Insights into betulinic acid as a promising molecule to fight the interkingdom biofilm Staphylococcus aureus-Candida albicans.

The demand for antibiofilm molecules has increased over several years due to their potential to fight biofilm-associated infections, such as those including the interkingdom Staphylococcus aureus-Candida albicans occurring in clinical settings worldwide. Recently, we identified a pentacyclic triterpenoid compound, betulinic acid, from invasive macrophytes, with interesting antibiofilm properties. The aim of the present study was to provide insights into the mechanism of action of betulinic acid against the clinically relevant bi-species S. aureus-C. albicans biofilms. Microscopy examinations, flow cytometry and crystal violet assays confirmed that betulinic acid was effective at damaging mature S. aureus-C. albicans biofilms or inhibiting their formation, reducing biofilm biomass by 70% on average and without microbicidal activity. The results suggested an action of betulinic acid on cell membranes, inducing changes in properties such as composition, hydrophobicity and fluidity as observed in C. albicans, which may hinder the early adhesion step, biofilm growth and the physical interactions of both microbial species. Further results of real-time polymerase chain reaction argued in favour of a reduction in S. aureus-C. albicans physical interaction due to betulinic acid by the modulation of biofilm-related gene expression, as observed in early stages of biofilm formation. This study revealed the potential of betulinic acid as a candidate agent for the prevention and treatment of S. aureus-C. albicans biofilm-related infections.

Valorization of Invasive Plant Extracts against the Bispecies Biofilm Staphylococcus aureus-Candida albicans by a Bioguided Molecular Networking Screening.

Invasive plants efficiently colonize non-native territories, suggesting a great production of bioactive metabolites which could be effective antibiofilm weapons. Our study aimed to look for original molecules able to inhibit bispecies biofilm formed by S. aureus and C. albicans. Extracts from five invasive macrophytes (Ludwigia peploides, Ludwigia grandiflora, Myriophyllum aquaticum, Lagarosiphon major and Egeria densa) were prepared and tested in vitro against 24 h old bispecies biofilms using a crystal violet staining (CVS) assay. The activities of the extracts reducing the biofilm total biomass by 50% or more were comparatively analyzed against each microbial species forming the biofilm by flow cytometry (FCM) and scanning electron microscopy. Extracts active against both species were fractionated. Obtained fractions were analyzed by UHPLC-MS/MS and evaluated by the CVS assay. Chemical and biological data were combined into a bioactivity-based molecular networking (BBMN) to identify active compounds. The aerial stem extract of L. grandiflora showed the highest antibiofilm activity (>50% inhibition at 50 µg∙mL−1). The biological, chemical and BBMN investigations of its fractions highlighted nine ions correlated with the antibiofilm activity. The most correlated compound, identified as betulinic acid (BA), inhibited bispecies biofilms regardless of the three tested couples of strains (ATCC strains: >40% inhibition, clinical isolates: ≈27% inhibition), confirming its antibiofilm interest.

Morphology and Ecology of Two New Amoebae, Isolated From a Thalassohaline Lake, Dziani Dzaha.

Dziani Dzaha is a hypersaline lake (Mayotte island), whose microbial community is dominated by photosynthetic microorganisms. Here, we describe two new free-living heteroloboseans. One belonging to the Pharyngomonas genus and the other, whose 18S rRNA gene sequence shares only 85% homology to its closest relatives Euplaesiobystra hypersalinica, was proposed as a new species of this genus being called Euplaesiobystra dzianiensis. Both strains were salt tolerant to 75‰ and grew between 25 and 37°C. Their distribution patterns varied seasonally and depended also on depth. Noticeably, both free-living amoebae isolates were able to graze on Arthrospira filaments, which are found within the same water layer. In conclusion, we document for the first time the presence and ecology of free-living amoebae in the thalassohaline lake Dziani Dzaha, and describe a new species of the Euplaesiobystra genus.

Characterization of antifungal compounds produced by lactobacilli in cheese-mimicking matrix: Comparison between active and inactive strains.

Biopreservation of dairy products by acid lactic bacteria appears as a promising alternative to either replace or reduce the use of chemical preservatives. This study aimed at the identification of bacteria preventing fungal spoilers growth in dairy products, and, at the understanding of their antifungal activity. First, antifungal activity of eighteen Lactobacillus strains was tested against five molds and four yeasts leading to selection of L. casei 7006 which had an activity against seven fungal targets. Then, challenge tests against C. lusistaniae 3668 in a cheese-mimicking matrix have been performed demonstrating that this strain was able to reduce strongly this yeast growth after 14 and 21 days storages at 7 °C. Antifungal compounds produced in cheese-mimicking matrix containing L. casei 7006 strain were quantified, then compared to the one prepared with an inactive strain (L. casei 6960) or without Lactobacillus strain. Three compounds were differently produced between cheeses with or without Lactobacillus strain after 21 days at 7 °C: lactic acid, benzoic acid and diacetyl. However, lactic acid concentrations were similar between the three cheeses after 14 days at 7 °C, but an antifungal activity was only associated to L. casei 7006 presence. Benzoic acid concentrations between cheese with L. casei 7006 and negative control L. casei 6960 were also the same. Among the antifungal molecules retrieved from these analyses, diacetyl was the most significantly overproduced in cheese containing L. casei 7006, thus this volatile was associated to the antifungal activity of this strain.

Encystment Induces Down-Regulation of an Acetyltransferase-Like Gene in Acanthamoeba castellanii.

Acanthamoeba castellanii is a ubiquitous free-living amoeba. Pathogenic strains are causative agents of Acanthamoeba keratitis and granulomatous amoebic encephalitis. In response to adverse conditions, A. castellanii differentiate into cysts, which are metabolically inactive and resistant cells. This process, also named encystment, involves biochemical and genetic modifications that remain largely unknown. This study characterizes the role of the ACA1_384820 Acanthamoeba gene during encystment. This gene encodes a putative N-acetyltransferase, belonging to the Gcn5-related N-acetyltransferase (GNAT) family. We showed that expression of the ACA1_384820 gene was down-regulated as early as two hours after induction of encystment in A. castellanii. Interestingly, overexpression of the ACA1_384820 gene affects formation of cysts. Unexpectedly, the search of homologs of ACA1_384820 in the Eukaryota gene datasets failed, except for some species in the Acanthamoeba genus. Bioinformatics analysis suggested a possible lateral acquisition of this gene from prokaryotic cells. This study enabled us to describe a new Acanthamoeba gene that is down-regulated during encystment.

Environmental Mycobacterium avium subsp. paratuberculosis Hosted by Free-Living Amoebae.

Mycobacterium avium subsp. paratuberculosis is responsible for paratuberculosis in animals. This disease, leading to an inflammation of the gastrointestinal tract, has a high impact on animal health and an important economic burden. The environmental life cycle of M. avium subsp. paratuberculosis is poorly understood and several studies suggest that free-living amoebae (FLA) might be a potential environmental host. FLA are protozoa found in water and soil that are described as reservoirs of pathogenic and non-pathogenic bacteria in the environment. Indeed, bacteria able to survive within these amoebae would survive phagocytosis from immune cells. In this study, we assessed the in vitro interactions between several strains of M. avium subsp. paratuberculosis and Acanthamoeba castellanii. The results indicate that the bacteria were able to grow within the amoeba and that they can survive for several days within their host. To explore the presence of M. avium subsp. paratuberculosis in environmental amoebae, we sampled water from farms positive for paratuberculosis. A M. avium subsp. paratuberculosis strain was detected within an environmental amoeba identified as related to the poorly described Rosculus genus. The bacterial strain was genotyped, showing that it was similar to previous infectious strains isolated from cattle. In conclusion, we described that various M. avium subsp. paratuberculosis strains were able to grow within amoebae and that these bacteria could be found on farm within amoebae isolated from the cattle environment. It validates that infected amoebae might be a reservoir and vector for the transmission of M. avium subsp. paratuberculosis.

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.

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.

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.

N-acetylglucosamine deacetylases modulate the anchoring of the gamma-glutamyl capsule to the cell wall of Bacillus anthracis.

Bacillus anthracis has a complex cell wall structure composed of a peptidoglycan (PG) layer to which major structures are anchored such as a neutral polysaccharide, an S-layer, and a poly-γ-D-glutamate (PDGA) capsule. Many of these structures have central roles in the biology of B. anthracis, particularly, in virulence. However, little attention has been devoted to structurally study the PG and how it is modified in the presence of these secondary cell wall components. We present here the fine structure of the PG of the encapsulated RPG1 strain harboring both pXO1 and pXO2 virulence plasmids. We show that B. anthracis has a high degree of cross-linking and its GlcNAc residues are highly modified by N-deacetylation. The PG composition is not dependent on the presence of either LPXTG proteins or the capsule. Using NMR analysis of the PG-PDGA complex, we provide evidence for the anchoring of the PDGA to the glucosamine residues. We show that anchoring of the PDGA capsule is impaired in two PG N-deacetylase mutants, Ba1961 and Ba3679. Thus, these multiple N-deactylase activities would constitute excellent drug targets in B. anthracis by simultaneously affecting its resistance to lysozyme and to phagocytosis impairing B. anthracis survival in the host.

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 ß-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.

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.

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.

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/.

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.

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.