Listeria monocytogenes CadC Regulates Cadmium Efflux and Fine-tunes Lipoprotein Localization to Escape the Host Immune Response and Promote Infection.

Listeria monocytogenes is a major intracellular human foodborne bacterial pathogen. We previously revealed L. monocytogenes cadC as highly expressed during mouse infection. Here we show that L. monocytogenes CadC is a sequence-specific, DNA-binding and cadmium-dependent regulator of CadA, an efflux pump conferring cadmium resistance. CadC but not CadA is required for L. monocytogenes infection in vivo. Interestingly, CadC also directly represses lspB, a gene encoding a lipoprotein signal peptidase whose expression appears detrimental for infection. lspB overexpression promotes the release of the LpeA lipoprotein to the extracellular medium, inducing tumor necrosis factor α and interleukin 6 expression, thus impairing L. monocytogenes survival in macrophages. We propose that L. monocytogenes uses CadC to repress lspB expression during infection to avoid LpeA exposure to the host immune system, diminishing inflammatory cytokine expression and promoting intramacrophagic survival and virulence. CadC appears as the first metal efflux pump regulator repurposed during infection to fine-tune lipoprotein processing and host responses.

Listeria monocytogenes encodes a functional ESX-1 secretion system whose expression is detrimental to in vivo infection.

Bacterial pathogenicity deeply depends on the ability to secrete virulence factors that bind specific targets on host cells and manipulate host responses. The Gram-positive bacterium Listeria monocytogenes is a human foodborne pathogen that remains a serious public health concern. To transport proteins across its cell envelope, this facultative intracellular pathogen engages a set of specialized secretion systems. Here we show that L. monocytogenes EGDe uses a specialized secretion system, named ESX-1, to secrete EsxA, a homolog of the virulence determinants ESAT-6 and EsxA of Mycobacterium tuberculosis and Staphylococcus aureus, respectively. Our data show that the L. monocytogenes ESX-1 secretion system and its substrates are dispensable for bacterial invasion and intracellular multiplication in eukaryotic cell lines. Surprisingly, we found that the EssC-dependent secretion of EsxA has a detrimental effect on L. monocytogenes in vivo infection.

PCR-based screening of targeted mutants for the fast and simultaneous identification of bacterial virulence factors.

Understanding the strategies used by pathogens to infect, survive, and proliferate in their hosts requires the identification of virulence factors. We developed PCR-based screening of targeted mutants to facilitate quick, simultaneous detection of multiple novel bacterial virulence genes. Based on direct PCR screening of pooled targeted mutants, this approach provides a fast and sensitive measure of virulence attenuation while significantly reducing the number of animals and time required. We demonstrate that the careful design of gene-specific primers allows the direct relative quantification of mixed mutants in infected mouse organs. Indeed, we show that the band intensity of the PCR product is directly related to the quantity of the corresponding strain in a pool of mutants. We applied the PCR-based screening of targeted mutants to the murine model of listeriosis and revealed new genes required for full pathogenicity of Listeria monocytogenes, a facultative human intracellular pathogen. PCR-based screening is a simple, useful, and fast technique to test pools of targeted bacterial mutants in vivo, without the requirements for a rigorous purification step, complicated PCR set-up, or special equipment. This approach can be adapted to other bacterial systems, constituting a significant advance in the field of infection biology.

The arsenal of virulence factors deployed by Listeria monocytogenes to promote its cell infection cycle.

Listeria monocytogenes is an intracellular Gram-positive pathogen and the etiological agent of listeriosis, a human food-borne disease potentially fatal for certain risk groups. The virulence of L. monocytogenes is supported by a highly complex and coordinated intracellular life cycle that comprises several crucial steps: host cell adhesion and invasion, intracellular multiplication and motility, and intercellular spread. The completion of each stage is dependent on the orchestrated activity of specialized bacterial factors, in turn tightly controlled by a specific set of regulators. Some virulence factors and modulators also assume an important role in bacterial resistance and evasion to host defense mechanisms. In the last years, the advent of genomics promoted an increasingly prolific identification and functional characterization of new Listeria virulence factors. In this review, we summarize the current knowledge on nearly 50 molecules deployed by L. monocytogenes to promote its cell infection cycle.

LapB, a novel Listeria monocytogenes LPXTG surface adhesin, required for entry into eukaryotic cells and virulence.

Attachment to mucosal surfaces is the initial event in the pathogenesis of the human foodborne pathogen Listeria monocytogenes. By use of comparative genomics, we identified a L. monocytogenes-specific gene, lapB, that encodes an LPXTG surface protein that is absent from nonpathogenic Listeria species. We showed that lapB expression is positively regulated by PrfA, the major transcriptional activator of the virulence genes of Listeria species, and is up-regulated in mouse spleens during infection. We demonstrated that LapB is an SrtA-anchored surface protein required for adhesion to and entry into mammalian cells and for virulence following intravenous or oral inoculation in mice. Our results highlight LapB as a new L. monocytogenes virulence adhesin with a function that is supported by its unique N-terminal domain through the probable interaction with a cellular receptor.

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

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.