New antibiotics are needed to combat rising resistance, with new Mycobacterium tuberculosis (Mtb) drugs of highest priority. Conventional whole-cell and biochemical antibiotic screens have failed. We developed a novel strategy termed PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) in which we screen compounds against pools of strains depleted for essential bacterial targets. We engineered strains targeting 474 Mtb essential genes and screened pools of 100-150 strains against activity-enriched and unbiased compounds libraries, measuring > 8.5-million chemical-genetic interactions. Primary screens identified > 10-fold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insight. We identified > 40 novel compounds targeting DNA gyrase, cell wall, tryptophan, folate biosynthesis, and RNA polymerase, as well as inhibitors of a novel target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating PROSPECT's ability to yield inhibitors against novel targets which would have eluded conventional drug discovery.

Mycobacterium tuberculosis can persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here that M. tuberculosis poly(P) accumulation is temporally linked to bacillary growth restriction. We also identify M. tuberculosis Rv1026 as a novel exopolyphosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression of Rv1026 (ppx2), we found that M. tuberculosis poly(P) accumulation leads to slowed growth and reduced susceptibility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electron microscopy, the ppx2 knockdown strain exhibited increased cell wall thickness, which was associated with reduced cell wall permeability to hydrophilic drugs rather than induction of drug efflux pumps or altered biofilm formation relative to the empty vector control. Transcriptomic and metabolomic analysis revealed a metabolic downshift of the ppx2 knockdown characterized by reduced transcription and translation and a downshift of glycerol-3-phosphate levels. In summary, poly(P) plays an important role in M. tuberculosis growth restriction and metabolic downshift and contributes to antibiotic tolerance through altered cell wall permeability.

The formation and maintenance of granulomas is central to the host response to Mycobacterium tuberculosis (Mtb) infection. It is widely accepted that the lungs of patients with tuberculosis (TB) usually contain multiple infection foci, and that the granulomas evolve and differentiate independently, resulting in considerable heterogeneity. Although gene expression profiles of human blood cells have been proposed as biomarkers of Mtb infection and/or active disease, the immune profiles of discrete lesion types has not been studied extensively. Using histology, immunopathology and genome-wide transcriptome analysis, we explored the immunological profile of human lung TB granulomas. We show that although the different granulomas share core similarities in their immunological/inflammatory characteristics, they also exhibit significant divergence. Despite similar numbers of CD68+ macrophages in the different lesions, the extent of immune reactivity, as determined by the density of CD3+ T cells in the macrophage rich areas, and the extent of fibrosis, shows considerable variation. Both quantitative and qualitative differences among significantly differentially expressed genes (SDEG) were noted in each of the lesion types studied. Further, network/pathway analysis of SDEG revealed differential regulation of inflammatory response, immune cell trafficking, and cell mediated immune response in the different lesions. Our data highlight the formidable challenges facing ongoing efforts to identify peripheral blood biomarkers due to the diversity of lesion types and complexity of local immune responses in the lung.

BackgroundPulmonary infection of humans by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), results in active disease in 5-10% of individuals, while asymptomatic latent Mtb infection (LTBI) is established in the remainder. The host immune responses that determine this differential outcome following Mtb infection are not fully understood. Using a rabbit model of pulmonary TB, we have shown that infection with the Mtb clinical isolate HN878 (a hyper-virulent W-Beijing lineage strain) leads to progressive cavitary disease similar to what is seen in humans with active TB. In contrast, infection with Mtb CDC1551 (a hyper-immunogenic clinical isolate) is efficiently controlled in rabbit lungs, with establishment of LTBI, which can be reactivated upon treatment with immune-suppressive drugs. We hypothesize that the initial interaction of Mtb with the cells of the host response in the lungs determine later outcome of infection.ResultsTo test this hypothesis, we used our rabbit model of pulmonary TB and infected the animals with Mtb HN878 or CDC1551. At 3 hours, with similar lung bacillary loads, HN878 infection caused greater accumulation of mononuclear and polymorphonuclear leukocytes (PMN) in the lungs, compared to animals infected with CDC1551. Using whole-genome microarray gene expression analysis, we delineated the early transcriptional changes in the lungs of HN878- or CDC1551-infected rabbits at this time and compared them to the differential response at 4 weeks of Mtb-infection. Our gene network and pathway analysis showed that the most significantly differentially expressed genes involved in the host response to HN878, compared to CDC1551, at 3 hours of infection, were components of the inflammatory response and STAT1 activation, recruitment and activation of macrophages, PMN, and fMLP (N-formyl-Methionyl-Leucyl-Phenylalanine)-stimulation. At 4 weeks, the CDC1551 bacillary load was significantly lower and the granulomatous response reduced compared to HN878 infection. Moreover, although inflammation was dampened in both Mtb infections at 4 weeks, the majority of the differentially expressed gene networks were similar to those seen at 3 hours.ConclusionsWe propose that differential regulation of the inflammation-associated innate immune response and related gene expression changes seen at 3 hours determine the long term outcome of Mtb infection in rabbit lungs.

Multidrug resistant organisms (MDROs) are a serious threat to human health 1,2. Fast, accurate antibiotic susceptibility testing (AST) is a critical need in addressing escalating antibiotic resistance, since delays in identifying MDROs increase mortality 3,4 and use of broad-spectrum antibiotics, further selecting for resistant organisms. Yet current growth-based AST assays, such as broth microdilution 5 , require several days before informing key clinical decisions. Rapid AST would transform the care of infected patients while ensuring that our antibiotic arsenal is deployed as efficiently as possible. Growth-based assays are fundamentally constrained in speed by doubling time of the pathogen, and genotypic assays are limited by the ever-growing diversity and complexity of bacterial antibiotic resistance mechanisms. Here, we describe a rapid assay for combined Genotypic and Phenotypic AST through RNA detection, GoPhAST-R, that classifies strains with 94-99% accuracy by coupling machine learning analysis of early antibiotic-induced transcriptional changes with simultaneous detection of key genetic resistance determinants to increase accuracy of resistance detection, facilitate molecular epidemiology, and enable early detection of emerging resistance mechanisms. This two-pronged approach provides phenotypic AST 24-36 hours faster than standard workflows, with <4 hour assay time on a pilot instrument for hybridization-based multiplexed RNA detection implemented directly from positive blood cultures.

Marginal deficiency of vitamin B-6 is common among segments of the population worldwide. Because pyridoxal 5′-phosphate (PLP) serves as a coenzyme in the metabolism of amino acids, carbohydrates, organic acids, and neurotransmitters, as well as in aspects of one-carbon metabolism, vitamin B-6 deficiency could have many effects. Healthy men and women (age: 20-40 y; n = 23) were fed a 2-day controlled, nutritionally adequate diet followed by a 28-day low-vitamin B-6 diet (<0.5 mg/d) to induce marginal deficiency, as reflected by a decline of plasma PLP from 52.6±14.1 (mean ± SD) to 21.5±4.6 nmol/L (P<0.0001) and increased cystathionine from 131±65 to 199±56 nmol/L (P<0.001). Fasting plasma samples obtained before and after vitamin B6 restriction were analyzed by 1H-NMR with and without filtration and by targeted quantitative analysis by mass spectrometry (MS). Multilevel partial least squares-discriminant analysis and S-plots of NMR spectra showed that NMR is effective in classifying samples according to vitamin B-6 status and identified discriminating features. NMR spectral features of selected metabolites indicated that vitamin B-6 restriction significantly increased the ratios of glutamine/glutamate and 2-oxoglutarate/glutamate (P<0.001) and tended to increase concentrations of acetate, pyruvate, and trimethylamine-N-oxide (adjusted P<0.05). Tandem MS showed significantly greater plasma proline after vitamin B-6 restriction (adjusted P<0.05), but there were no effects on the profile of 14 other amino acids and 45 acylcarnitines. These findings demonstrate that marginal vitamin B-6 deficiency has widespread metabolic perturbations and illustrate the utility of metabolomics in evaluating complex effects of altered vitamin B-6 intake.

Although it is accepted that the environment within the granuloma profoundly affects Mycobacterium tuberculosis (Mtb) and infection outcome, our ability to understand Mtb gene expression in these niches has been limited. We determined intragranulomatous gene expression in human-like lung lesions derived from nonhuman primates with both active tuberculosis (ATB) and latent TB infection (LTBI). We employed a non-laser-based approach to microdissect individual lung lesions and interrogate the global transcriptome of Mtb within granulomas. Mtb genes expressed in classical granulomas with central, caseous necrosis, as well as within the caseum itself, were identified and compared with other Mtb lesions in animals with ATB (n = 7) or LTBI (n = 7). Results were validated using both an oligonucleotide approach and RT-PCR on macaque samples and by using human TB samples. We detected approximately 2,900 and 1,850 statistically significant genes in ATB and LTBI lesions, respectively (linear models for microarray analysis, Bonferroni corrected, P < 0.05). Of these genes, the expression of approximately 1,300 (ATB) and 900 (LTBI) was positively induced. We identified the induction of key regulons and compared our results to genes previously determined to be required for Mtb growth. Our results indicate pathways that Mtb uses to ensure its survival in a highly stressful environment in vivo. A large number of genes is commonly expressed in granulomas with ATB and LTBI. In addition, the enhanced expression of the dormancy survival regulon was a key feature of lesions in animals with LTBI, stressing its importance in the persistence of Mtb during the chronic phase of infection.

Purpose – The purpose of this paper is to empirically examine the roles of self-esteem (SE), negative affect (NA), and consumer susceptibility to normative influence in the enactment of impulse buying behavior. Design/methodology/approach – A theoretical model is developed through an extensive review of literature. Survey research is conducted to collect the data from respondents. Structural equation modeling is performed to test the model and the hypotheses. Findings – The outcome of the study reveals that the act of impulsive buying is preceded by buying impulse (BI). BI is positively influenced by consumer susceptibility to normative influence and impulsive buying tendency (IBT). SE influences the generation of BI partially mediated by IBT. Research limitations/implications – The study is limited in its generalizability in terms of its geographic location, culture, and the context of product categories. Practical implications – The findings of the study have practical implications in developing marketing communications, merchandising, and personal selling strategies. Originality/value – In view of the contradictory empirical evidences in extant literature regarding the role NA the present study re-examines whether NA influences impulse buying. The study, conducted in the field setting also ascertains the external validity of the findings not tested in the prior research. Furthermore, in light of psychology literature, the relationship between SE and IBT was hypothesized and empirically established in the present study.