Accepted Manuscript Chemical communication in the gut: Effects of microbiota-generated metabolites on gastrointestinal bacterial pathogens Stefanie L. Vogt, Jorge Peña-Díaz, B.Brett Finlay PII:

S1075-9964(15)30019-6

DOI:

10.1016/j.anaerobe.2015.05.002

Reference:

YANAE 1442

To appear in:

Anaerobe

Received Date: 27 March 2015 Revised Date:

24 April 2015

Accepted Date: 4 May 2015

Please cite this article as: Vogt SL, Peña-Díaz J, Finlay BB, Chemical communication in the gut: Effects of microbiota-generated metabolites on gastrointestinal bacterial pathogens, Anaerobe (2015), doi: 10.1016/j.anaerobe.2015.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Chemical communication in the gut: Effects of microbiota-generated metabolites on

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Stefanie L. Vogta, Jorge Peña-Díaza,b, and B. Brett Finlaya,b,c#

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gastrointestinal bacterial pathogens

Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;

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and Departments of bMicrobiology and Immunology, and cBiochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

[email protected]

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[email protected]

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[email protected]

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#Corresponding author

Phone: +1 (604) 822-2210

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Abstract Gastrointestinal pathogens must overcome many obstacles in order to successfully colonize a host, not the least of which is the presence of the gut microbiota, the trillions of

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commensal microorganisms inhabiting mammals’ digestive tracts, and their products. It is well established that a healthy gut microbiota provides its host with protection from numerous

pathogens, including Salmonella species, Clostridium difficile, diarrheagenic Escherichia coli,

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and Vibrio cholerae. Conversely, pathogenic bacteria have evolved mechanisms to establish an infection and thrive in the face of fierce competition from the microbiota for space and nutrients.

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Here, we review the evidence that gut microbiota-generated metabolites play a key role in determining the outcome of infection by bacterial pathogens. By consuming and transforming dietary and host-produced metabolites, as well as secreting primary and secondary metabolites of their own, the microbiota define the chemical environment of the gut and often determine

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specific host responses. Although most gut microbiota-produced metabolites are currently uncharacterized, several well-studied molecules made or modified by the microbiota are known to affect the growth and virulence of pathogens, including short-chain fatty acids, succinate,

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mucin O-glycans, molecular hydrogen, secondary bile acids, and the AI-2 quorum sensing autoinducer. We also discuss challenges and possible approaches to further study of the

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chemical interplay between microbiota and gastrointestinal pathogens.

Keywords

Gastrointestinal Tract; Microbiota; Bacterial Pathogens; Metabolome; Microbial Interactions

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Abbreviations EHEC: enterohemorrhagic Escherichia coli SCFA: short-chain fatty acid

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T3S(S): type III secretion (system) SPI-1: Salmonella pathogenicity island 1 LEE: locus of enterocyte effacement

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LCM: low-complexity microbiota

AI(-2): autoinducer(-2) CAI-1: cholera autoinducer 1

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TCP: toxin-coregulated pilus

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QS: quorum sensing

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1. Introduction Bacterial pathogens face a difficult task in colonizing the mammalian digestive tract. Not only must they withstand assault from the host immune system and tolerate the presence of toxic

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chemicals such as hydrochloric acid and bile salts, pathogens must also contend with the resident gastrointestinal microbiota – the community of commensal microbes inhabiting the gut. The microbiota collectively includes all forms of microbial life, including viruses, archaea, and

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eukaryotes; however, bacteria are the most numerous and also the best studied. Considering that the human digestive tract is estimated to contain up to 1014 bacterial cells (Sommer and Bäckhed,

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2013), pathogens face fierce competition for available nutrients. In addition, some gut microbiota species engage in interbacterial warfare, using weapons such as bacteriocins and type VI secretion systems to kill competitors, including pathogens (Russell et al., 2014; Rea et al., 2010). In this review, we focus on a third way that the gut microbiota can affect the success of

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bacterial pathogens – through its impact on the gut metabolome.

The gut metabolome is the collection of all low-molecular-weight metabolites (~50 to 1500 Da) found in the gastrointestinal tract (Beebe et al., 2014). The composition of the gut

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metabolome is shaped by a complex interplay between the host, microbiota, diet, and xenobiotics such as drugs (Beebe et al., 2014). There are numerous ways in which the microbiota can affect

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the gut metabolome, such as by secreting products of bacterial metabolism, producing enzymes that modify host or dietary metabolites, or altering host metabolism through immune signalling (Beebe et al., 2014). Several recent studies have revealed the dramatic impact of the microbiota on the gut metabolome. When Matsumoto et al. (2012) compared the colonic luminal metabolome of germ-free (microbiota-lacking) and conventionally raised (microbiotapossessing) mice, they found that a majority (123/179) of detected metabolites were altered in

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abundance by the presence of the microbiota. Another study that compared the metabolomes of germ-free, conventional, and “humanized” mice (germ-free mice colonized with human microbiota) found significant differences even between conventional and humanized mice,

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indicating that some metabolic processes differ even between the highly related human and mouse gut microbiota (Marcobal et al., 2013a). In silico techniques have also been used to explore the metabolic potential of the gastrointestinal microbiota. Using available genome

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sequences of both the mammalian host and cultured gut microbiota species, Sridharan et al. (2014) identified a total of 3449 metabolic reactions predicted to occur in the gut, of which 1267

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were uniquely performed by microbiota. A similar in silico approach has been used to identify biosynthetic gene clusters present in human microbiota reference genome sequences (Donia et al., 2014). The authors found an average of 599 biosynthetic gene clusters in each gut metagenome dataset from the Human Microbiome Project, 519 of which appear to be relatively

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conserved between individuals (found in more than 50% of samples). In the vast majority of cases, the molecule produced by the enzymes encoded in the biosynthetic gene cluster is currently uncharacterized. Together, these studies show that the microbiota shapes the chemical

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environment of the gut, producing a multitude of characterized and uncharacterized metabolites

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whose effects on the host and other microbes are only beginning to be elucidated.

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2. Evidence for effects of gut microbiota metabolites on bacterial pathogens It has long been recognized that an intact gut microbiota protects the host against infection by gastrointestinal pathogens, a phenomenon known as colonization resistance (Buffie

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and Pamer, 2013; Kamada et al., 2013; Spees et al., 2013; Yurist-Doutsch et al., 2014). The microbiota can mediate these protective effects both through direct microbe-microbe interactions and through indirect effects on the host (such as modulating immune functions or altering the

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expression of host receptors for bacterial toxins). In this review, we focus on the direct effects of microbiota metabolites on bacterial pathogens; indirect effects of the microbiota on the immune

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system have recently been reviewed elsewhere (Abt and Artis, 2013; Brown et al., 2013). One line of evidence that microbiota metabolites affect the outcome of bacterial infection relates to the increased susceptibility of humans and animals treated with antibiotics to a variety of infectious agents (Keeney et al., 2014). For example, when Salmonella enterica serovar

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Typhimurium is administered orally to mice, it invades through the gut epithelium to cause a systemic, typhoid-like infection with minimal intestinal colonization. However, when mice are pre-treated with streptomycin, they become susceptible to gastroenteritis (intestinal inflammation

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and diarrhea), with high levels of S. Typhimurium colonization and inflammation in the cecum (Barthel et al., 2003; Ferreira et al., 2011; Sekirov et al., 2008). Although the mechanisms

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underlying this increased susceptibility to gastroenteritis are not fully understood, it is known that streptomycin treatment causes a drastic change in the gut metabolome. Untargeted mass spectrometry experiments found that a single dose of streptomycin caused changes in abundance of 87.8% of the more than 2000 metabolite features detected in mouse feces, with major changes in the levels of primary bile acid, steroid hormone, and eicosanoid hormone metabolites (Antunes et al., 2011). Pre-treatment with antibiotics including cefoperazone and clindamycin

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also facilitates Clostridium difficile colonization of the mouse gut (Reeves et al., 2011; Jump et al., 2014). Two recent studies showed major shifts in the abundance of bile acids, sugar alcohols, and short-chain fatty acids in the ceca and feces of mice treated with these antibiotics

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(Jump et al., 2014; Theriot et al., 2014). Further in vitro studies demonstrated that these

metabolites affect the growth of C. difficile. For example, the secondary bile acid deoxycholate, which was found to be less abundant in antibiotic-treated mice, inhibited C. difficile spore

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germination, while the primary bile acid taurocholate, which increased in abundance after

antibiotic treatment, promoted spore germination (Theriot et al., 2014). Furthermore, C. difficile

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could use some of the sugar alcohols whose abundance was dramatically increased by antibiotic treatment, such as mannitol and sorbitol, as a carbon source for growth in vitro (Theriot et al., 2014). Although the specific microbiota species driving changes in metabolite abundance were not identified in these studies, they provide strong evidence of a link between the microbiota, gut

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metabolism, and the outcome of pathogenesis.

Further evidence for effects of gut microbiota metabolites on bacterial pathogens arises from studies using individual cultured microbiota isolates. For example, Silva et al. (2001)

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screened human fecal samples for their ability to inhibit the growth of Vibrio cholerae in vitro. By screening microbiota isolates cultured from one inhibitory fecal sample, the authors identified

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a Peptostreptococcus sp. and a Lactobacillus sp. that both secreted unidentified metabolites that inhibited V. cholerae growth in vitro and in vivo in gnotobiotic mice. Two other studies focused on microbiota metabolites that inhibit virulence gene expression rather than pathogen growth. De Sablet et al. (2009) found that spent culture medium from human fecal microbiota repressed the expression of the enterohemorrhagic Escherichia coli (EHEC) stx2 gene, which encodes the Shiga toxin that causes haemolytic uremic syndrome and kidney failure in a fraction of EHEC-

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infected humans (Croxen et al., 2013). After screening individual culture supernatants from a collection of microbiota isolates, the authors determined that Bacteroides thetaiotaomicron secreted a small molecule (< 3 kDa) that strongly repressed stx2 expression. More recently,

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Antunes et al. (2014) used transcriptomics to identify S. Typhimurium genes activated or

repressed by metabolites present in an organic extract of human feces. Among the dozens of genes that were affected by the presence of these metabolites, the authors found upregulation of

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numerous motility and chemotaxis genes, while many of the downregulated genes were involved in invasion of host cells. This repression of invasion genes was a common trait among fecal

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extracts from different hosts, since the repression could be reproduced with feces from nine out of ten human donors and from two different mouse strains. The inhibitory effect was also observed with culture supernatants of individual gut microbiota isolates, particularly Clostridium citroniae and other representatives of the Lachnospiraceae, demonstrating that it is microbiota-

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generated metabolites and not host factors that are responsible for repressing Salmonella invasion gene expression. Even though none of these three studies identified the specific metabolite responsible for growth inhibition or virulence gene repression, together they establish

pathogens.

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that microbiota-secreted metabolites have appreciable effects on the behavior of a variety of

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In the following two sections, we describe cases where the individual metabolites that

affect bacterial pathogens have been identified – the first covers metabolites promoting infection, while the other covers those inhibiting infection. We recognize that the effects of these metabolites can vary according to their concentration and the pathogen examined; therefore, we have categorized the metabolites according to their best-studied effects.

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3. Microbiota metabolites promoting bacterial infections 3.1 Short-chain fatty acids Short-chain fatty acids (SCFAs) are among the most abundant products of bacterial

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fermentation in the gut, reaching concentrations of 50 to 150 mM in the human colon

(Macfarlane and Gibson, 1997). The principal SCFAs produced by gut microbiota are acetate, propionate, and butyrate, which are typically found in a 3:1:1 ratio (Louis et al., 2014;

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Macfarlane and Macfarlane, 2003). The major substrate for SCFA production is carbohydrates (both diet-derived and host-derived), although fermentation of amino acids also occurs, giving

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rise to SCFAs such as formate, valerate, and caproate (Macfarlane and Macfarlane, 2003). The different SCFAs vary in their spatial distribution throughout the mammalian digestive tract; acetate and propionate are found throughout the small and large intestines, while formate is found primarily in the ileum of the small intestine and butyrate is found mainly in the large

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intestine (cecum and colon) (Laerke and Jensen, 1999; Gantois et al., 2006; Garner et al., 2009). Acetate, the most abundant SCFA, is a fermentation product common to many microbiota species, since its production allows bacteria to generate ATP from acetyl-CoA by substrate-level

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phosphorylation (Fischbach and Sonnenburg, 2011). Most organisms produce a mixture of fermentation products that includes the relatively oxidized product acetate, plus one or more of

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lactate, ethanol, propionate, butyrate, or succinate (Louis et al., 2014). In general (although there are a few known exceptions, such as Coprococcus catus and Roseburia inulinivorans), organisms that produce propionate as a fermentation product do not produce butyrate, and vice versa (Reichardt et al., 2014). Propionate is produced by members of both of the most common gut phyla – the Bacteroidetes and the Firmicutes (Louis et al., 2014). There are three metabolic pathways that contribute to the production of propionate (Reichardt et al., 2014): (1) the

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succinate pathway of carbohydrate fermentation, commonly used by Bacteroidetes (discussed in more detail in section 3.2); (2) the acrylate pathway, used by some Lachnospiraceae and Negativicutes to ferment the substrate lactate; and (3) the propanediol pathway, used by

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Roseburia inulinivorans and other Lachnospiraceae to ferment deoxy sugars such as fucose and rhamnose. Finally, butyrate is produced by many Firmicutes commonly found in the gut,

including Fecalibacterium prausnitzii, Roseburia spp., Eubacterium rectale, and Anaerostipes

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spp. (Louis et al., 2014).

SCFAs are a double-edged sword for gut pathogens, with effects that can be either

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beneficial or inhibitory to their colonization depending on concentration and environmental pH. High concentrations of SCFAs and low external pH promote the accumulation of SCFAs in the bacterial cytoplasm; here, the SCFAs can exert various toxic effects such as dissipation of the proton motive force (Sun and O'Riordan, 2013). However, at lower concentrations, SCFAs can

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represent a useful cue for virulence gene regulation, allowing pathogens to determine their position within the gastrointestinal tract (Figure 1A). S. Typhimurium, for example, colonizes the distal ileum of mice, where it uses a type III secretion system (T3SS) encoded on Salmonella

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pathogenicity island 1 (SPI-1) to invade the host epithelium and cause systemic infection (Collazo and Galán, 1997; Galán and Curtiss, 1989; Carter and Collins, 1974). When S.

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Typhimurium is grown in media containing a mixture of acetate, propionate, and butyrate with concentrations matching those found in the distal ileum, expression of SPI-1 invasion genes increases (Lawhon et al., 2002). Formate, which is found at concentrations of approximately 8 mM in the distal ileum of mice but is undetectable in the cecum, also promotes expression of SPI-1 genes (Huang et al., 2008). However, growth in the presence of propionate and/or butyrate at concentrations comparable to those found in the colon inhibits SPI-1 gene expression

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A. colon ileum

Salmonella

acetate propionate butyrate

SPI-1 expression

Salmonella

SPI-1 expression

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propionate acetate formate

EHEC

invasion into host tissues

expression of LEE, Iha, flagella

B.

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cecum

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cecum

fermenters

fumarate

B. theta succinate

C. diff

H2

Citrobacter

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adherence to epithelium

butyrate

C. diff

Salmonella

proliferation

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adherence tissue damage host mortality

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proliferation

Salmonella

colon

Salmonella

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Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

sialic acid

proliferation

fucose

proliferation

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C. diff

C. diff

EHEC

B. theta

T3S,

growth outer mucus layer inner mucus layer

Figure 1. Microbiota metabolites promoting infection by bacterial pathogens. Diagrams depict effects of (A) short-chain fatty acids (Gantois et al., 2006; Herold et al., 2009; Huang et

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al., 2008; Hung et al., 2013; Lawhon et al., 2002; Nakanishi et al., 2009; Tobe et al., 2011), (B) succinate (Curtis et al., 2014; Ferreyra et al. 2014), (C) molecular hydrogen (Maier et al., 2013), and (D) mucin O-glycans (Ng et al., 2013; Pacheco et al., 2012). See section 3 of the text for

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details. In the diagrams, pathogens are depicted in red and microbiota species are depicted in green. Clostridium difficile and Bacteroides thetaiotaomicron are abbreviated as C. diff and B.

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theta, respectively.

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and invasion of tissue culture cells (Hung et al., 2013; Gantois et al., 2006; Lawhon et al., 2002). Monitoring the concentrations of different SCFAs therefore may help S. Typhimurium to determine its position within the gastrointestinal tract. In the distal ileum, which is believed to

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be the main site of systemic invasion in mice and humans (Hapfelmeier and Hardt, 2005), S. Typhimurium benefits from expressing its invasion genes and initiating infection. However, in the large intestine, invasion is unlikely to occur, so S. Typhimurium should adapt its gene

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expression to surviving environmental insults and/or preparing for transmission to a new host (Hung et al., 2013).

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EHEC also relies on SCFAs for virulence gene regulation, but unlike S. Typhimurium, EHEC’s preferred site of infection is the colon (Croxen et al., 2013). Accordingly, butyrate, which is more abundant in the colon than in the small intestine, significantly increases EHEC adherence to human tissue culture cells, while acetate and propionate have little effect on

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adherence (Nakanishi et al., 2009). The effect of butyrate on EHEC adherence is likely multifactorial. Butyrate promotes expression of the locus of enterocyte effacement (LEE), which encodes the T3SS crucial for intimate adherence of EHEC to epithelial cells (Nakanishi et al.,

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2009). In addition, a mixture of acetate, propionate, and butyrate in concentrations similar to those found in the colon stimulates expression of iha, encoding an outer membrane adhesin

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protein, while ileal concentrations of the SCFAs have no effect on iha expression (Herold et al., 2009). All three SCFAs, but especially propionate and butyrate, also increase production of flagella and motility (Tobe et al., 2011). EHEC flagella have been shown to play a role in adherence to bovine epithelial cells (Mahajan et al., 2009), and may also be involved in penetration of the mucus layer and/or host cell adherence during human infections (Tobe et al., 2011). These two examples of S. Typhimurium and EHEC illustrate how sensing SCFAs may

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allow pathogens to adapt their gene expression to different niches within the gut. Given the abundance of SCFAs throughout the intestine, it is likely that other gut pathogens also make use

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of this convenient positional cue.

3.2 Succinate

Succinate is an important intermediary metabolite among gut microbes. There are three

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known fermentation pathways by which propionate can be generated from carbohydrates; the pathway in which succinate is an intermediate is the most common among human gut microbiota

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(Reichardt et al., 2014). The succinate pathway is widespread among Bacteroides spp., and is also found in several organisms belonging to the Negativicutes class of the phylum Firmicutes, such as Veillonella parvula (Reichardt et al., 2014). In the Bacteroides version of the propionate fermentation pathway, ATP is generated using a primitive form of anaerobic respiration, where

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fumarate acts as a terminal electron acceptor and is converted to succinate (Fischbach and Sonnenburg, 2011). If environmental concentrations of carbon dioxide are low, Bacteroides converts the succinate into propionate and carbon dioxide, which it uses to generate more

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fumarate. However, if external carbon dioxide is available, the succinate produced by the reduction of fumarate is secreted as a waste product. Bacteroides species such as B. fragilis and

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B. ovatus therefore secrete substantial quantities of succinate under certain growth conditions (Macy et al., 1978; Macfarlane and Macfarlane, 2003). In spite of this, the concentration of succinate in the feces of healthy humans is typically low (Topping and Clifton, 2001), which is likely due to the presence of organisms such as Phascolarctobacterium succinatutens, which uses succinate as its only carbon source (Watanabe et al., 2012).

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The ability of succinate to promote infection is underscored by two recent studies focusing on the pathogens EHEC and C. difficile (Ferreyra et al., 2014; Curtis et al., 2014; Figure 1B). Both groups examined how pathogen gene expression is altered in the presence of B.

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thetaiotaomicron. When EHEC was co-cultured in vitro with B. thetaiotaomicron, expression of one-fifth of its genes was activated, including the LEE genes encoding its T3SS (Curtis et al., 2014). B. thetaiotaomicron also promotes T3SS gene expression in Citrobacter rodentium

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(Curtis et al., 2014), a natural pathogen of mice that also carries the LEE and is frequently used to model EHEC infections (Collins et al., 2014). When antibiotic-treated mice were colonized

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with B. thetaiotaomicron, then subsequently infected with C. rodentium, the mice lost more weight, showed greater tissue pathology, and succumbed to infection more quickly than control mice that were antibiotic-treated but not colonized with B. thetaiotaomicron. Cecal metabolomic analysis of the B. thetaiotaomicron-colonized mice found that levels of succinate and several

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other organic acids were elevated compared to the antibiotic-treated controls. Importantly, the addition of succinate to EHEC cultures in vitro increased type III secretion (T3S), demonstrating that succinate is one B. thetaiotaomicron-produced metabolite that promotes infection by EHEC

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and C. rodentium (Curtis et al., 2014).

To examine the effect of B. thetaiotaomicron on C. difficile gene expression, Ferreyra

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and colleagues (2014) infected germ-free mice with C. difficile alone or C. difficile and B. thetaiotaomicron in combination, then performed transcriptomics on cecal RNA from the two groups of mice. They found that, when mice were fed a polysaccharide-rich diet, the presence of B. thetaiotaomicron increased the expression of C. difficile genes involved in the conversion of succinate to butyrate. Similarly to the above study, metabolite analysis showed that succinate levels were increased in the ceca of mice that had been colonized with B. thetaiotaomicron,

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while co-infection with C. difficile led to an accumulation of butyrate, strongly suggesting that C. difficile uses succinate as an electron acceptor and converts it to butyrate during growth in vivo. In further support of this idea, the authors found that mutation of the succinate transporter-

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encoding gene Cd-CD2344 impaired C. difficile’s proliferation in mice colonized with B.

thetaiotaomicron. Although both of these studies made use of animals that were artificially

colonized with B. thetaiotaomicron, there is evidence that the abundance of succinate increases

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naturally when the gut microbiota is disrupted, thereby paving the way for infection by bacterial pathogens. Treatment with either the antibiotic streptomycin or the laxative polyethylene glycol

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increases cecal succinate levels in mice; both of these treatments also enhance infection by C. difficile (Ferreyra et al., 2014). Keeping succinate levels in check is therefore likely to be one important mechanism by which a healthy microbiota provides pathogen resistance.

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3.3 Hydrogen

Molecular hydrogen is an important currency of the gut microbial economy. Its production benefits primary fermenters, as it allows them to dispose of reducing equivalents,

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while its consumption represents a convenient energy source for hydrogen-oxidizing bacteria (Fischbach and Sonnenburg, 2011). This exchange of hydrogen is a mutually beneficial

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interaction, since accumulation of hydrogen in the gut would inhibit fermentation thermodynamically in the absence of hydrogen-utilizing bacteria (Carbonero et al., 2012). Many common gut inhabitants, including Roseburia spp., Ruminococcus spp., and Clostridium spp., have been shown to produce molecular hydrogen in vitro, and thus likely contribute to the abundance of H2 in the gut (Carbonero et al., 2012). Conversely, three major groups of hydrogen-utilizing bacteria are present in the human gut: the reductive acetogens (which produce

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acetate from CO2 and H2), the methanogenic archaea (which produce methane from CO2 and H2), and the sulphate-reducing bacteria (which produce hydrogen sulphide from SO42- and H2) (Carbonero et al., 2012).

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Not surprisingly, given the ubiquity of hydrogen-producing microbiota, many pathogenic bacteria have also evolved the ability to use molecular hydrogen as an energy source. The

genomes of pathogens including EHEC, Shigella, and Campylobacter jejuni encode one or more

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membrane-bound hydrogenases, which shuttle electrons from hydrogen molecules into the

electron transport chain (Maier, 2003). An elegant study recently demonstrated the importance

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of hydrogen as an energy source in S. Typhimurium’s ability to establish an infection (Maier et al., 2013; Figure 1C). The authors identified the hyb hydrogenase genes in a screen for S. Typhimurium mutants with an impaired ability to colonize the gut of low-complexity microbiota (LCM) mice. In competitive infections with wild-type Salmonella, the hyb mutant showed a

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colonization defect only during the first 24 hours of infection, suggesting that hydrogen respiration is critical for the initial invasion of the gut ecosystem. The authors confirmed that molecular hydrogen in the gut derives from microbial activity – experiments with an H2

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microsensor showed that LCM and conventional mice have abundant cecal hydrogen (>25 µM), while hydrogen is undetectable (

Chemical communication in the gut: Effects of microbiota-generated metabolites on gastrointestinal bacterial pathogens.

Gastrointestinal pathogens must overcome many obstacles in order to successfully colonize a host, not the least of which is the presence of the gut mi...
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