Antivirulence activity of the human gut metabolome

Abstract

The mammalian gut contains a complex assembly of commensal microbes termed microbiota. Although much has been learned about the role of these microbes in health, the mechanisms underlying these functions are ill defined. We have recently shown that the mammalian gut contains thousands of small molecules, most of which are currently unidentified. Therefore, we hypothesized that these molecules function as chemical cues used by hosts and microbes during their interactions in health and disease. Thus, a search was initiated to identify molecules produced by the microbiota that are sensed by pathogens. We found that a secreted molecule produced by clostridia acts as a strong repressor of Salmonella virulence, obliterating expression of the Salmonella pathogenicity island 1 as well as host cell invasion. It has been known for decades that the microbiota protects its hosts from invading pathogens, and these data suggest that chemical sensing may be involved in this phenomenon. Further investigations should reveal the exact biological role of this molecule as well as its therapeutic potential. Importance: Microbes can communicate through the production and sensing of small molecules. Within the complex ecosystem formed by commensal microbes living in and on the human body, it is likely that these molecular messages are used extensively during the interactions between different microbial species as well as with host cells. Deciphering such a molecular dialect will be fundamental to our understanding of host-microbe interactions in health and disease and may prove useful for the design of new therapeutic strategies that target these mechanisms of communication.

FIG 1

An extract from human feces is active against Salmonella. Salmonella was inoculated in LB broth with or without the addition of a dried ethyl acetate extract of human feces, and growth was monitored through measurements of optical density at 600 nm. Dried extracts were resuspended at a concentration that approximates the concentration present in feces (1×), given the weight of sample and volume of solvent used. (A) Squares represent cultures without the extract, whereas circles represent cultures containing the fecal extract. Results represent the averages of four independent measurements (n = 4), and bars (too small to be seen in most cases) show the standard errors of means. ML, mid-logarithmic growth phase; LL, late logarithmic growth phase; ES, early stationary growth phase. (B) To allow better visualization, the maximum optical density (9-h time point) achieved by each culture condition is shown. Results represent the averages of four independent measurements (n = 4), and bars show the standard errors of the means. ***, P < 0.0008.

FIG 2

The human gut metabolome contains a strong inhibitor of Salmonella invasion gene expression. (A) Salmonella was grown in LB broth with or without the addition of an extract from human feces at a concentration that approximates the concentration present in feces (1×), RNA was extracted, and hilA expression was assessed by real-time PCR. White bars, cultures without the fecal extract; gray bars, cultures with the fecal extract. ML, mid-logarithmic growth phase; LL, late logarithmic growth phase; ES, early stationary growth phase. Results represent the averages of four independent measurements (n = 4), except for measurements at ES in the presence of the extract, where 3 cultures were used (n = 3). Bars show the standard errors of the means. (B) Salmonella was grown in LB broth with various concentrations of the extract from human feces, RNA was extracted, and hilA expression was assessed by real-time PCR. Relative concentrations shown are in comparison with the concentration present in human feces, given the weight of sample and volume of solvent used. Results represent the averages of four independent measurements (n = 4), except for measurements at 0.5×, where 3 cultures were used (n = 3). Bars show the standard errors of the means. *, P < 0.02; **, P < 0.01; ***, P < 0.0004. (C) Schematic of the genetic locus responsible for host cell invasion by Salmonella, the Salmonella pathogenicity island 1 (SPI-1). Each arrow corresponds to a transcript, and the genes comprised within the transcript are indicated above the arrows. (D) Salmonella was grown in LB broth with or without the addition of an extract from human feces at a concentration that approximates the concentration present in feces (1×), RNA was extracted, and the expression of the indicated genes was assessed by real-time PCR. White bars, cultures without the fecal extract; gray bars, cultures with the fecal extract. Results represent the averages of four independent measurements (n = 4), and bars show the standard errors of the means. *, P < 0.02; **, P < 0.008.

FIG 3

Molecules from human feces strongly repress the invasion of cultured host cells by Salmonella. HeLa cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 1% GlutaMAX. Salmonella was grown in LB broth with or without the addition of an extract from human feces at a concentration that approximates the concentration present in feces (1×). Salmonella cultures were centrifuged, and cells were resuspended in phosphate-buffered saline and diluted in tissue culture medium. HeLa cells were infected at a multiplicity of infection of 10 for a total of 2 h at 37°C and 5% CO₂. Cells were washed with buffer and lysed, and serial dilutions of the lysates were plated on LB plates for bacterial enumeration. Each dot on the graph represents the average of the results of two wells using an individual bacterial culture, for a total of 6 independent measurements (n = 6). Bars show the averages of the results obtained. ***, P < 0.0001.

FIG 4

The inhibitory activity of the human gut metabolome is universal. Feces from 10 healthy subjects were extracted with ethyl acetate, and dried extracts were added to LB broth. Salmonella was grown in LB broth with or without the addition of the extracts at a concentration of approximately 0.25×, RNA was extracted, and hilA expression was assessed by real-time PCR. Results shown are the averages of three independent bacterial cultures (n = 3), except for donor 5, where 2 cultures were used (n = 2). *, P < 0.02; **, P < 0.004; ns, not significant (P > 0.05).

FIG 5

The mammalian gut microbiota is required for full inhibition of Salmonella invasion gene expression. (A) Molecules were extracted from feces of 129S1/SvImJ Nramp1^−/− mice using ethyl acetate, as described in the text. Animals were then treated with 20 mg of streptomycin through oral gavage, and feces were collected and extracted again, 24 h after treatment. Salmonella was grown in LB broth with or without the addition of the dried extracts, and hilA expression was tested through RT-PCR. Results shown are the averages of 5 to 6 measurements, and bars represent the standard errors of the means. (B) Feces from conventionally raised as well as germfree Swiss Webster mice were collected and extracted with ethyl acetate, as described in the text. Salmonella was grown in LB broth with or without the addition of the dried extracts, and hilA expression was tested through RT-PCR. Results shown are the averages of 3 to 5 measurements, and bars represent the standard errors of the means. Untr., samples collected before antibiotic treatment; Strep., samples collected after streptomycin treatment; MPF, murine-pathogen-free animals (conventionally raised); GF, germfree animals. *, P < 0.04; **, P < 0.003; ***, P < 0.002.

FIG 6

The mammalian gut microbiota is sufficient for production of the inhibitory activity. Feces from three healthy human donors were used to inoculate a bioreactor system run as a chemostat to culture microbial communities from the human gut. After appropriate incubation, effluents were collected and extracted with ethyl acetate. Dried extracts were then added to LB broth, and the medium was used to culture Salmonella. The expression of hilA in medium with or without the extracts was then monitored through RT-PCR. Results shown are the averages of 3 measurements, and bars represent the standard errors of the means. **, P < 0.01.

FIG 7

A human isolate of Clostridium citroniae produces strong activity against Salmonella invasion gene expression. Microbial isolates from a chemostat culture showing activity against hilA were tested individually for biological activity. Isolates were cultured under anaerobic conditions in Trypticase soy broth supplemented with menadione and hemin for at least 2 days, and the cultures were extracted with ethyl acetate. Dried extracts were added to LB broth, which was used to culture a Salmonella hilA::gfp reporter strain. GFP production was then monitored through flow cytometry. Results shown are the averages of three individual measurements (n = 3), except for the control culture, where six cultures were used (n = 6). Bars indicate the standard errors of the means.

FIG 8

Closely related Clostridium species are involved in the production of the inhibitory molecules. (A) Strains from diverse Clostridium species were tested for biological activity against hilA. Strains were cultured under anaerobic conditions in Trypticase soy broth supplemented with menadione and hemin for at least 2 days, and the cultures were extracted with ethyl acetate. Dried extracts were added to LB broth, which was used to culture a Salmonella hilA::gfp reporter strain. GFP production was then monitored through flow cytometry. Results shown are the averages of three individual measurements (n = 3), except for the control culture, where six cultures were used (n = 6). Bars indicate the standard errors of the means. (B) Production of the inhibitory molecule by select Clostridium strains was confirmed through RT-PCR targeting hilA. Results shown are the averages of 2 to 6 individual measurements, and bars show the standard errors of the means. *, P < 0.03; ns, not significant (P > 0.05).

FIG 9

The bioactive molecules are secreted. Strains of C. citroniae and C. aldenense that showed strong activity against hilA expression were cultured under anaerobic conditions in Trypticase soy broth supplemented with menadione and hemin for at least 2 days, and the cells were separated from the supernatants through centrifugation. Both the cells and the supernatants were then extracted with ethyl acetate, and dried extracts were added to LB broth, which was used to culture the Salmonella hilA::gfp reporter strain for measurements of hilA expression. Results shown are the averages of 3 individual measurements, and bars show the standard errors of the means. ***, P < 0.0003; ns, not significant (P > 0.05).