Transcriptional Profiling of Mycobacterium Tuberculosis During Infection: Lessons Learned

Infection with Mycobacterium tuberculosis, the causative agent of tuberculosis, is considered one of the biggest infectious disease killers worldwide. A significant amount of attention has been directed toward revealing genes involved in the virulence and pathogenesis of this air-born pathogen. With the advances in technologies for transcriptional profiling, several groups, including ours, took advantage of DNA microarrays to identify transcriptional units differentially regulated by M. tuberculosis within a host. The main idea behind this approach is that pathogens tend to regulate their gene expression levels depending on the host microenvironment, and preferentially express those needed for survival. Identifying this class of genes will improve our understanding of pathogenesis. In our case, we identified an in vivo expressed genomic island that was preferentially active in murine lungs during early infection, as well as groups of genes active during chronic tuberculosis. Other studies have identified additional gene groups that are active during macrophage infection and even in human lungs. Despite all of these findings, one of the lingering questions remaining was whether in vivo expressed transcripts are relevant to the virulence, pathogenesis, and persistence of the organism. The work of our group and others addressed this question by examining the contribution of in vivo expressed genes using a strategy based on gene deletions followed by animal infections. Overall, the analysis of most of the in vivo expressed genes supported a role of these genes in M. tuberculosis pathogenesis. Further, these data suggest that in vivo transcriptional profiling is a valid approach to identify genes required for bacterial pathogenesis

A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis

Twenty-four putative lipase/esterase genes of Mycobacterium tuberculosis H37Rv were expressed in Escherichia coli and assayed for long-chain triacylglycerol (TG) hydrolase activity. We show here that the product of Rv3097c (LIPY) hydrolyzed long-chain TG with high specific activity. LIPY was purified after solubilization from inclusion bodies; the enzyme displayed a K-m of 7.57 mM and V-max of 653.3 nmol/mg/min for triolein with optimal activity between pH 8.0 and pH 9.0. LIPY was inhibited by active serine-directed reagents and was inactivated at temperatures above 37 degrees C. Detergents above their critical micellar concentrations and divalent cations inhibited the activity of LIPY. The N-terminal half of LIPY showed sequence homology with the proline glutamic acid-polymorphic GC-rich repetitive sequences protein family of M. tuberculosis. The C-terminal half of LIPY possesses amino acid domains homologous with the hormone-sensitive lipase family and the conserved active-site motif GDSAG. LIPY shows low sequence identity with the annotated lipases of M. tuberculosis and with other bacterial lipases. We demonstrate that hypoxic cultures of M. tuberculosis, which had accumulated TG, hydrolyzed the stored TG when subjected to nutrient starvation. Under such conditions, lipY was induced more than all lipases, suggesting a central role for it in the utilization of stored TG. We also show that in the lipY-deficient mutant, TG utilization was drastically decreased under nutrient-deprived condition. Thus, LIPY may be responsible for the utilization of stored TG during dormancy and reactivation of the pathogen

A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen

Background: Mycobacterium tuberculosis (Mtb) becomes dormant and phenotypically drug resistant when it encounters multiple stresses within the host. Inability of currently available drugs to kill latent Mtb is a major impediment to curing and possibly eradicating tuberculosis (TB). Most in vitro dormancy models, using single stress factors, fail to generate a truly dormant Mtb population. An in vitro model that generates truly dormant Mtb cells is needed to elucidate the metabolic requirements that allow Mtb to successfully go through dormancy, identify new drug targets, and to screen drug candidates to discover novel drugs that can kill dormant pathogen. Methodology/Principal Findings: We developed a novel in vitro multiple-stress dormancy model for Mtb by applying combined stresses of low oxygen (5%), high CO2 (10%), low nutrient (10 % Dubos medium) and acidic pH (5.0), conditions Mtb is thought to encounter in the host. Under this condition, Mtb stopped replicating, lost acid-fastness, accumulated triacylglycerol (TG) and wax ester (WE), and concomitantly acquired phenotypic antibiotic-resistance. Putative neutral lipid biosynthetic genes were up-regulated. These genes may serve as potential targets for new antilatency drugs. The triacylglycerol synthase1 (tgs1) deletion mutant, with impaired ability to accumulate TG, exhibited a lesser degree of antibiotic tolerance and complementation restored antibiotic tolerance. Transcriptome analysis with microarray revealed the achievement of dormant state showing repression of energy generation, transcription and translation machineries an

Extractive Fermentation as A Novel Strategy for High Cell Mass Production of Hetero-Fermentative Probiotic Strain Limosilactobacillus reuteri

This study reports on a novel technique to enhance the high cell mass and viable cell counts of the heterofermentative probiotic strain, Limosilactobacillus reuteri. This is the first report on the cultivation of L. reuteri, which was incorporated with weak base anion-exchange resins to remove the accumulating lactic acid in the fermentation broth. Two anion-exchange resins—Amberlite IRA 67 and IRA 96—were found to have a high adsorption capacity with lactic acid. Batch fermentation and fed-batch cultivation were further analyzed using IRA 67 resins, as this application resulted in a higher maximum number of viable cells. The in situ application of anion-exchange resins was found to create shear stress, and thus, it does not promote growth of L. reuteri; therefore, an external and integrated resin column system was proposed. The viable cell count from batch fermentation, when incorporated with the integrated resin column, was improved by 71 times (3.89 × 1011 ± 0.07 CFU mL−1) compared with control batch fermentation (5.35 × 109 ± 0.32 CFU mL−1), without the addition of resins. The growth improvement was achieved due to the high adsorption rate of lactic acid, which was recorded by the integrated IRA 67 resin system, and coupled with the stirred tank bioreactor batch fermentation process

Cognitive Skills and DNA Methylation Are Correlating in Healthy and Novice College Students Practicing Preksha Dhyāna Meditation

The impact of different meditation protocols on human health is explored at the cognitive and cellular levels. Preksha Dhyana meditation has been observed to seemingly affect the cognitive performance, transcriptome, and methylome of healthy and novice participant practitioners. In this study, we performed correlation analyses to investigate the presence of any relationships in the changes in cognitive performance and DNA methylation in a group of college students practicing Preksha Dhyāna (N = 34). Nine factors of cognitive performance were assessed at baseline and 8 weeks postintervention timepoints in the participants. Statistically significant improvements were observed in six of the nine assessments, which were predominantly relating to memory and affect. Using Illumina 850 K microarray technology, 470 differentially methylated sites (DMS) were identified between the two timepoints (baseline and 8 weeks), using a threshold of p-value \u3c 0.05 and methylation levels beyond −3% to 3% at every site. Correlation analysis between the changes in performance on each of the nine assessments and every DMS unveiled statistically significant positive and negative relationships at several of these sites. The identified DMS were in proximity of essential genes involved in signaling and other important metabolic processes. Interestingly, we identified a set of sites that can be considered as biomarkers for Preksha meditation improvements at the genome level

Effects of Combining Meditation Techniques on Short-Term Memory, Attention, and Affect in Healthy College Students.

Meditation refers to a family of self-regulation practices that focuses on training attention and awareness to foster psycho-emotional well-being and to develop specific capacities such as calmness, clarity, and concentration. We report a prospective convenience-controlled study in which we analyzed the effect of two components of Preksha Dhyāna – buzzing bee sound meditation (Mahapran dhvani) and color meditation (leśyā dhyāna) on healthy college students. Mahapran and leśya dhyāna are two Preksha Dhyāna practices that are based on sound and green color, respectively. The study population represents a suitable target as college students experience different stress factors during the school year. This study measures the individual and combined effects of two techniques (one focusing on sound and one focusing on color), on short-term memory, attention, and affect, in novice meditators. We used a battery of cognitive, performance, and compared results with baseline and control values. We found improved cognition, especially attention, short-term memory, and affect in terms of positivity and reduced negativity. Overall, the two techniques produced variable benefits and subjects showed improved scores over baseline for short-term memory, cognitive function, and overall wellbeing. Further studies are required to understand underlying mechanisms for the observed differences between the two techniques and to elucidate mechanisms underlying the more pronounced and global benefits observed with the combined techniques. These results underscore a need to examine individual components of meditation practices in order to individualize treatment approaches for attention disorders in young adults

The Gut Microbiome Alterations in Pediatric Patients with Functional Abdominal Pain Disorders

In this prospective longitudinal study, we enrolled 54 healthy pediatric controls and 28 functional abdominal pain disorders (FAPDs) pediatric patients (mean age was 11 ± 2.58 years old). Fecal samples and symptom questionnaires were obtained from all participants over the course of the year. Clinical data assessment showed that FAPDs patients were more symptomatic than the control group. Microbiome analysis revealed that Phylum Bacteroidetes was higher in FAPDs compared to the control group (p < 0.05), while phylum Firmicutes was lower in FAPDs (p < 0.05). In addition, Verrucomicrobiota was higher in the control group than the FAPDs (p < 0.05). At the genus level the relative abundance of 72 bacterial taxa showed statistically significant differences between the two groups and at the school term levels. In the control group, Shannon diversity, Observed_species, and Simpson were higher than the FAPDs (p < 0.05), and beta diversity showed differences between the two groups (PERMANOVA = 2.38; p = 0.002) as well. Using linear discriminant analysis effect size (LEfSe), Enterobacteriaceae family and Megaspherae showed increased abundances in vacation term (LDA score > 2.0, LEfSe, p < 0.05). In the FAPDs group, the severity of symptoms (T-scores) correlated with 11 different taxa bacterial relative abundances using Pearson′s correlation and linear regression analyses. Our data showed that gut microbiome is altered in FAPDs compared to the control. Differences in other metrics such as alpha- and beta diversity were also reported between the two groups. Correlation of the severity of the disease (T-scores) correlated with gut microbiome. Finally, our findings support the use of Faecalibacterium/Bacteroides ratio as a potential diagnostic biomarker for FAPDs

Gut Microbiome Remains Static in Functional Abdominal Pain Disorders Patients Compared to Controls: Potential for Diagnostic Tools

Background: Functional Abdominal Pain disorders (FAPDs) are a group of heterogeneous gastrointestinal disorders with unclear pathophysiology. In children, FAPDs are more common in the winter months than summer months. The possible influence of school stressors has been proposed. Previously, our group showed differences in bacterial relative abundances and alpha diversity in the gut microbiome and its relationship with stressors in a cross-sectional evaluation of children suffering from FAPDs compared to a healthy control group. We present longitudinal data to assess whether the gut microbiome changes over school terms in the control and FAPDs groups. Methods: The longitudinal study included children with FAPDs (n = 28) and healthy controls (n = 54). Gastrointestinal symptoms, as well as stool microbiome, were assessed in both groups. Stool samples were serially collected from all participants during both the school term and summer vacation. The stool samples were subjected to total genomic extraction, 16S rRNA amplicon sequencing, and bioinformatics analysis. The gut microbiome was compared at school and during vacation. Other metrics, alpha diversity, and beta diversity, were also compared between the two school terms in every group. Results: In the healthy group, there were differences in microbiome composition between school terms and summer vacation. Conversely, we found no differences in the FAPDs group between the two terms. The healthy control group revealed differences (p-value < 0.05) in 55 bacterial species between the school term and vacation. Several of the differentially abundant identified bacteria were involved in short-chain fatty acids production (SCFAs), inflammation reduction, and gut homeostasis. Alpha diversity metrics, such as the Shannon index, were different in the control group and remained unchanged in the FAPDs group. Conclusion: Although preliminary, our findings suggest that the gut microbiome is static in FAPDs. This compares with a more dynamic healthy gut microbiome. Further studies are warranted to corroborate this and understand the interplay between stress, symptoms, and a less diverse and static microbiome. Future studies will also account for different variables such as diet and other patient demographic criteria that were missing in the current study

Induction of a Novel Class of Diacylglycerol Acyltransferases and Triacylglycerol Accumulation in Mycobacterium tuberculosis as It Goes into a Dormancy-Like State in Culture

Mycobacterium tuberculosis enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the host's immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy. The dormant pathogen can survive for decades till the host's immune system is weakened and active tuberculosis develops. Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known. We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids. Little is known about the biosynthesis of TG in M. tuberculosis. We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase (tgs) when expressed in Escherichia coli showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes. We show that several tgs genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of O(2) and also by NO treatment, which is known to induce dormancy-associated genes. The gene (Rv3130c) that shows the highest TGS activity when expressed in E. coli shows the highest induction by hypoxia and NO treatment. Biochemical evidence shows that TG synthesis and accumulation occur under both conditions. We conclude that TG may be a form of energy storage for use during long-term dormancy. Therefore, TG synthesis may be an appropriate target for novel antilatency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis