The human opportunistic pathogen Serratia marcescens is a bacterium with a broad host range, and represents a growing problem for public health. Serratia marcescens kills Caenorhabditis elegans after colonizing the nematode's intestine. We used C.elegans to screen a bank of transposon-induced S.marcescens mutants and isolated 23 clones with an attenuated virulence. Nine of the selected bacterial clones also showed a reduced virulence in an insect model of infection. Of these, three exhibited a reduced cytotoxicity in vitro, and among them one was also markedly attenuated in its virulence in a murine lung infection model. For 21 of the 23 mutants, the transposon insertion site was identified. This revealed that among the genes necessary for full in vivo virulence are those that function in lipopolysaccharide (LPS) biosynthesis, iron uptake and hemolysin production. Using this system we also identified novel conserved virulence factors required for Pseudomonas aeruginosa pathogenicity. This study extends the utility of C.elegans as an in vivo model for the study of bacterial virulence and advances the molecular understanding of S.marcescens pathogenicity.

Pseudomonas aeruginosa is a frequently encountered pathogen that is involved in acute and chronic lung infections. Lectin-mediated bacterium-cell recognition and adhesion are critical steps in initiating P. aeruginosa pathogenesis. This study was designed to evaluate the contributions of LecA and LecB to the pathogenesis of P. aeruginosa-mediated acute lung injury. Using an in vitro model with A549 cells and an experimental in vivo murine model of acute lung injury, we compared the parental strain to lecA and lecB mutants. The effects of both LecA-and Lec B-specific lectin-inhibiting carbohydrates (␣-methyl-galactoside and ␣-methyl-fucoside, respectively) were evaluated. In vitro, the parental strain was associated with increased cytotoxicity and adhesion on A549 cells compared to the lecA and lecB mutants. In vivo, the P. aeruginosa-induced increase in alveolar barrier permeability was reduced with both mutants. The bacterial burden and dissemination were decreased for both mutants compared with the parental strain. Coadministration of specific lectin inhibitors markedly reduced lung injury and mortality. Our results demonstrate that there is a relationship between lectins and the pathogenicity of P. aeruginosa. Inhibition of the lectins by specific carbohydrates may provide new therapeutic perspectives.

Enteroaggregative Escherichia coli (EAEC) is a pathogen implicated in several infant diarrhea or diarrheal outbreaks in areas of endemicity. Although multiple genes involved in EAEC pathogenesis have been identified, the overall mechanism of virulence is not well understood. Recently, a novel secretion system, called type VI secretion (T6S) system (T6SS), has been identified in EAEC and most animal or plant gram-negative pathogens. T6SSs are multicomponent cell envelope machines responsible for the secretion of at least two putative substrates, Hcp and VgrG. In EAEC, two copies of T6S gene clusters, called sci-1 and sci-2, are present on the pheU pathogenicity island. In this study, we focused our work on the sci-1 gene cluster. The Sci-1 apparatus is probably composed of all, or a subset of, the 21 gene products encoded on the cluster. Among these subunits, some are shared by all T6SSs identified to date, including a ClpV-type AAA ؉ ATPase (SciG) and an IcmF (SciS) and an IcmH (SciP) homologue, as well as a putative lipoprotein (SciN). In this study, we demonstrate that sciN is a critical gene necessary for T6S-dependent secretion of the Hcp-like SciD protein and for biofilm formation. We further show that SciN is a lipoprotein, as shown by the inhibition of its processing by globomycin and in vivo labeling with [ 3 H]palmitic acid. SciN is tethered to the outer membrane and exposed in the periplasm. Sequestration of SciN at the inner membrane by targeting the ؉2 residue responsible for lipoprotein localization (Gly2Asp) fails to complement an sciN mutant for SciD secretion and biofilm formation. Together, these results support a model in which SciN is an outer membrane lipoprotein exposed in the periplasm and essential for the Sci-1 apparatus function.

Significance Entry of bacteria into host cells critically depends on their proper engulfment by the plasma membrane. So far, actin polymerization has been described as a major driving force in this process. However, our study reveals that the interaction of the bacterial surface lectin LecA with the host cell glycosphingolipid Gb3 is fully sufficient to promote engulfment of Pseudomonas aeruginosa , whereas actin polymerization is dispensable. Hence, the formation of a “lipid zipper” represents a previously unidentified mechanism of bacterial uptake and demonstrates that bacterial pathogens have evolved lipid-based invasion strategies that may function in addition to protein receptor-based ones. Furthermore, by identifying the LecA/Gb3 interaction as the major invasion-promoting factor, our study provides new targets for drugs that may prevent bacterial invasion and dissemination.