In vivo transcriptional profiling of Listeria monocytogenes and mutagenesis identify new virulence factors involved in infection

Abstract

Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host-pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, approximately 20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence.

Figure 1. Macroarray validations.

(A) Analysis of the impact of the in vitro culture conditions used as reference. The expression of known and potential virulence factors was analyzed in BHI at 37°C in exponential (BHI log) or stationary (BHI stat) growth phase, or in minimal medium in exponential growth phase (MM log) by real-time RT-PCR, and normalized to expression in mouse spleen. (B) Validation of macroarray data by real-time RT-PCR. Fold changes in in vivo gene expression 48 h p.i. compared to that in BHI were measured by macroarray and real-time RT-PCR, log transformed and compared for correlation analysis. (C) Analysis of the effect of the RNA extraction method on L. monocytogenes gene expression. RNAs from bacteria grown in BHI were prepared using the standard and adapted procedures for RNA extraction. The relative expression of potential virulence genes, cold shock genes and known virulence genes was determined by real-time RT-PCR.

Figure 2. Venn diagrams showing the distribution of the up and down regulated genes at the three in vivo infection time points.

Figure 3. Differentially regulated genes of L. monocytogenes EGDe obtained from temporal transcriptome profiling experiments, classified in functional categories.

Figure 4. In vivo expression of virulence genes.

Expression during mouse spleen infection of the 31 known virulence genes differentially regulated in vivo. A peak of expression was observed for the majority of these virulence genes 48 h p.i. All measurements are relative to culture in exponential phase in BHI. Genes were selected for this analysis when their expression deviated from BHI by at least a factor of 2.0 in at least one time point. The image was produced as described in Materials and Methods. Each gene is represented by a single row of colored boxes; each time point is represented by a single column.

Figure 5. In vivo characterization of new Listeria mutants.

BALB/c mice were intravenously inoculated with 10⁴ CFUs. The number of bacteria in the spleen (A) and liver (B) of mice was determined at 72 h post-infection. A prsA mutant was constructed and used as control. Five mice for each bacterial strain. Statistically significant differences are indicated as compared to wild type strain: ** = P<0.01, *** = P<0.001.

Figure 6. In vitro behavior of L. monocytogenes mutants.

(A) Growth curves of L. monocytogenes EGDe strains in BHI at 37°C with shaking. (B) Intracellular behavior of L. monocytogenes EGDe strains in J774 cultured cells.

Figure 7. In vivo complementation of new Listeria mutants.

BALB/c mice were intravenously inoculated with 10⁴ CFUs. The number of bacteria in the spleen (A) and liver (B) of mice was determined at 72 h post-infection. Five mice for each bacterial strain. Statistically significant differences are indicated as compared to the corresponding mutant for complemented strains: * = P<0.05, ** = P<0.01, *** = P<0.001.