Short Chain Fatty Acids Modulate the Growth and Virulence of Pathosymbiont Escherichia coli and Host Response

Short chain fatty acids (SCFA), principally acetate, propionate, and butyrate, are produced by fermentation of dietary fibers by the gut microbiota. SCFA regulate the growth and virulence of enteric pathogens, such as enterohemorrhagic E. coli (EHEC), Klebsiella and Salmonella. We sought to investigate the impact of SCFA on growth and virulence of pathosymbiont E. coli associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC), and their role in regulating host responses to bacterial infection in vitro. We found that under ileal conditions (pH = 7.4; 12 mM total SCFA), SCFA significantly (p < 0.05) potentiate the growth and motility of pathosymbiont E. coli. However, under colonic conditions (pH = 6.5; 65 to 123 mM total SCFA), SCFA significantly (p < 0.05) inhibit growth in a pH dependent fashion (up to 60%), and down-regulate virulence gene expression (e.g., fliC, fimH, htrA, chuA, pks). Functional analysis reveals that colonic SCFA significantly (p < 0.05) inhibit E. coli motility (up to 95%), infectivity (up to 60%), and type 1 fimbria-mediated agglutination (up to 50%). In addition, SCFA significantly (p < 0.05) inhibit the activation of NF-κB, and IL-8 production by epithelial cells. Our findings provide novel insights on the role of the regional chemical microenvironment in regulating the growth and virulence of pathosymbiont E. coli and opportunities for therapeutic intervention.


Introduction
Short-chain fatty acids (SCFA), primarily acetate, propionate, and butyrate, are produced by microbial fermentation of undigested carbohydrates and dietary fibers [1,2]. The amount and type of SCFA in the intestine are influenced by dietary intake, particularly non-digestible carbohydrates, protein and fat [3,4], and the composition of the gut microbiota [3,5]. The main producers of SCFA are Firmicutes and Bacteroidetes, the two most abundant phyla in human intestine [2,4]. Bacteroidetes produce mainly acetate and propionate, while Firmicutes produce butyrate [6,7]. Acetate is the most abundant SCFA in the gut and is produced from acetyl-CoA through glycolysis; butyrate and propionate are produced from both carbohydrate metabolism (glycolysis) and metabolisms of fatty and GC dog colon D + - [24] a NC101 induces IBD and cancer in IL10-/-mice. b Because of their cytotoxicity, these strains were not tested for AIEC characteristics.
2.1.1. Ileal SCFA (i-SCFA) Promote E. coli Growth SCFA are detected in the distal ileum at total concentrations of 10 to 20 mM [10]. The pH in the distal ileum is 7.4 in health and disease [45,46]. The influence of SCFA on E. coli growth under the ileal conditions (12 mM total SCFA, pH = 7.4; see Table 1) was examined in complex medium (Luria-Bertani, or LB) and chemically defined medium M9 (see Materials and Methods). To simulate the enteric luminal environment, bacteria were grown under microaerophilic conditions for 24 h at 37 • C. i-SCFA enhanced (p < 0.05) the growth of E. coli ( Figure 1A,B) in both media: 5/9 strains in LB ( Figure 1A) and 16/19 strains in M9 medium ( Figure 1B). The degree of stimulation ranged from 10 to 60% and was strain specific. Three of the four mouse strains (CUMSL1, CUMSL6, and CUMT8) Antibiotics 2020, 9,462 4 of 20 grew much better (>50%; p < 0.05) in the presence of i-SCFA compared to NaCl controls ( Figure 1B). In addition, i-SCFA stimulated-E. coli growth was media independent, and unaffected by the origins and disease association of E. coli. Antibiotics 2020, 9, x FOR PEER REVIEW 4 of 19 ileal conditions (12 mM total SCFA, pH = 7.4; see Table 1) was examined in complex medium (Luria-Bertani, or LB) and chemically defined medium M9 (see Materials and Methods). To simulate the enteric luminal environment, bacteria were grown under microaerophilic conditions for 24 h at 37 °C. i-SCFA enhanced (p < 0.05) the growth of E. coli ( Figure 1A,B) in both media: 5/9 strains in LB ( Figure 1A) and 16/19 strains in M9 medium ( Figure 1B). The degree of stimulation ranged from 10 to 60% and was strain specific. Three of the four mouse strains (CUMSL1, CUMSL6, and CUMT8) grew much better (>50%; p < 0.05) in the presence of i-SCFA compared to NaCl controls ( Figure 1B). In addition, i-SCFA stimulated-E. coli growth was media independent, and unaffected by the origins and disease association of E. coli.

Colonic SCFA (c-SCFA) inhibit E. coli Growth
Colonic concentrations of SCFA range from 60-150 mM in a healthy gut [9,10]. The physiological pH of the colon is between 5.6 and 6.7 [45,46]. To investigate the effect of SCFA on E. coli growth under the colonic conditions, we evaluated total SCFA at a concentration of 123 mM (65 mM acetate, 29 mM propionate, and 29 mM butyrate) and pH = 6.5 to simulate the in vivo milieu [10]. In both complex (LB, Figure 2A) and chemically defined (M9, Figure 2B) media, c-SCFA inhibited the growth of E. coli (p < 0.05) from human, mice, and dogs (except canine GC CUKD2 in M9 media, p = 0.194). The degree of inhibition varied by strain and was independence of media type. Non-pathogenic DH5α, canine GC-AIEC CUDC1 and CUDL1, and murine AIEC CUMSL1 and CUMSL6 were highly sensitive to c-SCFA ( Figure 2B), with average growth inhibited >80% (p < 0.05) under the same conditions.

Colonic SCFA (c-SCFA) inhibit E. coli Growth
Colonic concentrations of SCFA range from 60-150 mM in a healthy gut [9,10]. The physiological pH of the colon is between 5.6 and 6.7 [45,46]. To investigate the effect of SCFA on E. coli growth under the colonic conditions, we evaluated total SCFA at a concentration of 123 mM (65 mM acetate, 29 mM propionate, and 29 mM butyrate) and pH = 6.5 to simulate the in vivo milieu [10]. In both complex (LB, Figure 2A) and chemically defined (M9, Figure 2B) media, c-SCFA inhibited the growth of E. coli (p < 0.05) from human, mice, and dogs (except canine GC CUKD2 in M9 media, p = 0.194). The degree of inhibition varied by strain and was independence of media type. Non-pathogenic DH5α, canine GC-AIEC CUDC1 and CUDL1, and murine AIEC CUMSL1 and CUMSL6 were highly sensitive to c-SCFA ( Figure 2B), with average growth inhibited >80% (p < 0.05) under the same conditions. Antibiotics 2020, 9, x FOR PEER REVIEW 5 of 19 Figure 2. Colonic SCFA inhibit E. coli growth in vitro. E. coli were cultured in LB (A) or M9 (B) media containing either 123 mM NaCl (Control) or SCFA at pH = 6.5. Growth conditions were the same as for Figure 1. Data from three independent experiments; Mean ± SE. * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant.
Further analysis with different C-and N-sources in M9 background revealed that the inhibition of E. coli growth by c-SCFA was independent of C-and N-sources (data not shown).
2.1.3. Inhibition of Growth by c-SCFA is pH-Dependent Colonic pH ranges from 5.6 near the cecum to 6.6 in the left colon vs 7.4 in the distal ileum [46]. Colonic SCFA inhibit E. coli growth in vitro. E. coli were cultured in LB (A) or M9 (B) media containing either 123 mM NaCl (Control) or SCFA at pH = 6.5. Growth conditions were the same as for Figure 1. Data from three independent experiments; Mean ± SE. * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant. Antibiotics 2020, 9, 462 5 of 20 Further analysis with different C-and N-sources in M9 background revealed that the inhibition of E. coli growth by c-SCFA was independent of C-and N-sources (data not shown).

Inhibition of Growth by c-SCFA is pH-Dependent
Colonic pH ranges from 5.6 near the cecum to 6.6 in the left colon vs 7.4 in the distal ileum [46]. To determine the influence of regional pH on the inhibitory effect of c-SCFA on E. coli growth, we used buffered-LB broth (containing 100 mM HEPES for pH = 7.4, 100 mM MOPS for pH = 6.5, or 100 mM PIPES for pH = 6.2 medium, respectively) ± c-SCFA. At pH = 7.4, none of the 10 E. coli strains were inhibited by c-SCFA ( Figure 3A), rather 50% grew better in the presence of c-SCFA (p < 0.05) ( Figure 3A). In contrast, the growth of pathosymbiont E. coli strains were inhibited (p < 0.05) at pH ≤ 6.5 ( Figure 3B,C), with the exception of AIEC CU576-1 (p = 0.061) in medium pH = 6.5 ( Figure 3B). The growth of symbiont E. coli CUT75 was less affected at pH = 6.5 (p = 0.119) but was reduced to 79% of control (p < 0.01) at pH = 6.2 ( Figure 3C). Colonic SCFA inhibit E. coli growth in vitro. E. coli were cultured in LB (A) or M9 (B) media containing either 123 mM NaCl (Control) or SCFA at pH = 6.5. Growth conditions were the same as for Figure 1. Data from three independent experiments; Mean ± SE. * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant.
Further analysis with different C-and N-sources in M9 background revealed that the inhibition of E. coli growth by c-SCFA was independent of C-and N-sources (data not shown).

c-SCFA Inhibit Virulence Gene Expression in E. coli
Based on their effects on E. coli growth (Figures 2-4), we speculated that c-SCFA may also impact virulence gene expression in pathosymbiont E. coli. We evaluated a panel of 11 virulence genes (Table  3) in CD-AIEC and CRC-pks -/+ E. coli ± c-SCFA. To achieve an adequate yield of bacteria for total RNA isolation (see Methods) at mid-log phase, we used SCFA at sub-maximal inhibitory concentration (65 mM vs.123 mM) with the same molar ratio (Table 1). Virulence gene expression, determined by qRT-PCR (Table 3; Primers in Table 4), showed that c-SCFA treatment down regulated numerous virulence genes (p < 0.05), especially those associated with motility (fliC), adhesion and invasion (fimH, ompC, yfgL, and lpfA), stress (htrA) and genotoxicity (pks) for majority of E. coli strains. For instance, the motility gene fliC was down regulated in 11/15 E. coli strains (p < 0.05), and the adhesin gene fimH in 9/15 strains (p < 0.05) ( Table 3).

c-SCFA Inhibit Virulence Gene Expression in E. coli
Based on their effects on E. coli growth (Figures 2-4), we speculated that c-SCFA may also impact virulence gene expression in pathosymbiont E. coli. We evaluated a panel of 11 virulence genes (Table 3) in CD-AIEC and CRC-pks −/+ E. coli ± c-SCFA. To achieve an adequate yield of bacteria for total RNA isolation (see Methods) at mid-log phase, we used SCFA at sub-maximal inhibitory concentration (65 mM vs.123 mM) with the same molar ratio (Table 1). Virulence gene expression, determined by qRT-PCR (Table 3; Primers in Table 4), showed that c-SCFA treatment down regulated numerous virulence genes (p < 0.05), especially those associated with motility (fliC), adhesion and invasion (fimH, ompC, yfgL, and lpfA), stress (htrA) and genotoxicity (pks) for majority of E. coli strains. For instance, the motility gene fliC was down regulated in 11/15 E. coli strains (p < 0.05), and the adhesin gene fimH in 9/15 strains (p < 0.05) ( Table 3).

SCFA Modulate E. coli Motility
Bacterial motility is involved in the virulence of pathogens [47] and directly correlates with the ability of AIEC to invade epithelial cells [28]. Recent studies of CD-E. coli have linked motility to the AIEC pathotype [28,48]. The flagellin protein FliC plays an essential role in bacterial motility. The functional consequence of down regulation of fliC by c-SCFA (Table 3) was evaluated by motility assays on sloppy agar containing different concentrations of total SCFA ( Figure 5A-D). Non-motile, non-AIEC symbiont CUT75 was excluded from the analysis. The motility of CD-associated E. coli was reduced significantly (p < 0.05) compared to the NaCl controls at pH 6.5, even at 60 mM level of c-SCFA ( Figure 5A,D), with the exception of AIEC CU578-1 (p = 0.215). At 120 mM, c-SCFA reduced E. coli motility to <5% ( Figure 5A,D), except for AIEC CU24LW-1 (<25%). Prototypical AIEC LF82 was greatly impacted ( Figure 5A), with a >90% reduction of motility at 60 mM c-SCFA. We observed similar dose-dependent inhibition for CRC-E. coli ( Figure 5B) under the same assay conditions. At 30 mM c-SCFA, more than 30% of inhibition was obtained across all strains ( Figure 5B), while the motility of CRC-E. coli was completely inhibited at 123 mM c-SCFA (data not shown).
Antibiotics 2020, 9, x FOR PEER REVIEW 8 of 19 functional consequence of down regulation of fliC by c-SCFA (Table 3) was evaluated by motility assays on sloppy agar containing different concentrations of total SCFA ( Figure 5A-D). Non-motile, non-AIEC symbiont CUT75 was excluded from the analysis. The motility of CD-associated E. coli was reduced significantly (p < 0.05) compared to the NaCl controls at pH 6.5, even at 60 mM level of c-SCFA ( Figure 5A,D), with the exception of AIEC CU578-1 (p = 0.215). At 120 mM, c-SCFA reduced E. coli motility to <5% ( Figure 5A,D), except for AIEC CU24LW-1 (<25%). Prototypical AIEC LF82 was greatly impacted ( Figure 5A), with a >90% reduction of motility at 60 mM c-SCFA. We observed similar dose-dependent inhibition for CRC-E. coli ( Figure 5B) under the same assay conditions. At 30 mM c-SCFA, more than 30% of inhibition was obtained across all strains ( Figure 5B), while the motility of CRC-E. coli was completely inhibited at 123 mM c-SCFA (data not shown). In contrast, i-SCFA stimulated (p < 0.05) E. coli motility ( Figure 5C, and the left column in Figure  5D), particularly the AIEC CU576-1 and CU578-1. At the physiological concentrations (6 to 12 mM), i-SCFA stimulated the motility of AIEC CU576-1 up to 1.5 fold, and CU578-1 up to 2.5 fold (p < 0.05).

c-SCFA Inhibit Type 1 Pili FimH-Mediated Yeast Agglutination
E. coli type I fimbrial protein, FimH, can bind to the mannose residues on yeast cell surface, and subsequently initiate yeast cell agglutination [49]. This activity of FimH also mediates the adherence and colonization of E. coli to the intestinal epithelial cells [50][51][52] and stimulates TLR4 [53]. We visualized the functional consequences of c-SCFA on fimH gene expression with yeast agglutination assays. After pretreatment with 123 mM c-SCFA or NaCl (control) in LB broth at pH 6.5, E. coli were mixed with equal amount of yeast cells. Pretreatment of E. coli with c-SCFA reduced agglutination (Table 5 and Figure 6) compared to NaCl controls. c-SCFA induced a 2-fold or greater reduction in yeast cell agglutination for the majority (5/8) of E. coli strains ( Table 4). The biggest reduction was seen with CD-AIEC CU541-15 and CRC-pks -HM288 (Table 5; Figure 6). In contrast, i-SCFA stimulated (p < 0.05) E. coli motility ( Figure 5C, and the left column in Figure 5D), particularly the AIEC CU576-1 and CU578-1. At the physiological concentrations (6 to 12 mM), i-SCFA stimulated the motility of AIEC CU576-1 up to 1.5 fold, and CU578-1 up to 2.5 fold (p < 0.05).

c-SCFA Inhibit Type 1 Pili FimH-Mediated Yeast Agglutination
E. coli type I fimbrial protein, FimH, can bind to the mannose residues on yeast cell surface, and subsequently initiate yeast cell agglutination [49]. This activity of FimH also mediates the adherence and colonization of E. coli to the intestinal epithelial cells [50][51][52] and stimulates TLR4 [53]. We visualized the functional consequences of c-SCFA on fimH gene expression with yeast agglutination assays. After pretreatment with 123 mM c-SCFA or NaCl (control) in LB broth at pH 6.5, E. coli were mixed with equal amount of yeast cells. Pretreatment of E. coli with c-SCFA reduced agglutination (Table 5 and Figure 6) compared to NaCl controls. c-SCFA induced a 2-fold or greater reduction in yeast cell agglutination for the majority (5/8) of E. coli strains ( Table 4). The biggest reduction was seen with CD-AIEC CU541-15 and CRC-pks − HM288 (Table 5; Figure 6). * The yeast agglutination scores were made based on the degree of aggregation in Figure 6. Antibiotics 2020, 9, x FOR PEER REVIEW 9 of 19 Figure 6. c-SCFA inhibit yeast agglutination by E. coli. E. Coli were grown in LB media (pH = 6.5) with either 123 mM NaCl (control) or SCFA at 37 °C for 18 h. After centrifugation, the cell pellets were resuspended in PBS, followed by mix with equal volume of yeast suspension at the same optical density (OD 600 = 5.2) (see Methods).

c-SCFA Inhibit E. coli Adhesion and Invasion of Intestinal Epithelial Cells
c-SCFA down regulated a number of virulence genes (fimH, ompC, nlpL, and lpfA) involved in the process of adhesion and invasion [54][55][56][57] (Table 3), suggesting that c-SCFA would reduce the infection of intestinal epithelial cells by E. coli. In the presence of 65 mM c-SCFA, the ability of E. coli to adhere to and invade Caco-2 epithelial cells was significantly reduced (p < 0.05; Figure 7A,B). The impact of c-SCFA was greater (p = 0.0073) on invasion than adhesion compared with controls ( Figure  7A vs Figure 7B), particularly for CD-AIEC CU524-2, CU541-1, CU541-15, CU576-1, CU578-1, and Figure 6. c-SCFA inhibit yeast agglutination by E. coli. E. Coli were grown in LB media (pH = 6.5) with either 123 mM NaCl (control) or SCFA at 37 • C for 18 h. After centrifugation, the cell pellets were resuspended in PBS, followed by mix with equal volume of yeast suspension at the same optical density (OD 600 = 5.2) (see Methods).

c-SCFA Inhibit E. coli Adhesion and Invasion of Intestinal Epithelial Cells
c-SCFA down regulated a number of virulence genes (fimH, ompC, nlpL, and lpfA) involved in the process of adhesion and invasion [54][55][56][57] (Table 3), suggesting that c-SCFA would reduce the infection of intestinal epithelial cells by E. coli. In the presence of 65 mM c-SCFA, the ability of E. coli to adhere to and invade Caco-2 epithelial cells was significantly reduced (p < 0.05; Figure 7A,B). The impact of c-SCFA was greater (p = 0.0073) on invasion than adhesion compared with controls ( Figure 7A vs. Figure 7B), particularly for CD-AIEC CU524-2, CU541-1, CU541-15, CU576-1, CU578-1, and LF82. Under identical conditions, the average inhibition of c-SCFA was 71% for adhesion and 93% for invasion ( Figure 7A vs Figure 7B).  Hek-Blue cells were infected with E. coli in the presence of 123 mM NaCl or SCFA at pH 6.5 (see Methods). At 24 h post infection, the supernatant of the infected cells was used to detect the reporter protein (secreted alkaline phosphatase, SEAP) production. (B). Caco-2 cells were infected by E. coli for 3 h in the presence of either NaCl or c-SCFA (65 mM) at pH = 6.5. The supernatant of infected Caco-2 cells was used for IL-8 detection using ELISA method. Data from three independent experiments; Mean ± SE. *p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant. [13]. IL-8 is a pivotal chemokine produced by the gut epithelial cells during pathogen infection, and its production is down stream of the NF-kB signal transduction pathway [59]. We measured the levels of IL-8 produced by Caco-2 cells after infection by E. coli in the presence or absence of c-SCFA under colonic conditions. c-SCFA inhibited IL-8 secretion (p < 0.05) induced by all E. coli strains tested ( Figure 8B), including CD-AIEC and CRC-associated E. coli. The inhibition ranged from 20 to 60% depending on strains ( Figure 8B).

Discussion
Intestinal SCFA, predominantly acetate, propionate, and butyrate, are by-products of bacterial fermentation [1]. Concentrations of SCFA are 10-fold higher in the colon than the ileum, with concordant differences in luminal pH of 7.4 in the ileum and 5.6-6.7 in the colon [9,10,45]. We sought to determine the effects of SCFA in the context of region-specific variations in the chemical microenvironment (SCFA and pH) on the growth and virulence of pathosymbiont E. coli isolated

c-SCFA Inhibit NF-kB Signal Transduction
The nuclear factor, NF-κB, is a family of regulators controlling multiple cellular inflammatory responses through signal transduction. It is present in all types of cells, and can be induced by pathogen infection [58]. To determine the effect of c-SCFA on host epithelial responses to E. coli infection, we used HEK-Blue KD-TLR5 cells to detect the NF-κB pathway activation [59]. In the presence of 123 mM c-SCFA, the activation of NF-κB pathway by most E. coli strains was inhibited (p < 0.05), except for non-pathogenic CD-E. coli CUT75 and the pks-negative CRC-E. coli HM288 ( Figure 8A). The inhibition ranged from 17 to 70% compared to controls for all, except non-pathogenic E. coli CUT75, which was minimally able to activate NF-κB.

c-SCFA Inhibit IL-8 Secretion by Epithelial Cells
SCFA interact with intestinal epithelial cells and modulate immune responses in the gut [13]. IL-8 is a pivotal chemokine produced by the gut epithelial cells during pathogen infection, and its production is down stream of the NF-kB signal transduction pathway [59]. We measured the levels of IL-8 produced by Caco-2 cells after infection by E. coli in the presence or absence of c-SCFA under colonic conditions. c-SCFA inhibited IL-8 secretion (p < 0.05) induced by all E. coli strains tested ( Figure 8B), including CD-AIEC and CRC-associated E. coli. The inhibition ranged from 20 to 60%

c-SCFA Inhibit IL-8 Secretion by Epithelial Cells
SCFA interact with intestinal epithelial cells and modulate immune responses in the gut [13]. IL-8 is a pivotal chemokine produced by the gut epithelial cells during pathogen infection, and its production is down stream of the NF-κB signal transduction pathway [59]. We measured the levels of IL-8 produced by Caco-2 cells after infection by E. coli in the presence or absence of c-SCFA under colonic conditions. c-SCFA inhibited IL-8 secretion (p < 0.05) induced by all E. coli strains tested ( Figure 8B), including CD-AIEC and CRC-associated E. coli. The inhibition ranged from 20 to 60% depending on strains ( Figure 8B).

Discussion
Intestinal SCFA, predominantly acetate, propionate, and butyrate, are by-products of bacterial fermentation [1]. Concentrations of SCFA are 10-fold higher in the colon than the ileum, with concordant differences in luminal pH of 7.4 in the ileum and 5.6-6.7 in the colon [9,10,45]. We sought to determine the effects of SCFA in the context of region-specific variations in the chemical microenvironment (SCFA and pH) on the growth and virulence of pathosymbiont E. coli isolated from people with Crohn's disease (AIEC pathotype) and CRC (pks genotoxicity), dogs with granulomatous colitis (AIEC pathotype), and mice with intestinal inflammation (AIEC pathotype). We also examined the impact of SCFA on host-E. coli inflammatory responses.
We found that SCFA affect the growth of pathosymbiont E. coli in a concentration and pH dependent fashion, with colonic [SCFA] at colonic pH suppressing growth, and ileal [SCFA] at ileal pH favoring growth. The effect was largely independent of E. coli pathotype, disease association, species of origin, and type of media, supporting a direct effect of SCFA and pH. The concentrations and proportions of SCFA and pH levels we used to model the ileal and colonic microenvironment were selected to be physiologically relevant for people: i-SCFA (12 mM with a molar ratio of 8:2.5:1.5 for acetate, propionate, and butyrate, respectively), c-SCFA (60 to 123 mM with a ratio of 65:29:29 for acetate, propionate, and butyrate, respectively), and pH = 6.2-7.4 [9,10,40]. MIC values for acetate, propionate, and butyrate were 20 to 40, 10, 10 mM, respectively. To further simulate the enteric microenvironment, E. coli were cultured in microaerophilic conditions at 37 • C.
A mechanistic understanding of the interactions between the chemical microenvironment (e.g., SCFA, pH, bile acids), the resident microbiota, and host immune responses in the intact GI tract in health and disease remains to be elucidated. Lower fecal concentrations of butyrate and propionate in patients with IBD vs. controls [60], and acetate in CD vs. UC [60], may reflect decreased microbial production, increased utilization, or a combination of these processes. Region-specific differences in the chemical and microbial microenvironment in the ileum and colon may underlie the phenotypic variation of IBD. Crohn's ileitis is consistently linked to dysbiosis characterized by an overabundance of E. coli and depletion of Firmicutes (e.g., Faecalibacterium prausnitzii) [18,61], and E. coli with an AIEC pathotype have been more frequently isolated from ileal (36.4%) than colonic (3.6%) mucosa of CD patients in some studies [18,62]. Our finding that i-SCFA (12 mM, pH = 7.4) promote the growth of AIEC whereas c-SCFA (123 mM, pH = 6.5) suppress it, suggest region specific differences in the chemical environment may influence colonization by pathosymbiont E. coli.
SCFA propionate and acetate have recently been implicated in regulating the growth, colonization, and virulence of AIEC [28][29][30]63]. Propionate-adapted AIEC LF82 more proficiently colonized the colon and ileum, but not the cecum, of mice fed propionate (at levels selected to simulating human gut: 20 mM) than non-propionate adapted LF82 [30]. Acetate utilization has also been linked to enhanced colonization by AIEC NRG857 (LF82-like, B2 O83) in mice, and E. coli from CD patients were better able to grow on acetate (K-acetate 0.4% w/v in M9 media), but not complex media, than E. coli from healthy controls [28].
In contrast to the growth enhancing effects of propionate and acetate, we found that propionate at ≥5 mM and acetate ≥10mM suppressed the growth of AIEC LF82 at colonic pH = 6.5. These differences may reflect the pH dependency of the effects of SCFA we observed, with stimulation of growth with c-SCFA at pH = 7.4 and repression at pH = 6.5. However, since the previous studies were conducted in mice, which have a mean intestinal pH of mice < pH = 5.2, and regional pH = 4.8-5.2 in the ileum, 4.4-4.6 cecum and 4.4-5.02 colon [64], substantially lower than that of humans and the conditions we simulated, it is difficult to reconcile these different outcomes. Differences in methodology, such as growth in the microaerophilic conditions and composition of media, may play a role.
In addition to the effects of SCFA on bacterial growth, their regional concentration and composition throughout the gastrointestinal tract may serve as environmental cues that differentially regulate motility and virulence gene expression [15,65]. We found that i-SCFA (12 mM, pH = 7.4) promote the motility of pathosymbiont E. coli, whereas c-SCFA (120 mM, pH = 6.5) suppress motility, virulence gene expression, adhesion and invasion of cultured cells, and pro-inflammatory responses. Our findings parallel those with EHEC and Salmonella. For example, genes involved in EHEC flagella biosynthesis and motility are upregulated by SCFA simulating the small intestine, but down regulated by SCFA simulating the large intestine [15]. Similarly, the expression of virulence genes in Salmonella encoding invasion of epithelial cells and survival within macrophages are increased by ileal SCFA, but inhibited by colonic SCFA [65].
Transcriptional analysis of E. coli grown in c-SCFA revealed consistent down-regulation of virulence genes involved in motility (fliC), adhesion and invasion (fimH, ompC, lpfA, and nlpL), iron acquisition (chuA), stress protein (dsbA and htrA), and colibactin protein (pks). Reductions in transcription correlated with reduced functions, e.g., reduced fliC gene transcription with decreased motility; reductions in fimH, yfgL, ompC and nlpL with decreased yeast agglutination, adhesion and invasion of intestinal epithelial cells. c-SCFA also reduced the ability of CD-and CRC-E. coli induced activation of NF-kB, and the secretion of IL-8 by Caco-2 epithelial cells. NF-κB regulates a large array of genes associated with immune and inflammatory responses [58] and controls in part the secretion of IL-8, which is upregulated in patients with IBD and CRC [66,67]. Our findings support a direct role of the chemical microenvironment (SCFA, pH) in modulating crosstalk between pathosymbiont E. coli and the epithelium and pro-inflammatory signaling.
The efficacy of SCFA against enteropathogens is exemplified by the use of propionate to suppress Salmonella associated disease in poultry [68,69]. In the context of the colonic environment (65-123 mM SCFA, pH = 6.5), we found that SCFA mixtures containing 29 mM propionate markedly suppressed parameters associated with virulence of pathosymbiont E. coli associated with intestinal inflammation across species. However, in the context of the ileal environment, we found that i-SCFA (containing 2.5 mM propionate, 8 mM acetate, 1.5 mM butyrate) stimulated growth and motility of pathosymbiont E. coli, including LF82. Previous studies have shown that propionate can enhance the ability of AIEC to adhere to (2/5 AIEC strains) and invade (3/5 AIEC) Caco-2 cells [30], and increase transcription of the eut operon and ability to utilize ethanolamine [29], which is linked to virulence in a number of enteropathogens [70,71]. Motility has been reported to correlate with the degree of invasion in vitro by AIEC, murine colonization by AIEC NRG857, and the isolation of E. coli from CD patients vs healthy controls [28]. These findings point to complex multifactorial interactions of the metabolism, growth, and virulence of pathosymbiont E. coli, region specific luminal microenvironment and host.
Our investigations of the effects of SCFA on virulence extended to the genotoxic effects of E. coli associated with CRC. The polyketide synthase gene (pks) is responsible for the formation of colibactin, which is mutagenic [35,72]. We found that c-SCFA down-regulate pks transcription in E. coli NC101, which induces inflammation-associated CRC in mice [35], and E. coli isolated from patients with CRC [43]. These findings suggest that the chemical environment of the healthy colon may restrict the ability of pks+ CRC-E. coli to grow and produce colibactin. Recent studies in patients with CRC reveal a correlation between the loss of Bifidobacterium and reduced levels of total SCFA, especially butyrate, in CRC patients vs healthy controls [73]. While it remains to be established if reduced SCFA leads to proliferation and colibactin production by E. coli, it suggests the potential for therapeutic intervention with SCFA, which are known to be protective against the development of CRC [2,12].
The mechanism by which SCFA inhibit bacterial growth is postulated as intracellular acidification caused by uptake of these free acids at acidic pH [17,74]. At acidic pH, undissociated SCFA can freely diffuse through cell membrane and concentrate in bacterial cytoplasm, resulting in reduction of the intracellular pH [17]. Acidified bacterial cells have reduced transmembrane potentials and disrupted cellular biological activities (such as DNA replication), thereby exhibiting low growth phenotype. This explains the results that c-SCFA inhibit E. coli growth only at pH ≤ 6.5, but not at pH = 7.4. Sorbara et al. reported that E. coli and Klebsiella failed to replicate at internal pH = 7 or 7.25, respectively, and an internal pH = 6.75 or 6.5 is bactericidal [17], indicating the importance of luminal pH of the host in bacterial fitness. In the same report [17], the authors also found that at concentrations ≥10 mM, acetate, butyrate, and propionate (>10 mM) were able to reduce the intracellular pH of K. pneumonia and E. coli to pH < 6.7 at low medium pH (pH = 5.75), which resulted in slow growth of these bacteria. These results raise the speculation that when the levels of c-SCFA are reduced due to inflammation-associated loss of commensal bacteria, the luminal pH would rise, and ultimately the luminal microenvironment would change concurrently. These changes could be in favor of pathosymbiont (like AIEC) growth and virulence gene expression, and consequently potentiate inflammation in the intestine.

Bacterial Strains
In this study, we used 22 E. coli strains from different origins, including 1 laboratory strain (DH5α), 13 human, 4 mouse, and 4 dog strains ( Table 2). The human strains were from the intestinal mucosa of patients with IBD (9 strains) [32,41,42] and CRC (4 strains) [72]. All the IBD strains, except CUT75, have an AIEC pathotype. CUT75 is a non-pathogenic strain from a CD patient [32] and was used as a non-AIEC control in this study. Prototypical AIEC LF82 (kindly provided by Arlette Darfeuille-Michaud) [40] was used as a positive control for AIEC. The 4 CRC-associated E. coli strains ( Table 2) were kindly provided by Dr. Jonathan Rhodes [72]. The mouse strain NC101 was isolated from the feces of a healthy mouse, and it induces CRC in IL10-/-AOM treated and monocolonized mice [33,74]. CUMSL1 and CUMSL6 were isolated in our laboratory from Agr2 −/− mouse ileum provided by Dr. Steven Lipkin. CUMT8 was isolated from mouse ileitis tissue in our laboratory [25]. The 4 dog strains CUDC1, CUDLU1, CUKD1 and CUKD2 were isolated in our laboratory from dog colons with granulomatous colitis (GC) [24,38]. All E. coli strains were stored at −70 • C.

Bacterial Culture
A single colony from a fresh Luria-Bertani (LB) agar plate was used to prepare a stock culture for each experiment. Two types of liquid culture media were used in this study, LB and M9 minimal media. M9 minimal medium was made of 1 × M9 salts ( (20 mM) was used as the carbon source. All E. coli strains were first grown in LB broth overnight, then diluted 1:100 in media (LB or M9) supplemented with SCFA or NaCl at concentrations specified under each condition. For all controls, equal amount of NaCl was added in place of the SCFA used in the experiments described.

Chemicals and Stock Solutions
Sodium acetate, sodium propionate, sodium butyrate, and M9 salts were purchased from Sigma-Aldrich (St. Louis, MO). For consistency and reproducibility, total SCFA solutions were premixed as 1 M stock solutions with a molar ratio of 65:29:29 for acetate, propionate, and butyrate, respectively for c-SCFA, or 8:2.5:1.5 for acetate, propionate, and butyrate, respectively for i-SCFA.

Standardized Growth Analysis
E. coli were grown in LB broth overnight at 37 • C with shaking. Overnight cultures were diluted 1:100 into fresh LB broth or M9 medium containing NaCl (control) or SCFA at specified concentration in a 100 well-plate (Growth Curve, USA). On top of the growth medium in each well, 75 µL of mineral oil was gently added to achieve a microaerophilic growth environment. Growth of E. coli was monitored Antibiotics 2020, 9,462 15 of 20 24 to 48 h at 37 • C in a BioScreen C system (Growth Curve, USA). The OD 600 was taken every 15 min by the machine. Growth curves were generated with OD 600 as the function of time. For easier comparison between SCFA-treated and untreated control samples, the area under each growth curve (AUC) was calculated with Graphpad Prism7.03.

Transcriptional Analysis of Virulence Genes
E. coli was grown in media with either c-SCFA (65 mM) or NaCl (65 mM, Control) to mid log phase. Total RNA was extracted using the Qiagen RNAProtect-RNeasy Kit per manufacturer's protocol. Total RNA was treated with TURBO DNA-Free Kit (Ambion), followed by a two-step qRT-PCR analysis, using Qiagen's QuantiTect Reverse Transcription Kit and QuantiNova SYBR Green PCR Kit. Eleven genes associated with virulence of IBD-and CRC-associated E. coli (see Table 3) were selected for transcriptional analysis with or without SCFA treatment. Primers for these virulence genes are listed in Table 4. E. coli mdH was used as the reference gene. Each qPCR reaction contained 1 µL of cDNA, 0.7 µL of each forward and reverse primers (10 µM), 5 µL of 2× SYBR Green Master Mix, 1 µL of QN ROX Reference Dye and 2.3 µL of nuclease-free water to make the total volume of 10 µL. The reaction was run with ABI7000 (Applied Biosystems). The comparative quantification (∆C t ) method was used to determine the up-or down-regulated genes. The relative change of a targeted gene expression was calculated by using the equation RQ = 2 −∆∆CT .

Motility Assay
E. coli was grown overnight at 37 • C in LB broth. Soft agar plates (1% tryptone, 0.5% NaCl, 0.25% agar) were prepared the day before assay. Sterile 1 M SCFA or NaCl stock solution was added into the agar right before pouring the plates. The control plates contain the same amount of NaCl as the total SCFA. There were three replicates per treatment. The overnight cultures of E. coli were transferred (3 µL) on to the center of each plate, followed by incubation of the plates at 37 • C for 10 h. E. coli motility was quantified by measuring the diameter of the circular swarming area formed by the growing motile bacteria. E. coli T75 and HM334 were found to be non-motile and excluded from this assay.

Yeast Agglutination Assay
E. coli was cultured in LB ± 123 mM c-SCFA for 18 h at 37 • C. After centrifugation at 3000× g for 15 min at 4 • C, the pellet was resuspended in PBS at OD 600 = 5.2. Yeast cells were suspended in PBS at OD 600 = 5.2 on the day of assay. The E. coli suspension (100 µL) was mixed with equal volume of the yeast cell suspension in a well of 48-well plate. The plate was kept on ice and rocked for 30 to 60 min at 20 rpm.

E. coli Adhesion and Invasion of Cultured Epithelial Cells
E. coli was cultured overnight in LB at 37 • C with shaking. Bacterial pellets were re-suspended in PBS before dilution in cell culture media ±65 mM c-SCFA to an m.o.i (multiplicity of infection) of 10. Caco-2 cells were infected with bacteria using the same procedures as described by Zhang et al. [59]. At 3 h post infection, cells were washed 3× with PBS, and lysed with 1% Triton X-100. Serial dilutions of the lysates were made in PBS and plated on LB agar. The total number of colonies recovered was used to calculate the number of adherent bacteria. For invasion assays, cells were treated with gentamicin (100 µg mL −1 ) for one hour after initial infection and 3× wash with PBS to kill extracellular bacteria. Cells were then washed 3× after gentamicin treatment, lysed, and plated as described above.

NF-kB Activation Assay
HEK-Blue KD-TLR5 cells were used to detect the induction of NF-kB by E. coli infection, as previously described by Zhang et al. [59]. Briefly, cells were seeded in 96-well plates at a density of 5 × 10 4 cells per well. E. coli was diluted into fresh cell medium containing either 123 mM NaCl (control) or SCFA at an m.o.i of 200 as 10× inocula, followed by addition of this inoculum (10 µL) into each well containing 100 µL of medium for a final m.o.i of 20. At 3 h post infection, the cell medium was carefully removed from each well, and replaced with 100 µL of fresh medium containing gentamycin (200 µg mL −1 ). At 24 h post infection, the spent medium was collected, and centrifuged at 12,000 rpm for 5 min to remove any particulate matter. QUANTI-Blue Kit (InvivoGen, San Diego, CA, USA) was used to detect the reporter protein SEAP (secreted alkaline phosphatase) following the manufacturer's instructions. The SEAP activity was detected as optical density at 620 nm.

Proinflammatory Cytokine IL-8 Secretion
Supernatants of Caco-2 (at 3 h post infection) cell cultures were collected and centrifuged to remove any cells or cell debris. The concentrations of IL-8 secreted by Caco-2 cells were analyzed by ELISA methods, using the Human IL-8 Antibody Pair Kit (Invitrogen) as per the manufacturer's instructions.

Statistical Analysis
Differences in growth, gene expression, motility, adhesion, invasion, and cytokine production between control and SCFA-treated samples were analyzed by 2-way ANOVA with Dunnett's test for multiple comparisons. All statistical analyses were performed with GraphPad Prism 7.03 software and p < 0.05 was considered significant.

Conclusions
In conclusion, our data reveal a multifaceted and previously unrecognized role of the regional chemical microenvironment (SCFA and pH) on growth and virulence of IBD-and CRC-associated E. coli, and on pro-inflammatory pathosymbiont-host interactions. Our findings provide novel insights and opportunities for therapeutic intervention in people and companion animals centered on restraining the growth and virulence of pathosymbiont E. coli through modification of the luminal SCFA and pH.