How Listeria monocytogenes organizes its surface for virulence

Listeria monocytogenes is a Gram-positive pathogen responsible for the manifestation of human listeriosis, an opportunistic foodborne disease with an associated high mortality rate. The key to the pathogenesis of listeriosis is the capacity of this bacterium to trigger its internalization by non-phagocytic cells and to survive and even replicate within phagocytes. The arsenal of virulence proteins deployed by L. monocytogenes to successfully promote the invasion and infection of host cells has been progressively unveiled over the past decades. A large majority of them is located at the cell envelope, which provides an interface for the establishment of close interactions between these bacterial factors and their host targets. Along the multistep pathways carrying these virulence proteins from the inner side of the cytoplasmic membrane to their cell envelope destination, a multiplicity of auxiliary proteins must act on the immature polypeptides to ensure that they not only maturate into fully functional effectors but also are placed or guided to their correct position in the bacterial surface. As the major scaffold for surface proteins, the cell wall and its metabolism are critical elements in listerial virulence. Conversely, the crucial physical support and protection provided by this structure make it an ideal target for the host immune system. Therefore, mechanisms involving fine modifications of cell envelope components are activated by L. monocytogenes to render it less recognizable by the innate immunity sensors or more resistant to the activity of antimicrobial effectors. This review provides a state-of-the-art compilation of the mechanisms used by L. monocytogenes to organize its surface for virulence, with special focus on those proteins that work "behind the frontline", either supporting virulence effectors or ensuring the survival of the bacterium within its host.We apologize to authors whose relevant work could not be cited owing to space limitations. Research in the group of Molecular Microbiology is funded by the project "NORTE-07-0124-FEDER-000002-Host-Pathogen Interactions" co-funded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referencia Estrategico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER), the Operational Competitiveness Programme (COMPETE) and FCT (Fundacdo para a Ciencia e Tecnologia), and by projects ERANet Pathogenomics LISTRESS ERA-PTG/0003/2010, PTDC/SAU-MIC/111581/2009FCOMP-FEDER, PTDC/BIA-BCM/100088/2008FCOMP-01-0124-FEDER-008860 and PTDC/BIA-BCM/111215/2009FCOMP-01-0124-FEDER-014178. Filipe Carvalho was supported by FCT doctoral grant SFRH1BD16182512009, and Sandra Sousa by the Ciencia 2008 and FCT-Investigator programs (COMPETE, POPH, and FCT)

Importance of the Conserved Residues in the Peptidoglycan Glycosyltransferase Module of the Class A Penicillin-binding Protein 1b of Escherichia coli*S⃞

The peptidoglycan glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs catalyze glycan chain elongation of the bacterial cell wall. These enzymes belong to the GT51 family, are characterized by five conserved motifs, and have some fold similarity with the phage λ lysozyme. In this work, we have systematically modified all the conserved amino acid residues of the GT module of Escherichia coli class A PBP1b by site-directed mutagenesis and determined their importance for the in vivo and in vitro activity and the thermostability of the protein. To get an insight into the GT active site of this paradigm enzyme, a model of PBP1b GT domain was constructed based on the available crystal structures (PDB codes 2OLV and 2OLU). The data show that in addition to the essential glutamate residues Glu233 of motif 1 and Glu290 of motif 3, the residues Phe237 and His240 of motif 1 and Gly264, Thr267, Gln271, and Lys274 of motif 2, all located in the catalytic cavity of the GT domain, are essential for the in vitro enzymatic activity of the PBP1b and for its in vivo functioning. Thus, the first three conserved motifs contain most of the residues that are required for the GT activity of the PBP1b. The residues Asp234, Phe237, His240, Thr267, and Gln271 are proposed to maintain the structure of the active site and the positioning of the catalytic Glu233

Discrete and overlapping functions of peptidoglycan synthases in growth, cell division and virulence of L

Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with β-lactam antibiotics. These target Listeria's five high molecular weight penicillin-binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi-functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross-linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)-PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod-shape and PBP B2 for cross-wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased β-lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross-linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence