Anne-Béatrice Blanc-Potard

The Salmonella selC locus contains a pathogenicity island mediating intramacrophage survival

Pathogenicity islands are chromosomal clusters of horizontally acquired virulence genes that are often found at tRNA loci. The selC tRNA locus of Escherichia coli has served as the site of integration of two distinct pathogenicity islands which are responsible for converting benign strains into uro‐ and enteropathogens. Because virulence genes are targeted to the selC locus of E.coli, we investigated the homologous region of the Salmonella typhimurium chromosome for the presence of horizontally acquired sequences. At this site, we identified a 17 kb DNA segment that is both unique to Salmonella and necessary for virulence. This segment harbors a gene, mgtC, that is required for intramacrophage survival and growth in low Mg2+ media. The mgtC locus is regulated by the PhoP/PhoQ two‐component system, a major regulator of virulence functions present in both pathogenic and non‐pathogenic bacterial species. Cumulatively, our experiments indicate that the ability to replicate in low Mg2+ environments is necessary for Salmonella virulence, and suggest that a similar mechanism is responsible for the dissemination and acquisition of pathogenicity islands in enteric bacteria.

Peptide-assisted degradation of the Salmonella MgtC virulence factor

MgtC is a virulence factor common to several intracellular pathogens that is required for intramacrophage survival and growth in magnesium‐depleted medium. In Salmonella enterica, MgtC is coexpressed with the MgtB magnesium transporter and transcription of the mgtCB operon is induced by magnesium deprivation. Despite the high level of mgtCB transcriptional induction in magnesium‐depleted medium, the MgtC protein is hardly detected in a wild‐type Salmonella strain. Here, we show that downregulation of MgtC expression is dependent on a hydrophobic peptide, MgtR, which is encoded by the mgtCB operon. Our results suggest that MgtR promotes MgtC degradation by the FtsH protease, providing a negative regulatory feedback. Bacterial two‐hybrid assays demonstrate that MgtR interacts with the inner‐membrane MgtC protein. We identified mutant derivatives of MgtR and MgtC that prevent both regulation and interaction between the two partners. In macrophages, overexpression of the MgtR peptide led to a decrease of the replication rate of Salmonella. This study highlights the role of peptides in bacterial regulatory mechanisms and provides a natural antagonist of the MgtC virulence factor.

Hydrophobic peptides: novel regulators within bacterial membrane

Identification of short coding sequences is challenging, both experimentally and in silico, and functional natural peptides (< 50 amino acids) have to a large extent been overlooked in Gram‐negative bacteria. Recent results have converged to highlight the role of hydrophobic peptides that form a novel class of active molecules in Escherichia coli and Salmonella enterica serovar Typhimurium. These peptides can play a regulatory role by interacting with protein partners at the inner membrane and by modulating protein partner activity or stability. Genome‐wide analyses in both bacterial species have identified several conserved short open reading frames encoding a single transmembrane segment. We discuss the known and predicted membrane‐associated peptides and the tools for their identification. Besides the identification of novel regulatory networks, characterization of peptides with a single transmembrane helix segment and proteins that interact with them provides a powerful opportunity to study interactions between alpha helices within biological membranes. In addition, some bioactive membrane peptides could provide a basis for engineering membrane protein antagonists.

Requirement of MgtC for Brucella suis Intramacrophage Growth: a Potential Mechanism Shared by Salmonella enterica and Mycobacterium tuberculosis for Adaptation to a Low-Mg2+ Environment

ABSTRACT A Brucella suis mgtC mutant is defective for growth within macrophages and in low-Mg2+ medium. These phenotypes are strikingly similar to those observed with mgtC mutants from Salmonella enterica and Mycobacterium tuberculosis, two other pathogens that proliferate within phagosomes. MgtC appears as a remarkable virulence factor that would have been acquired by distantly related intracellular pathogens to contribute to the adaptation to a low-Mg2+ environment in the phagosome.

Afa/Dr Diffusely Adhering Escherichia coli C1845 Infection Promotes Selective Injuries in the Junctional Domain of Polarized Human Intestinal Caco-2/TC7 Cells

ABSTRACT The Afa/Dr diffusely adhering Escherichia coli (DAEC) C1845 strain harboring the F1845 fimbrial adhesin interacts with the brush border-associated CD55 molecule and promotes elongation of brush border microvilli resulting from rearrangement of the F-actin network. This phenomenon involves the activation of a cascade of signaling coupled to the glycosylphosphatidylinositol-anchored receptor of the F1845 adhesin. We provide evidence that infection of the polarized human intestinal cell line Caco-2/TC7 by strain C1845 is followed by an increase in the paracellular permeability for [3H]mannitol without a decrease of the transepithelial resistance of the monolayers. Alterations in the distribution of tight-junction (TJ)-associated occludin and ZO-1 protein are observed, whereas the distribution of the zonula adherens-associated E-cadherin is not affected. Using the recombinantE. coli strains HB101(pSSS1) and -(pSSS1C) expressing the F1845 fimbrial adhesin, we demonstrate that the adhesin-CD55 interaction is not sufficient for the induction of structural and functional TJ lesions. Moreover, using the actin filament-stabilizing agent Jasplakinolide, we demonstrate that the C1845-induced functional alterations in TJs are independent of the C1845-induced apical cytoskeleton rearrangements. The results indicated that pathogenic factor(s) other than F1845 adhesin may be operant in Afa/Dr DAEC C1845.

Pyelonephritogenic diffusely adhering Escherichia coli EC7372 harboring Dr-II adhesin carries classical uropathogenic virulence genes and promotes cell lysis and apoptosis in polarized epithelial caco-2/TC7 cells.

ABSTRACT Diffusely adhering Escherichia coli (DAEC) strains expressing adhesins of the Afa/Dr family bind to epithelial cells in a diffuse adherence pattern by recognizing a common receptor, the decay-accelerating factor (CD55). Recently, a novel CD55-binding adhesin, named Dr-II, was identified from the pyelonephritogenic strain EC7372. In this report, we show that despite the low level of sequence identity between Dr-II and other members of the Afa/Dr family, EC7372 induces pathophysiological effects similar to those induced by other Afa/Dr DAEC strains on the polarized epithelial cell line Caco-2/TC7. Specifically, the Dr-II adhesin was sufficient to promote CD55 and CD66e clustering around adhering bacteria and apical cytoskeleton rearrangements. Unlike other Afa/Dr DAEC strains, EC7372 expresses a functional hemolysin that promotes a rapid cellular lysis. In addition, cell death by apoptosis or necrosis was observed in EC7372-infected Caco-2/TC7 cells, depending on infection time. Our results indicate that EC7372 harbors a pathogenicity island (PAI) similar to the one described for the pyelonephritogenic strain CFT073, which carries bothhly and pap operons. Cumulatively, our findings indicate that strain EC7372 can be considered a prototype of a subclass of Afa/Dr DAEC isolates that have acquired a PAI harboring several classical uropathogenic virulence genes.

Molecular evolution of Salmonella enterica serovar Typhimurium and pathogenic Escherichia coli: from pathogenesis to therapeutics.

Salmonella enterica serovar Typhimurium (S. Typhimurium) and certain Escherichia coli are human pathogens that have evolved through the acquisition of multiple virulence determinants by horizontal gene transfer. Similar genetic elements, as pathogenicity islands and virulence plasmids, have driven molecular evolution of virulence in both species. In addition, the contribution of prophages has been recently highlighted as a reservoir for pathogenic diversity. Characterization of horizontally acquired virulence genes has several clinical implications. First, identification of virulence determinants that have a sporadic distribution and are specifically associated with a pathotype and/or a pathology can be useful markers for risk assessment and diagnosis. Secondly, virulence factors widely distributed in pathogenic strains, but absent from non-pathogenic bacteria, are interesting targets for the development of novel antimicrobial chemotherapies and vaccines. Here, we summarize the horizontally acquired virulence factors of S. Typhimurium, enterohemorrhagic E. coli O157:H7 and uropathogenic E. coli, and we describe their use in novel therapeutic approaches.

Identification of a Haarlem Genotype-Specific Single Nucleotide Polymorphism in the mgtC Virulence Gene of Mycobacterium tuberculosis

ABSTRACT MgtC is a virulence factor common to several intracellular pathogens, including Mycobacterium tuberculosis, that might have been acquired through horizontal gene transfer. In the present study, we investigated the polymorphism of mgtC in clinical isolates representative of the main epidemic groups of M. tuberculosis. MgtC appears to have a low polymorphism rate in M. tuberculosis that consists exclusively of nonsynonymous mutations. We identified a single nucleotide polymorphism (SNP) at mgtC codon 182 (mgtC182) specifically associated with the Haarlem genotype. A simple PCR assay, called the “on/off switch assay,” using phosphorothioate-modified primers and Pfu polymerase allowed us to distinguish Haarlem from non-Haarlem strains based on the mgtC182 SNP. The amino acid change (H182R) associated with the mgtC182 SNP in Haarlem strains does not appear to procure a selective advantage. Our results offer a simple and rapid tool to distinguish between Haarlem and non-Haarlem strains. In addition, the on/off switch assay, which allows the detection of SNPs on chromosomal DNA and M. tuberculosis cultures, provides a novel approach for the screening of known SNPs in M. tuberculosis.

A class of gyrase mutants of Salmonella typhimurium show quinolone-like lethality and require rec functions for viability.

We have identified a new class of DNA gyrase mutants of Salmonella typhimurium that show chronic derepression of the SOS regulon. Thus, these mutants mimic the response of wild‐type cells to gyrase inhibitors of the quinolone family. SOS induction by conditional lethal mutations gyrA208 or gyrB652, like that mediated by quinolones, is completely dependent on the function of the recB gene product. Introduction of recA or recB null mutations into these strains exacerbates their temperature‐sensitive phenotype and prevents growth at the otherwise permissive temperature of 37°C. Selection of suppressors that concomitantly restore growth at 37°C and SOS induction in a recB− background yielded mutations that relieve the RecB requirement for homologous recombination; namely, sbcB mutations as well as mutations at a new locus that was named sbcE. Such mutations also restore SOS induction in quinolone‐treated gyr+recB− strains. These findings indicate that Rec functions are needed for growth of the gyrase mutants at 37°C and suggest that recombinational repair intermediates constitute the SOS‐inducing signal in the mutants as well as in quinolone‐treated wild‐type bacteria. Unlike quinolones, however, the gyr mutations described in this study do not cause detectable accumulation of ‘cleavable’ gyrase–DNA complexes in plasmid or chromosomal DNA. Yet gyrA208 (the only allele tested) was found to trigger RecB‐mediated reckless degradation of chromosomal DNA in recA− cells at restrictive temperatures. Indirect evidence suggests that double‐stranded DNA ends, entry sites for the RecBCD enzyme, are generated in the gyr mutants by the breakage of DNA‐replication forks. We discuss how this could occur and how recombinational rescue of collapsed replication forks could account for cell survival (and SOS induction) in the gyr mutants as well as in quinolone‐treated bacteria.

Representational Difference Analysis between Afa/Dr Diffusely Adhering Escherichia coli and Nonpathogenic E. coli K-12

ABSTRACT Diffusely adhering Escherichia coli strains harboring Afa/Dr adhesins (Afa/Dr DAEC) have been associated with diarrhea and urinary tract infections (UTIs). The present work is the first extensive molecular study of a Afa/Dr DAEC strain using the representational difference analysis technique. We have searched for DNA sequences present in strain C1845, recovered from a diarrheagenic child, but absent from a nonpathogenic K-12 strain. Strain C1845 harbors part of a pathogenicity island (PAICFT073) and several iron transport systems found in other E. coli pathovars. We did not find genes encoding factors known to subvert host cell proteins, such as type III secretion system or effector proteins. Several C1845-specific sequences are homologous to putative virulence genes or show no homology with known sequences, and we have analyzed their distribution among Afa/Dr and non-Afa/Dr clinical isolates and among strains from the E. coli Reference Collection. Three C1845-specific sequences (MO30, S109, and S111) have a high prevalence (77 to 80%) among Afa/Dr strains and a low prevalence (12 to 23%) among non-Afa/Dr strains. In addition, our results indicate that strain IH11128, an Afa/Dr DAEC strain recovered from a patient with a UTI, is genetically closely related to strain C1845.