| Abstract |
Bacillus anthracis is a Gram-positive, spore forming bacterium and the etiological agent of anthrax, a lethal disease sporadically affecting humans and animals. The cell wall of B. anthracis is composed of peptidoglycan, polysaccharides, proteins and a poly-γ-D-glutamic acid capsule.
Bacterial lipoproteins are a functionally diverse class of peripheral membrane proteins in Gram-positive bacteria, with important roles in substrate binding for ABC transporters, adhesion, antibiotic, lantibiotic and bacteriocin resistance and phage superinfection, cell envelope homeostasis, protein secretion, folding and localization, redox and sensory processes, including signaling in sporulation and germination.
Polysaccharide deacetylases belong to Carbohydrate Esterase Family 4 (CE4). Interestingly, the genomes of Bacillus sp., and especially of B. cereus sensu lato, including B. anthracis contain multiple putative polysaccharide deacetylase genes with high sequence homologies. The physiological role of five polysaccharide deacetylases in B. anthracis has been recently elucidated.
The structures of CE4 enzymes from various bacterial species have been determined and they all contain a conserved NodB homology domain and adopt a (α/β)8 barel fold. Most of the structures contain a divalent cation in the active site bound in a His-His-Asp triad. The catalytic machinery is completed by an aspartic acid and a histidine which act as the catalytic base and catalytic acid respectively.
BA0330 and BA0331 from B. anthracis are predicted as putative lipoproteins and polysaccharide deacetylases and share 55% sequence identity. Furthermore, BA0330 shares 91% identity with its corresponding homologue BC0361 from B. cereus, while a homologue of BA0331, which is present in all B. anthracis strains, is missing in many B. cereus strains including B. cereus ATCC 14579. BA0331 is mainly expressed during exponential phase, but is secreted at lower amounts during the stationary phase, in both the avirulent B. anthracis UM23C1-2 (pXO1-, pXO2-) and the wild-type virulent Vollum strain.
In this study we employed biochemical and genetic (knockout) analysis and protein localization to elucidate the biological roles of BA0330 and BA0331 from the avirulent B. anthracis UM23C1-2 strain. Although both proteins lack deacetylase activity towards commonly used deacetylase substrates, the construction of both single Δba0330 and Δba0331 and double Δba0330Δba0331 mutants revealed a significant role for the two proteins in maintaining cell wall integrity.
Electron Microscopy revealed that the mutant cells exhibited aberrant phenotypes. In Δba0330 cells a partial detachment of the membrane from the cell wall was observed, probably due to a weakened interaction between the two layers. Furthermore Δba0330 cells exhibited impaired growth rate compared to wild type cells, when challenged with increased NaCl concentrations, indicating that BA0330 contributes to the adaptation of the bacterium to high salt stress. On the contrary, Δba0331 cells had normal peptidoglycan-membrane connection but were unable to maintain the typical bacilli shape, exhibiting a distorted cell shape, a finding which was confirmed by both Transmission and Scanning Electron Microscopy. To examine the importance of the putative deacetylase activity of the proteins in vivo, a point mutation was introduced in ba0330 and ba0331 to replace key catalytic residues. The distorted phenotypes of Δba0330 and Δba0331 were restored when the mutant cells were complemented with the point mutated BA0330 and BA0331 proteins respectively.
Both proteins were located in the cell envelope but exhibited different localization patterns: BA0330 is localized in the cell periphery and enhanced at the septa and BA0331 in distinct foci with lower representation at the septa. Both BA0330 and BA0331 interact with peptidoglycan, thus reinforcing their implication in stabilizing the cell wall of B. anthracis. Furthermore, both proteins may affect the function of autolysins, since Δba0330 and to a lesser extent Δba0331 mutant strains showed decreased autolysis.
Although BA0330 and BA0331 contain most of the catalytic and zinc binding residues conserved in five catalytic motifs of enzymatically and structurally characterized CE4 esterases, the recently solved structure of BA0330 revealed that the catalytic site differs from that typically found in polysaccharide deacetylases and could explain the apparent lack of activity of this protein.
To our knowledge this is the first report of lipoproteins implicated in maintenance of cell wall integrity in Gram-positive bacteria.
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