Evidence of chitosanase involvement in the protection of bacteria against the antimicrobial activity of the chitosan

Chitosan, a biopolymer composed of [béta]-(1,4)-linked D-glucosamine and N-acetyl-D-glucosamine residues has multiple industrial applications. Recently, chitosan has gained great interest due to its antimicrobial activity. Chitosan has antimicrobial activity against a wide range of target organisms such as bacteria, fungi and viruses. This antimicrobial activity is based on its cationic character, and is mediated by the chitosan's positively charged amino groups interactions with negatively charged residues in the bacterial cell wall. Enzymes with chitosanase activity catalyzing the hydrolysis of glycoside linkages in chitosan are found in many organisms, including bacteria, fungi, and plants. In the last three decades, chitosanases have been intensively studied as tools for biotechnological transformation of chitosan. However, less is known about their physiological functions in chitosanase-producing microorganisms. Previous reports have characterized chitosanases as metabolic enzymes allowing bacteria to use chitosan as carbon and nitrogen sources. The aim of this research project was to examine chitosanases significance as possible resistance factors against the antimicrobial effect of chitosan. Our work, as well as previous studies realized in our laboratory, showed that expression of a heterologous chitosanase gene in the Gram-negative bacterium Escherichia coli (naturally devoid of chitosanase activity) increases the level of resistance against chitosan. Interestingly, the resistance level to chitosan was influenced by the relative activity of the heterologous chitosanase. The expression of inactive heterologous chitosanase did not confer any resistance to chitosan supporting our hypothesis that chitosanases may have a role in the protection against the antimicrobial effect of chitosan. In order to obtain more direct evidence sustaining our hypothesis, we inactivated the chitosanase gene from Streptomyces lividans TK24. Hence, we developed a new system for gene disruption and replacement in Streptomyces with cytosine deaminase as negative selection marker. The disruption of the chitosanase gene in S. lividans TK24 resulted in an increased susceptibility of the mutant strain towards the toxic effect of chitosan. Our in vivo experiments showed that, in the presence of chitosan, growth of this mutant strain as well as its ability for xylose uptake were impaired compared to the wildtype strain. This represents the first genetical proof for the protective role of a chitosanase against the bactericidal effect of chitosan. In our quest to discover chitosanases with new characteristics, we determined the biochemical properties of the chitosanase CsnA from Streptomyces coelicolor A3(2). Our studies revealed that CsnA was, in many aspects, very similar to the chitosanase CsnN174 from Streptomyces sp. N174. An interesting feature of the CsnA is its secretion. The signal peptide of the CsnA has a Tat-dependent motif. The CsnA is the first studied chitosanase to be secreted via the Tat pathway. These studies also contributed to a better understanding of the chitosanase secretion. Evidence concerning the role of chitosanases in the protection of bacteria against the bactericidal effect of chitosan was also brought by the study of cell localization of the exo-[béta]-D-glucosaminidase (CsxA) from Amycolatopsis orientalis. CsxA has a carbohydrate-binding module (CBM35) with an unusual affinity This module appended to CsxA recognizes as substrate glucuronic acid, a component of the Gram-positive bacterie cell wall. Thereby, we analyzed by epifluorescence and confocal microscopy the cellular localization of the CsxA-CBM35 in Amycolatopsis orientalis cells grown in the presence of chitosan

Uncovering the Prevalence and Diversity of Integrating Conjugative Elements in Actinobacteria

Horizontal gene transfer greatly facilitates rapid genetic adaptation of bacteria to shifts in environmental conditions and colonization of new niches by allowing one-step acquisition of novel functions. Conjugation is a major mechanism of horizontal gene transfer mediated by conjugative plasmids and integrating conjugative elements (ICEs). While in most bacterial conjugative systems DNA translocation requires the assembly of a complex type IV secretion system (T4SS), in Actinobacteria a single DNA FtsK/SpoIIIE-like translocation protein is required. To date, the role and diversity of ICEs in Actinobacteria have received little attention. Putative ICEs were searched for in 275 genomes of Actinobacteria using HMM-profiles of proteins involved in ICE maintenance and transfer. These exhaustive analyses revealed 144 putative FtsK/SpoIIIE-type ICEs and 17 putative T4SS-type ICEs. Grouping of the ICEs based on the phylogenetic analyses of maintenance and transfer proteins revealed extensive exchanges between different sub-families of ICEs. 17 ICEs were found in Actinobacteria from the genus Frankia, globally important nitrogen-fixing microorganisms that establish root nodule symbioses with actinorhizal plants. Structural analysis of ICEs from Frankia revealed their unexpected diversity and a vast array of predicted adaptive functions. Frankia ICEs were found to excise by site-specific recombination from their host's chromosome in vitro and in planta suggesting that they are functional mobile elements whether Frankiae live as soil saprophytes or plant endosymbionts. Phylogenetic analyses of proteins involved in ICEs maintenance and transfer suggests that active exchange between ICEs cargo-borne and chromosomal genes took place within the Actinomycetales order. Functionality of Frankia ICEs in vitro as well as in planta lets us anticipate that conjugation and ICEs could allow the development of genetic manipulation tools for this challenging microorganism and for many other Actinobacteria

Mechanisms of regulation of haptoglobin by C/EBPs

Les cellules épithéliales intestinales participent à une réponse inflammatoire aiguë (RIA) lors de l'inflammation de l'intestin. Nous avons démontré que la RIA induit l'expression de gènes encodant les protéines de la réponse inflammatoire. La modulation de la synthèse d'une molécule pro-inflammatoire est une stratégie essentielle dans la régulation du processus inflammatoire caractéristique de plusieurs maladies comme les maladies inflammatoire intestinales. Il a été démontré que différents inhibiteurs comme les inhibiteurs du protéasome peuvent réprimer l'action de molécules pro-inflammatoires. Par exemple, le MG132 est un inhibiteur très spécifique de la dégradation protéique par le système biquitine/protéasome. MG 132 module négativement l'activation de gènes spécifiques impliqués dans la RIA via l'inhibition de NF-KB. La famille des facteurs de transcription C/EBP (CCAAT/enhancer binding protein) est aussi impliquée dans la régulation de gènes de réponse inflammatoire dans les cellules épithéliales intestinales. Dans cette étude, nous avons vérifié si MG132 modifie l'expression des cibles de C/EBP ou la stabilité des isoformes de C/EBP. Dans les cellules épithéliales intestinales, nous avons démontré par Northem blot que MG132 cause une diminution de l'induction IL-1 dépendante du gène encodant la protéine de la réponse inflammatoire haptoglobine. L'effet de MG132 sur l'expression de l'haptoglobine est transcriptionnel, tel que démontré par transfections transitoires. MG132 entraîne une augmentation de l'activité de liaison des C/EBPs à l'ADN au site consensus HaptoA ainsi qu'un changement dans la mobilité du complexe C/EBP, peut être dû à la formation d'une nouvelle forme de C/EBPf) de 26 kD. Cette forme paraît ne pas être le résultat d'un clivage causé par les caspases. Par des études de Western blot, nous avons montré que MG 132 entraîne une stabilisation des isoformes C/EBP.B et C/EBPô. Ces résultats suggèrent qu'en plus d'une modulation des gènes régularisés par NF-KB, MG132 peut diminuer l'expression des gènes régularisés par les C/EBPs. De plus, la stabilité de C/EBP{J et C/EBPô est contrôlée par une voie protéasomedépendante.Abstract: Effect of the proteasome inhibitor MG132 on the inflammatory response in intestinal epithelial cells. Intestinal epithelial cells participate in the acute phase response (APR) during intestinal inflammation. We have shown that acute phase protein (APP) genes are induced during the intestinal APR. One way of repressing the activity of proinflammatory molecules is by the use of different inhibitors such as proteasome inhibitors. MG132 is a highly specific inhibitor of protein degradation via the ubiquitin/proteasome system and negatively modulates the activation of specific genes involved in the intestinal acute phase response via NF-[kappa]B inhibition. Effect of C/EBP[alpha] mutant overexpression on haptoglobin expression in intestinal epithelial cells. The transcription factor C/EBP[alpha] plays an important role in the regulation of haptoglobin expression in intestinal epithelial cells, but the specific regions involved in this regulation were unknown. To determine which regions of the C/EBP[alpha] isoform are involved in the control of haptoglobin expression, we generated IEC-6 rat intestinal epithelial cell lines stably expressing different C/EBP[alpha] mutants, by infection with the retroviral vector pBabepuro."--Résumé abrégé par UMI

Antibiofilm and antibacterial effects of specific chitosan molecules on Staphylococcus aureus isolates associated with bovine mastitis.

Staphylococcus aureus is one of the major pathogens causing bovine intramammary infections (IMIs) and mastitis. Mastitis is the primary cause for the use of antibiotics in dairy farms but therapeutic failure is often observed. One of the reasons for the lack of effectiveness of antibiotic therapy despite the observed susceptibility of bacterial isolates in vitro are bacterial biofilms. In this study, we used chitosan of well-defined molecular weight (0.4-0.6, 1.3, 2.6 and 4.0 kDa) and investigated their antibiofilm and antibacterial activities in in vitro and in vivo models related to S. aureus IMIs. A chitosan of at least 6 units of glucosamine was necessary for maximum antibacterial activity. The 2.6 and 4.0 kDa forms were able to prevent biofilm production by the biofilm hyperproducer strain S. aureus 2117 and a bovine MRSA (methicillin-resistant S. aureus). The intramammary administration of the 2.6 kDa chitosan showed no adverse effects in mice or in cows, as opposed to the slight inflammatory effect observed in mammary glands with the 4.0 kDa derivative. The 2.6 kDa chitosan killed bacteria embedded in pre-established biofilms in a dose-dependent manner with a >3 log10 reduction in CFU at 4 mg/ml. Also, the 2.6 kDa chitosan could prevent the persistence of the internalized MRSA into the mammary epithelial cell line MAC-T. An in vitro checkerboard assay showed that the 2.6 kDa chitosan produced a synergy with the macrolide class of antibiotics (e.g., tilmicosin) and reduced the MIC of both molecules by 2-8 times. Finally, the intramammary administration of the 2.6 kDa chitosan alone (P<0.01) or in combination with tilmicosin (P<0.0001) reduced the colonization of mammary glands in a murine IMI model. Our results suggest that the use of chitosan alone or in combination with a low dose of a macrolide could help reduce antibiotic use in dairy farms

Cytosine Deaminase as a Negative Selection Marker for Gene Disruption and Replacement in the Genus Streptomyces and Other Actinobacteria ▿ †

We developed a novel negative selection system for actinobacteria based on cytosine deaminase (CodA). We constructed vectors that include a synthetic gene encoding the CodA protein from Escherichia coli optimized for expression in Streptomyces species. Gene disruption and the introduction of an unmarked in-frame deletion were successfully achieved with these vectors