| Abstract |
Bacillus anthracis, a Gram-positive, spore-forming bacterium, is a potential bioterrorism weapon. Ingestion or inhalation of B. anthracis spores can lead to anthrax disease in mammalian hosts which, in most cases, proves to be fatal.
Presently used antibiotics to combat B. anthracis infections exhibit low potency against B. anthracis strains that show antimicrobial resistance, necessitating the need to identify new targets and to develop novel antibacterial drugs against this pathogen.
Putative and known polysaccharide deacetylases (PDAs) from B. anthracis are validated drug targets due to their key roles in bacterial physiology. PDAs have been implicated in lysozyme resistance and also participate in distinct cellular functions including cell division/elongation, cell shape maintenance and osmotic stability, biofilm formation and adhesion/invasion to host cells. PDAs are members of the carbohydrate esterase family 4 (CE4) which includes acetylxylan esterases, chitin deacetylases, chitooligosaccharide deacetylases and peptidoglycan (PG) deacetylases.
There is an unusual occurrence of eleven putative PDAs in B. anthracis. Except from the characterized active PDAs, B. anthracis also possesses putative pseudoPDAs. Pseudoenzymes are defined as enzyme homologues that are predicted to retain protein expression, subcellular localization and typical protein folding but lack catalytic activity. Different roles of pseudoenzymes have been reported including regulation of their active homologues, interaction with the substrates of their enzyme homologues, binding and targeting of other proteins to specific cellular locations, modulation of protein ubiquitination and signal transduction.
Here we elucidated the physiological role of the putative PDAs BA1836 and BA3943 from B. anthracis by employing biochemical and genetic (knockout) analysis.
The ba1836 gene is expressed upon entrance into the stationary phase of growth and enhanced during the early stages of sporulation. Δba1836 knockout strain had normal growth rate, did not exhibit any significant alterations in PG composition of stationary phase cells and was not sensitive to lysozyme, but showed a defect in cell separation. Strikingly, the Δba1836 mutant strain exhibited a severe delay in spore development although mature spores were ultimately developed and exhibited normal morphology. Finally, digestion of mutant spore PG with muramidase produced similar but less muropeptides compared to PG from wild type spores, and mutant spores exhibited a lower germination rate.
Our in vitro data demonstrated that BA3943 is a pseudoenzyme consistent with the absence of three conserved, essential, catalytic residues. In addition, BA3943 lacks hydroxylation at the Cα of a highly conserved Pro residue of the active site, which has been recently shown to enhance the enzymatic activity of PDAs from B. anthracis. The crystal structure revealed the presence of a lysine-rich domain at the N-terminus of the pseudoPDA. The lysine-rich domain is unique and to our knowledge it has not been identified in any other enzyme family. Site-directed mutagenesis at the three residues of the active site of BA3943 resulted in restoration of both deacetylase activity and hydroxylation levels.
The ba3943 gene is expressed upon entrance into the engulfment stage of sporulation. Interestingly, the Δba3943 mutant strain exhibited a significant competition deficit under sporulation-inducing conditions, suggesting that BA3943 is actively involved in sporulation. Nevertheless, Δba3943 mature spores were ultimately developed, exhibited normal morphology and could efficiently sporulate and germinate. Finally, a Δba3943 knockout strain had normal growth rate, did not exhibit any significant alterations in PG composition of stationary phase cells or spores and was not sensitive to lysozyme, but showed a defect in cell separation.
To our knowledge BA1836 has a unique, sporulation/germination-specific role among the functions of other PDAs, while BA3943 is the first characterized pseudoPDA which affects the sporulation process in B. anthracis. Our data provide information towards a more complete understanding of the complex processes of sporulation and germination in this bacterial pathogen.
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