Microbiota Metabolites in Health and Disease.

Metabolism is one of the strongest drivers of interkingdom interactions-including those between microorganisms and their multicellular hosts. Traditionally thought to fuel energy requirements and provide building blocks for biosynthetic pathways, metabolism is now appreciated for its role in providing metabolites, small-molecule intermediates generated from metabolic processes, to perform various regulatory functions to mediate symbiotic relationships between microbes and their hosts. Here, we review recent advances in our mechanistic understanding of how microbiota-derived metabolites orchestrate and support physiological responses in the host, including immunity, inflammation, defense against infections, and metabolism. Understanding how microbes metabolically communicate with their hosts will provide us an opportunity to better describe how a host interacts with all microbes-beneficial, pathogenic, and commensal-and an opportunity to discover new ways to treat microbial-driven diseases.

Cooperative Metabolic Adaptations in the Host Can Favor Asymptomatic Infection and Select for Attenuated Virulence in an Enteric Pathogen.

Pathogen virulence exists on a continuum. The strategies that drive symptomatic or asymptomatic infections remain largely unknown. We took advantage of the concept of lethal dose 50 (LD50) to ask which component of individual non-genetic variation between hosts defines whether they survive or succumb to infection. Using the enteric pathogen Citrobacter, we found no difference in pathogen burdens between healthy and symptomatic populations. Iron metabolism-related genes were induced in asymptomatic hosts compared to symptomatic or naive mice. Dietary iron conferred complete protection without influencing pathogen burdens, even at 1000× the lethal dose of Citrobacter. Dietary iron induced insulin resistance, increasing glucose levels in the intestine that were necessary and sufficient to suppress pathogen virulence. A short course of dietary iron drove the selection of attenuated Citrobacter strains that can transmit and asymptomatically colonize naive hosts, demonstrating that environmental factors and cooperative metabolic strategies can drive conversion of pathogens toward commensalism.

When the Gut Gets Tough, the Enterocytes Get Going.

It is assumed that collateral damage from the immune system drives intestinal epithelial cell (IEC) expulsion during enteric infections. In this issue of Immunity, Zhai et al. (2018) describe how Drosophila's canonical immune deficiency (Imd) pathway programs IEC delamination in the gut.

Role of respiratory NADH oxidation in the regulation of Staphylococcus aureus virulence.

The success of Staphylococcus aureus as a pathogen is due to its capability of fine-tuning its cellular physiology to meet the challenges presented by diverse environments, which allows it to colonize multiple niches within a single vertebrate host. Elucidating the roles of energy-yielding metabolic pathways could uncover attractive therapeutic strategies and targets. In this work, we seek to determine the effects of disabling NADH-dependent aerobic respiration on the physiology of S. aureus. Differing from many pathogens, S. aureus has two type-2 respiratory NADH dehydrogenases (NDH-2s) but lacks the respiratory ion-pumping NDHs. Here, we show that the NDH-2s, individually or together, are not essential either for respiration or growth. Nevertheless, their absence eliminates biofilm formation, production of α-toxin, and reduces the ability to colonize specific organs in a mouse model of systemic infection. Moreover, we demonstrate that the reason behind these phenotypes is the alteration of the fatty acid metabolism. Importantly, the SaeRS two-component system, which responds to fatty acids regulation, is responsible for the link between NADH-dependent respiration and virulence in S. aureus.

Listeria monocytogenes MenI Encodes a DHNA-CoA Thioesterase Necessary for Menaquinone Biosynthesis, Cytosolic Survival, and Virulence.

Listeria monocytogenes is a Gram-positive, intracellular pathogen that is highly adapted to invade and replicate in the cytosol of eukaryotic cells. Intermediate metabolites in the menaquinone biosynthesis pathway are essential for the cytosolic survival and virulence of L. monocytogenes, independent of the production of menaquinone (MK) and aerobic respiration. Determining which specific intermediate metabolite(s) are essential for cytosolic survival and virulence has been hindered by the lack of an identified 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) thioesterase essential for converting DHNA-CoA to DHNA in the MK synthesis pathway. Using the recently identified Escherichia coli DHNA-CoA thioesterase as a query, homology sequence analysis revealed a single homolog in L. monocytogenes, LMRG_02730 Genetic deletion of LMRG_02730 resulted in an ablated membrane potential, indicative of a nonfunctional electron transport chain (ETC) and an inability to aerobically respire. Biochemical kinetic analysis of LMRG_02730 revealed strong activity toward DHNA-CoA, similar to its E. coli homolog, further demonstrating that LMRG_02730 is a DHNA-CoA thioesterase. Functional analyses in vitro, ex vivo, and in vivo using mutants directly downstream and upstream of LMRG_02730 revealed that DHNA-CoA is sufficient to facilitate in vitro growth in minimal medium, intracellular replication, and plaque formation in fibroblasts. In contrast, protection against bacteriolysis in the cytosol of macrophages and tissue-specific virulence in vivo requires the production of 1,4-dihydroxy-2-naphthoate (DHNA). Taken together, these data implicate LMRG_02730 (renamed MenI) as a DHNA-CoA thioesterase and suggest that while DHNA, or an unknown downstream product of DHNA, protects the bacteria from killing in the macrophage cytosol, DHNA-CoA is necessary for intracellular bacterial replication.

Mutation of the Transcriptional Regulator YtoI Rescues Listeria monocytogenes Mutants Deficient in the Essential Shared Metabolite 1,4-Dihydroxy-2-Naphthoate (DHNA).

Listeria monocytogenes, a Gram-positive, facultative intracellular pathogen, survives and replicates in the cytosol of host cells. Synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an intermediate of menaquinone biosynthesis, is essential for cytosolic survival of L. monocytogenes independent from its role in respiration. Here, we demonstrate that DHNA is essential for virulence in a murine model of listeriosis due to both respiration-dependent and -independent functions. In addition, DHNA can be both secreted and utilized as an extracellular shared metabolite to promote cytosolic survival inside host macrophages. To understand the role(s) of DHNA in L. monocytogenes intracellular survival and virulence, we isolated DHNA-deficient (ΔmenD strain) suppressor mutants that formed plaques in monolayers of fibroblasts. Five ΔmenD suppressor (mds) mutants additionally rescued at least 50% of the cytosolic survival defect of the parent ΔmenD mutant. Whole-genome sequencing revealed that four of the five suppressor mutants had independent missense mutations in a putative transcriptional regulator, ytoI (lmo1576). Clean deletion and complementation in trans confirmed that loss of ytoI could restore plaquing and cytosolic survival of DHNA-deficient L. monocytogenes RNA-seq transcriptome analysis revealed five genes (lmo0944, lmo1575, lmo1577, lmo2005, and lmo2006) expressed at a higher level in the ΔytoI strain than in the wild-type strain, whereas two genes (lmo1917 and lmo2103) demonstrated lower expression in the ΔytoI mutant. Intriguingly, the majority of these genes are involved in controlling pyruvate flux. Metabolic analysis confirmed that acetoin, acetate, and lactate flux were altered in a ΔytoI mutant, suggesting a critical role for regulating these metabolic programs. In conclusion, we have demonstrated that, similar to findings in select other bacteria, DHNA can act as a shared resource, and it is essential for cytosolic survival and virulence of L. monocytogenes Furthermore, we have identified a novel transcriptional regulator in L. monocytogenes and determined that its metabolic regulation is implicated in cytosolic survival of L. monocytogenes.

The Listeria monocytogenes PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence.

Obstacles to bacterial survival and replication in the cytosol of host cells, and the mechanisms used by bacterial pathogens to adapt to this niche are not well understood. Listeria monocytogenes is a well-studied Gram-positive foodborne pathogen that has evolved to invade and replicate within the host cell cytosol; yet the mechanisms by which it senses and responds to stress to survive in the cytosol are largely unknown. To assess the role of the L. monocytogenes penicillin-binding-protein and serine/threonine associated (PASTA) kinase PrkA in stress responses, cytosolic survival and virulence, we constructed a ΔprkA deletion mutant. PrkA was required for resistance to cell wall stress, growth on cytosolic carbon sources, intracellular replication, cytosolic survival, inflammasome avoidance and ultimately virulence in a murine model of Listeriosis. In Bacillus subtilis and Mycobacterium tuberculosis, homologues of PrkA phosphorylate a highly conserved protein of unknown function, YvcK. We found that, similar to PrkA, YvcK is also required for cell wall stress responses, metabolism of glycerol, cytosolic survival, inflammasome avoidance and virulence. We further demonstrate that similar to other organisms, YvcK is directly phosphorylated by PrkA, although the specific site(s) of phosphorylation are not highly conserved. Finally, analysis of phosphoablative and phosphomimetic mutants of YvcK in vitro and in vivo demonstrate that while phosphorylation of YvcK is irrelevant to metabolism and cell wall stress responses, surprisingly, a phosphomimetic, nonreversible negative charge of YvcK is detrimental to cytosolic survival and virulence in vivo. Taken together our data identify two novel virulence factors essential for cytosolic survival and virulence of L. monocytogenes. Furthermore, our data demonstrate that regulation of YvcK phosphorylation is tightly controlled and is critical for virulence. Finally, our data suggest that yet to be identified substrates of PrkA are essential for cytosolic survival and virulence of L. monocytogenes and illustrate the importance of studying protein phosphorylation in the context of infection.

Listeria monocytogenes cytosolic metabolism promotes replication, survival, and evasion of innate immunity.

Listeria monocytogenes, the causative agent of listeriosis, is an intracellular pathogen that is exquisitely evolved to survive and replicate in the cytosol of eukaryotic cells. Eukaryotic cells typically restrict bacteria from colonising the cytosol, likely through a combination of cell autonomous defences, nutritional immunity, and innate immune responses including induction of programmed cell death. This suggests that L. monocytogenes and other professional cytosolic pathogens possess unique metabolic adaptations, not only to support replication but also to facilitate resistance to host-derived stresses/defences and avoidance of innate immune activation. In this review, we outline our current understanding of L. monocytogenes metabolism in the host cytosol and highlight major metabolic processes which promote intracellular replication and survival.

Pseudomonas putida F1 has multiple chemoreceptors with overlapping specificity for organic acids.

Previous studies have demonstrated that Pseudomonas putida strains are not only capable of growth on a wide range of organic substrates, but also chemotactic towards many of these compounds. However, in most cases the specific chemoreceptors that are involved have not been identified. The complete genome sequences of P. putida strains F1 and KT2440 revealed that each strain is predicted to encode 27 methyl-accepting chemotaxis proteins (MCPs) or MCP-like proteins, 25 of which are shared by both strains. It was expected that orthologous MCPs in closely related strains of the same species would be functionally equivalent. However, deletion of the gene encoding the P. putida F1 orthologue (locus tag Pput_4520, designated mcfS) of McpS, a known receptor for organic acids in P. putida KT2440, did not result in an obvious chemotaxis phenotype. Therefore, we constructed individual markerless MCP gene deletion mutants in P. putida F1 and screened for defective sensory responses to succinate, malate, fumarate and citrate. This screen resulted in the identification of a receptor, McfQ (locus tag Pput_4894), which responds to citrate and fumarate. An additional receptor, McfR (locus tag Pput_0339), which detects succinate, malate and fumarate, was found by individually expressing each of the 18 genes encoding canonical MCPs from strain F1 in a KT2440 mcpS-deletion mutant. Expression of mcfS in the same mcpS deletion mutant demonstrated that, like McfR, McfS responds to succinate, malate, citrate and fumarate. Therefore, at least three receptors, McfR, McfS, and McfQ, work in concert to detect organic acids in P. putida F1.

Cooperation between physiological defenses and immune resistance produces asymptomatic carriage of a lethal bacterial pathogen.

Animals have evolved two defense strategies to survive infections. Antagonistic strategies include mechanisms of immune resistance that operate to sense and kill invading pathogens. Cooperative or physiological defenses mediate host adaptation to the infected state, limiting physiological damage and disease, without killing the pathogen, and have been shown to cause asymptomatic carriage and transmission of lethal pathogens. Here we demonstrate that physiological defenses cooperate with the adaptive immune response to generate long-term asymptomatic carriage of the lethal enteric murine pathogen, Citrobacter rodentium. Asymptomatic carriage of genetically virulent C. rodentium provided immune resistance against subsequent infections. Host immune protection was dependent on systemic antibody responses and pathogen virulence behavior, rather than the recognition of specific virulent factor antigens. Finally, we demonstrate that an avirulent strain of C. rodentium in the field has background mutations in two genes that are important for LPS structure. Our work reveals novel insight into how asymptomatic infections can arise mechanistically with immune resistance, mediating exclusion of phenotypically virulent enteric pathogen to promote asymptomatic carriage.

Cooperation between physiological defenses and immune resistance produces asymptomatic carriage of a lethal bacterial pathogen.

Animals evolved two defense strategies to survive infections. Antagonistic strategies include immune resistance mechanisms that operate to kill invading pathogens. Cooperative or physiological defenses mediate host adaptation to the infected state, limiting physiological damage and disease, without killing the pathogen, and have been shown to cause asymptomatic carriage and transmission of lethal pathogens. Here, we demonstrate that physiological defenses cooperate with the adaptive immune response to generate long-term asymptomatic carriage of the lethal enteric murine pathogen, Citrobacter rodentium. Asymptomatic carriage of genetically virulent C. rodentium provided immune resistance against subsequent infections. Immune protection was dependent on systemic antibody responses and pathogen virulence behavior rather than the recognition of specific virulent antigens. Last, we demonstrate that an avirulent strain of C. rodentium in the field has background mutations in genes that are important for LPS structure. Our work reveals insight into how asymptomatic infections can arise mechanistically with immune resistance, mediating exclusion of phenotypically virulent enteric pathogen to promote asymptomatic carriage.

Emergence of novel genomic regulatory regions associated with light-organ development in the bobtail squid.

Light organs (LO) with symbiotic bioluminescent bacteria are hallmarks of many bobtail squid species. These organs possess structural and functional features to modulate light, analogous to those found in coleoid eyes. Previous studies identified four transcription factors and modulators (SIX, EYA, PAX6, DAC) associated with both eyes and light organ development, suggesting co-option of a highly conserved gene regulatory network. Using available topological, open chromatin, and transcriptomic data, we explore the regulatory landscape around the four transcription factors as well as genes associated with LO and shared LO/eye expression. This analysis revealed several closely associated and putatively co-regulated genes. Comparative genomic analyses identified distinct evolutionary origins of these putative regulatory associations, with the DAC locus showing a unique topological and evolutionarily recent organization. We discuss different scenarios of modifications to genome topology and how these changes may have contributed to the evolutionary emergence of the light organ.

A Genetic Screen Reveals that Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA), but Not Full-Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival.

Through unknown mechanisms, the host cytosol restricts bacterial colonization; therefore, only professional cytosolic pathogens are adapted to colonize this host environment. Listeria monocytogenes is a Gram-positive intracellular pathogen that is highly adapted to colonize the cytosol of both phagocytic and nonphagocytic cells. To identify L. monocytogenes determinants of cytosolic survival, we designed and executed a novel screen to isolate L. monocytogenes mutants with cytosolic survival defects. Multiple mutants identified in the screen were defective for synthesis of menaquinone (MK), an essential molecule in the electron transport chain. Analysis of an extensive set of MK biosynthesis and respiratory chain mutants revealed that cellular respiration was not required for cytosolic survival of L. monocytogenes but that, instead, synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an MK biosynthesis intermediate, was essential. Recent discoveries showed that modulation of the central metabolism of both host and pathogen can influence the outcome of host-pathogen interactions. Our results identify a potentially novel function of the MK biosynthetic intermediate DHNA and specifically highlight how L. monocytogenes metabolic adaptations promote cytosolic survival and evasion of host immunity.IMPORTANCE Cytosolic bacterial pathogens, such as Listeria monocytogenes and Francisella tularensis, are exquisitely evolved to colonize the host cytosol in a variety of cell types. Establishing an intracellular niche shields these pathogens from effectors of humoral immunity, grants access to host nutrients, and is essential for pathogenesis. Through yet-to-be-defined mechanisms, the host cytosol restricts replication of non-cytosol-adapted bacteria, likely through a combination of cell autonomous defenses (CADs) and nutritional immunity. Utilizing a novel genetic screen, we identified determinants of L. monocytogenes cytosolic survival and virulence and identified a role for the synthesis of the menaquinone precursor 1,4-dihydroxy-2-naphthoate (DHNA) in cytosolic survival. Together, these data begin to elucidate adaptations that allow cytosolic pathogens to survive in their intracellular niches.

Integrating grant-funded research into the undergraduate biology curriculum using IMG-ACT.

It has become clear in current scientific pedagogy that the emersion of students in the scientific process in terms of designing, implementing, and analyzing experiments is imperative for their education; as such, it has been our goal to model this active learning process in the classroom and laboratory in the context of a genuine scientific question. Toward this objective, the National Science Foundation funded a collaborative research grant between a primarily undergraduate institution and a research-intensive institution to study the chemotactic responses of the bacterium Pseudomonas putida F1. As part of the project, a new Bioinformatics course was developed in which undergraduates annotate relevant regions of the P. putida F1 genome using Integrated Microbial Genomes Annotation Collaboration Toolkit, a bioinformatics interface specifically developed for undergraduate programs by the Department of Energy Joint Genome Institute. Based on annotations of putative chemotaxis genes in P. putida F1 and comparative genomics studies, undergraduate students from both institutions developed functional genomics research projects that evolved from the annotations. The purpose of this study is to describe the nature of the NSF grant, the development of the Bioinformatics lecture and wet laboratory course, and how undergraduate student involvement in the project that was initiated in the classroom has served as a springboard for independent undergraduate research projects.