Cold-Shock Domain Family Proteins (Csps) Are Involved in Regulation of Virulence, Cellular Aggregation, and Flagella-Based Motility in Listeria monocytogenes

Abstract

Cold shock-domain family proteins (Csps) are highly conserved nucleic acid binding proteins regulating the expression of various genes including those involved in stress resistance and virulence in bacteria. We show here that Csps are involved in virulence, cell aggregation and flagella-based extracellular motility of Listeria monocytogenes. A L. monocytogenes mutant deleted in all three csp genes (ΔcspABD) is attenuated with respect to human macrophage infection as well as virulence in a zebrafish infection model. Moreover, this mutant is incapable of aggregation and fails to express surface flagella or exhibit swarming motility. An evaluation of double csp gene deletion mutant (ΔcspBD, ΔcspAD and ΔcspAB) strains that produce single csp genes showed that there is redundancy as well as functional differences among the three L. monocytogenes Csps in their contributions to virulence, cellular aggregation, flagella production, and swarming motility. Protein and mRNA expression analysis further showed impaired expression of key virulence and motility genes in the csp mutants. Our observations at protein and mRNA level suggest Csp-dependent expression regulation of these genes at transcriptional and post-transcriptional levels. In a mutant lacking all csp genes (ΔcspABD) as well as those possessing single csp genes (ΔcspBD, ΔcspAD, and ΔcspAB) we detected reduced levels of proteins or activity as well as transcripts from the prfA, hly, mpl, and plcA genes suggesting a Csp-dependent transcriptional regulation of these genes. These csp mutants also had reduced or completely lacked ActA proteins and cell surface flagella but contained elevated actA and flaA mRNA levels compared to the parental wild type strain suggesting Csp involvement in post-transcriptional regulation of these genes. Overall, our results suggest that Csps contribute to the expression regulation of virulence and flagella-associated genes thereby promoting host pathogenicity, cell aggregation and flagella-based motility processes in L. monocytogenes.

Keywords: Csps; Listeria monocytogenes; cellular aggregation; flagella; swarming motility; virulence.

Figure 1

Csp loss impairs survival and growth of L. monocytogenes in human macrophages. (A) Bar charts depicting percentage change in intracellular bacterial CFUs for the WT and csp mutants of L. monocytogenes EGDe at 6 (t₆) and 24 (t₂₄) h post infection (hpi) of human derived THP-1 macrophages. The percentage changes (CFUs) are expressed relative to the intracellular bacteria CFUs determined at time point 0 (t₀). Data showing the means and standard deviations derived from three independent biological experiments are presented. ^*p < 0.05 based on one-way ANOVA and Tukey post-hoc test comparison relative to the WT strain as well as between the different csp mutants. (B) Representative images from the fluorescent microscopic analysis based monitoring of the intracellular fates of GFP expressing WT and csp mutants of L. monocytogenes EGDe strain during THP-1 macrophage infections at 0(t₀), 6(t₆), and 24(t₂₄) hpi.

Figure 2

Csp loss attenuates virulence and reduces bacterial loads of L. monocytogenes in zebrafish embryos. (A) Survival curves were generated for zebrafish embryos (n = 5 per L. monocytogenes strain and time-point) infected (100 CFU per embryo) with WT and csp mutants of L. monocytogenes EGDe and monitored over 3 days. Zebrafish embryos rapidly succumbed to the WT strain, while those infected with the csp mutants died later or were not killed at all. Survival curves and trends were significantly different (log-rank test p < 0.05) between WT and different csp mutant strains. (B) Bacterial load quantifications were conducted at 1 and 2 days post infection in zebrafish embryos (n = 30) infected with WT and csp mutant strains. Data showing the mean and standard deviations of three independent biological experiments are presented. ^*p < 0.05 based on one-way ANOVA and Tukey post-hoc test comparison relative to the WT strain.

Figure 3

Impact of Csp on the expression of virulence proteins. (A) Representative image from the qualitative assessment of PI-PLC activity (turbid zone around streaked bacteria) WT and csp mutants of L. monocytogenes EGDe grown on ALOA plates. (B,C) Representative Western blot images and densitometry based quantification of the PrfA, LLO, Mpl, ActA proteins in BHI cultured (16 h at 37°C and 150 rpm) and THP-1 macrophage grown (6 hpi) WT and csp mutants of L. monocytogenes EGDe. Virulence proteins densities were normalized to P60 levels and expressed as a percentage relative to WT strain levels. Data showing the means and standard deviations of three independent biological experiments are presented. Bars denoting relative protein abundance levels that share a letter are not significantly different, whereas those marked with different letters are significantly different compared to each other (p < 0.05 based on one-way ANOVA and Tukey post-hoc test pairwise comparison of all the strains).

Figure 4

Impact of Csp loss on virulence gene mRNA levels. Quantification of plcA, prfA, hly, mpl, and actA mRNAs using qRT-PCR in L. monocytogenes EGDe WT and csp mutant strains that were cultured in BH broth (16 h at 37°C and 150 rpm) and grown in THP-1 macrophage (6 hpi). Relative quantities (RQ) of plcA, prfA, hly, and actA mRNA levels were normalized to 16S rRNA and expressed relative to those in an EGDe WT strain based mRNA calibrator sample. Data showing the means and standard deviations of three independent biological experiments are presented. Bars denoting relative mRNA abundance levels that share a letter are not significantly different, whereas those marked with different letters are significantly different compared to each other (P < 0.05 based on one-way ANOVA and Tukey post-hoc test pairwise comparison of all the strains).

Figure 5

Aggregation analysis in stationary phase BHI cultures of L. monocytogenes EGDe WT and csp mutant strains incubated for 24 h at 37°C without shaking. (A) Bacterial sedimentation observed at 24 h and (B) time resolved aggregation kinetics based on changes in the culture supernatant OD₆₀₀ during 24 h of static incubation.

Figure 6

Impact of Csp loss on swarming motility, surface flagellation and flaA mRNA levels. Representative images of swarming motility assessment from bacteria cultures spotted on 0.25% BHI agar and incubated at 25°C for 48 h and transmission electron microscopy analysis for bacterium-associated flagella, as well as qRT-PCR quantification of flaA mRNA in EGDe WT and csp mutants cultivated to stationary phase in BHI at 25°C. Relative quantity (RQ) of flaA mRNA levels normalized 16S rRNA and expressed relative to an EGDe WT strain derived mRNA calibrator sample are shown. Data showing the means (bars) and standard deviations (error bars) of three independent biological experiment are presented. ^*p < 0.05 based on one-way ANOVA and Tukey post-hoc test comparison relative to the WT strain. Presence or lack of flagella on cell surfaces is highlighted by red arrows.