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 (AmenD strain) suppressor mutants that formed plaques in monolayers of fibroblasts. Five AmenD suppressor (mds) mutants additionally rescued at least 50% of the cytosolic survival defect of the parent AmenD mutant. Whole-genome sequencing revealed that four of the five suppressor mutants had independent missense mutations in a putative transcriptional regulator, ytol (Imo1576). Clean deletion and complementation in trans confirmed that loss of ytol could restore plaguing and cytosolic survival of DHNA-deficient L. monocytogenes. RNAseq transcriptome analysis revealed five genes (lmo0944, lmo1575, Imo1577, lmo2005, and lmo2006) expressed at a higher level in the Delta ytol strain than in the wild-type strain, whereas two genes (lmo1917 and lmo2103) demonstrated lower expression in the Delta ytol 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 Delta ytol 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.

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.

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 1000x 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.

We report that dietary iron protects mice from infection by Citrobacter rodentium. Iron induces a state of insulin resistance and increases glucose availability in the gut, thereby attenutating C. rodentium virulence. Additionally the pathogen appears to be driven towards a long-term commensal state. Here, we identify mutations in persistent and avirulent Citrobacter rodentium isolates from mice given an iron supplemented diet.

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 alpha-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 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-naphthoylcoenzyme 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.

The accumulation of Fe and Mn in seasonally stratified drinking water reservoirs adversely impacts water quality. To control issues with Fe and Mn at the source, some drinking water utilities have deployed hypolimnetic oxygenation systems to create well-oxygenated conditions in the water column that are favorable for the oxidation, and thus removal, of Fe and Mn. However, in addition to being controlled by dissolved oxygen (DO), Fe and Mn concentrations are also influenced by pH and metal-oxidizing microorganisms. We studied the response of Fe and Mn concentrations to hypolimnetic oxygenation in a shallow drinking water reservoir in Vinton, Virginia, USA by sequentially activating and deactivating an oxygenation system over two summers. We found that maintaining well-oxygenated conditions effectively prevented the accumulation of soluble Fe in the hypolimnion. However, while the rate of Mn oxidation increased under well-oxygenated conditions, soluble Mn still accumulated in the slightly acidic to neutral (pH 5.6 to 7.5) hypolimnion. In parallel, we conducted laboratory incubation experiments, which showed that the presence of Mn-oxidizing microorganisms increased the rate of Mn oxidation in comparison with rates under oxic, abiotic conditions. Combined, our field and laboratory results demonstrate that increasing DO concentrations in the water column is important for stimulating the oxidation of Fe and Mn, but that the successful management of Mn is also tied to the activity of Mn-oxidizing organisms in the water column and favorable (neutral to alkaline) pH. (C) 2016 Elsevier Ltd. All rights reserved.

Physical and chemical gradients across ecosystems, such as stream-to-lake continua within human-made reservoirs, provide valuable opportunities to examine how organisms respond to changing environments. We quantified the rate of dinoflagellate recruitment across a small reservoir to test the hypothesis that organisms are controlled by different factors along a reservoir continuum. We predicted that recruitment would be tightly coupled with reservoir physics in the riverine zone and closely related to water chemistry in the lacustrine zone. For the dominant dinoflagellate genus in the reservoir, Peridinium, recruitment from the sediments accounted for a median of 16% of increases in pelagic cell abundance throughout the summer. As predicted, Peridinium recruitment rates at the riverine site were correlated with physical variables, while at the lacustrine site, recruitment rates were highly correlated with water chemistry (e.g. nutrient ratios and dissolved oxygen). Recruitment patterns of the second most common genus, Gymnodinium, were not correlated with environmental variables, though Gymnodinium's much lower densities suggest that its dynamics were controlled by other factors. Our results reveal that the physical-biological coupling controlling algal recruitment, which can play a large role in pelagic population growth and bloom formation, can vary substantially on a spatial gradient within even a small reservoir.

The magnitude of lateral dissolved inorganic carbon (DIC) export from terrestrial ecosystems to inland waters strongly influences the estimate of the global terrestrial carbon dioxide (CO2) sink. At present, no reliable number of this export is available, and the few studies estimating the lateral DIC export assume that all lakes on Earth function similarly. However, lakes can function along a continuum from passive carbon transporters (passive open channels) to highly active carbon transformers with efficient in-lake CO2 production and loss. We developed and applied a conceptual model to demonstrate how the assumed function of lakes in carbon cycling can affect calculations of the global lateral DIC export from terrestrial ecosystems to inland waters. Using global data on in-lake CO2 production by mineralization as well as CO2 loss by emission, primary production, and carbonate precipitation in lakes, we estimated that the global lateral DIC export can lie within the range of 0.70(-0.31)(+0.27) 1.52(-0.90)(+1.09) Pg C yr(-1) depending on the assumed function of lakes. Thus, the considered lake function has a large effect on the calculated lateral DIC export from terrestrial ecosystems to inland waters. We conclude that more robust estimates of CO2 sinks and sources will require the classification of lakes into their predominant function. This functional lake classification concept becomes particularly important for the estimation of future CO2 sinks and sources, since in-lake carbon transformation is predicted to be altered with climate change.