Phage-Loaded Biomimetic Apatite Powder With Antibiofilm Activity to Treat Bone-Associated Infections.

For decades, calcium phosphate (CaP)-based ceramics have been used for coating of bone and joint substitutes after arthroplasty due to their biocompatible properties. Infections following orthopedic replacement occur in 1%-5% of cases, causing serious complications. Biofilm formation either on the biomaterial's surface or on patient's tissues greatly enhances the resistance against antibiotic treatments and can induce a chronic infection, emphasizing the need for novel antimicrobial delivery systems. In this study, we established a protocol enabling bacteriophage loading during the synthesis of a CaP-based powder. The resulting biomaterial proved to be noncytotoxic against human osteoblastic cells and able to significantly inhibit 24-h matured S. aureus biofilm cultures or even completely eradicate it after 5 days of contact. Additional S. aureus biofilm assays with a freeze-dried material using two different excipients showed that sucrose had a protective role against Remus bacteriophage treatment of S. aureus biofilms, whereas lactose-freeze-dried powder maintained the antibiofilm activity.

Emergence of the Dickeya genus involved duplication of the OmpF porin and the adaptation of the EnvZ-OmpR signaling network.

Genome evolution, and more specifically gene duplication, is a key process shaping host-microorganism interaction. The conserved paralogs usually provide an advantage to the bacterium to thrive. If not, these genes become pseudogenes and disappear. Here, we show that during the emergence of the genus Dickeya, the gene encoding the porin OmpF was duplicated. Our results show that the ompF2 expression is deleterious to the virulence of Dickeya dadantii, the agent causing soft rot disease. Interestingly, ompF2 is regulated while ompF is constitutive but activated by the EnvZ-OmpR two-component system. In vitro, acidic pH triggers the system. The pH measured in four eudicotyledons increased from an initial pH of 5.5 to 7 within 8 h post-infection. Then, the pH decreased to 5.5 at 10 h post-infection and until full maceration of the plant tissue. Yet, the production of phenolic acids by the plant's defenses prevents the activation of the EnvZ-OmpR system to avoid the ompF2 expression even though environmental conditions should trigger this system. We highlight that gene duplication in a pathogen is not automatically an advantage for the infectious process and that, there was a need for our model organism to adapt its genetic regulatory networks to conserve these duplicated genes. IMPORTANCE Dickeya species cause various diseases in a wide range of crops and ornamental plants. Understanding the molecular program that allows the bacterium to colonize the plant is key to developing new pest control methods. Unlike other enterobacterial pathogens, Dickeya dadantii, the causal agent of soft rot disease, does not require the EnvZ-OmpR system for virulence. Here, we showed that during the emergence of the genus Dickeya, the gene encoding the porin OmpF was duplicated and that the expression of ompF2 was deleterious for virulence. We revealed that while the EnvZ-OmpR system was activated in vitro by acidic pH and even though the pH was acidic when the plant is colonized, this system was repressed by phenolic acid (generated by the plant's defenses). These results provide a unique- biologically relevant-perspective on the consequence of gene duplication and the adaptive nature of regulatory networks to retain the duplicated gene.

Reliable Approach for Pure Yeast Cell Wall Protein Isolation from Saccharomyces cerevisiae Yeast Cells.

Saccharomyces cerevisiae yeast is a fungus presenting a peripheral organelle called the cell wall. The cell wall protects the yeast cell from stress and provides means for communication with the surrounding environment. It has a complex molecular structure, composed of an internal part of cross-linked polysaccharides and an external part of mannoproteins. These latter are very interesting owing to their functional properties, dependent on their molecular features with massive mannosylations. Therefore, the molecular characterization of mannoproteins is a must relying on the optimal isolation and preparation of the cell wall fraction. Multiple methods are well reported for yeast cell wall isolation. The most applied one consists of yeast cell lysis by mechanical disruption. However, applying this classical approach to S288C yeast cells showed considerable contamination with noncell wall proteins, mainly comprising mitochondrial proteins. Herein, we tried to further purify the yeast cell wall preparation by two means: ultracentrifugation and Triton X-100 addition. While the first strategy showed limited outcomes in mitochondrial protein removal, the second strategy showed optimal results when Triton X-100 was added at 5%, allowing the identification of more mannoproteins and significantly enriching their amounts. This promising method could be reliably implemented on the lab scale for identification of mannoproteins and molecular characterization and industrial processes for "pure" cell wall isolation.

What do we know about osmoadaptation of Yersinia pestis?

The plague agent Yersinia pestis mainly spreads among mammalian hosts and their associated fleas. Production of a successful mammal-flea-mammal life cycle implies that Y. pestis senses and responds to distinct cues in both host and vector. Among these cues, osmolarity is a fundamental parameter. The plague bacillus lives in a tightly regulated environment in the mammalian host, while osmolarity fluctuates in the flea gut (300-550 mOsM). Here, we review the mechanisms that enable Y. pestis to perceive fluctuations in osmolarity, as well as genomic plasticity and physiological adaptation of the bacterium to this stress.

Modified Galacto- or Fuco-Clusters Exploiting the Siderophore Pathway to Inhibit the LecA- or LecB-Associated Virulence of Pseudomonas aeruginosa.

Galacto- and fuco-clusters conjugated with one to three catechol or hydroxamate motifs were synthesised to target LecA and LecB lectins of Pseudomonas aeruginosa (PA) localised in the outer membrane and inside the bacterium. The resulting glycocluster-pseudosiderophore conjugates were evaluated as Trojan horses to cross the outer membrane of PA by iron transport. The data suggest that glycoclusters with catechol moieties are able to hijack the iron transport, whereas those with hydroxamates showed strong nonspecific interactions. Mono- and tricatechol galactoclusters (G1C and G3C) were evaluated as inhibitors of infection by PA in comparison with the free galactocluster (G0). All of them exhibited an inhibitory effect between 46 to 75 % at 100 µM, with a higher potency than G0. This result shows that LecA localised in the outer membrane of PA is involved in the infection mechanism.

3D printed bioceramic for phage therapy against bone nosocomial infections.

This study provides a new therapeutic response to postoperative joint and bone infections. Alone or in combination with antibiotics, phage therapy has many advantages, including accurate targeting of pathogenic bacteria. In addition, a decrease in harmful side effects can improve the healing process. Integrating the bacteriophage directly into the graft product will improve the antibacterial spread over the site of the surgery. The phage cocktail-filled ceramics are an innovative device for localized and curative phage therapy (in prosthetic replacement surgery, for example) in bone and joint surgery. Calcium phosphate-based ceramics were synthesized and shaped by stereolithography (3D) before loading by a phage cocktail to lyse a heterospecific bacterial population. In addition, the device makes possible the protection of osteoblastic cells against Staphylococcus aureus infection during their colonization on the ceramic material and prevents the formation of biofilm on the surface of biomaterials.

Structural and Proteomic Changes in Viable but Non-culturable Vibrio cholerae.

Aquatic environments are reservoirs of the human pathogen Vibrio cholerae O1, which causes the acute diarrheal disease cholera. Upon low temperature or limited nutrient availability, the cells enter a viable but non-culturable (VBNC) state. Characteristic of this state are an altered morphology, low metabolic activity, and lack of growth under standard laboratory conditions. Here, for the first time, the cellular ultrastructure of V. cholerae VBNC cells raised in natural waters was investigated using electron cryo-tomography. This was complemented by a comparison of the proteomes and the peptidoglycan composition of V. cholerae from LB overnight cultures and VBNC cells. The extensive remodeling of the VBNC cells was most obvious in the passive dehiscence of the cell envelope, resulting in improper embedment of flagella and pili. Only minor changes of the peptidoglycan and osmoregulated periplasmic glucans were observed. Active changes in VBNC cells included the production of cluster I chemosensory arrays and change of abundance of cluster II array proteins. Components involved in iron acquisition and storage, peptide import and arginine biosynthesis were overrepresented in VBNC cells, while enzymes of the central carbon metabolism were found at lower levels. Finally, several pathogenicity factors of V. cholerae were less abundant in the VBNC state, potentially limiting their infectious potential. This study gives unprecedented insight into the physiology of VBNC cells and the drastically altered presence of their metabolic and structural proteins.

The EnvZ-OmpR Two-Component Signaling System Is Inactivated in a Mutant Devoid of Osmoregulated Periplasmic Glucans in Dickeya dadantii.

Osmoregulated periplasmic glucans (OPGs) are general constituents of alpha-, beta-, and gamma-Proteobacteria. This polymer of glucose is required for full virulence of many pathogens including Dickeya dadantii (D. dadantii). The phytopathogenic enterobacterium D. dadantii causes soft-rot disease in a wide range of plants. An OPG-defective mutant is impaired in environment sensing. We previously demonstrated that (i) fluctuation of OPG concentration controlled the activation level of the RcsCDB system, and (ii) RcsCDB along with EnvZ/OmpR controlled the mechanism of OPG succinylation. These previous data lead us to explore whether OPGs are required for other two-component systems. In this study, we demonstrate that inactivation of the EnvZ/OmpR system in an OPG-defective mutant restores full synthesis of pectinase but only partial virulence. Unlike for the RcsCDB system, the EnvZ-OmpR system is not controlled by OPG concentration but requires OPGs for proper activation.

A Sensitive and Rapid Method to Determin the Adhesion Capacity of Probiotics and Pathogenic Microorganisms to Human Gastrointestinal Mucins.

Mucus is the habitat for the microorganisms, bacteria and yeast that form the commensal flora. Mucins, the main macromolecules of mucus, and more specifically, the glycans that cover them, play essential roles in microbial gastrointestinal colonization. Probiotics and pathogens must also colonize mucus to have lasting positive or deleterious effects. The question of which mucin-harboured glycan motifs favour the adhesion of specific microorganisms remains very poorly studied. In the current study, a simple test based on the detection of fluorescent-labeled microorganisms raised against microgram amounts of mucins spotted on nitrocellulose was developed. The adhesion of various probiotic, commensal and pathogenic microorganisms was evaluated on a panel of human purified gastrointestinal mucins and compared with that of commercially available pig gastric mucins (PGM) and of mucins secreted by the colonic cancer cell line HT29-MTX. The latter two proved to be very poor indicators of adhesion capacity on intestinal mucins. Our results show that the nature of the sialylated cores of O-glycans, determined by MALDI MS-MS analysis, potentially enables sialic acid residues to modulate the adhesion of microorganisms either positively or negatively. Other identified factors affecting the adhesion propensity were O-glycan core types and the presence of blood group motifs. This test should help to select probiotics with enhanced adhesion capabilities as well as deciphering the role of specific mucin glycotopes on microbial adhesion.

The Chlamydomonas mex1 mutant shows impaired starch mobilization without maltose accumulation.

The MEX1 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that affects starch metabolism. Mutation of MEX1 causes a slow-down in the mobilization of storage polysaccharide. Cosegregation and functional complementation analyses were used to assess the involvement of the Mex1 protein in starch degradation. Heterologous expression experiments performed in Escherichia coli and Arabidopsis thaliana allowed us to test the capacity of the algal protein in maltose export. In contrast to the A. thaliana mex1 mutant, the mutation in C. reinhardtii does not lead to maltose accumulation and growth impairment. Although localized in the plastid envelope, the algal protein does not transport maltose efficiently across the envelope, but partly complements the higher plant mutant. Both Mex orthologs restore the growth of the E. coli ptsG mutant strain on glucose-containing medium, revealing the capacity of these proteins to transport this hexose. These findings suggest that Mex1 is essential for starch mobilization in both Chlamydomonas and Arabidopsis, and that this protein family may support several functions and not only be restricted to maltose export across the plastidial envelope.

Osmoregulated Periplasmic Glucans.

Among all the systems developed by enterobacteria to face osmotic stress, only osmoregulated periplasmic glucans (OPGs) were found to be modulated during osmotic fluxes. First detected in 1973 by E.P. Kennedy's group in a study of phospholipid turnover in Escherichia coli, OPGs have been shown across alpha, beta, and gamma subdivisions of the proteobacteria. Discovery of OPG-like compounds in the epsilon subdivision strongly suggested that the presence of periplasmic glucans is essential for almost all proteobacteria. This article offers an overview of the different classes of OPGs. Then, the biosynthesis of OPGs and their regulation in E. coli and other species are discussed. Finally, the biological role of OPGs is developed. Beyond structural function, OPGs are involved in pathogenicity, in particular, by playing a role in signal transduction pathways. Recently, OPG synthesis proteins have been suggested to control cell division and growth rate.

Scout-MRM: Multiplexed Targeted Mass Spectrometry-Based Assay without Retention Time Scheduling Exemplified by Dickeya dadantii Proteomic Analysis during Plant Infection.

Targeted mass spectrometry of a surrogate peptide panel is a powerful method to study the dynamics of protein networks, but chromatographic time scheduling remains a major limitation for dissemination and implementation of robust and large multiplexed assays. We unveil a Multiple Reaction Monitoring method (Scout-MRM) where the use of spiked scout peptides triggers complex transition lists, regardless of the retention time of targeted surrogate peptides. The interest of Scout-MRM method regarding the retention time independency, multiplexing capability, reproducibility, and putative interest in facilitating method transfer was illustrated by a 782-peptide-plex relative assay targeting 445 proteins of the phytopathogen Dickeya dadantii during plant infection.

The opgC gene is required for OPGs succinylation and is osmoregulated through RcsCDB and EnvZ/OmpR in the phytopathogen Dickeya dadantii.

Osmoregulated periplasmic glucans (OPGs) are a family of periplasmic oligosaccharides found in the envelope of most Proteobacteria. They are required for virulence of zoo- and phyto-pathogens. The glucose backbone of OPGs is substituted by various kinds of molecules depending on the species, O-succinyl residues being the most widely distributed. In our model, Dickeya dadantii, a phytopathogenic bacteria causing soft rot disease in a wide range of plant species, the backbone of OPGs is substituted by O-succinyl residues in media of high osmolarity and by O-acetyl residues whatever the osmolarity. The opgC gene encoding a transmembrane protein required for the succinylation of the OPGs in D. dadantii was found after an in silico search of a gene encoding a protein with the main characteristics recovered in the two previously characterized OpgC of E. coli and R. sphaeroides, i.e. 10 transmembrane segments and one acyl-transferase domain. Characterization of the opgC gene revealed that high osmolarity expression of the succinyl transferase is controlled by both the EnvZ-OmpR and RcsCDB phosphorelay systems. The loss of O-succinyl residue did not affect the virulence of D. dadantii, suggesting that only the glucose backbone of OPGs is required for virulence.

New insights into the biological role of the osmoregulated periplasmic glucans in pathogenic and symbiotic bacteria.

This review emphasizes the biological roles of the osmoregulated periplasmic glucans (OPGs). Osmoregulated periplasmic glucans occur in almost all α-, ß- and γ-Proteobacteria. This polymer of glucose is required for full virulence. The roles of the OPGs are complex and vary depending on the species. Here, we outline the four major roles of the OPGs through four different pathogenic and one symbiotic bacterial models (Dickeya dadantii, Salmonella enterica, Pseudomonas aeruginosa, Brucella abortus and Sinorhizobium meliloti). When periplasmic, the OPGs are a part of the signal transduction pathway and indirectly regulate genes involved in virulence. The OPGs can also be secreted. When outside of the cell, they interact directly with antibiotics to protect the bacterial cell or interact with the host cell to facilitate the invasion process. When OPGs are not found, as in the ε-Proteobacteria, OPG-like oligosaccharides are present. Their presence strengthens the evidence that OPGs play an important role in virulence.

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis.

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥ 37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary.

Mannose-centered aromatic galactoclusters inhibit the biofilm formation of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA) is a major public health care issue due to its ability to develop antibiotic resistance mainly through adhesion and biofilm formation. Therefore, targeting the bacterial molecular arsenal involved in its adhesion and the formation of its biofilm appears as a promising tool against this pathogen. The galactose-binding LecA (or PA-IL) has been described as one of the PA virulence factors involved in these processes. Herein, the affinity of three tetravalent mannose-centered galactoclusters toward LecA was evaluated with five different bioanalytical methods: HIA, ELLA, SPR, ITC and DNA-based glycoarray. Inhibitory potential towards biofilms was then assessed for the two glycoclusters with highest affinity towards LecA (Kd values of 157 and 194 nM from ITC measurements). An inhibition of biofilm formation of 40% was found for these galactoclusters at 10 µM concentration. Applications of these macromolecules in anti-bacterial therapy are therefore possible through an anti-adhesive strategy.

The two-component system CpxAR is essential for virulence in the phytopathogen bacteria Dickeya dadantii†EC3937.

The CpxAR two-component system is present in many Proteobacteria. It controls expression of genes required to maintain envelope integrity in response to environmental injury. Consequently, this two-component system was shown to be required for virulence of several zoo-pathogens, but it has never been investigated in phyto-pathogens. In this paper, we investigate the role of the CpxAR two-component system in vitro and in vivo in Dickeya dadantii, an enterobacterial phytopathogen that causes soft-rot disease in a large variety of plant species. cpxA null mutant displays a constitutively phosphorylated CpxR phenotype as shown by direct analysis of phosphorylation of CpxR by a Phos-Tag retardation gel approach. Virulence in plants is completely abolished in cpxA or cpxR mutants of D. dadantii. In planta, CpxAR is only activated at an early stage of the infection process as shown by Phos-Tag and gene fusion analyses. To our knowledge, this is the first time that the timing of CpxAR phosphorelay activation has been investigated during the infection process by direct monitoring of response regulator phosphorylation.

Increased phosphorylation of the RcsB regulator of the RcsCDB phosphorelay in strains of Dickeya dadantii devoid of osmoregulated periplasmic glucans revealed by Phos-tag gel analysis.

Osmoregulated periplasmic glucans (OPGs) are general constituents of many proteobacteria. OPGs are important factors required for full virulence in many pathogens including Dickeya dadantii. D. dadantii causes the soft-rot disease in a wide range of plant species. The pleiotropic phenotype of opg-negative strains includes total loss of virulence and motility, and is linked to the constitutive activation of the RcsCDB phosphorelay, deduced from expression analysis of genes of the RcsCDB regulon. The constitutive activation of the RcsCDB phosphorelay in an opg-negative strain was demonstrated by direct analysis of the phosphorylation level of the RcsB regulator protein in vivo by using a Phos-tag retardation gel approach, and was correlated with the phenotype and the expression of motility genes. Data revealed a low level of RcsB phosphorylated form in the wild-type strain and a slight increase of phosphorylation in opgG mutant strains sufficient to induce the pleiotropic phenotype observed.

Inactivation of pecS restores the virulence of mutants devoid of osmoregulated periplasmic glucans in the phytopathogenic bacterium Dickeya dadantii.

Dickeya dadantii is a phytopathogenic enterobacterium that causes soft rot disease in a wide range of plant species. Maceration, an apparent symptom of the disease, is the result of the synthesis and secretion of a set of plant cell wall-degrading enzymes (PCWDEs), but many additional factors are required for full virulence. Among these, osmoregulated periplasmic glucans (OPGs) and the PecS transcriptional regulator are essential virulence factors. Several cellular functions are controlled by both OPGs and PecS. Strains devoid of OPGs display a pleiotropic phenotype including total loss of virulence, loss of motility and severe reduction in the synthesis of PCWDEs. PecS is one of the major regulators of virulence in D. dadantii, acting mainly as a repressor of various cellular functions including virulence, motility and synthesis of PCWDEs. The present study shows that inactivation of the pecS gene restored virulence in a D. dadantii strain devoid of OPGs, indicating that PecS cannot be de-repressed in strains devoid of OPGs.

Biosynthesis of osmoregulated periplasmic glucans in Escherichia coli: the phosphoethanolamine transferase is encoded by opgE.

Osmoregulated periplasmic glucans (OPGs) are oligosaccharides found in the periplasm of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this backbone may be substituted by various kinds of molecules depending on the species. In E. coli, OPG are substituted by phosphoglycerol and phosphoethanolamine derived from membrane phospholipids and by succinyl residues. In this study, we describe the isolation of the opgE gene encoding the phosphoethanolamine transferase by a screen previously used for the isolation of the opgB gene encoding the phosphoglycerol transferase. Both genes show structural and functional similarities without sequence similarity.