Valeriy Poroyko

Strain-resolved community genomic analysis of gut microbial colonization in a premature infant

The intestinal microbiome is a critical determinant of human health. Alterations in its composition have been correlated with chronic disorders, such as obesity and inflammatory bowel disease in adults, and may be associated with neonatal necrotizing enterocolitis in premature infants. Increasing evidence suggests that strain-level genomic variation may underpin distinct ecological trajectories within mixed populations, yet there have been few strain-resolved analyses of genotype–phenotype connections in the context of the human ecosystem. Here, we document strain-level genomic divergence during the first 3 wk of life within the fecal microbiota of an infant born at 28-wk gestation. We observed three compositional phases during colonization, and reconstructed and intensively curated population genomic datasets from the third phase. The relative abundance of two Citrobacter strains sharing ~99% nucleotide identity changed significantly over time within a community dominated by a nearly clonal Serratia population and harboring a lower abundance Enterococcus population and multiple plasmids and bacteriophage. Modeling of Citrobacter strain abundance suggests differences in growth rates and host colonization patterns. We identified genotypic variation potentially responsible for divergent strain ecologies, including hotspots of sequence variation in regulatory genes and intergenic regions, and in genes involved in transport, flagellar biosynthesis, substrate metabolism, and host colonization, as well as differences in the complements of these genes. Our results demonstrate that a community genomic approach can elucidate gut microbial colonization at the resolution required to discern medically relevant strain and species population dynamics, and hence improve our ability to diagnose and treat microbial community-mediated disorders.

Redefining the Role of Intestinal Microbes in the Pathogenesis of Necrotizing Enterocolitis

Neonatal necrotizing enterocolitis (NEC) remains an important cause of morbidity and mortality among very low birth weight infants. It has long been suspected that intestinal microbes contribute to the pathogenesis of NEC, but the details of this relationship remain poorly understood. Recent advances in molecular biology and enteric microbiology have improved our ability to characterize intestinal microbes from infants with NEC and from healthy unaffected newborns. The lack of diversity within the neonatal intestine makes it possible to study gut microbial communities at a high level of resolution not currently possible in corresponding studies of the adult intestinal tract. Here, we summarize clinical and laboratory evidence that supports the hypothesis that NEC is a microbe-mediated disorder. In addition, we detail recent technologic advances that may be harnessed to perform high-throughput, comprehensive studies of the gut microbes of very low birth weight infants. Methods for characterizing microbial genotype are discussed, as are methods of identifying patterns of gene expression, protein expression, and metabolite production. Application of these technologies to biological samples from affected and unaffected newborns may lead to advances in the care of infants who are at risk for the unabated problem of NEC.

Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1

During host injury, Pseudomonas aeruginosa can be cued to express a lethal phenotype within the intestinal tract reservoir—a hostile, nutrient scarce environment depleted of inorganic phosphate. Here we determined if phosphate depletion activates a lethal phenotype in P. aeruginosa during intestinal colonization. To test this, we allowed Caenorhabditis elegans to feed on lawns of P. aeruginosa PAO1 grown on high and low phosphate media. Phosphate depletion caused PAO1 to kill 60% of nematodes whereas no worms died on high phosphate media. Unexpectedly, intense redness was observed in digestive tubes of worms before death. Using a combination of transcriptome analyses, mutants, and reporter constructs, we identified 3 global virulence systems that were involved in the “red death” response of P. aeruginosa during phosphate depletion; they included phosphate signaling (PhoB), the MvfR–PQS pathway of quorum sensing, and the pyoverdin iron acquisition system. Activation of all 3 systems was required to form a red colored PQS+Fe3+ complex which conferred a lethal phenotype in this model. When pyoverdin production was inhibited in P. aeruginosa by providing excess iron, red death was attenuated in C. elegans and mortality was decreased in mice intestinally inoculated with P. aeruginosa. Introduction of the red colored PQS+Fe3+ complex into the digestive tube of C. elegans or mouse intestine caused mortality associated with epithelial disruption and apoptosis. In summary, red death in C. elegans reveals a triangulated response between PhoB, MvfR–PQS, and pyoverdin in response to phosphate depletion that activates a lethal phenotype in P. aeruginosa.

Lymphotoxin regulates commensal responses to enable diet-induced obesity

Microbiota are essential for weight gain in mouse models of diet-induced obesity (DIO), but the pathways that cause the microbiota to induce weight gain are unknown. We report that mice deficient in lymphotoxin, a key molecule in gut immunity, were resistant to DIO. Ltbr−/− mice had different microbial community composition compared to their heterozygous littermates, including an overgrowth of segmented filamentous bacteria (SFB). Furthermore, cecal transplantation conferred leanness to germ-free recipients. Housing Ltbr−/− mice with their obese siblings rescued weight gain in Ltbr−/− mice, demonstrating the communicability of the obese phenotype. Ltbr−/− mice lacked interleukin 23 (IL-23) and IL-22, which can regulate SFB. Mice deficient in these pathways also resisted DIO, demonstrating that intact mucosal immunity guides diet-induced changes to the microbiota to enable obesity.

Gut microbial gene expression in mother-fed and formula-fed piglets.

Background Effects of diet on the structure and function of gut microbial communities in newborn infants are poorly understood. High-resolution molecular studies are needed to definitively ascertain whether gut microbial communities are distinct in milk-fed and formula-fed infants. Methodology/Principal Findings Pyrosequencing-based whole transcriptome shotgun sequencing (RNA-seq) was used to evaluate community wide gut microbial gene expression in 21 day old neonatal piglets fed either with sows milk (mother fed, MF; n = 4) or with artificial formula (formula fed, FF; n = 4). Microbial DNA and RNA were harvested from cecal contents for each animal. cDNA libraries and 16S rDNA amplicons were sequenced on the Roche 454 GS-FLX Titanium system. Communities were similar at the level of phylum but were dissimilar at the level of genus; Prevotella was the dominant genus within MF samples and Bacteroides was most abundant within FF samples. Screened cDNA sequences were assigned functional annotations by the MG-RAST annotation pipeline and based upon best-BLASTX-hits to the NCBI COG database. Patterns of gene expression were very similar in MF and FF animals. All samples were enriched with transcripts encoding enzymes for carbohydrate and protein metabolism, as well as proteins involved in stress response, binding to host epithelium, and lipopolysaccharide metabolism. Carbohydrate utilization transcripts were generally similar in both groups. The abundance of enzymes involved in several pathways related to amino acid metabolism (e.g., arginine metabolism) and oxidative stress response differed in MF and FF animals. Conclusions/Significance Abundant transcripts identified in this study likely contribute to a core microbial metatranscriptome in the distal intestine. Although microbial community gene expression was generally similar in the cecal contents of MF and FF neonatal piglets, several differentially abundant gene clusters were identified. Further investigations of gut microbial gene expression will contribute to a better understanding of normal and abnormal enteric microbiology in animals and humans.

The Mu Opioid Receptor Promotes Opioid and Growth Factor-Induced Proliferation, Migration and Epithelial Mesenchymal Transition (EMT) in Human Lung Cancer

Recent epidemiologic studies implying differences in cancer recurrence based on anesthetic regimens raise the possibility that the mu opioid receptor (MOR) can influence cancer progression. Based on our previous observations that overexpression of MOR in human non-small cell lung cancer (NSCLC) cells increased tumor growth and metastasis, this study examined whether MOR regulates growth factor receptor signaling and epithelial mesenchymal transition (EMT) in human NSCLC cells. We utilized specific siRNA, shRNA, chemical inhibitors and overexpression vectors in human H358 NSCLC cells that were either untreated or treated with various concentrations of DAMGO, morphine, fentanyl, EGF or IGF. Cell function assays, immunoblot and immunoprecipitation assays were then performed. Our results indicate MOR regulates opioid and growth factor-induced EGF receptor signaling (Src, Gab-1, PI3K, Akt and STAT3 activation) which is crucial for consequent human NSCLC cell proliferation and migration. In addition, human NSCLC cells treated with opioids, growth factors or MOR overexpression exhibited an increase in snail, slug and vimentin and decrease ZO-1 and claudin-1 protein levels, results consistent with an EMT phenotype. Further, these effects were reversed with silencing (shRNA) or chemical inhibition of MOR, Src, Gab-1, PI3K, Akt and STAT3 (p<0.05). Our data suggest a possible direct effect of MOR on opioid and growth factor-signaling and consequent proliferation, migration and EMT transition during lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings.

Pseudomonas aeruginosa virulence expression is directly activated by morphine and is capable of causing lethal gut-derived sepsis in mice during chronic morphine administration.

Objective: This study was designed to examine the effect of morphine administration on the intestinal mucus barrier and determine its direct effect on the virulence and lethality of Pseudomonas aeruginosa, one of the most frequent pathogens to colonize the gut of critically ill patients. Background Data: Surgical injury is associated with significant exposure of host tissues to morphine from both endogenous release and its use as a potent analgesic agent. Morphine use in surgical patients exposed to extreme physiologic stress is well established to result in increased infection risk. Although morphine is a known immunosuppressant, whether it directly induces virulence expression and lethality in microbes that colonize the human gut remains unknown. Methods: Mice were implanted with a slow release morphine or placebo pellet with and without intestinal inoculation of P. aeruginosa created by direct cecal injection. Mucus production and epithelial integrity was assessed in cecal tissue via Alcian blue staining and histologic analysis. In vivo and in vitro P. aeruginosa virulence expression was examined using reporter strains tagged to the epithelial barrier disrupting protein PA-I lectin. P. aeruginosa chemotaxis toward morphine was also assayed in vitro. Finally, the direct effect of morphine to induce PA-I lectin expression was determined in the absence and presence of methylnaltrexone, a &mgr; opioid receptor antagonist. Results: Mice intestinally inoculated with P. aeruginosa and implanted with a morphine pellet demonstrated significant suppression of intestinal mucus, disrupted intestinal epithelium, and enhanced mortality; whereas exposure of mice to either systemic morphine or intestinal P. aeruginosa alone enhanced intestinal mucus without mortality, suggesting a shift in P. aeruginosa during morphine exposure to a mucus suppressing, barrier disrupting, and lethal phenotype. Direct exposure of P. aeruginosa to morphine in vitro confirmed that morphine can transform P. aeruginosa to a more virulent phenotype that is attenuated in part by methylnaltrexone. Conclusions: Morphine administration shifts intestinal P. aeruginosa to express a virulent phenotype and may play a role in its ability to causes lethal gut-derived sepsis in a susceptible host.

Total Parenteral Nutrition–Associated Lamina Propria Inflammation in Mice Is Mediated by a MyD88-Dependent Mechanism

Enteral nutrient deprivation via total parenteral nutrition (TPN) administration leads to local mucosal inflammatory responses, but the underlying mechanisms are unknown. Wild-type (WT) and MyD88−/− mice underwent jugular vein cannulation. One group received TPN without chow, and controls received standard chow. After 7 d, we harvested intestinal mucosally associated bacteria and isolated small-bowel lamina propria (LP) cells. Bacterial populations were analyzed using 454 pyrosequencing. LP cells were analyzed using quantitative PCR and multicolor flow cytometry. WT, control mucosally associated microbiota were Firmicutes-dominant, whereas WT TPN mice were Proteobacteria-domiant. Similar changes were observed in MyD88−/− mice with TPN administration. UniFrac analysis showed divergent small bowel and colonic bacterial communities in controls, merging toward similar microbiota (but distinct from controls) with TPN. The percentage of LP T regulatory cells significantly decreased with TPN in WT mice. F4/80+CD11b+CD11cdull/− macrophage–derived proinflammatory cytokines significantly increased with TPN. These proinflammatory immunologic changes were significantly abrogated in MyD88−/− TPN mice. Thus, TPN administration is associated with significant expansion of Proteobacteria within the intestinal microbiota and increased proinflammatory LP cytokines. Additionally, MyD88 signaling blockade abrogated decline in epithelial cell proliferation and epithelial barrier function loss.

Gram Negative Bacteria Are Associated with the Early Stages of Necrotizing Enterocolitis

Introduction Necrotizing enterocolitis (NEC) affects 5–10% of infants born weighing less than 1500 g. Most models of NEC recapitulate late-stage disease with gut necrosis and elevated inflammatory mediators. Evaluation of NEC at earlier, less lethal stages of disease will allow investigation of initial disease triggers and may advance our understanding of temporal relationships between factors implicated in NEC pathogenesis. In this manuscript, we describe our investigation of early NEC and test the hypothesis that bacteria and inflammatory mediators differ between animals with early NEC and disease free animals. Methods On DOL7 C3HeB/FeJ pups were fed liquid formula with 1×104 Streptococcus thoraltensis, Serratia marcescens, and Pseudomonas aeruginosa every 3 h. To initiate NEC, pups underwent asphyxia (100% N2 for 90 s) and hypothermia (4°C for 10 min) after feeding. Pups were euthanized at 72 h. Intestines were collected for histologic NEC scoring and DNA/RNA extraction. Bacterial populations were identified by 16S rRNA pyrosequencing and principal component analysis (PCA). RNA isolates underwent QRT-PCR for Toll-like Receptor 4 (TLR4) and inducible nitric oxide synthase (iNOS). Results Despite histologic, intestinal damage in mice with NEC, no gross necrosis was observed suggesting early disease. QRT-PCR yielded no difference between groups in TLR4 or iNOS mRNA levels. PCA demonstrated relative clustering of microbial communities based on presence or absence of NEC. 16S pyrosequencing demonstrated similar phyla between groups (Firmicutes and Proteobacteria predominated in all animals). However, the colonic microbiota of animals with NEC had more Citrobacter (p<0.01), Klebsiella (p<0.05), and Tatumella (p<0.05), while that of animals without NEC had more Streptococcus (p<0.01) and Enterococcus (p<0.01). Conclusion Citrobacter, Klebsiella, and Tatumella are associated with NEC. Differential colonic bacteria were identified despite the lack of inflammatory mediator elevation traditionally associated with NEC. This suggests a temporal relationship between bacteria and inflammatory mediators such that alterations in gut microbiota are associated with early NEC, while inflammatory mediator elevation is associated with advanced NEC.

Chronic sleep disruption alters gut microbiota, induces systemic and adipose tissue inflammation and insulin resistance in mice.

Chronic sleep fragmentation (SF) commonly occurs in human populations, and although it does not involve circadian shifts or sleep deprivation, it markedly alters feeding behaviors ultimately promoting obesity and insulin resistance. These symptoms are known to be related to the host gut microbiota. Mice were exposed to SF for 4 weeks and then allowed to recover for 2 weeks. Taxonomic profiles of fecal microbiota were obtained prospectively, and conventionalization experiments were performed in germ-free mice. Adipose tissue insulin sensitivity and inflammation, as well as circulating measures of inflammation, were assayed. Effect of fecal water on colonic epithelial permeability was also examined. Chronic SF-induced increased food intake and reversible gut microbiota changes characterized by the preferential growth of highly fermentative members of Lachnospiraceae and Ruminococcaceae and a decrease of Lactobacillaceae families. These lead to systemic and visceral white adipose tissue inflammation in addition to altered insulin sensitivity in mice, most likely via enhanced colonic epithelium barrier disruption. Conventionalization of germ-free mice with SF-derived microbiota confirmed these findings. Thus, SF-induced metabolic alterations may be mediated, in part, by concurrent changes in gut microbiota, thereby opening the way for gut microbiome-targeted therapeutics aimed at reducing the major end-organ morbidities of chronic SF.