The Production of Listeriolysin O and Subsequent Intracellular Infections by Listeria monocytogenes Are Regulated by Exogenous Short Chain Fatty Acid Mixtures

Abstract

Listeria monocytogenes is a foodborne pathogen capable of secreting listeriolysin O (LLO), a pore-forming toxin encoded by the hly gene. While the functions of LLO have been studied extensively, how the production of LLO is modulated by the intestinal environment, devoid of oxygen and enriched in short chain fatty acids (SCFAs), is not completely understood. Using L. monocytogenes strain 10403s, we found that hly transcription was moderately decreased by aerobic SCFA exposures but significantly increased by anaerobic SCFA exposures. Moreover, aerobic, but not anaerobic, exposure to low levels of SCFAs resulted in a significantly higher LLO activity. These results demonstrated that transcriptional and post-transcriptional regulations of LLO production were separately modulated by SCFAs and were responsive to oxygen levels. Examining isogenic mutants revealed that PrfA and SigB play a role in regulating LLO production in response to SCFAs. Effects of SCFAs were also present in the cardiotropic strain 07PF0776 but distinctly different from those in strain 10403s. For both strains, prior exposures to SCFAs altered intracellular infections in Caco-2 and RAW264.7 cells and the plaque sizes in L fibroblasts, a result confirming the ability of L. monocytogenes to adapt to SCFAs in ways that impact its subsequent infection outcomes.

Keywords: LLO; Listeria monocytogenes; aerobic; anaerobic; hly; short chain fatty acids.

Figure 1

Fatty acid composition (in percentages) varies in L. monocytogenes strain 10403s grown in BHI supplemented with or without M_lo or M_hi SCFA mixtures. Averages of 2 independent replicates were plotted. “Straight” indicates straight chain fatty acids. “Iso” and “Anteiso” indicate iso- and anteiso-branched chain fatty acids. Details of specific fatty acids are listed in Table 3 and Table 4.

Figure 2

Supplementations of SCFAs alter hly transcription levels and supernatant LLO activity. Using an hly transcriptional reporter, relative fluorescence units (RFU) were obtained from GUS reporter assays and normalized to sample protein concentrations (µg/mL) to assess the level of hly transcription in bacteria grown in BHI supplemented with or without SCFA mixtures (A), acetate (B), propionate (C), or butyrate (D). Averages of 9 replicates from three independent experiments were plotted with error bars representing SEM. Significant differences between samples are indicated by: * p < 0.05, ** p < 0.01, *** p < 0.001. The impact of aerobic or anaerobic SCFA supplementations on supernatant listeriolysin O (LLO) activity (E,F). “BD” represents values below detection or a level lower than half complete lysis, thus unable to calculate hemolytic units. For anaerobic cultures, hemolytic assay absorbance values, normalized by culture optical densities, were shown to highlight differences among samples (F). Averages of three replicates were plotted with error measurements indicating SD. Results were representative of at least three independent experiments. Significant difference between samples are indicated by: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Supplementations of SCFAs alter supernatant LLO activity in the PrfA* (A) and ΔsigB (B) mutants of strain 10403s. The impact of aerobic or anaerobic SCFA supplementations on supernatant LLO activity was determined in overnight cultures. Hemolytic units were normalized by overnight culture optical densities and then compared to the no supplementation control in each strain. “BD” represents values below detection or a level lower than half complete lysis, where hemolytic units could not be calculated. Averages of three independent experiments (n = 9) were plotted with error measurements indicating SEM. Significant difference between samples are indicated by: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Supplementations of SCFA mixtures alter supernatant LLO activity in strains 10403s and 07PF0776. The impact of aerobic (A) or anaerobic (B) SCFA supplementations on supernatant LLO activity was determined in both strain 10403s and the cardiotropic strain 07PF0776. Hemolytic units were normalized by culture optical densities. “BD” represents values below detection or a level lower than half complete lysis, where hemolytic units could not be calculated. Averages of three replicates were plotted with error measurements indicating SD. * p < 0.05, ** p < 0.01, *** p < 0.001. BD signifies below detection. Results represent 3 independent experiments.

Figure 5

Supplementations of SCFA mixtures alter L. monocytogenes invasion in non-polarized Caco-2 intestinal epithelial. The impact of aerobic or anaerobic pre-exposure to SCFAs on L. monocytogenes epithelial cell invasion was determined for strain 10403s (A), the cardiotropic strain 07PF0776 (B), and the mutants PrfA* (C) and ΔsigB (D). PrfA* and ΔsigB are isogenic strains of wildtype strain 10403s. Cells were infected with a MOI of 10 and lysed at 2 hpi to determine L. monocytogenes invasion. Averages of three replicates were plotted with error measurements indicating SD. * p < 0.05, ** p < 0.01, *** p < 0.001. BD signifies below detection. Results represent 2 independent experiments.

Figure 6

Supplementations of SCFA mixtures alter L. monocytogenes intracellular survival in RAW264.7 macrophages. The impact of aerobic or anaerobic pre-exposure to SCFAs on L. monocytogenes intracellular survival in macrophages was determined for strain 10403s (A), the cardiotropic strain 07PF0776 (B), and the mutants PrfA* (C) and ΔsigB (D). PrfA* and ΔsigB are isogenic strains of wildtype strain 10403s. Cells were infected with a MOI of 10 and lysed at 2 hpi to determine L. monocytogenes early intracellular survival. Averages of three replicates were plotted with error measurements indicating SD. * p < 0.05, ** p < 0.01, *** p < 0.001. BD signifies below detection. Results represent 3 independent experiments.

Figure 7

SCFA pre-exposures by L. monocytogenes prior to infections alter plaque sizes in infected L fibroblasts. The impact of aerobic or anaerobic SCFA pre-exposures by L. monocytogenes on plaque sizes in subsequent infections in L-cell monolayers was determined for both strains 10403s (A) and 07PF0776 (B) and 10430s isogenic mutants PrfA* (C) and ΔsigB (D). Plaques were visualized two days post infection. Percent plaque sizes were calculated based on comparisons to the average plaque sizes by cells infected by bacteria without SCFA pre-exposures. Averages of 30 plaques were plotted with error measurements indicating SD. Results represent 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.