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Antivirulence activity of the human gut metabolome.

Antunes LC, McDonald JA, Schroeter K, Carlucci C, Ferreira RB, Wang M, Yurist-Doutsch S, Hira G, Jacobson K, Davies J, Allen-Vercoe E, Finlay BB - MBio (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.

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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).
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fig8: 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).

Mentions: The results described above established that the inhibitory activity is produced by the intestinal microbiota in the absence of any host factors. In order to determine if individual microbial isolates can produce the bioactive molecule or if a community of microbes is required for such activity, we screened individual isolates from one of the chemostat microbial communities used (donor A, Table 2) for specific strains with inhibitory activity against hilA. This was done using a reporter strain containing a fusion between the promoter of hilA and gfp in pFPV25 (28, 29). The bacterial strains were grown in culture medium, as described in Materials and Methods, and extracted with ethyl acetate. Extracts were dried and resuspended in Luria-Bertani (LB) broth. Salmonella was then grown in LB medium supplemented with these extracts or with an ethyl acetate extract of culture medium alone to the late logarithmic growth phase, and green fluorescent protein (GFP) production was tested using flow cytometry. As can be seen in Fig. 7, most microbial isolates showed little to no inhibitory activity against hilA. However, a specific strain of Clostridium citroniae caused strong inhibition of invasion gene expression. Therefore, this determined not only that the microbiota is involved in the production of the active molecule but also that a single microbial species can produce the biological activity in the laboratory. We tested several other C. citroniae strains as well as strains of closely related species for inhibitory activity using this reporter system and found that multiple strains of C. citroniae were active. In addition, multiple isolates of Clostridium aldenense also produced active molecules, suggesting that a closely related clade within the Clostridiales cluster XIVa (otherwise known as the Lachnospiraceae family) is involved in this phenomenon (Fig. 8A). We also determined hilA mRNA levels using RT-PCR for selected C. citroniae and C. aldenense strains and confirmed that extracts from cultures of these microbes showed strong inhibitory effects on the expression of Salmonella invasion genes (Fig. 8B).


Antivirulence activity of the human gut metabolome.

Antunes LC, McDonald JA, Schroeter K, Carlucci C, Ferreira RB, Wang M, Yurist-Doutsch S, Hira G, Jacobson K, Davies J, Allen-Vercoe E, Finlay BB - MBio (2014)

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).
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Related In: Results  -  Collection

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fig8: 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).
Mentions: The results described above established that the inhibitory activity is produced by the intestinal microbiota in the absence of any host factors. In order to determine if individual microbial isolates can produce the bioactive molecule or if a community of microbes is required for such activity, we screened individual isolates from one of the chemostat microbial communities used (donor A, Table 2) for specific strains with inhibitory activity against hilA. This was done using a reporter strain containing a fusion between the promoter of hilA and gfp in pFPV25 (28, 29). The bacterial strains were grown in culture medium, as described in Materials and Methods, and extracted with ethyl acetate. Extracts were dried and resuspended in Luria-Bertani (LB) broth. Salmonella was then grown in LB medium supplemented with these extracts or with an ethyl acetate extract of culture medium alone to the late logarithmic growth phase, and green fluorescent protein (GFP) production was tested using flow cytometry. As can be seen in Fig. 7, most microbial isolates showed little to no inhibitory activity against hilA. However, a specific strain of Clostridium citroniae caused strong inhibition of invasion gene expression. Therefore, this determined not only that the microbiota is involved in the production of the active molecule but also that a single microbial species can produce the biological activity in the laboratory. We tested several other C. citroniae strains as well as strains of closely related species for inhibitory activity using this reporter system and found that multiple strains of C. citroniae were active. In addition, multiple isolates of Clostridium aldenense also produced active molecules, suggesting that a closely related clade within the Clostridiales cluster XIVa (otherwise known as the Lachnospiraceae family) is involved in this phenomenon (Fig. 8A). We also determined hilA mRNA levels using RT-PCR for selected C. citroniae and C. aldenense strains and confirmed that extracts from cultures of these microbes showed strong inhibitory effects on the expression of Salmonella invasion genes (Fig. 8B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.

Show MeSH
Related in: MedlinePlus