A Novel Tool to Identify Bactericidal Compounds against Vulnerable Targets in Drug-Tolerant M. tuberculosis found in Caseum.

Caseous necrosis is a hallmark of tuberculosis (TB) pathology and creates a niche for drug-tolerant persisters within the host. Cavitary TB and high bacterial burden in caseum require longer treatment duration. An in vitro model that recapitulates the major features of Mycobacterium tuberculosis (Mtb) in caseum would accelerate the identification of compounds with treatment-shortening potential. We have developed a caseum surrogate model consisting of lysed and denatured foamy macrophages. Upon inoculation of Mtb from replicating cultures, the pathogen adapts to the lipid-rich matrix and gradually adopts a nonreplicating state. We determined that the lipid composition of ex vivo caseum and the surrogate matrix are similar. We also observed that Mtb in caseum surrogate accumulates intracellular lipophilic inclusions (ILI), a distinctive characteristic of quiescent and drug-tolerant Mtb. Expression profiling of a representative gene subset revealed common signatures between the models. Comparison of Mtb drug susceptibility in caseum and caseum surrogate revealed that both populations are similarly tolerant to a panel of TB drugs. By screening drug candidates in the surrogate model, we determined that the bedaquiline analogs TBAJ876 and TBAJ587, currently in clinical development, exhibit superior bactericidal against caseum-resident Mtb, both alone and as substitutions for bedaquiline in the bedaquiline-pretomanid-linezolid regimen approved for the treatment of multidrug-resistant TB. In summary, we have developed a physiologically relevant nonreplicating persistence model that reflects the distinct metabolic and drug-tolerant state of Mtb in caseum. IMPORTANCE M. tuberculosis (Mtb) within the caseous core of necrotic granulomas and cavities is extremely drug tolerant and presents a significant hurdle to treatment success and relapse prevention. Many in vitro models of nonreplicating persistence have been developed to characterize the physiologic and metabolic adaptations of Mtb and identify compounds active against this treatment-recalcitrant population. However, there is little consensus on their relevance to in vivo infection. Using lipid-laden macrophage lysates, we have designed and validated a surrogate matrix that closely mimics caseum and in which Mtb develops a phenotype similar to that of nonreplicating bacilli in vivo. The assay is well suited to screen for bactericidal compounds against caseum-resident Mtb in a medium-throughput format, allowing for reduced reliance on resource intensive animal models that present large necrotic lesions and cavities. Importantly, this approach will aid the identification of vulnerable targets in caseum Mtb and can accelerate the development of novel TB drugs with treatment-shortening potential.

The evolving biology of Mycobacterium tuberculosis drug resistance.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb) is an ancient disease that has remained a leading cause of infectious death. Mtb has evolved drug resistance to every antibiotic regimen ever introduced, greatly complicating treatment, lowering rates of cure and menacing TB control in parts of the world. As technology has advanced, our understanding of antimicrobial resistance has improved, and our models of the phenomenon have evolved. In this review, we focus on recent research progress that supports an updated model for the evolution of drug resistance in Mtb. We highlight the contribution of drug tolerance on the path to resistance, and the influence of heterogeneity on tolerance. Resistance is likely to remain an issue for as long as drugs are needed to treat TB. However, with technology driving new insights and careful management of newly developed resources, antimicrobial resistance need not continue to threaten global progress against TB, as it has done for decades.

A multi-epitope fusion antigen candidate vaccine for Enterotoxigenic Escherichia coli is protective against strain B7A colonization in a rabbit model.

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's and travelers' diarrhea. Developing effective vaccines against this heterologous group has proven difficult due to the varied nature of toxins and adhesins that determine their pathology. A multivalent candidate vaccine was developed using a multi-epitope fusion antigen (MEFA) vaccinology platform and shown to effectively elicit broad protective antibody responses in mice and pigs. However, direct protection against ETEC colonization of the small intestine was not measured in these systems. Colonization of ETEC strains is known to be a determining factor in disease outcomes and is adhesin-dependent. In this study, we developed a non-surgical rabbit colonization model to study immune protection against ETEC colonization in rabbits. We tested the ability for the MEFA-based vaccine adhesin antigen, in combination with dmLT adjuvant, to induce broad immune responses and to protect from ETEC colonization of the rabbit small intestine. Our results indicate that the candidate vaccine MEFA antigen elicits antibodies in rabbits that react to seven adhesins included in its construction and protects against colonization of a challenge strain that consistently colonized naïve rabbits.

Experimental and Computational Workflow for RNA Sequencing in Mycobacterium tuberculosis : From Total RNA to Differentially Expressed Genes.

RNA sequencing (RNAseq) in bacteria has become a transformative tool for many applications, including the identification of mechanisms that contribute to pathogenesis, environmental adaptation, and drug response. The kinds of analysis outputs achievable from RNA-seq depend heavily on several key technical parameters during the sample preparation, sequencing, and data processing steps. In this chapter, we will describe the process of preparing Mycobacterium tuberculosis samples into sequencing libraries, selecting the appropriate sequencing platform, and performing data processing compatible with gene expression quantification. We will also discuss how each parameter could affect outcomes. The protocols described below produce consistently high yields. This chapter should inform on the technical considerations that impact sequencing output and enable the reader to decide on the best parameters to implement based on their own experimental goals.

Vibrio fischeri DarR Directs Responses to d-Aspartate and Represents a Group of Similar LysR-Type Transcriptional Regulators.

Mounting evidence suggests that d-amino acids play previously underappreciated roles in diverse organisms. In bacteria, even d-amino acids that are absent from canonical peptidoglycan (PG) may act as growth substrates, as signals, or in other functions. Given these proposed roles and the ubiquity of d-amino acids, the paucity of known d-amino-acid-responsive transcriptional control mechanisms in bacteria suggests that such regulation awaits discovery. We found that DarR, a LysR-type transcriptional regulator (LTTR), activates transcription in response to d-Asp. The d-Glu auxotrophy of a Vibrio fischerimurI::Tn mutant was suppressed, with the wild-type PG structure maintained, by a point mutation in darR This darR mutation resulted in the overexpression of an adjacent operon encoding a putative aspartate racemase, RacD, which compensated for the loss of the glutamate racemase encoded by murI Using transcriptional reporters, we found that wild-type DarR activated racD transcription in response to exogenous d-Asp but not upon the addition of l-Asp, l-Glu, or d-Glu. A DNA sequence typical of LTTR-binding sites was identified between darR and the divergently oriented racD operon, and scrambling this sequence eliminated activation of the reporter in response to d-Asp. In several proteobacteria, genes encoding LTTRs similar to DarR are linked to genes with predicted roles in d- and/or l-Asp metabolism. To test the functional similarities in another bacterium, darR and racD mutants were also generated in Acinetobacter baylyi In V. fischeri and A. baylyi, growth on d-Asp required the presence of both darR and racD Our results suggest that multiple bacteria have the ability to sense and respond to d-Asp.IMPORTANCE d-Amino acids are prevalent in the environment and are generated by organisms from all domains of life. Although some biological roles for d-amino acids are understood, in other cases, their functions remain uncertain. Given the ubiquity of d-amino acids, it seems likely that bacteria will initiate transcriptional responses to them. Elucidating d-amino acid-responsive regulators along with the genes they control will help uncover bacterial uses of d-amino acids. Here, we report the discovery of DarR, a novel LTTR in V. fischeri that mediates a transcriptional response to environmental d-Asp and underpins the catabolism of d-Asp. DarR represents the founding member of a group of bacterial homologs that we hypothesize control aspects of aspartate metabolism in response to d-Asp and/or to d-Asp-containing peptides.

Ammonia-based feedforward and feedback aeration control in activated sludge processes.

Aeration control at wastewater treatment plants based on ammonia as the controlled variable is applied for one of two reasons: (1) to reduce aeration costs, or (2) to reduce peaks in effluent ammonia. Aeration limitation has proven to result in significant energy savings, may reduce external carbon addition, and can improve denitrification and biological phosphorus (bio-P) performance. Ammonia control for limiting aeration has been based mainly on feedback control to constrain complete nitrification by maintaining approximately one to two milligrams of nitrogen per liter of ammonia in the effluent. Increased attention has been given to feedforward ammonia control, where aeration control is based on monitoring influent ammonia load. Typically, the intent is to anticipate the impact of sudden load changes, and thereby reduce effluent ammonia peaks. This paper evaluates the fundamentals of ammonia control with a primary focus on feedforward control concepts. A case study discussion is presented that reviews different ammonia-based control approaches. In most instances, feedback control meets the objectives for both aeration limitation and containment of effluent ammonia peaks. Feedforward control, applied specifically for switching aeration on or off in swing zones, can be beneficial when the plant encounters particularly unusual influent disturbances.

Mechanical induction of cough in Idiopathic Pulmonary Fibrosis.

BACKGROUND: Patients with idiopathic pulmonary fibrosis (IPF) frequently develop a dry, irritating cough which often proves refractory to anti-tussive therapies. The precise pathogenetic mechanisms responsible for this cough are unknown. We hypothesised that changes in nerves modulating mechanical sensitivity in areas of interstitial fibrosis might lead to enhanced cough response to mechanical stimulation of the chest in IPF. METHODS: We studied 27 non-smoking subjects with IPF (63% male), mean (SD) age 71.7 (7) years and 30 healthy non-smokers. Quality of life (Leicester Cough Questionnaire), cough symptom scores and cough severity scores (visual analog scales) were recorded. Percussion stimulation was applied over the posterior lung base, upper anterior chest and manubrium sternum at sequential frequencies (20 Hertz (Hz), 40 Hz and 60 Hz) for up to 60 seconds and repeated twice at two minute intervals. The number of subjects achieving two and five-cough responses, total cough counts and cough latency were recorded. In separate experiments, the effect of mechanical stimulation on the pattern of breathing was determined in eight IPF subjects and five control subjects. RESULTS: In patients with IPF, we demonstrated strong correlations between subjective cough measurements, particularly the cough symptom score and Leicester Cough Questionnaire (r = -0.86; p < 0.001). Mechanical percussion induced a true cough reflex in 23/27 (85%) IPF subjects, but only 5/30 (17%) controls (p < 0.001). More patients with IPF reached the two-cough response at a lower frequency (20 Hz) posteriorly than at other positions. Highest mean cough totals were seen with stimulation at or above 40 Hz. Mechanical stimulation had no effect on respiratory rate but increased tidal volume in four (50%) subjects with IPF, particularly at higher frequencies. It was associated with increased urge to cough followed by a true cough reflex. CONCLUSIONS: This study demonstrates that patients with IPF show enhanced cough reflex sensitivity to mechanical stimulation of the chest wall whilst normal individuals show little or no response. The observation that low frequency stimulation over the lung base, where fibrosis is most extensive, induces cough in more patients than at other sites supports the hypothesis that lung distortion contributes to the pathogenesis of cough in IPF.