ActA is required for crossing of the fetoplacental barrier by Listeria monocytogenes.

The facultative intracellular bacterial pathogen Listeria monocytogenes induces severe fetal infection during pregnancy. Little is known about the molecular mechanisms allowing the maternofetal transmission of bacteria. In this work, we studied fetoplacental invasion by infecting mice with various mutants lacking virulence factors involved in the intracellular life cycle of L. monocytogenes. We found that the placenta was highly susceptible to bacteria, including avirulent bacteria, such as an L. monocytogenes mutant with an hly deletion (DeltaLLO) and a nonpathogenic species, Listeria innocua, suggesting that permissive trophoblastic cells, trapping bacteria, provide a protective niche for bacterial survival. The DeltaLLO mutant, which is unable to escape the phagosomal compartment of infected cells, failed to grow in the trophoblast tissue and to invade the fetus. Mutant bacteria with inlA and inlB deletion (DeltaInlAB) grew in the placenta and fetus as well as did the wild-type virulent stain (EGDwt), indicating that in the murine model, internalins A and B are not involved in fetoplacental invasion by L. monocytogenes. Pregnant mice were then infected with an actA deletion (DeltaActA) strain, a virulence-attenuated mutant that is unable to polymerize actin and to spread from cell to cell. With the DeltaActA mutant, fetal infection occurs, but with a significant delay and restriction, and it requires a placental bacterial load 2 log units higher than that for the wild-type virulent strain. Definitive evidence for the role of ActA was provided by showing that a actA-complemented DeltaActA mutant was restored in its capacity to invade fetuses. ActA-mediated cell-to-cell spreading plays a major role in the vertical transmission of L. monocytogenes to the fetus in the murine model.

Lessons from signature-tagged mutagenesis on the infectious mechanisms of pathogenic bacteria.

Studies on the genetic basis of bacterial pathogenicity have been undertaken for almost 30 years, but the development of new genetic tools in the past 10 years has considerably increased the number of identified virulence factors. Signature-tagged mutagenesis (STM) is one of the most powerful general genetic approaches, initially developed by David Holden and colleagues in 1995, which has now led to the identification of hundreds of new genes requested for virulence in a broad range of bacterial pathogens. We have chosen to present in this review, the most recent and/or most significant contributions to the understanding of the molecular mechanisms of bacterial pathogenicity among over 40 STM screens published to date. We will first briefly review the principle of the method and its major technical limitations. Then, selected studies will be discussed where genes implicated in various aspects of the infectious process have been identified (including tropism for specific host and/or particular tissues, interactions with host cells, mechanisms of survival and persistence within the host, and the crossing of the blood brain barrier). The examples chosen will cover intracellular as well as extracellular Gram-negative and Gram-positive pathogens.

Identification of the agr locus of Listeria monocytogenes: role in bacterial virulence.

Listeria monocytogenes is a gram-positive facultative intracellular food-borne pathogen that can cause severe infections in humans and animals. We have recently adapted signature-tagged transposon mutagenesis (STM) to identify genes involved in the virulence of L. monocytogenes. A new round of STM allowed us to identify a new locus encoding a protein homologous to AgrA, the well-studied response regulator of Staphylococcus aureus and part of a two-component system involved in bacterial virulence. The production of several secreted proteins was modified in the agrA mutant of L. monocytogenes grown in broth, indicating that the agr locus influenced protein secretion. Inactivation of agrA did not affect the ability of the pathogen to invade and multiply in cells in vitro. However, the virulence of the agrA mutant was attenuated in the mouse (a 10-fold increase in the 50% lethal dose by the intravenous route), demonstrating for the first time a role for the agr locus in the virulence of L. monocytogenes.

Capacity of ivanolysin O to replace listeriolysin O in phagosomal escape and in vivo survival of Listeria monocytogenes.

Listeriolysin O (LLO, hly-encoded) is a major virulence factor secreted by the pathogen Listeria monocytogenes. The amino acid sequence of LLO shows a high degree of similarity with that of ivanolysin O (ILO), the cytolysin secreted by the ruminant pathogen Listeria ivanovii. Here, it was tested whether ILO could functionally replace LLO by expressing the gene encoding ILO under the control of the hly promoter, in an hly-deleted strain of L. monocytogenes. It is shown that ILO allows efficient phagosomal escape of L. monocytogenes in both macrophages and hepatocytes. Moreover, expression of ILO is not cytotoxic and promotes normal intracellular multiplication. In vivo, the ILO-expressing strain can multiply and persist for several days in the liver of infected mice but is unable to survive in the spleen. This work underscores the key role played by the cytolysin in the virulence of pathogenic Listeria.

Functional assembly of two membrane-binding domains in listeriolysin O, the cytolysin of Listeria monocytogenes.

Listeriolysin O (LLO) is a major virulence factor secreted by the pathogenic Listeria monocytogenes and acts as pore-forming cytolysin. Based on sequence similarities between LLO and perfringolysin (PFO), the cytolysin from Clostridium perfringens of known crystallographic structure, two truncated LLO proteins were produced: LLO-d123, comprising the first three predicted domains, and LLO-d4, the last C-terminal domain. The two proteins were efficiently secreted into the culture supernatant of L. monocytogenes and were able to bind to cell membranes. Strikingly, when expressed simultaneously, the two secreted domains LLO-d123 and LLO-d4 reassembled into a haemolytically active form. Two in-frame linker insertions were generated in the hinge region between the d123 and d4 domains. In both cases, the insertion created a major cleavage site for proteolytic degradation and abolished cytolytic activity, which might suggest that the region connecting d123 and d4 participates in the interaction between the two portions of the monomer.