Use of a continuous-flow anaerobic culture to characterize enteric virulence gene expression - PubMed (original) (raw)

Use of a continuous-flow anaerobic culture to characterize enteric virulence gene expression

Fernando Ruiz-Perez et al. Infect Immun. 2004 Jul.

Abstract

We developed an in vitro culture method to characterize the expression of bacterial genes under conditions mimicking the colonic environment. Our culture system (the intestinal simulator) comprised a continuous-flow anaerobic culture which was inoculated with fecal samples from healthy volunteers. As a test organism, we employed enteroaggregative Escherichia coli (EAEC), an emerging diarrheal pathogen that is thought to cause infection in both the small and large intestines. After the simulator culture achieved equilibrium conditions, we inoculated the system with prototype EAEC strain 042 and assessed the expression of three EAEC virulence-related genes. We focused particularly on expression of aggR, which encodes a global transcriptional regulator of EAEC virulence factors, and two AggR-regulated genes. By using real-time quantitative reverse transcription-PCR, we showed that aggR expression in the simulator is increased 3- to 10-fold when 042 is grown under low-pH (5.5 to 6.0) conditions, compared with results with neutral pH (7.0). Interestingly, however, this effect was seen only when the strain was grown in the presence of commensal bacteria. We also found that expression of aggR is 10- to 20-fold higher at low NaCl concentrations, and this effect was also observed only in the presence of commensal bacteria. Using coculture and conditioned-media experiments, we identified specific strains of Enterococcus and Clostridium that upregulated aggR expression; in contrast, strains of Lactobacillus and Veillonella downregulated aggR expression. Our data provide new insights into regulation of virulence genes in EAEC and suggest the utility of intestinal simulation cultures in characterizing enteric gene regulation.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Main bacterial populations present in precultures of stool samples and after 7 days' growth in the simulator. The simulator culture was inoculated with 60 ml of precultured stool from a single subject and was incubated under continuous flow for 7 days. The dominant culturable bacterial populations were determined by biochemical analysis. The results for three representative experiments (S-1, S-2, and S-3) are shown.

FIG. 2.

FIG. 2.

Dominant culturable bacterial populations present in the simulator at different pH setpoints. EAEC 042 was inoculated in the intestinal simulator, and the system was set to maintain pH 7.0, 6.5, 6.0, or 5.5 for 7 days at each pH, at which time dominant bacterial populations were enumerated. Three representative experiments are shown, using stools from different subjects.

FIG. 3.

FIG. 3.

Expression of aggR, aafD, and aap from EAEC 042 grown at different pH setpoints in the simulator culture. (A) After equilibration of the system for 72 h, EAEC 042 was inoculated at a final concentration 107 CFU/ml, and incubation was continued for 4 to 18 h. Real-time qRT-PCR was performed for EAEC virulence genes as described in Materials and Methods. Data are reported in arbitrary units of transcript abundance normalized against cat transcript abundance (quantity of target gene/quantity of cat gene). Three representative experiments (E-1, E-2, and E-3) are shown (A). For E-1, samples were only withdrawn 18 h after 042 inoculation. For E-2 and E-3, fermenter samples before EAEC 042 inoculation were included as negative controls. (B) Effect of increasing and decreasing pH on cat, aggR, aafD, and aap expression in the simulator system. The pH setpoint was increased from 5.5 to a maximum of 7.0 and then reduced to 6.0. The system spent at least 24 h at each setpoint. Real- Time qRT-PCR was performed as for Fig. 3A. Data are expressed in arbitrary units of transcript abundance normalized against cat transcript abundance.

FIG. 4.

FIG. 4.

Levels of aggR, aafD, and aap expression in EAEC 042 grown at pH 7.0 (A) or 6.0 (B) in the intestinal simulator. EAEC 042 was grown in the intestinal simulator at the respective pH setpoint, and the expression levels of the three virulence genes were compared directly by real-time qRT-PCR. Data are expressed in arbitrary units of transcript abundance normalized against cat.

FIG. 5.

FIG. 5.

Effect of the NaCl concentration on EAEC virulence gene expression in the intestinal simulator. (A) EAEC 042 was inoculated once at the beginning of the experiment and grown at different NaCl concentrations for 7 days in the simulator system including fecal flora. (B) EAEC 042 was inoculated anaerobically in simulator medium under continuous flow at various NaCl concentrations, in the absence of fecal commensals. After overnight growth at the osmotic setpoint, total RNA was isolated and cDNA was synthesized and analyzed for cat, aggR, aafD, and aap expression by real-time qRT-PCR. Data represent transcript abundance from three separate experiments normalized against cat transcript.

FIG. 6.

FIG. 6.

Expression of EAEC virulence genes at various pH setpoints in the absence of commensal flora. Experiments were performed as for Fig. 3, except that no fecal bacteria were added to the system.

FIG. 7.

FIG. 7.

Effects of commensal flora on aggR expression. EAEC 042 was anaerobically cocultured (A) with specific strains of intestinal bacteria in fermenter medium at pH 6.0 or cultured in media preconditioned (B) by growth of the same bacterial strains. After 18 h of growth in either system, the level of aggR expression was determined by real-time qRT-PCR. Data are expressed as fold increases or decreases of aggR transcript in coculture or preconditioned medium, normalized against aggR abundance in pure cultures of 042 or medium preconditioned by growth of 042.

Similar articles

Cited by

References

    1. Alander, M., I. De Smet, L. Nollet, W. Verstraete, A. Von Wright, and T. Mattila-Sandholm. 1999. The effect of probiotic strains on the microbiota of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). Int. J. Food Microbiol. 46:71-79. - PubMed
    1. Behrens, M., J. Sheikh, and J. P. Nataro. 2002. Regulation of the overlapping pic/set locus in Shigella flexneri and enteroaggregative Escherichia coli. Infect. Immun. 70:2915-2925. - PMC - PubMed
    1. Benjamin, M. M., and A. R. Datta. 1995. Acid tolerance of enterohemorrhagic Escherichia coli. Appl. Environ. Microbiol. 61:1669-1672. - PMC - PubMed
    1. Berg, Rodney, D. 1996. The indigenous gastrointestinal microflora. Trends Microbiol. 4:430-435. - PubMed
    1. De Boever, P., B. Deplancke, and W. Verstraete. 2000. Fermentation by gut microbiota cultured in a simulator of the human intestinal microbial ecosystem is improved by supplementing a soygerm powder. J. Nutr. 130:2599-2606. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources