The alternative sigma factor sigma B and the virulence gene regulator PrfA both regulate transcription of Listeria monocytogenes internalins

Abstract

Some Listeria monocytogenes internalins are recognized as contributing to invasion of mammalian tissue culture cells. While PrfA is well established as a positive regulator of L. monocytogenes virulence gene expression, the stress-responsive sigma(B) has been recognized only recently as contributing to expression of virulence genes, including some that encode internalins. To measure the relative contributions of PrfA and sigma(B) to internalin gene transcription, we used reverse transcription-PCR to quantify transcript levels for eight internalin genes (inlA, inlB, inlC, inlC2, inlD, inlE, inlF, and inlG) in L. monocytogenes 10403S and in isogenic Delta prfA, Delta sigB, and Delta sigB Delta prfA strains. Strains were grown under defined conditions to produce (i) active PrfA, (ii) active sigma(B) and active PrfA, (iii) inactive PrfA, and (iv) active sigma(B) and inactive PrfA. Under the conditions tested, sigma(B) and PrfA contributed differentially to the expression of the various internalins such that (i) both sigma(B) and PrfA contributed to inlA and inlB transcription, (ii) only PrfA contributed to inlC transcription, (iii) only sigma(B) contributed to inlC2 and inlD transcription, and (iv) neither sigma(B) nor PrfA contributed to inlF and inlG transcription. inlE transcript levels were undetectable. The important role for sigma(B) in regulating expression of L. monocytogenes internalins suggests that exposure of this organism to environmental stress conditions, such as those encountered in the gastrointestinal tract, may activate internalin transcription. Interplay between sigma(B) and PrfA also appears to be critical for regulating transcription of some virulence genes, including inlA, inlB, and prfA.

FIG. 1.

Normalized, log-transformed transcript levels of opuCA and gadA (A) and of prfA and plcA (B) for L. monocytogenes parent strain 10403S (wild type) as well as isogenic ΔsigB, ΔprfA, and ΔsigB ΔprfA strains cultured under four different conditions, including (i) growth in BHI with 0.2% charcoal for 120 min, (ii) growth in BHI with 25 mM cellobiose for 120 min, (iii) growth in BHI with 0.2% charcoal for 120 min with exposure to 0.3 M NaCl for the last 10 min, and (iv) growth in BHI with 25 mM cellobiose for 120 min with exposure to 0.3 M NaCl for the last 10 min. Data represent average normalized and log-transformed transcript levels for three independent replicates (i.e., three RNA isolations performed on different days); error bars represent one standard deviation each. Tukey's multiple-comparison procedure was used to determine whether transcript levels for a gene and a given condition differ among the four different strains; bars labeled with different letters indicate that the transcript levels differ significantly (P < 0.05), while bars labeled with the same letter indicate that the transcript levels do not differ significantly.

FIG. 2.

Normalized, log-transformed transcript levels of inlA and inlB for L. monocytogenes parent strain 10403S (wild type) as well as isogenic ΔsigB, ΔprfA, and ΔsigB ΔprfA strains cultured under the four different conditions outlined in Materials and Methods and in the legend for Fig. 1. Data represent average normalized and log-transformed transcript levels for three independent replicates (i.e., three RNA isolations performed on different days); error bars represent one standard deviation each. Tukey's multiple-comparison procedure was used to determine whether transcript levels for a gene and a given condition differ among the four different strains; bars labeled with different letters indicate that the transcript levels differ significantly (P < 0.05), while bars labeled with the same letter indicate that the transcript levels do not differ significantly.

FIG. 3.

Normalized, log-transformed transcript levels for inlC for L. monocytogenes 10403S parent strain (wild type) as well as isogenic ΔsigB, ΔprfA, and ΔsigB ΔprfA strains cultured under the four different conditions outlined in Materials and Methods and in the legend for Fig. 1. Data represent average normalized and log-transformed transcript levels for three independent replicates (i.e., three RNA isolations performed on different days); error bars represent one standard deviation each. Tukey's multiple-comparison procedure was used to determine whether transcript levels for a gene and a given condition differ among the four different strains; bars labeled with different letters indicate that the transcript levels differ significantly (P < 0.05), while bars labeled with the same letter indicate that the transcript levels do not differ significantly. NS indicates that no individual transcript levels were significantly different by Tukey's multiple-comparison procedure.

FIG. 4.

Normalized, log-transformed transcript levels of inlC2 and inlD for L. monocytogenes parent strain 10403S (wild type) as well as isogenic ΔsigB, ΔprfA, and ΔsigB ΔprfA strains cultured under the four different conditions outlined in Materials and Methods and the legend for Fig. 1. Data represent average normalized and log-transformed transcript levels for three independent replicates (i.e., three RNA isolations performed on different days); error bars represent one standard deviation each. Tukey's multiple-comparison procedure was used to determine whether transcript levels for a gene and a given condition differ among the four different strains; bars labeled with different letters indicate that the transcript levels differ significantly (P < 0.05), while bars labeled with the same letter indicate that the transcript levels do not differ significantly.

FIG. 5.

Surface localization of L. monocytogenes InlB by immunoelectron microscopy using whole cells of L. monocytogenes 10403S (A) and the ΔprfA (B), ΔsigB (C), and ΔinlB (D) strains. Arrows indicate the bacterial cell surface and point towards the cytoplasm. The average numbers of gold particles per μm² for each strain were estimated to be 126 ± 38 (10403S); 91 ± 16 (ΔprfA strain); 32 ± 12 (ΔsigB strain); and 2 ± 5 (ΔinlB strain).