Proteomic Profiling of Burkholderia thailandensis During Host Infection Using Bio-Orthogonal Noncanonical Amino Acid Tagging (BONCAT)

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively, and are often fatal to humans and animals. Owing to the high fatality rate, potential for spread by aerosolization, and the lack of efficacious therapeutics, B. pseudomallei and B. mallei are considered biothreat agents of concern. In this study, we investigate the proteome of Burkholderia thailandensis, a closely related surrogate for the two more virulent Burkholderia species, during infection of host cells, and compare to that of B. thailandensis in culture. Studying the proteome of Burkholderia spp. during infection is expected to reveal molecular mechanisms of intracellular survival and host immune evasion; but proteomic profiling of Burkholderia during host infection is challenging. Proteomic analyses of host-associated bacteria are typically hindered by the overwhelming host protein content recovered from infected cultures. To address this problem, we have applied bio-orthogonal noncanonical amino acid tagging (BONCAT) to B. thailandensis, enabling the enrichment of newly expressed bacterial proteins from virtually any growth condition, including host cell infection. In this study, we show that B. thailandensis proteins were selectively labeled and efficiently enriched from infected host cells using BONCAT. We also demonstrate that this method can be used to label bacteria in situ by fluorescent tagging. Finally, we present a global proteomic profile of B. thailandensis as it infects host cells and a list of proteins that are differentially regulated in infection conditions as compared to bacterial monoculture. Among the identified proteins are quorum sensing regulated genes as well as homologs to previously identified virulence factors. This method provides a powerful tool to study the molecular processes during Burkholderia infection, a much-needed addition to the Burkholderia molecular toolbox

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Heterogeneity of Discrete Biogeographical Nutrient-Niches Dictate Intestinal Microbial Homeostasis

The gut microbiota is comprised of trillions of diverse and densely populated microbial species. Recent understanding has linked the maintenance of microbial homeostasis to regulation of other organ system to subsequently impact the overall health of the host. The coexistence of these microbial species requires many different nutrient niches to support microbial diversity and limit overgrowth of any particular species. However, foodborne pathogens, like Salmonella enterica serovar Typhimurium, overcome this competitive environment by triggering sweeping physiological changes to establish new niches for outgrowth. Here, we present novel evidence that nutrient-niches in the gut are subdivided into discrete host cell-derived microhabitats. We use Salmonella and E. coli, to show how 1. different bacteria exhibit biogeographical localization to these distinct luminal nutrient-niches and 2. pathogens modulate the host to promote pro-inflammatory landscapes and outcompete commensal microbial members.In chapter 1, we introduce the host factors that shape the competitive environment of the gut microbiota, known as habitat filters. The pressures exerted by the host are driving forces for commensal microbial members to evolve better protective strategies by occupying existing niches. Thus, habitat filters maintain the longitudinal and cross-sectional heterogeneity of the microbiota to promote microbial diversity and gut homeostasis.In chapter 2, we show that critical resources in the large intestine, such as nitrate, aren’t defined by only global availability, but separated by different host cells into distinct biogeographical microhabitats for gut microbes. We show that commensal Escherichia coli and pathogenic Salmonella enterica serovar Typhimurium both utilized nitrate for intestinal growth, but each accessed this resource in a distinct biogeographical niche. Commensal E. coli utilized epithelial-derived nitrate, whereas nitrate in the niche occupied by S. Typhimurium was derived from phagocytes recruited by its virulence factors. Surprisingly, avirulent S. Typhimurium was unable to utilize epithelial-derived nitrate, because the chemotaxis receptors McpB and McpC excluded the pathogen from the niche occupied by E. coli. In contrast, E. coli invaded the niche constructed by S. Typhimurium virulence factors and conferred colonization resistance by competing for nitrate. Thus, nutrient-niches are not defined solely by critical resources but can be further subdivided by the host biogeographically into distinct microhabitats, thereby driving the effects of habitat filters and generating new niche opportunities for distinct bacterial species. In chapter 3, we show Salmonella changes the intestinal landscape to introduce a nutrient-niche that benefits the pathogen while suppressing commensal microbiota . While Salmonella benefits from gut inflammation, commensal members of the microbiota often cannot survive this robust immune response. However, the specifics of this reduction of commensal microbial members are not well understood. We show that phagocyte infiltration during Salmonella outgrowth is correlated to the depletion of Clostridia, a commensal microbe that is known to inhibit Salmonella. Chemical inhibition of reactive species and depletion of phagocytes during Salmonella infection both mitigate Clostridia depletion. We also show replicate this phenomenon in a non-infectious piroxicam-accelerated IL10-/- model for colitis. These findings demonstrate phagocyte-derived reactive species reduce Clostridia numbers during gut inflammation and suggest new therapeutic directions for mitigating both infectious and non-infectious gut colitis. Overall, we provide evidence that the host limits access to valuable host-derived nutrients by subdividing availability of these critical resources into discrete biogeographical niches. This heterogeneity of nutrient-niches maintains homeostasis in the large intestine and supports gut commensals ability to prevent infection and drives enteric pathogens to evolve new strategies to overcome colonization resistance and habitat filters

Anaerobic Respiration of NOX1-Derived Hydrogen Peroxide Licenses Bacterial Growth at the Colonic Surface

The colonic microbiota exhibits cross-sectional heterogeneity, but the mechanisms that govern its spatial organization remain incompletely understood. Here we used Citrobacter rodentium, a pathogen that colonizes the colonic surface, to identify microbial traits that license growth and survival in this spatial niche. Previous work showed that during colonic crypt hyperplasia, type III secretion system (T3SS)-mediated intimate epithelial attachment provides C. rodentium with oxygen for aerobic respiration. However, we find that prior to the development of colonic crypt hyperplasia, T3SS-mediated intimate attachment is not required for aerobic respiration but for hydrogen peroxide (H2O2) respiration using cytochrome c peroxidase (Ccp). The epithelial NADPH oxidase NOX1 is the primary source of luminal H2O2 early after C. rodentium infection and is required for Ccp-dependent growth. Our results suggest that NOX1-derived H2O2 is a resource that governs bacterial growth and survival in close proximity to the mucosal surface during gut homeostasis

Medical Students’ Exposure to the Humanities Correlates with Positive Personal Qualities and Reduced Burnout: A Multi-Institutional U.S. Survey

Background: Literature, music, theater, and visual arts play an uncertain and limited role in medical education. One of the arguments often advanced in favor of teaching the humanities refers to their capacity to foster traits that not only improve practice, but might also reduce physician burnout—an increasing scourge in today’s medicine. Yet, research remains limited. Objective: To test the hypothesis that medical students with higher exposure to the humanities would report higher levels of positive physician qualities (e.g., wisdom, empathy, self-efficacy, emotional appraisal, spatial skills), while reporting lower levels of negative qualities that are detrimental to physician well-being (e.g., intolerance of ambiguity, physical fatigue, emotional exhaustion, and cognitive weariness). Design: An online survey. Participants: All students enrolled at five U.S. medical schools during the 2014–2015 academic year were invited by email to take part in our online survey. Main Measures: Students reported their exposure to the humanities (e.g., music, literature, theater, visual arts) and completed rating scales measuring selected personal qualities. Key Results: In all, 739/3107 medical students completed the survey (23.8%). Regression analyses revealed that exposure to the humanities was significantly correlated with positive personal qualities, including empathy (p \u3c 0.001), tolerance for ambiguity (p \u3c 0.001), wisdom (p \u3c 0.001), emotional appraisal (p = 0.01), self-efficacy (p = 0.02), and spatial skills (p = 0.02), while it was significantly and inversely correlated with some components of burnout (p = 0.01). Thus, all hypotheses were statistically significant, with effect sizes ranging from 0.2 to 0.59. Conclusions: This study confirms the association between exposure to the humanities and both a higher level of students’ positive qualities and a lower level of adverse traits. These findings may carry implications for medical school recruitment and curriculum design. “[Science and humanities are] twin berries on one stem, grievous damage has been done to both in regarding [them]... in any other light than complemental.” (William Osler, Br Med J. 1919;2:1–7)

Endogenous Enterobacteriaceae underlie variation in susceptibility to Salmonella infection

Lack of reproducibility is a prominent problem in biomedical research. An important source of variation in animal experiments is the microbiome, but little is known about specific changes in the microbiota composition that cause phenotypic differences. Here, we show that genetically similar laboratory mice obtained from four different commercial vendors exhibited marked phenotypic variation in their susceptibility to Salmonella infection. Faecal microbiota transplant into germ-free mice replicated donor susceptibility, revealing that variability was due to changes in the gut microbiota composition. Co-housing of mice only partially transferred protection against Salmonella infection, suggesting that minority species within the gut microbiota might confer this trait. Consistent with this idea, we identified endogenous Enterobacteriaceae, a low-abundance taxon, as a keystone species responsible for variation in the susceptibility to Salmonella infection. Protection conferred by endogenous Enterobacteriaceae could be modelled by inoculating mice with probiotic Escherichia coli, which conferred resistance by using its aerobic metabolism to compete with Salmonella for resources. We conclude that a mechanistic understanding of phenotypic variation can accelerate development of strategies for enhancing the reproducibility of animal experiments

Host cells subdivide nutrient niches into discrete biogeographical microhabitats for gut microbes.

Changes in the microbiota composition are associated with many human diseases, but factors that govern strain abundance remain poorly defined. We show that a commensal Escherichia coli strain and a pathogenic Salmonella enterica serovar Typhimurium isolate both utilize nitrate for intestinal growth, but each accesses this resource in a distinct biogeographical niche. Commensal E. coli utilizes epithelial-derived nitrate, whereas nitrate in the niche occupied by S. Typhimurium is derived from phagocytic infiltrates. Surprisingly, avirulent S. Typhimurium was shown to be unable to utilize epithelial-derived nitrate because its chemotaxis receptors McpB and McpC exclude the pathogen from the niche occupied by E. coli. In contrast, E. coli invades the niche constructed by S. Typhimurium virulence factors and confers colonization resistance by competing for nitrate. Thus, nutrient niches are not defined solely by critical resources, but they can be further subdivided biogeographically within the host into distinct microhabitats, thereby generating new niche opportunities for distinct bacterial species