Tungstate treatment of the dysbiosis associated with gastrointestinal inflammation

#Over 90% of the cells in the human body are microbes, the majority of which reside in bacterial communities (microbiota) that inhabit the large intestine. Recent advances in high-throughput microbiota sequencing (metagenomics) provide a powerful tool for profiling the previously hidden microbial diversity in the gut. For example, metagenomic analysis shows that the large intestine is host to a diverse bacterial community whose structure, at the phylum level, is maintained through unknown mechanisms. The bacterial species dominating the microbiota in the large bowel are strict anaerobes, which lack the ability to respire oxygen and rely on fermentation of complex polysaccharides for growth (Mahowald et al., Proc. Natl. Acad. Sci. U.S.A., 106:5859-5864 (2009)). Towering above all other are obligate anaerobic bacteria belonging to the phyla Bacteroidetes (class Bacteroidia) and Firmicutes (class Clostridia) (Eckburg et al., Science, 308:1635-1638 (2005)). The dominance of obligate anaerobic bacteria belonging to the classes Bacteroidia and Clostridia is a conserved feature of bacterial communities inhabiting the large intestine of both humans and mice (Eckburg et al., Science, 308:1635-1638 (2005); Ley et al., Proc. Natl. Acad. Sci. U.S.A., 102:11070-11075 (2005)).

#However, conditions of gastrointestinal inflammation can lead to a microbial imbalance (dysbiosis) characterized by phylum-level changes in the microbiota composition, including a marked decrease in the representation of obligate anaerobic bacteria and an increased relative abundance of facultative anaerobic bacteria. For example, acute intestinal inflammation triggered by pathogenic Enterobacteriaceae (class Gammaproteobacteria, phylum Proteobacteria), such as Salmonella enterica or Citrobacter rodentium, is accompanied by changes in the bacterial community structure that are marked by an outgrowth of the respective facultative anaerobic pathogen (Barman et al., Infect. Immun., 76:907-915 (2008); Lupp et al, Cell Host Microbe, 2:119-129 (2007); Stecher et al., PLoS Biol., 5:2177-2189 (2007)). Similarly, a reduced relative abundance of strictly anaerobic members of the classes Bacteroidia and Clostridia, and a concomitant increased relative abundance of facultative anaerobic commensal bacteria belonging to the family Enterobacteriaceae or to the class Bacilli (phylum Firmicutes) is seen in individuals with inflammatory bowel disease (IBD) (Baumgart et al., ISME J., 1:403-418 (2007); Frank et al., Proc. Natl. Acad. Sci U.S.A., 104:13780-13785 (2007); Giaffer et al., J. Med. Microbiol., 35:238-243 (1991); Gophna et al., J. Clin. Microbiol., 44:4136-4141 (2006); Krook et al., J. Clinical Pathology, 34:645-650 (1981); Seksik et al., Gut, 52:237-242 (2003); Walker et al., ISME J., 5:220-230 (2011)).

#A marked decrease in the representation of obligate anaerobic Bacteroidia and Clostridia and an increased relative abundance of facultative anaerobic Enterobacteriaceae can also be observed in mice when colitis is induced chemically (Lupp et al., Cell Host Microbe, 2:119-129 (2007)) or through genetically engineered immune defects (Garrett et al., Cell Host Microbe, 8:292-300 (2010)). The dense bacterial communities inhabiting the distal gut compete fiercely for a limited quantity of diet-derived or host mucus-derived carbohydrate available for fermentation (reviewed in, e.g., Fischbach and Sonnenburg, Cell Host Microbe, 10:336-347 (2011); Koropatkin et al., Nat. Rev. Microbiology, 10:323-335 (2012)). Changes in the diet can alter the microbial community structure on the species level; however, the dominance of obligate anaerobic Clostridia and Bacteroidia over the facultative anaerobic Enterobacteriaceae remains untouched (Faith et al., Science, 333:101-104 (2011); Martinez et al., PLoS One, 5:e15046; Sonnenburg et al., Cell, 141:1241-1252 (2010); Walker et al., ISME J., 5:220-230 (2011); Wu et al., Science, 334:105-108 (2011)).

#FIG. 1 illustrates a schematic diagram of inflammation dependent anaerobic respiration of Enterobacteriaceae.

#FIG. 2 shows that anaerobic respiration boosts growth of E. coli during colitis. Mice were mock-treated or treated with dextran sulfate sodium (DSS) or DSS+aminoguanidine (AG) and infected with equal mixtures of the indicated E. coli strains. Five days after infection, the ratio of wild-type (wt) and mutant recovered from colon contents was determined (competitive index). Bars represent geometric means±standard error.

#FIGS. 3A-B show characterization of nitrate respiration-deficient mutants. FIG. 3A shows detection of nitrate reductase activity in E. coli strains. FIG. 3B shows competitive indices for anaerobic growth in mucin broth with (+) or without (−) nitrate (n=3). Bars represent geometric means±standard error. **, P<0.01.

#I. Introduction

#The lumen of the distal gut is a fairly anaerobic environment. Traces of oxygen present in this habitat are readily consumed by facultative anaerobic bacteria (e.g., Enterobacteriaceae), which constitute a small fraction (approximately 0.1%) of the microbiota (Eckburg et al., Science, 308:1635-1638 (2005)). The amount of available oxygen seems to limit the growth of Enterobactericeae in this environment, because elevated oxygen levels increase their relative abundance. For example, the ileostomy of small bowel transplant patients provides a portal that allows oxygen to reach the otherwise anaerobic distal ileum. An increase in the relative abundance of Enterobacteriaceae is observed in close proximity to the ileostomy and the microbial community returns to its normal composition after surgical closure of the ileostomy (Hartman et al., Proc. Natl. Acad. Sci. U.S.A., 106:17187-17192 (2009)). Thus, once the available oxygen is consumed, Enterobacteriaceae are apparently poorly equipped to compete with obligate anaerobic bacteria for high-energy nutrients to support their growth by fermentation.

#Intestinal inflammation is accompanied by the release of antimicrobials, a host defense mechanism designed to eradicate microbes from tissue or from close vicinity to the epithelium. Some antimicrobials function in nutrient withholding by interfering with microbial acquisition of trace elements, such as iron or zinc. For example, upon stimulation with IL-22 epithelial cells release the antimicrobial lipocalin-2 into the intestinal lumen (Raffatellu et al., Cell Host Microbe, 5:476-486 (2009)). Lipocalin-2 binds enterobactin, thereby preventing bacteria from using this siderophore for iron acquisition (Berger et al., Proc. Natl. Acad. Sci. U.S.A., 103:1834-1839 (2006), Flo et al., Nature, 432:917-921 (2004), Goetz et al., Mol. Cell., 10:1033-1043 (2002)). The release of lipocalin-2 can provide a selective advantage for enteric pathogens that possess specific lipocalin-2 resistance mechanisms (Raffatellu et al., Cell Host Microbe, 5:476-486 (2009)). However, many commensal Enterobacteriaceae are susceptible to lipocalin-2, suggesting that release of this antimicrobial is not likely responsible for the phylum-level changes in microbial communities associated with gut inflammation.