| Abstract |
The development of new antibacterial agents to combat the rapidly increasing antibiotic resistance is a priority area in antiinfectives research. Peptidoglycan deacetylases (PGNG-dacs) are required for bacterial evasion to lysozyme and innate immune responses. Interestingly, there is an unusual occurrence of multiple putative polysaccharide deacetylases in Bacillus anthracis. The envelope of B. anthracis is a dynamic organelle composed of peptidoglycan (PG), a pyruvylated neutral polysaccharide (PS) which anchors SLH-harboring proteins including the S-layer proteins Sap and EA1 and a poly-γ-D-glutamic (PDGA) capsule. Our results provide novel and fundamental insights on the function of polysaccharide deacetylases in a major bioterrorism agent and a framework for the design of novel antibacterial agents targeting enzymes involved in the modification of crucial cell wall polysaccharides for cell growth and virulence.
PGNG-dacs are members of Carbohydrate esterase family 4 (CE4 as defined in the CAZY database http://www.cazy.org/CE4_bacteria.html). Today ten PGNG-dacs and/or their corresponding genes from several bacteria including major pathogens such as B. cereus, S. pneumoniae and L. monocytogenes have been identified and characterized. B. cereus sensu lato strains including B. anthracis contain ten polysaccharide deacetylase homologues. Five of these homologues have been proposed to be PGNG-dacs.
We have employed a combined biochemical and genetic (knock-out) analysis in order to elucidate the biological roles of the five putative PGNG-dac’s. The intrinsic biochemical properties of each deacetylase did not reveal any clear hypothesis for their individual role in B. anthracis. To assign such function, we constructed single mutants of all five putative deacetylase encoding genes from B. anthracis and compared their major phenotype characteristics. The combination of different phenotypes of each mutant allowed us to propose a role for BA1961, BA1977 and BA3679 in PG metabolism. Instead, BA2944 and BA5436 appear to be involved at two distinct steps in the synthesis of the neutral polysaccharide. BA1977 exhibit similar properties to presently characterized PGNG-dacs (lysozyme sensitivity, reduced virulence of their mutants in mice models) indicating that PG N- deacetylation could be a general mechanism used by bacteria to evade the host immune system. Inhibition of PGNG-dacs would enable the host organism to lyse invading bacteria with lysozyme that is inactive against bacteria with functional enzymes. Transmission electron microscopy (TEM) of the 7702 Δba1961 and 7702 Δba3679 mutant strains reveal local thickenings of their PG mainly in the septa and show a defect in cell separation and a reduced efficiency of spore formation compared to the wild type. While little is known about PG biosynthesis in B. anthracis, two enzymatic systems are thought to act in B. subtilis, one for lateral cell wall growth and a second for division/septation. If an analogous organization exists in B. anthracis our data suggest that BA1961 and BA3679 would interact predominantly with complexes at the septum while BA1977 would associate with lateral PG synthesis. Finally, BA5436 and BA2944, dο not act on peptidoglycan. In fact, in the 7702 Δba5436 mutant strain, we were unable to detect the neutral polysaccharide in the cell wall nor the S-layer proteins associated to it. HPLC analysis of 7702 Δba2944 mutant polysaccharide reveal a shift of the peak corresponding to PS of the parental 7702 strain suggesting the polysaccharide as substrate of BA2944. Our results suggest that BA2944 is required to deacetylate the trisaccharide repeating unit, →6)-α-d-GlcNAc-(1→4)-β-d-ManNAc-(1→4)-β-d-GlcNAc-(1→, the building block for the synthesis of the neutral polysaccharide, while BA5436 would modify the proposed linkage unit GlcNAc-ManNAc. Experiments aiming to further elucidate the biological roles of BA5436 and BA2944 are in progress.
PGNG-dacs are validated antibiotic targets as demonstrated by the reduced virulence of mutant strains of various pathogens and RΔba1977 in this study. The identification of additional PGNG-dacs BA1961 and BA3679 in B. anthracis with different suggested biological role, offers new targets for the design of inhibitors against this bioterrorism agent. Furthermore, elucidation of the structural requirements for S-layer assembly in Gram+ bacteria may lead to numerous translational applications in industrial processes exploiting the SLH domain and synthetic pyruvylated polysaccharides.
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Polysaccharide deacetylases from Bacillus anthracis
