
==== Front
Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38502690
202400226
10.1073/pnas.2400226121
research-articleResearch ArticlemicrobioMicrobiology423
Biological Sciences
Microbiology
Glucuronic acid confers colonization advantage to enteric pathogens
Rosay Thibaut a b https://orcid.org/0000-0001-6945-4850

Jimenez Angel G. b
Sperandio Vanessa vsperandio@wisc.edu
a b 1 https://orcid.org/0000-0003-0819-7904

aDepartment of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706
bDepartment of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390
1To whom correspondence may be addressed. Email: vsperandio@wisc.edu.
Edited by Caroline Harwood, University of Washington, Seattle, WA; received January 4, 2024; accepted February 26, 2024

19 3 2024
26 3 2024
19 9 2024
121 13 e240022612104 1 2024
26 2 2024
Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

To successfully colonize their host, enteric pathogens evolved to sense their immediate surroundings to tightly regulate virulence expression and metabolism to overcome colonization resistance due to the gut microbiota. Here, we show that Citrobacter rodentium utilizes glucuronic acid released by the microbiota from neurotransmitters and xenobiotics glucuronidated by the host during intestinal colonization as a carbon source. Pharmacological inhibition of microbial β-glucuronidases decreases the levels of this sugar and offers protection from colonization by enteric pathogens without affecting the host and the commensal microbiota.

Glucuronidation is a detoxification process to eliminate endo- and xeno-biotics and neurotransmitters from the host circulation. Glucuronosyltransferase binds these compounds to glucuronic acid (GlcA), deactivating them and allowing their elimination through the gastrointestinal (GI) tract. However, the microbiota produces β-glucuronidases that release GlcA and reactivate these compounds. Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium sense and utilize galacturonic acid (GalA), an isomer of GlcA, to outcompete the microbiota promoting gut colonization. However, the role of GlcA in pathogen colonization has not been explored. Here, we show that treatment of mice with a microbial β-glucuronidase inhibitor (GUSi) decreased C. rodentium’s colonization of the GI tract, without modulating bacterial virulence or host inflammation. Metagenomic studies indicated that GUSi did not change the composition of the intestinal microbiota in these animals. GlcA confers an advantage for pathogen expansion through its utilization as a carbon source. Congruently mutants unable to catabolize GlcA depict lower GI colonization compared to wild type and are not sensitive to GUSi. Germfree mice colonized with a commensal E. coli deficient for β-glucuronidase production led to a decrease of C. rodentium tissue colonization, compared to animals monocolonized with an E. coli proficient for production of this enzyme. GlcA is not sensed as a signal and doesn’t activate virulence expression but is used as a metabolite. Because pathogens can use GlcA to promote their colonization, inhibitors of microbial β-glucuronidases could be a unique therapeutic against enteric infections without disturbing the host or microbiota physiology.

glucuronic acid
Citrobacter rodentium
enteric pathogens
microbiota glucuronidase
HHS | NIH | NIAID | Division of Intramural Research (DIR, NIAID) 100006492 AI053067 Vanessa Sperandio HHS | NIH | NIAID | Division of Intramural Research (DIR, NIAID) 100006492 AI154597 Vanessa Sperandio HHS | NIH | NIAID | Division of Intramural Research (DIR, NIAID) 100006492 AI155398 Vanessa Sperandio
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pmcAttaching and effacing (A/E) enteric pathogens cause outbreaks of diarrhea worldwide (1). The common feature of A/E pathogens is that they harbor the locus of enterocyte effacement (LEE) pathogenicity island. The LEE contains 41 genes encoding proteins necessary for the assembly of a type 3 secretion system (T3SS) (Fig. 1I). This system is a syringe-like apparatus that injects effectors (proteins that mimic or highjack mammalian function) directly into host cells leading to effacement of the enterocyte microvilli and actin accumulation beneath the adherent bacterium (2, 3). It also plays a role in the regulation of host cell immune responses (4). Members of this family include enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. EPEC is the predominant agent of infantile diarrhea in developing countries (1). EHEC causes bloody diarrhea and also produces Shiga toxin, which can lead to hemolytic uremic syndrome and high levels of morbidity and mortality (2). C. rodentium is a murine A/E pathogen widely used as surrogate animal model of EPEC and EHEC, since these pathogens strictly infect humans and do not colonize mice (5).

Fig. 1. Commensal E. coli β-glucuronidase modulates pathogen colonization. (A) The gut microbiota was depleted with treatment of four antibiotics (ampicillin, neomycin, metronidazole, and vancomycin), and mice are recolonized with a commensal E. coli strain HS or its mutant for β-glucuronidase ΔuidA, then infected with C. rodentium (DBS100). (B) Fecal shedding of C. rodentium. (C and D) Cecum tissue colonization at days 5 and 7, respectively. (E) Germfree mice were recolonized with E. coli HS or ΔuidA 7 d before infection with C. rodentium, tissue colonization was evaluated 7 d post-infection. (F) Fecal shedding of C. rodentium. (G and H) C. rodentium cecum tissue and content colonization, respectively. (I) Schematic depiction of the LEE pathogenicity island with genes used for qRT-PCR assessment of expression of each LEE operon (LEE1-5). (J) qRT-PCR of LEE-encoded genes of attached bacteria. (K–M) qRT-PCR of cytokines genes. Error bars represent SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001, Mann–Whitney U test.

To colonize the intestine, A/E pathogens must overcome the microbiota colonization resistance (6). Pathogens sense and respond to a variety of environmental cues, including metabolites and signals derived from both the microbiota and the host, as well as dietary byproducts (7, 8). In the GI tract, carbon sources are a limited resource that can prevent pathogen expansion. Hence, pathogens evolved to use alternative carbon sources. Among these are the sugar acids, hexuronates, which promote EHEC and C. rodentium gut colonization (9). Galacturonic acid (GalA) is used by these pathogens as a carbon source in the gut, aiding their initial expansion. GalA is also sensed as a signal through the ExuR regulator, which in the absence of this sugar, acts as a transcriptional activator of the LEE. Importantly, the ExuR–GalA regulation plays a key role in the establishment and progression of C. rodentium murine infection (10). GalA and Glucuronic acid (GlcA) share the same catabolism pathway (10), however, the role of GlcA in enteric pathogenesis remains unknown.

GlcA can be derived from plant polysaccharides and diet (11). Most of the GlcA is derived from the host and is used in the glucuronidation of endo- and xeno-biotics and neurotransmitters. Glucuronidation is a detoxification process that leads to the inactivation and elimination of these compounds from the circulation (12). It also plays an important role in drug metabolism, decreasing their activity (13). Uracylglucosyltransferases (UGTs) link GlcA to these molecules inactivating them and making them highly hydrophilic to facilitate their elimination through urine or bile (12). Upon reaching the GI tract through bile secretion, glucuronidated compounds are reactivated by the microbiota produced β-glucuronidases, encoded by the uidA gene, which also releases GlcA that can be used as a carbon source (14). EHEC and C. rodentium do not produce β-glucuronidases. The neurotransmitter norepinephrine is deactivated by glucuronidation, and a previous study showed that its activity in the GI tract was directly linked to the presence of microbial β-glucuronidases. It is notable that norepinephrine is mostly found in its glucuronidated state in the gut of germfree mice (15). Norepinephrine can regulate EHEC and C. rodentium virulence expression, through the QseC regulator activating LEE expression (16). Additionally, serotonin, which also plays a role in LEE regulation by inhibiting LEE expression trough CpxA (17), is also glucuronidated (18). Altogether, β-glucuronidases could potentially regulate virulence by modulating LEE expression, as well as GlcA availability as a carbon source necessary for EHEC colonization (9).

Here, we show that microbiota β-glucuronidase activity liberates GlcA that is used as a carbon source by C. rodentium within the murine GI tract, promoting colonization. GlcA does not influence LEE regulation nor changes microbiota composition or host physiology. Consequently, targeting microbial β-glucuronidases could be a unique strategy to prevent enteric pathogen colonization, and an alternative to antibiotic treatment without affecting the host and microbiota.

Results

Microbial β-Glucuronidases Modulate Pathogen Murine Colonization.

To address the role of microbial β-glucuronidases in the colonization of the GI tract by C. rodentium, we first employed a mono-colonization approach. Initially, the microbiota was depleted by antibiotics and mice were recolonized with commensal E. coli HS wild-type (WT) or ΔuidA HS (does not produce β-glucuronidase), the day before infection (Fig. 1A). We note that both strains colonize these animals at similar levels (SI Appendix, Fig. S1A). There was only a significant decrease of C. rodentium fecal shedding in the absence of β-glucuronidases on day 6 post infection (Fig. 1B), and a nonsignificant decrease was observed on tissue colonization at day 5 (Fig. 1C), which disappeared by day 7 post infection (Fig. 1D). This can be explained by recolonization by the microbiota post-antibiotic treatment that leads to the restoration of β-glucuronidase activity. We then employed germfree mice to avoid commensal recolonization and assess the role of microbial β-glucuronidases in C. rodentium colonization of the GI tract. Germfree mice were first colonized with E. coli WT HS or ΔuidA for seven days to allow for stable colonization (SI Appendix, Fig. S1C) and then infected with C. rodentium (Fig. 1E). We followed colonization through fecal shedding and recolonization with both WT and ΔuidA E. coli remained stable even after infection with C. rodentium (SI Appendix, Fig. S1C). Fecal shedding of C. rodentium was not decreased in the absence of E. coli β-glucuronidase activity (Fig. 1F). Tissue colonization was significantly decreased in the absence of E. coli β-glucuronidase (Fig. 1G), while there was no decrease of the C. rodentium load in cecum content (Fig. 1H). LEE gene expression of tissue attached bacteria was unchanged in the presence of WT HS or ΔuidA (Fig. 1 I and J), as was expression of inflammation markers on tissues (Fig. 1 K–M). Inflammation was increased in C. rodentium-infected animals compared to uninfected controls as expected (Fig. 1 K–M).

To investigate the impact of the complex microbiota deglucuronidase activity in C. rodentium murine colonization, we chose a pharmacological approach employing a specific inhibitor of bacterial enzymes (GUSi), as previously described (19). Briefly, mice were administrated GUSi through drinking water and gavaged the day before infection to spike GUSi activity (Fig. 2A). We observed that GUSi administration induces a significant decrease of C. rodentium fecal shedding (Fig. 2B) and cecal and colon tissue colonization (Fig. 2C and SI Appendix, Fig. S2). A decrease is also observed in cecum content (Fig. 2D) but it is not significant, as observed in GF mice (Fig. 1H). This could be explained by a gradient of availability of glucuronidated compounds in the GI tract, with higher concentration closer to the tissue since these compounds are released by the host. As observed with monocolonization with E. coli proficient or defective for β-glucuronidase activity (Fig. 1), expression of the LEE genes was similar in bacteria attached to the tissue or in the cecum content of animals treated or untreated with GUSi (Fig. 2 E and F).

Fig. 2. Microbiota β-glucuronidases modulate pathogen colonization. (A) Starting 2 d before infection, mice are treated with the microbial β-glucuronidase inhibitor (GUSi) or vehicle administered through water (replaced every 2 d, yellow circles) and gavaged with 20 µg the day prior to infection. (B) C. rodentium fecal shedding in GUSi treated and untreated animals. (C) Cecum tissue colonization by C. rodentium GUSi treated and untreated animals. (D) C. rodentium in cecum content of GUSi treated and untreated animals. (E) qRT-PCR of LEE-encoded genes of attached bacteria. (F) qRT-PCR of LEE-encoded genes of luminal bacteria. (G) Histological representation (10×) of cecum and (H) score 7 d post C. rodentium infection (n = 9). (I–K) qRT-PCR of cytokines genes from cecal tissue. Each symbol represents an individual mouse. Error bars represent SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001, Mann–Whitney U test.

Inflammation increases host susceptibility to colonization by enteric pathogens (20). Consequently, we assessed whether inhibition of β-glucuronidase activity affected inflammation. Histological analysis of cecum tissue was performed and scored for eight criteria: submucosal edema, goblet cell depletion, epithelial hyperplasia, mononuclear infiltration, bacterial attachment, epithelial integrity, hemorrhage, and hyperemia (Fig. 2 G and H). Inflammation was only increased in the presence of C. rodentium (Fig. 2 G and H), confirming that the inhibitor itself didn’t have any inflammatory effects (19). Infected mice treated with GUSi presented less tissue inflammation (Fig. 2H), but this was not due to changes in LEE gene expression and can probably be attributed to decreased tissue colonization. Cytokine expression is also not altered by GUSi, and as expected, it is only enhanced upon C. rodentium infection (Fig. 2 I–K). We also investigated whether GUSi promoted a shift in the microbiota composition, which could lead to an increase in colonization resistance (20). β-glucuronidase inhibition did not lead to any significant modification of cecal microbiota composition at the level of Phylum, Class, and Order (SI Appendix, Fig. S3). There were some differences observed at the level of species only for infected mice, not when vehicle and inhibitor treatment were compared (SI Appendix, Table S2). C. rodentium was decreased in GUSi treated compared to control mice, confirming our previous results. Altogether, these data indicate that a decrease in microbial β-glucuronidases results in decreased tissue colonization by C. rodentium, but this is not the result of differences in virulence gene expression, host inflammation, or changes in the microbiota membership.

Glucuronic Acid Metabolism Impacts C. rodentium Colonization.

The sugar acid GalA aids the initial expansion of C. rodentium in the intestine due to its utilization as a carbon source (10). GalA is an isomer of GlcA, but the role of GlcA in pathogen gut colonization has not been explored. β-glucuronidases could be responsible for releasing GlcA in the gut, which could give a metabolic advantage for C. rodentium colonization. The catabolic pathway for GalA and GlcA is partially shared (Fig. 3A). To better understand the role of sugar acid catabolism in C. rodentium intestinal colonization, we generated an ΔuxaC mutant. In the Ashwell pathway, the uxaC gene encodes for an uronate isomerase, which is the first step in the catabolic pathway for both GalA and GlcA utilization. The uxuB gene encodes an enzyme that is specific to the catabolism of GlcA (Fig. 3A and SI Appendix, Fig. S4A). C. rodentium utilizes both GalA and GlcA as sugar sources. C. rodentium ΔuxuB, which specifically can’t utilize GlcA as a carbon source, but can still use GalA, has reduced ability to colonize cecum tissue and expand in the lumen compared to WT (Fig. 3 B and C). However, its colonization is not affected by GUSi, unlike colonization by the WT, indicating that ΔuxuB is not sensitive to GUSi (Fig. 3 B and C). Virulence expression remains mostly unaffected by the inhibitor, and LEE expression is also not modulated in ΔuxuB compared to WT (Fig. 3 D and E). Inflammation also does not change by GUSi treatment in animals infected with either the WT or ΔuxuB as shown by cytokines expression (Fig. 3 F–H). We note that ler and tir expression was increased for GUSi treatment in ΔuxuB, but only for attached bacteria, and no differences in attachment and inflammation were observed so this does not appear to be biologically significant (Fig. 3). Similar results were obtained with a ΔuxaC C. rodentium, which is unable to utilize both GlcA and GalA (SI Appendix, Fig. S4). Because both knock-out strains depict lower murine gut colonization, do not affect LEE gene expression, and are not susceptible to GUSi, this colonization defect is probably due to their inability to utilize GlcA as a carbon source.

Fig. 3. Glucuronic acid utilization in C. rodentium intestinal colonization. (A) Hexuronates catabolism pathway. (B and C) C. rodentium cecum tissue and content colonization, respectively. (D and E) qRT-PCR of LEE-encoded genes of attached bacteria and bacteria in the cecum tissue and the lumen, respectively. (F–H) qRT-PCR of cytokines genes of cecum tissue. Error bars represent SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001, Mann–Whitney U test.

Global Transcriptomic Analysis of Host and C. rodentium.

To investigate whether host pathways play a role in the microbial β-glucuronidases impact on C. rodentium gut colonization, we performed host transcriptomics during infection on cecum tissue. Four groups of animals were compared (Fig. 4A): vehicle non-infected (A), GUSi treated and non-infected (B), infected with C. rodentium DBS100 plus vehicle (C), and infected with C. rodentium DBS100 and treated with GUSi (D). PCoA analyses (Fig. 4B) show that GUSi treatment doesn’t impact host transcription, and the biggest differences observed between experimental groups is within the uninfected and infected animals. In infected mice treaded with GUSi, the variance between infected and uninfected animals correlates with the decrease in colonization and lower tissue inflammation. Analysis of differentially expressed genes (Fig. 4C) indicate that GUSi treatment doesn’t affect host tissue physiology, since only one gene is up-regulated. The major pathway modulated in infected animals (Fig. 4D) appears to be the immune response to infection, and it is mostly up-regulated with the control (uninfected) vs. infection, and the GUSi-treated infection only shows a mild response due to lower pathogen colonization. These data confirm that GUSi treatment lowers pathogen colonization. To assess whether GlcA affect the C. rodentium’s transcriptome, we also performed RNAseq in GlcA treated and untreated C. rodentium in vitro (Fig. 4E), using low concentrations of this sugar acid that do not promote growth changes due to its use as a metabolite, and to rule out any signaling roles GlcA may have, as we have already shown that GlcA doesn’t act as a signal toward LEE gene expression (10). Transcriptomic analysis has further confirmed this phenotype (Fig. 4H). We also observed that there is no significant difference in the variance analysis between GlcA treated and untreated C. rodentium (Fig. 4F). Only one gene, zraP, appears to be up-regulated by GlcA (Fig. 4G). zraP is the third member of the zraSR two-component system, which is involved in zinc stress responses (21). However, none of the genes under the control of the zra system are differentially expressed in response to GlcA, hence this may not be physiologically relevant. Consequently, these data suggest that GlcA is not perceived as a signal at low concentrations by C. rodentium, since it does not alter the transcriptome. Altogether, these results confirm that the inhibition of microbial β-glucuronidases doesn’t affect the host physiology and prevents C. rodentium expansion without regulation of its virulence repertoire.

Fig. 4. Host and Citrobacter rodentium transcriptomic analysis. (A) Cecum tissue was collected at day 7 post infection for host transcriptomic analysis. (B) PCoA analasis, A- control, B- GUSi, C- C. rodentium DBS100, D- C. rodentium DBS100+GUSi. (C) Number of genes significantly differentially expressed (FDR < 0.5, FC > 2), B-A control vs. GUSi, C-A control vs. DBS100, D-C DBS100+GUSi vs. DBS100. (D) Pathway analysis on host tissue from RNAseq gene expression analysis: A- control, B- GUSi, C- C. rodentium DBS100, D- C. rodentium DBS100+GUSi. (E) Transcriptomic analysis of invitro effect of Glucuronic acid at low concentration on C. rodentium. (F) PCoA analysis of DBS100 exposed or not to GlcA100 µM. (G) Volcano plot of differentially expressed gene between DBS100 exposed or not to GlcA100 µM (FDR < 0.5, FC > 2). (H) LEE gene expression log2 fold change after exposure to GlcA100 µM.

Discussion

The rise of multi antibiotic-resistant bacterial infections, combined with the impact antibiotics have in the resident microbiota membership, urges the search for alternative treatments for infectious diseases. Here, we report that inhibition of microbial β-glucuronidases produced by the GI microbiota leads to decreased pathogen expansion and colonization. This may offer an alternative to antibiotic treatment. Importantly, this treatment does not impact host physiology and gene expression, as well as the composition of the microbiota.

The finding that pathogens can synchronize the expression of their virulence factors after sensing different signals, led to the emergence of anti-virulence approaches to combat infections. One such approach targets the QseC sensor kinase of EHEC and C. rodentium, which senses the neurotransmitters epinephrine and norepinephrine leading to increased expression of the T3SS (16). QseC inhibitors prevent the expression of the T3SS and constitute a promising anti-virulence approach (22). The availability of active epinephrine and norepinephrine in the gut is dictated by the microbiota. Epinephrine and norepinephrine are deactivated by the host through glucuronidation, and the microbiota encodes for β-glucuronidases (encoded by the uidA gene) that deconjugate GlcA from epinephrine and norepinephrine, rendering these signals active in the gut (15). Serotonin is another neurotransmitter, which has opposite effects to epinephrine and norepinephrine on bacterial pathogenesis, by decreasing LEE gene expression (17). Serotonin is also deactivated by glucuronidation, being reactivated in the gut by microbial β-glucuronidases (15) (18). In addition to modulation of neurotransmitter activity in the GI tract, microbiota-derived β-glucuronidases also release GlcA. Of note neither EHEC nor C. rodentium have a functional uidA gene and do not have deglucuronidase activity, relying on the microbiota for this process.

β-glucuronisases produced by commensal E. coli as well as by a complete microbiota promote C. rodentium intestinal colonization (Figs. 1 and 2). Inhibition of this microbial-derived enzyme does not change the composition of the intestinal microbiota (SI Appendix, Fig. S3). Because epinephrine and norepinephrine activate QseC to increase LEE gene expression and virulence in both EHEC and C. rodentium (16), we reasoned that there would be a decrease in LEE gene expression in the absence of deglucuronidation. However, surprisingly, there was no effect on LEE gene expression in C. rodentium in the intestines of animals treated or not with GUSi or colonized with either HS WT or ΔuidA (in germfree mice) (Figs. 1 and 2). These data suggest that the effect observed in C. rodentium colonization by deglucuronidation is not due to differences in LEE expression. A potential explanation is that LEE expression is differentially modulated by several neurotransmitters that are deactivated when glucuronidated and are deglucuronidated by the microbiota. For instance, while epinephrine and norepinephrine increase LEE gene expression through QseC (16), serotonin, which inhibits LEE expression through CpxA, is also glucuronidated (18).

It is noticeable that the microbiota-encoded glucuronidases also free GlcA from glucuronidated compounds in the intestine, and sugar acids aid the initial expansion of C. rodentium in the intestine due to their utilization as a carbon source (10). A C. rodentium ΔuxaC mutant (can’t use both GalA and GlcA as a carbon source) presents decreased intestinal colonization in competition experiments with WT in the presence of an intact microbiota. However, upon pharmacological inhibition of the microbiota glucuronidase, this competition advantage from WT disappears (SI Appendix, Fig. S4). To uncouple the metabolism of GalA and GlcA, we also constructed a ΔuxuB C. rodentium mutant (can use GalA, but exclusively can’t use GlcA as a carbon source). Similarly, to the ΔuxaC mutant, a C. rodentium ΔuxuB mutant presents decreased intestinal colonization compared to WT and is also irresponsive to GUSi (Fig. 3). Consequently, we have uncovered a different collaboration between the intestinal microbiota and a pathogen through the deglucuronidation activity of the microbiota. This activity frees GlcA in the gut, which promotes pathogen expansion.

As previously mentioned, C. rodentium doesn’t harbor the uid operon encoding the β-glucuronidase (23), and it is notable that several enteric pathogens accumulated mutations in this operon, being unable to produce a functional β-glucuronidase. EHEC and EPEC possess the complete operon (24, 25), but both acquired mutations in the uid genes, preventing production of this enzyme. Consequently, they rely on the microbiota β-glucuronidase activity to access GlcA to facilitate intestinal colonization. Salmonella, another major enteric pathogen, is also unable to produce β-glucuronidases, and possesses a pathogenicity island dedicated to its catabolism (26). SPI-13 contains genes highly similar to the E. coli UxuAB-UxaC system, which are important for C. rodentium gut colonization. These data suggest that GlcA utilization may be important for multiple enteric pathogens and that the utilization of GUSi to lower its availability in the gut could have a broader spectrum to treat these infections.

Here, we described another example of microbiota-pathogen “collaboration.” The release of GlcA by the microbiota β-glucuronidase activity allows its utilization as a carbon source in the murine intestine aiding in pathogen expansion (Fig. 5). The relationships among host, microbiota and enteric pathogens are complex and multi-layered. It involves a high level of integration between metabolic circuits and virulence gene expression. This research is a step forward to define the molecular mechanisms that govern how commensal species impact virulence of an intestinal pathogen. It has fundamental implications for how differential species compositions and metabolic/enzymatic functions of the microbiota may affect disease outcome and susceptibility to pathogens.

Fig. 5. Schematic model of β-glucuronidase inhibition on C. rodentium colonization. Xenobiotics are detoxified from the circulation in the liver through glucuronidation. Uracylglycosyltransferases (UGTs) bind GlcA to these molecules, allowing their elimination through the bile duct to the GI tract. When these compounds reach the GI, the presence of β-glucuronidases produced by the microbiota allows the liberation of free GlcA. The pathogen C. rodentium (Cr) can use the free sugar acid to increase its colonization. Inhibition of microbial β-glucuronidases with GUSi leads to decreased Cr expansion.

Materials and Methods

Bacterial Strains, Plasmids, and Growth and Culture Condition.

All strains and plasmids used in this study are listed in SI Appendix, Table S1. Bacterial overnight cultures were grown in LB with antibiotics shaking at 37 °C. Growth curves were realized in low glucose DMEM (Gibco) with or without 100 μM glucuronic acid in microaerophilic conditions at 37 °C with 5% CO2.

Recombinant DNA Techniques.

All primers used for qRT-PCR and mutant generation can be found in SI Appendix, Table S1. Knockout strains were constructed using the λ-red method (27). All mutants were whole-genome sequenced to ensure that there are no secondary mutations.

qRT-PCR.

RNA from three biological replicates was extracted using Trizol chloroform extraction. Samples were DNase treated with InvitroGen Turbo DNA-free kit, and purified by SPRi (RNAClean XP). qRT-PCR was performed as follows. Briefly, 2 μg of diluted extracted RNA was converted to cDNA using superscript IV reverse transcriptase, random primers, DTT, and dNTPs. Validated Primers (SI Appendix, Table S1) and SYBR Green were added to the cDNA and the mix run in Quantstudio 6 flex (Applied Biosystems). Data were collected using QuantStudio Real-Time PCR Software v1.3, normalized to endogenous rpoA levels for C. rodentium, and gapdh levels for mice and analyzed using the comparative critical threshold (CT) method. For all the experiments, error bars indicate SEM. A P-value of less than 0.05 was considered significant.

Microbiota Depletion and Recolonization.

SPF female C57B6/J mice 8 wk of age purchased from Jackson were used. Mice were orally administered a combination of four antibiotics, ampicillin, neomycin, metronidazole, and vancomycin (5 mg of each) via oral gavage for 3 d to deplete the gut microbiota. Fecal pellets were collected before and after antibiotic treatment to confirm depletion of the gut microbiota. Feces were resuspended in PBS and plated on brain heart infusion (BHI)-blood agar plates containing no antibiotics. Colony counts were performed after 48 h incubation at 37 °C under both aerobic and anaerobic conditions. Twenty-four hours after stopping antibiotic administration, SPF mice were recolonized by oral gavage with 109CFU of HS or ΔuidA resuspended in PBS and infected the following day with 108CFU of C. rodentium DBS100. Mice were checked daily for colonization. Stools and tissues were collected for analysis of CFU, inflammation, and LEE gene expression. To assess tissue colonization, mice were killed on different days, and the cecum tissue and content were collected and separated in 3. Samples for RNA analysis were snap-frozen in liquid nitrogen and stored at −80 °C until use. Samples for histopathology were conserved in formalin 10%. Samples for CFU counting were weighted and diluted for counting. RNAs from mice tissues were extracted using the previously described method. RNAs from feces were isolated from individual mouse using RNeasy Power Microbiome kit (Qiagen) as per the manufacturer’s instructions. qRT-PCR was performed as described earlier. Significance was determined by the unpaired Mann–Whitney U test. A P-value of less than 0.05 was considered significant.

Microbial β-Glucuronidases Inhibition (Fig. 1E).

SPF female C57B6/J mice 8 wk of age purchased from Jackson were used. Microbial β-glucuronidase inhibition was performed by an initial oral gavage of 20 µg of Glucuronidase Inhibitor (GUSi, Sigma-Aldrich, #347423) (19). GUSi was resuspended PBS with 2% DMSO for oral gavage. GUSi was then administrated to mice through drinking water at 5 µg/mL with 0.1% DMSO for the rest of the experiment, and water was replaced every other day. Mice were infected with 108 CFU of DBS100 or ΔuxuB resuspended in PBS, or PBS alone. Mice were checked daily for colonization. To look at tissue colonization, mice were killed, and the cecum tissue and content were collected and analyzed as previously described. Significance was determined either by unpaired Mann–Whitney U test. A P-value of less than 0.05 was considered significant.

Gnotobiotic Animal Experiments (Fig. 3D).

Gnotobiotic C57B6/J Mice between 8 and 10 wk of age were obtained from Taconic Biosciences. Gnotobiotic commensals colonization was performed by oral gavage with 108 CFU of E.coli HS, ΔuidA mutant, or PBS 7 d before infection with 108 CFU of DBS100 or PBS. Samples were collected and analyzed as described above.

Animal Experiments Disclaimer.

Research involving animals has been approved by the University of Texas Southwestern and University of Wisconsin Institutional Animal Care and Use Committee. Animal colonization was checked daily and samples were collected as previously described above.

Histology and Scoring.

Cecal tip was fixed in 10% neutral buffered formalin. Histological inflammation scores (0 to 20) of hematoxylin and eosin (H&E) stained sections were blindly assessed as previously described (28). Briefly, inflammation was assessed based on the following histopathological features: epithelial hyperplasia (0 to 4), submucosal edema (0 to 4), goblet cell depletion (0 to 4), epithelial integrity (0 to 4), and polymononuclear (PMN) cell and inflammatory monocyte infiltration (0 to 4). Data are expressed as the sum of these individual scores (0 to 20). Histological inflammation scores and representative images corresponding to all animals were assigned parallel during the same experimental evaluation.

16S Sequencing and Analysis.

For 16S rRNA microbiota profiling, genomic DNA was isolated from feces that were collected after the mice were killed, snap-frozen, maintained at −80 °C, and processed as in ref. 29. Briefly, the hypervariable region V3 and V4 of bacterial 16S rRNA gene were captured using the Illumina Nextera protocol and a single amplicon of about 460 bp was amplified as described in the Illumina protocol. The PCR products were cleaned using Agencourt AmpureXP beads from Beckman Counter Genomics. Illumina adaptor and barcode sequences were ligated to the amplicons to attach them to MiSeqDx flow cell and for multiplexing. Quality and quantity of each sequencing library were assessed using Bioanlyzer and picogreen measurements, respectively. About 6 pM of pooled libraries were loaded onto a MiSeqDX flow cell and sequenced using PE300 (Paired end 300 bp) v3 kit. Raw fastq files were demultiplexed based on unique barcodes and assessed for quality. Samples with more than 50 K QC pass sequencing reads were used for downstream 16S operational taxonomic units (OTU) analysis. Results were analyzed using the OmicsBox software Metagenomics tools. Statistical analysis was performed as described by Robinson et al. (30).

In Vivo RNA-Seq Library Preparation and Analysis.

RNAs were extracted from mice cecum tissue with Trizol chloroform extraction. Samples were DNase treated with InvitroGene Turbo DNA-free kit and purified by SPRi (RNAClean XP). Sequencing was run at the Microbial ‘Omics Core, Broad Institute, Cambridge, MA. A modified RNAtag-Seq protocol was used to generate Illumina strand-specific cDNA libraries. Mammalian ribosomal RNA is depleted using the RiboCop kit from Lexogen. The data analysis was conducted using the software iDEP 0.96. Gene expression was considered significant for gene with fold change >2 and FDR < 0.05. (RNA-seq data have been deposited at the European Nucleotide Archive, accession GSE252823.)

In Vitro RNA-Seq Library Preparation and Analysis.

Briefly, RNAs extracted as previously described from four biological replicates were used to perform RNA-seq experiments. Sequencing was run by SEQcenter. RNA libraries were prepared using Illumina’s Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit and 10 bp unique dual indices (UDI). Sequencing was done on a NovaSeq X Plus, producing paired-end 150 bp reads. Demultiplexing, quality control, and adapter trimming were performed with bcl-convert (v4.1.5). Reads were mapped to the C. rodentium ICC168 genome (National Center for Biotechnology Information NC_013716). The data analysis was conducted using the software iDEP 0.96. Gene expression was considered significant for genes with fold change >2 and FDR < 0.05. (RNA-seq data have been deposited at the European Nucleotide Archive, accession GSE252543.)

Supplementary Material

Appendix 01 (PDF)

This project was funded by NIH grants AI053067, AI154597, and AI155398. Schematic figures were created using https://Biorender.com.

Author contributions

T.R. and V.S. designed research; T.R. and A.G.J. performed research; T.R. and A.G.J. contributed new reagents/analytic tools; T.R. and V.S. analyzed data; and T.R. and V.S. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

RNA-Seq data have been deposited in European Nucleotide Archive (GSE252543 (31) and GSE252823 (32). All other data are included in the manuscript and/or SI Appendix.

Supporting Information

This article is a PNAS Direct Submission.
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