Elisabeth Pellegrini

Formation of D-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes

The dlt operon of Gram‐positive bacteria comprises four genes (dltA, dltB, dltC and dltD) that catalyse the incorporation of D‐alanine residues into the cell wall‐associated lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Listeria monocytogenes and constructed a D‐Ala‐deficient LTA mutant by inactivating the first gene (dltA) of this operon. The DltA− mutant did not show any morphological alterations and its growth rate was similar to that of the wild‐type strain. However, it exhibited an increased susceptibility to the cationic peptides colistin, nisin and polymyxin B. The virulence of the DltA− mutant was severely impaired in a mouse infection model (4 log increase in the LD50) and, in vitro, the adherence of the mutant to various cell lines (murine bone marrow‐derived macrophages and hepatocytes and a human epithelial cell line) was strongly restricted, although the amounts of surface proteins implicated in virulence (ActA, InlA and InlB) remains unaffected. We suggest that the decreased adherence of the DltA− mutant to non‐phagocytic and phagocytic cells might be as a result of the increased electronegativity of its charge surface and/or the presence at the bacterial surface of adhesins possessing altered binding activities. These results show that the D‐alanylation of the LTAs contributes to the virulence of the intracellular pathogen L. monocytogenes.

Aromatase in the brain of teleost fish: expression, regulation and putative functions.

Unlike that of mammals, the brain of teleost fish exhibits an intense aromatase activity due to the strong expression of one of two aromatase genes (aromatase A or cyp19a1a and aromatase B or cyp19a1b) that arose from a gene duplication event. In situ hybridization, immunohistochemistry and expression of GFP (green fluorescent protein) in transgenic tg(cyp19a1b-GFP) fish demonstrate that aromatase B is only expressed in radial glial cells (RGC) of adult fish. These cells persist throughout life and act as progenitors in the brain of both developing and adult fish. Although aromatase B-positive radial glial cells are most abundant in the preoptic area and the hypothalamus, they are observed throughout the entire central nervous system and spinal cord. In agreement with the fact that brain aromatase activity is correlated to sex steroid levels, the high expression of cyp19a1b is due to an auto-regulatory loop through which estrogens and aromatizable androgens up-regulate aromatase expression. This mechanism involves estrogen receptor binding on an estrogen response element located on the cyp19a1b promoter. Cell specificity is achieved by a mandatory cooperation between estrogen receptors and unidentified glial factors. Given the emerging roles of estrogens in neurogenesis, the unique feature of the adult fish brain suggests that, in addition to classical functions on brain sexual differentiation and sexual behaviour, aromatase expression in radial glial cells could be part of the mechanisms authorizing the maintenance of a high proliferative activity in the brain of fish.

Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish.

Compared with other vertebrates, the brain of adult teleost fish exhibits two unique features: it exhibits unusually high neurogenic activity and strongly expresses aromatase, a key enzyme that converts aromatizable androgens into estrogens. Until now, these two features, high neurogenic and aromatase activities, have never been related to each other. Recently, it was shown that aromatase is expressed in radial glial cells of the forebrain and not in neurons. Here, we further document that Aromatase B is never detected in cells expressing the markers of postmitotic neurons, Hu and acetylated tubulin. By using a combination of bromodeoxyuridine (BrdU) treatment and immunohistochemical techniques, we demonstrate for the first time to our knowledge that aromatase‐positive radial cells actively divide to generate newborn cells in many forebrain regions. Such newborn cells can further divide, as shown by BrdU‐proliferating cell nuclear antigen double staining. We also demonstrate that, over time, newborn cells move away from the ventricles, most likely by migrating along the radial processes. Finally, by using antisera to Hu and acetylated tubulin, we further document that some of the newborn cells derived from radial glia differentiate into neurons. These data provide new evidence for the mechanism of neurogenesis in the brain of adult fish. In addition, given that estrogens are well‐known neurotrophic and neuroprotective factors affecting proliferation, apoptosis, migration, and differentiation, the expression of aromatase in the neural stem cells of the adult strongly demonstrates that the fish brain is an outstanding model for studying the effects of estrogens on adult neurogenesis and brain repair. J. Comp. Neurol. 501:150–167, 2007.

The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes

We identified the stress‐induced ClpP of Listeria monocytogenes and demonstrated its crucial role in intracellular survival of this pathogen. ClpP is a 21.6 kDa protein belonging to a family of proteases highly conserved in prokaryotes and eukaryotes. A clpP‐deleted mutant enabled us to demonstrate that ClpP is involved in proteolysis and is required for growth under stress conditions. Intramacrophage survival of this mutant was strongly restricted, thus resulting in loss of virulence for the mouse. The activity of listeriolysin O, a major virulence factor implicated in bacterial escape from phagosomes of macrophages, was much reduced in the clpP mutant under stress conditions. Direct evidence for the role of ClpP in the intracellular parasitism was obtained by showing that virulence and haemolytic activity were fully restored by complementation of the mutant. These results suggest that ClpP is involved in the rapid adaptive response of intracellular pathogens during the infectious process.

OppA of Listeria monocytogenes, an Oligopeptide-Binding Protein Required for Bacterial Growth at Low Temperature and Involved in Intracellular Survival

ABSTRACT We identified a new oligopeptide permease operon in the pathogenListeria monocytogenes. This opp operon consists of five genes (oppA, oppB, oppC, oppD, andoppF) and displays the same genetic organization as those of several bacterial species. The first gene of this operon,oppA, encodes a 62-kDa protein sharing 33% identity with OppA of Bacillus subtilis and is expressed predominantly during exponential growth. The function of oppA was studied by constructing an oppA deletion mutant. The phenotype analysis of this mutant revealed that OppA mediates the transport of oligopeptides and is required for bacterial growth at low temperature. The wild-type phenotype was restored by complementing the mutant withoppA. We also found that OppA is involved in intracellular survival in macrophages and in bacterial growth in organs of mice infected with L. monocytogenes, although the level of virulence was not altered in the mutant. These results show the major role of OppA in the uptake of oligopeptides and the pleiotropic effects of this oligopeptide-binding protein on the behavior of this pathogen in the environment and in its host.

CovS/CovR of group B streptococcus: a two-component global regulatory system involved in virulence

In this study, we carried out a detailed structural and functional analysis of a Streptococcus agalactiae (GBS) two‐component system which is orthologous to the CovS/CovR (CsrS/CsrR) regulatory system of Streptococcus pyogenes. In GBS, covR and covS are part of a seven gene operon transcribed from two promoters that are not regulated by CovR. A ΔcovSR mutant was found to display dramatic phenotypic changes such as increased haemolytic activity and reduced CAMP activity on blood agar. Adherence of the ΔcovSR mutant to epithelial cells was greatly increased and analysis by transmission electron microscopy revealed the presence at its surface of a fibrous extracellular matrix that might be involved in these intercellular interactions. However, the ΔcovSR mutant was unable to initiate growth in RPMI and its viability in human normal serum was greatly impaired. A major finding of this phenotypic analysis was that the CovS/CovR system is important for GBS virulence, as a 3 log increase of the LD50 of the mutant strain was observed in the neonate rat sepsis model. The pleiotropic phenotype of the ΔcovSR mutant is in full agreement with the large number of genes controlled by CovS/CovR as seen by expression profiling analysis, many of which encode potentially secreted or cell surface‐associated proteins: 76 genes are repressed whereas 63 were positively regulated. CovR was shown to bind directly to the regulatory regions of several of these genes and a consensus CovR recognition sequence was proposed using both DNase I footprinting and computational analyses.

Attenuated virulence of Streptococcus agalactiae deficient in D-alanyl-lipoteichoic acid is due to an increased susceptibility to defensins and phagocytic cells

D‐alanylation of lipoteichoic acid (LTA), allows Gram‐positive bacteria to modulate their surface charge, regulate ligand binding and control the electromechanical properties of the cell wall. In this study, the role of D‐alanyl LTA in the virulence of the extracellular pathogen Streptococcus agalactiae was investigated. We demonstrate that a DltA– isogenic mutant displays an increased susceptibility to host defence peptides such as human defensins and animal‐derived cationic peptides. Accordingly, the mutant strain is more susceptible to killing by mice bone marrow‐derived macrophages and human neutrophils than the wild‐type strain. In addition, the virulence of the DltA– mutant is severely impaired in mouse and neonatal rat models. This mutant was eliminated more rapidly than the wild‐type strain from the lung of three‐week‐old mice inoculated intranasally and, consequently, is unable to induce a pneumonia. Finally, after intravenous injection of three‐week‐old mice, the survival of the DltA– mutant is markedly reduced in the blood in comparison to that of the wild‐type strain. We hypothesize that the decreased virulence of the DltA– mutant is a consequence of its increased susceptibility to cationic antimicrobial peptides and to killing by phagocytes. These results demonstrate that the D‐alanylation of LTA contributes to the virulence of S. agalactiae.

Identification of a ClpC ATPase required for stress tolerance and in vivo survival of Listeria monocytogenes

We identified a new chromosomal locus involved in the virulence of the facultative intracellular pathogen Listeria monocytogenes. This locus displays the same genetic organization as that of the clpC/mecB locus of Bacillus subtilis. It contains a thermoregulated operon of four genes, whose transcription is upregulated at 42°C. The last gene of this operon is clpC, which encodes a protein of 826 amino acid residues, identified as a ClpC ATPase, sharing a strong peptide sequence identity (78%) with ClpC/MecB of B. subtilis. Tn917 insertions inactivating the entire operon, or only clpC, gave mutants highly susceptible to stress, including iron limitation, elevated temperatures and high osmolarity. The virulence of these mutants was severely impaired in the mouse. A clpC insertional mutant was also restricted in its capacity to grow in bone‐marrow‐derived macrophages. These results demonstrate that the ClpC ATPase of L. monocytogenes is a general stress protein involved in intracellular growth and in vivo survival of this pathogen in host tissues.

Lipoproteins Are Critical TLR2 Activating Toxins in Group B Streptococcal Sepsis

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Δlgt and the Δlsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Δlgt and Δlsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Δlgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Δlgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.

Contribution of Mn-Cofactored Superoxide Dismutase (SodA) to the Virulence of Streptococcus agalactiae

ABSTRACT Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide, which, in turn, is metabolized by catalases and/or peroxidases. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and hence play a role in the pathogenesis of certain bacteria. We previously demonstrated that group B streptococci (GBS) possess a single Mn-cofactored superoxide dismutase (SodA). To analyze the role of this enzyme in the pathogenicity of GBS, we constructed a sodA-disrupted mutant of Streptococcus agalactiae NEM316 by allelic exchange. This mutant was subsequently cis complemented by integration into the chromosome of pAT113/Sp harboring the wild-typesodA gene. The SOD specific activity detected by gel analysis in cell extracts confirmed that active SODs were present in the parental and complemented strains but absent in thesodA mutant. The growth rates of these strains in standing cultures were comparable, but the sodA mutant was extremely susceptible to the oxidative stress generated by addition of paraquat or hydrogen peroxide to the culture medium and exhibited a higher mutation frequency in the presence of rifampin. In mouse bone marrow-derived macrophages, the sodA mutant showed an increased susceptibility to bacterial killing by macrophages. In a mouse infection model, after intravenous injection the survival of thesodA mutant in the blood and the brain was markedly reduced in comparison to that of the parental and complemented strains whereas only minor effects on survival in the liver and the spleen were observed. These results suggest that SodA plays a role in GBS pathogenesis.