RNA Helicase Important for Listeria monocytogenes Hemolytic Activity and Virulence Factor Expression

Abstract

RNA helicases have been shown to be important for the function of RNA molecules at several levels, although their putative involvement in microbial pathogenesis has remained elusive. We have previously shown that Listeria monocytogenes DExD-box RNA helicases are important for bacterial growth, motility, ribosomal maturation, and rRNA processing. We assessed the importance of the RNA helicase Lmo0866 (here named CshA) for expression of virulence traits. We observed a reduction in hemolytic activity in a strain lacking CshA compared to the wild type. This phenomenon was less evident in strains lacking other RNA helicases. The reduced hemolysis was accompanied by lower expression of major listerial virulence factors in the ΔcshA strain, mainly listeriolysin O, but also to some degree the actin polymerizing factor ActA. Reduced expression of these virulence factors in the strain lacking CshA did not, however, correlate with a decreased level of the virulence regulator PrfA. When combining the ΔcshA knockout with a mutation creating a constitutively active PrfA protein (PrfA*), the effect of the ΔcshA knockout on LLO expression was negated. These data suggest a role for the RNA helicase CshA in posttranslational activation of PrfA. Surprisingly, although the expression of several virulence factors was reduced, the ΔcshA strain did not demonstrate any reduced ability to infect nonphagocytic cells compared to the wild-type strain.

FIG 1

Hemolytic activity at pH 5.5. The indicated strains were grown at 37°C to an OD₆₀₀ of 0.9 in pH-adjusted BHI (pH 5.5) before the supernatant was removed. Filtered supernatants were added at a 1:1 ratio to a 10% suspension of red blood cells, followed by incubation for 3 h at 37°C. The absorbance was measured at 541 nm. The figure shows the hemolytic activity of the indicated strains in percentages relative to the wild type (100%) with the standard deviations. All samples were compared to the wild type using a two-tailed Student t test (**, P < 0.01; ***, P < 0.001).

FIG 2

Hemolytic activity at different pH levels. The indicated strains were grown at 37°C to an OD₆₀₀ of 0.9 in BHI of pH 7.6 (A) or pH 5.5 (B) before the supernatant was removed. Filtered supernatants were added to a 10% suspension of red blood cells at a 1:1 ratio and incubated for 3 h at 37°C. The absorbance was measured at 541 nm. The figure shows the hemolytic activity of the indicated strains relative to the wild type (100%) with the standard deviations. All samples were compared to the wild type using a two-tailed Student t test (***, P < 0.001).

FIG 3

Virulence factor expression in different strain backgrounds. (A) The indicated strains were grown at 37°C in pH-adjusted BHI (pH 5.5) to an OD₆₀₀ of 0.9 before protein extraction, SDS-PAGE separation, and Western blot analysis. In the upper panel, the expression levels of LLO were analyzed using LLO-specific antibodies in samples of protein precipitated by trichloroacetic acid from filtered culture supernatants. In the middle panel, the expression levels of ActA were examined by extraction of boiled bacterial cells in Laemmli buffer using ActA-specific antibodies. In the lower panel, the PrfA level was examined from whole-cell fractions using PrfA-specific antibodies. (B) The expression of LLO, ActA, and PrfA, respectively, was quantified from panel A, and the results are shown relative to the wild type (WT) for EGDe or ΔcshA or to the WT::pIMK3 strain for WT::pIMK3, ΔcshA::pIMK3, or ΔcshA::pcshA, respectively. WT and WT::pIMK3 were arbitrarily set to 1.0. Error bars show the standard deviations. Statistics show two-tailed Student t test determinations (*, P < 0.05; ***, P < 0.001).

FIG 4

Glutathione levels and virulence factor expression in various strain backgrounds. (A) Wild-type or ΔcshA strains were grown at 37°C in pH-adjusted BHI (pH 5.5) to an OD₆₀₀ of 0.9 before the intracellular glutathione concentration was determined. The glutathione concentration in the wild-type strain was arbitrarily set to 100% (n = 3). For statistics, the sample from the ΔcshA strain was compared to the WT using a two-tailed Student t test (ns, not significant). (B) The indicated strains were grown at 37°C in pH-adjusted BHI (pH 5.5) until an OD₆₀₀ of ∼0.9 before protein extraction, SDS-PAGE separation, and Western blot analysis. The expression levels of LLO (upper panel) or P60 (control, lower panel) were analyzed, using LLO- or P60-specific antibodies, in samples of protein precipitated by trichloroacetic acid from filtered culture supernatant (n = 3). (C) LLO expression (from panel A) was quantified in the indicated strains and related to P60 expression. LLO expression in EGDe was arbitrarily set to 1. Error bars show the standard deviations. For statistics, all samples were compared to EGDe using a two-tailed Student t test (*, P < 0.05; ***, P < 0.001; ns, not significant).

FIG 5

Infection assay. (A and C) Caco-2 cells were infected with wild-type, ΔprfA, Δhly, or ΔcshA strains for the indicated time points before cells were lysed, and bacteria were plated and counted. The infectivity of the ΔprfA, Δhly, and ΔcshA strains are shown relative to the wild-type strain (100%). Error bars show the standard deviations. All samples were compared to the wild-type using a two-tailed Student t test (***, P < 0.001). (B and D) Phase-contrast and fluorescence microscopy. The right panels show Caco-2 cells infected with the indicated strains for 1 h (B) or 8 h (D) and stained for Listeria (green) or cell nuclei (blue). The left panels show phase-contrast images of right panels. Bars, 10 μm.

FIG 6

Infection assay. (A and C) J774 cells were infected with wild-type, ΔprfA, or ΔcshA strains for the indicated time points before cells were lysed, and bacteria were plated and counted. The infectivity of the ΔprfA and ΔcshA strains is shown relative to the wild-type strain (100%). Error bars show the standard deviations. All samples were compared to the wild type using a two-tailed Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B and D) Phase-contrast and fluorescence microscopy. The right panels show J774 cells infected with the indicated strains for 1 h (B) or 8 h (D) and stained for Listeria (green) or cell nuclei (blue). The left panels show phase-contrast images of right panels. Bars, 10 μm.