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.

A Localized Phage-Based Antimicrobial System: Effect of Alginate on Phage Desorption from ß-TCP Ceramic Bone Substitutes.

Tricalcium phosphate (TCP) is a prosthetic material commonly used as a bone substitute to repair osteoarticular diseases and injuries. In this type of bone reconstruction surgery, antibiotics remain the common preventive and therapeutic treatment for bacterial infection. Nevertheless, the emergence of multi-resistant strains requires complimentary or alternative treatments. Today, one of the promising alternative approaches is phage therapy. Phages are bacterial viruses that have several advantages over chemotherapy, such as the specificity of bacterial strain, the absence of side effects, and a rapid response. In this work, we studied the impact of alginate hydrogels for overlaying λvir-phage-loaded ß-TCP ceramic bone substitutes, delaying the phage desorption. The results show that the use of a 1% alginate-CaCl2 hydrogel overlapping the ß-TCP ceramic pellets leads to higher initial phage concentration on the material and extends the released time of phages to two weeks when compared with control pellets. These alginate-coated biomaterials also generate faster bacterial lysis kinetics and could therefore be a good practical prosthetic device for bone and joint surgeries by allowing local treatment of bacterial infections with phage therapy for a longer period of time.

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.

New antibacterial microporous CaP materials loaded with phages for prophylactic treatment in bone surgery.

Hydroxyapatite and beta-tricalcium phosphate (ß-TCP) are materials commonly used in bone repair. The most important problem occurring in bone repair surgery is bacterial infection which is usually overcome by treatment with antibiotics. Currently, emergence of multidrug resistant strains has led to development of alternative treatments such as phage therapy. Phages are bacterial viruses with several advantages over chemotherapy such as specificity of bacterial strain, no side effects and fast response. This study evaluates the possibility of loading hydroxyapatite and ß-tricalcium phosphate ceramics used as bone substitutes with phages and their antibacterial activity against Escherichia coli K12. The majority of phages were retained in dense and microporous HA and ß-TCP samples during at least 6 days suggesting the occurrence of strong interaction between phages and ceramics, which did not prevent bacterial attachment and lysis. This study has shown for the first time that phage loaded ceramics could be used in prophylactic treatments.

The virulence of a Dickeya dadantii 3937 mutant devoid of osmoregulated periplasmic glucans is restored by inactivation of the RcsCD-RcsB phosphorelay.

Dickeya dadantii is a pectinolytic phytopathogen enterobacterium that causes soft rot disease on a wide range of plant species. The virulence of D. dadantii involves several factors, including the osmoregulated periplasmic glucans (OPGs) that are general constituents of the envelope of proteobacteria. In addition to the loss of virulence, opg-negative mutants display a pleiotropic phenotype, including decreased motility and increased exopolysaccharide synthesis. A nitrosoguanidine-induced mutagenesis was performed on the opgG strain, and restoration of motility was used as a screen. The phenotype of the opg mutant echoes that of the Rcs system: high level activation of the RcsCD-RcsB phosphorelay is needed to activate exopolysaccharide synthesis and to repress motility, while low level activation is required for virulence in enterobacteria. Here, we show that mutations in the RcsCDB phosphorelay system restored virulence and motility in a D. dadantii opg-negative strain, indicating a relationship between the Rcs phosphorelay and OPGs.

Proteomic analysis of a non-virulent mutant of the phytopathogenic bacterium Erwinia chrysanthemi deficient in osmoregulated periplasmic glucans: change in protein expression is not restricted to the envelope, but affects general metabolism.

Osmoregulated periplasmic glucans (OPGs) are general constituents of the envelope of Gram-negative bacteria. They are required for full virulence of bacterial phytopathogens such as Pseudomonas syringae, Xanthomonas campestris and Erwinia chrysanthemi. E. chrysanthemi is a pectinolytic gamma-proteobacterium that causes soft rot disease on a wide range of plant species. In addition to the loss of virulence, opg mutants exhibit a pleiotropic phenotype that affects motility, bile-salt resistance, exoenzyme secretion, exopolysaccharide synthesis and membrane lipid composition. This is believed to be the first proteomic analysis of an OPG-defective mutant of E. chrysanthemi and it revealed that, in addition to the effects described, catabolic enzyme synthesis was enhanced and there was a greater abundance of some proteins catalysing the folding and degradation of proteins needed for various stress responses. Thus, in the opg mutant strain, loss of virulence was the result of a combination of envelope structure changes and cellular metabolism modifications.

GC/MS identification and quantification of constituents of bacterial lipids and glycoconjugates obtained after methanolysis as heptafluorobutyrate derivatives.

In previous articles [Anal. Biochem. 284 (2000) 201; J. Lipid Res. 43 (2002) 794], we reported that the GC/MS identification and quantification of nearly all constituents of glycolipids could be obtained on the same sample in a single GC/MS analysis as heptafluorobutyrate derivatives of the products liberated using acid-catalyzed methanolysis. The same type of data could be obtained on glycoproteins and proteoglycans [Biochemistry 42 (2003) 8342]. These experiments were performed on material from higher organisms, and there was no evidence that bacteria-specific constituents could also be identified and quantified. The current article reports that the GC/MS analysis of compounds liberated by acid-catalyzed methanolysis as heptafluorobutyrate derivatives allows the simultaneous qualitative and quantitative determinations of pentoses, deoxyhexoses, hexoses, hexosamines, uronic acids, Kdo, Mur, heptose, Kdn, and neuraminic acid as well as of most fatty acids (including hydroxylated fatty acids). This approach provides a way of obtaining fingerprints of bacterial constituents and quantification of the overall effect of gene inactivation or of culture conditions.