Willy Aucher

CodY regulation is required for full virulence and heme iron acquisition in Bacillus anthracis

Capsule and toxin are the major virulence factors of Bacillus anthracis. The B. anthracis pleiotropic regulator CodY activates toxin gene expression by post‐translationally regulating the accumulation of the global regulator AtxA. However, the role of CodY on B. anthracis capsulation and virulence of encapsulated strains has been unknown. The role of CodY in B. anthracis virulence was studied in mouse and guinea pig models. Spore outgrowth and dissemination of the vegetative cells was followed in mice by bioluminescent imaging. We also determined the state of capsulation and the iron requirement for growth of the codY mutant. In all models tested, the codY mutant strain was strongly attenuated compared to the wild‐type strain and, in mice, also compared to the atxA strain. The disruption of codY did not affect either ex vivo or in vivo capsulation, whereas atxA deletion affected ex vivo capsulation only. The disruption of codY led to a delayed initiation of dissemination but similar kinetics of subsequent spread of the bacilli. The codY mutant cannot grow on heme iron as sole iron source, whereas the parental and complemented strains can. The lack of CodY‐mediated transcription weakens virulence by controlling iron acquisition and synthesis of toxin, but without modifying capsulation.—Château, A., van Schaik, W., Six, A., Aucher, W., Fouet, A. CodY regulation is required for full virulence and heme iron acquisition in Bacillus anthracis. FASEB J. 25, 4445–4456 (2011). www.fasebj.org

Phosphatidylglycerol::Prolipoprotein Diacylglyceryl Transferase (Lgt) of Escherichia coli Has Seven Transmembrane Segments, and Its Essential Residues Are Embedded in the Membrane

Lgt of Escherichia coli catalyzes the transfer of an sn-1,2-diacylglyceryl group from phosphatidylglycerol to prolipoproteins. The enzyme is essential for growth, as demonstrated here by the analysis of an lgt depletion strain. Cell fractionation demonstrated that Lgt is an inner membrane protein. Its membrane topology was determined by fusing Lgt to β-galactosidase and alkaline phosphatase and by substituted cysteine accessibility method (SCAM) studies. The data show that Lgt is embedded in the membrane by seven transmembrane segments, that its N terminus faces the periplasm, and that its C terminus faces the cytoplasm. Highly conserved amino acids in Lgt of both Gram-negative and Gram-positive bacteria were identified. Lgt enzymes are characterized by a so-called Lgt signature motif in which four residues are invariant. Ten conserved residues were replaced with alanine, and the activity of these Lgt variants was analyzed by their ability to complement the lgt depletion strain. Residues Y26, N146, and G154 are absolutely required for Lgt function, and R143, E151, R239, and E243 are important. The results demonstrate that the majority of the essential residues of Lgt are located in the membrane and that the Lgt signature motif faces the periplasm.

Legionella pneumophila: The Paradox of a Highly Sensitive Opportunistic Waterborne Pathogen Able to Persist in the Environment

Legionella pneumophila, the major causative agent of Legionnaires’ disease, is found in freshwater environments in close association with free-living amoebae and multispecies biofilms, leading to persistence, spread, biocide resistance, and elevated virulence of the bacterium. Indeed, legionellosis outbreaks are mainly due to the ability of this bacterium to colonize and persist in water facilities, despite harsh physical and chemical treatments. However, these treatments are not totally efficient and, after a lag period, L. pneumophila may be able to quickly re-colonize these systems. Several natural compounds (biosurfactants, antimicrobial peptides…) with anti-Legionella properties have recently been described in the literature, highlighting their specific activities against this pathogen. In this review, we first consider this hallmark of Legionella to resist killing, in regard to its biofilm or host-associated life style. Then, we focus more accurately on natural anti-Legionella molecules described so far, which could provide new eco-friendly and alternative ways to struggle against this important pathogen in plumbing.

Influence of Amino Acid Substitutions in the Leader Peptide on Maturation and Secretion of Mesentericin Y105 by Leuconostoc mesenteroides

By site-specific mutagenesis, the hydrophobic conserved amino acids and the C-terminal GG doublet of the leader peptide of pre-mesentericin Y105 were demonstrated to be critical for optimal secretion of mesentericin Y105, as well as for the maturation of the pre-bacteriocin by the protease portion of the ABC transporter MesD.

Characterization of the Sortase Repertoire in Bacillus anthracis

LPXTG proteins, present in most if not all Gram-positive bacteria, are known to be anchored by sortases to the bacterial peptidoglycan. More than one sortase gene is often encoded in a bacterial species, and each sortase is supposed to specifically anchor given LPXTG proteins, depending of the sequence of the C-terminal cell wall sorting signal (cwss), bearing an LPXTG motif or another recognition sequence. B. anthracis possesses three sortase genes. B. anthracis sortase deleted mutant strains are not affected in their virulence. To determine the sortase repertoires, we developed a genetic screen using the property of the gamma phage to lyse bacteria only when its receptor, GamR, an LPXTG protein, is exposed at the surface. We identified 10 proteins that contain a cell wall sorting signal and are covalently anchored to the peptidoglycan. Some chimeric proteins yielded phage lysis in all sortase mutant strains, suggesting that cwss proteins remained surface accessible in absence of their anchoring sortase, probably as a consequence of membrane localization of yet uncleaved precursor proteins. For definite assignment of the sortase repertoires, we consequently relied on a complementary test, using a biochemical approach, namely immunoblot experiments. The sortase anchoring nine of these proteins has thus been determined. The absence of virulence defect of the sortase mutants could be a consequence of the membrane localization of the cwss proteins.

Differences in mesentericin secretion systems from two Leuconostoc strains

Leuconostoc mesenteroides Y105 and L. mesenteroides FR52 produce both mesentericin Y105 and B105, in equal amounts. The mesentericin operons of L. mesenteroides FR52 and Y105 which are involved in mesentericin Y105 and B105 production, were both sequenced and compared. Differences were limited to the two genes, mesD and mesE, which encode the dedicated transport system of mesentericin Y105. Analysis of mesentericin non-producing mutants and complementation experiments demonstrated that the major role of the membrane fusion protein, MesE, was in bacteriocin secretion for both strains. Moreover, the secretion machinery MesDE was demonstrated to be capable of transportation and maturation of the two pre-bacteriocins, mesentericin Y105 and B105. We also demonstrate that although MesDEs from strains Y105 and FR52 have significant sequence differences, both transporters were capable of assuring secretion of either bacteriocin.

A Strategy for Interaction Site Prediction between Phospho-binding Modules and their Partners Identified from Proteomic Data

Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/.

Legionella pneumophila prevents proliferation of its natural host Acanthamoeba castellanii

Legionella pneumophila is a ubiquitous, pathogenic, Gram-negative bacterium responsible for legionellosis. Like many other amoeba-resistant microorganisms, L. pneumophila resists host clearance and multiplies inside the cell. Through its Dot/Icm type IV secretion system, the bacterium injects more than three hundred effectors that modulate host cell physiology in order to promote its own intracellular replication. Here we report that L. pneumophila prevents proliferation of its natural host Acanthamoeba castellanii. Infected amoebae could not undergo DNA replication and no cell division was observed. The Dot/Icm secretion system was necessary for L. pneumophila to prevent the eukaryotic proliferation. The absence of proliferation was associated with altered amoebal morphology and with a decrease of mRNA transcript levels of CDC2b, a putative regulator of the A. castellanii cell cycle. Complementation of CDC28-deficient Saccharomyces cerevisiae by the CDC2b cDNA was sufficient to restore proliferation of CDC28-deficient S. cerevisiae and suggests for the first time that CDC2b from A. castellanii could be functional and a bona fide cyclin-dependent kinase. Hence, our results reveal that L. pneumophila impairs proliferation of A. castellanii and this effect could involve the cell cycle protein CDC2b.

Development of a new method for the detection of lactic acid bacteria capable of protecting ham against Enterobacteriaceae

Aims:  Challenge trials seem to be the best assessment approach to evaluate the potency of food protective cultures. However, this method is time consuming and often difficult to implement. Here, we describe the development of the ‘sequential culturing method’, a new method for the screening of strains as protective cultures.

Fate of Eight Different Polymers under Uncontrolled Composting Conditions: Relationships Between Deterioration, Biofilm Formation, and the Material Surface Properties

With the ever-increasing volume of polymer wastes and their associated detrimental impacts on the environment, the plastic life cycle has drawn increasing attention. Here, eight commercial polymers selected from biodegradable to environmentally persistent materials, all formulated under a credit card format, were incubated in an outdoor compost to evaluate their fate over time and to profile the microbial communities colonizing their surfaces. After 450 days in compost, the samples were all colonized by multispecies biofilms, these latest displaying different amounts of adhered microbial biomass and significantly distinct bacterial and fungal community compositions depending on the substrate. Interestingly, colonization experiments on the eight polymers revealed a large core of shared microbial taxa, predominantly composed of microorganisms previously reported from environments contaminated with petroleum hydrocarbons or plastics debris. These observations suggest that biofilms may contribute to the alteration process of all the polymers studied. Actually, four substrates, independently of their assignment to a polymer group, displayed a significant deterioration, which might be attributed to biologically mediated mechanisms. Relevantly, the deterioration appears strongly associated with the formation of a high-cell density biofilm onto the polymer surfaces. The analysis of various surface properties revealed that roughness and hydrophilicity are likely prominent parameters for driving the biological interactions with the polymers.