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

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

MIC values of antibiotics and chitosan (2.6 kDa) in a checkerboard assay against <i>S</i>. <i>aureus</i> ATCC 29213.

<p>MIC values of antibiotics and chitosan (2.6 kDa) in a checkerboard assay against <i>S</i>. <i>aureus</i> ATCC 29213.</p

Relative innocuity of different forms of chitosan in cows.

<p>Each quarter of cow’s udder has received 500 mg of chitosan (2.6 kDa or 4.0 kDa) or saline as the negative control. Milk samples and somatic cell counts (SCC) were determined 12 and 3 hours before the instillation of chitosan. After the intramammary instillation, milk samples were aseptically collected from cows at several points in time to evaluate inflammation by determining the SCC (A) and milk yields (B). Symbols represent the means and vertical lines the standard deviation. Data were analyzed by ANOVA using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). For the first experiment, time was used as a repeated effect and treatment (cow) was used as the subject. Orthogonal contrasts were performed to compare the effect of each treatment to control. No difference was observed between saline and the 2.6 kDa chitosan (SCC and quarter milk yield). Significant differences were observed between saline and 4.0 kDa for SCC: *, <i>P</i><0.05; **, <i>P</i>< 0.01; ***, <i>P</i><0.0001.</p

Antibiofilm activity of the different forms of chitosan against MRSA 1158c and the biofilm hyperproducer strain 2117.

<p>(A) CHOS, (B) 1.3 kDa, (C) 2.6 kDa, and (D) 4.0 kDa chitosan. Bars represent the means and vertical lines the standard deviation (SD). Data were obtained from three independent experiments. Significant differences in comparison to the untreated control (0 mg/ml) are shown by asterisks. Statistical analysis was performed using Kruskal-Wallis test (non-parametric one way ANOVA) with Dunn’s multiple comparison test: ns, non significant; *, <i>P</i><0.05.</p

Preformed biofilms of <i>S</i>. <i>aureus</i> strains exposed to the 2.6 kDa chitosan.

<p>(A) Reduction of a preformed biofilm on pegs following exposure to increasing concentrations of chitosan. (B) Bactericidal effect of chitosan on preformed biofilms. The CFU/peg after 24 h of biofilm formation was evaluated for control pegs (CTRL 24 h) and this represented the inoculum at the onset of treatment, which occurred at 24 h for another 24 h of incubation. The CFU/peg obtained for the untreated pegs after the total incubation period served as the reference (CTRL 48 h) for treatment efficacy. Data were obtained from three independent experiments. Significant differences in comparison to the untreated controls (0 mg/ml in A, and CTRL 48 h in B) are shown. Statistical analysis was performed using non-parametric one way ANOVA: **, <i>P</i><0.005; ***, <i>P</i><0.001; ****, <i>P</i><0.0001.</p