of (), which may enable the pathogen to withstand treatment. While antibiotic-treated has been evaluated in experiments it is unclear if and how long-term treatment with diverse antibiotics with varying treatment-shortening activity (sterilizing activity) affects physiologic processes differently. Here, we used SEARCH-TB, a pathogen-targeted RNA-sequencing platform, to characterize the transcriptome in the BALB/c high-dose aerosol infection mouse model following 4 weeks of treatment with three sterilizing and three non-sterilizing antibiotics.

Nitric oxide induces the distinct invisibility phenotype of Mycobacterium tuberculosis.

During infection Mycobacterium tuberculosis (Mtb) forms physiologically distinct subpopulations that are recalcitrant to treatment and undetectable using standard diagnostics. These difficult to culture or differentially culturable (DC) Mtb are revealed in liquid media, their revival is often stimulated by resuscitation-promoting factors (Rpf) and prevented by Rpf inhibitors. Here, we investigated the role of nitric oxide (NO) in promoting the DC phenotype.

Emergence of antibiotic-specific phenotypes during prolonged treatment of mice.

A major challenge in tuberculosis (TB) therapeutics is that antibiotic exposure leads to changes in the physiologic state of () which may enable the pathogen to withstand treatment. While antibiotic-treated have been evaluated in short-term experiments, it is unclear if and how long-term treatment with diverse antibiotics with varying treatment-shortening activity (sterilizing activity) affect physiologic states differently. Here, we used SEARCH-TB, a pathogen-targeted RNA-sequencing platform, to characterize the transcriptome in the BALB/c high-dose aerosol infection mouse model following 4-week treatment with three sterilizing and three non-sterilizing antibiotics.

Rv0100: An essential acyl carrier protein from M. tuberculosis important in dormancy.

We have identified an acyl-carrier protein, Rv0100, that is up-regulated in a dormancy model. This protein plays a critical role in the fatty acid biosynthesis pathway, which is important for energy storage and cell wall synthesis in Mycobacterium tuberculosis (MTB). Knocking out the Rv0100 gene resulted in a significant reduction of growth compared to wild-type MTB in the Wayne model of non-replicating persistence.

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

A major reason that curing tuberculosis requires prolonged treatment is that drug exposure changes bacterial phenotypes. The physiologic adaptations of that survive drug exposure have been obscure due to low sensitivity of existing methods in drug-treated animals. Using the novel SEARCH-TB RNA-seq platform, we elucidated phenotypes in mice treated for with the global standard 4-drug regimen and compared them with the effect of the same regimen .

Population heterogeneity in and .

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 - smooth, rough, and an intermediate morphotype that predominated under typical laboratory conditions.

Lung microenvironments harbor 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.

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

The sigmoid E model was used to describe the rRNA synthesis ratio (RS ratio) response of Mycobacterium tuberculosis to antimicrobial concentration. RS-E 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-EC is the concentration needed to achieve 90% of the RS-E, which may guide dose selection to achieve a maximal RS ratio effect .

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.

Reproducibility of the Ribosomal RNA Synthesis Ratio in Sputum and Association with Markers of Mycobacterium tuberculosis Burden.

There is a critical need for improved pharmacodynamic markers for use in human tuberculosis (TB) drug trials. Pharmacodynamic monitoring in TB has conventionally used culture or molecular methods to enumerate the burden of Mycobacterium tuberculosis organisms in sputum. A recently proposed assay called the rRNA synthesis (RS) ratio measures a fundamentally novel property, how drugs impact ongoing bacterial rRNA synthesis.

A Role for Sigma Factor C in Copper Nutritional Immunity.

Sigma factor C (SigC) contributes to virulence in various animal models, but the stress response coordinated by this transcription factor was undefined. The results presented here indicate that SigC prevents copper starvation. Whole genome expression studies demonstrate short-term (4-h) induction of , controlled from a tetracycline-inducible promoter, upregulates and genes in the nonribosomal peptide synthase () operon.

Transcriptional portrait of M. bovis BCG during biofilm production shows genes differentially expressed during intercellular aggregation and substrate attachment.

Mycobacterium tuberculosis and M. smegmatis form drug-tolerant biofilms through dedicated genetic programs. In support of a stepwise process regulating biofilm production in mycobacteria, it was shown elsewhere that lsr2 participates in intercellular aggregation, while groEL1 was required for biofilm maturation in M.

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112.

Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H-NS-like regulator Lsr2 at threonine 112.

Antibiotic Lethality and Membrane Bioenergetics.

A growing body of research suggests bacterial metabolism and membrane bioenergetics affect the lethality of a broad spectrum of antibiotics. Electrochemical gradients spanning energy-transducing membranes are the foundation of the chemiosmotic hypothesis and are essential for life; accordingly, their dysfunction appears to be a critical factor in bacterial death. Proton flux across energy-transducing membranes is central for cellular homeostasis as vectorial proton translocation generates a proton motive force used for ATP synthesis, pH homeostasis, and maintenance of solute gradients.

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

is a strict aerobe capable of prolonged survival in the absence of oxygen. We investigated the ability of anaerobic to counter challenges to internal pH homeostasis in the absence of aerobic respiration, the primary mechanism of proton efflux for aerobic bacilli. Anaerobic populations were markedly impaired for survival under a mildly acidic pH relative to standard culture conditions.

Mycobacterium tuberculosis Phosphate Uptake System Component PstA2 Is Not Required for Gene Regulation or Virulence.

The Mycobacterium tuberculosis genome encodes two complete high-affinity Pst phosphate-specific transporters. We previously demonstrated that a membrane-spanning component of one Pst system, PstA1, was essential both for M. tuberculosis virulence and for regulation of gene expression in response to external phosphate availability.

Sputum is a surrogate for bronchoalveolar lavage for monitoring Mycobacterium tuberculosis transcriptional profiles in TB patients.

Pathogen-targeted transcriptional profiling in human sputum may elucidate the physiologic state of Mycobacterium tuberculosis (M. tuberculosis) during infection and treatment. However, whether M.

Adaptation of Mycobacterium tuberculosis to Impaired Host Immunity in HIV-Infected Patients.

Background: It is unknown whether immunosuppression influences the physiologic state of Mycobacterium tuberculosis in vivo. We evaluated the impact of host immunity by comparing M. tuberculosis and human gene transcription in sputum between human immunodeficiency virus (HIV)-infected and uninfected patients with tuberculosis.

Bedaquiline and Pyrazinamide Treatment Responses Are Affected by Pulmonary Lesion Heterogeneity in 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.

In vitro models that utilize hypoxia to induce non-replicating persistence in Mycobacteria.

The Wayne model and Rapid Anaerobic Dormancy model are widely used methods to analyze the response of Mycobacterium tuberculosis to hypoxia and anaerobiosis. In these models tubercle bacilli are grown in sealed tubes in which bacilli aerobic respiration produces a temporal oxygen gradient. The gradual depletion of oxygen results in a non-replicating persistent culture capable of extended microaerobic and anaerobic survival.

Transcriptional Adaptation of Drug-tolerant Mycobacterium tuberculosis During Treatment of Human Tuberculosis.

Background: Treatment initiation rapidly kills most drug-susceptible Mycobacterium tuberculosis, but a bacterial subpopulation tolerates prolonged drug exposure. We evaluated drug-tolerant bacilli in human sputum by comparing messenger RNA (mRNA) expression of drug-tolerant bacilli that survive the early bactericidal phase with treatment-naive bacilli.

Methods: M.

Nitric oxide from IFNγ-primed macrophages modulates the antimicrobial activity of β-lactams against the intracellular pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella.

Our investigations show that nonlethal concentrations of nitric oxide (NO) abrogate the antibiotic activity of β-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to β-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics.