Acinetobacter baumannii is gram-negative conditionally pathogenic bacteria, which cause nosocomial infections, such as pneumonia, wound and catheter-related urinary tract infections, peritonitis, meningitis, endocarditis, and bloodstream infections. Treatment of the infections is complicated by the ability of the bacteria to acquire and accumulate various mechanisms of antibiotic resistance, which poses a serious public health problem. One of the virulence factors of A. baumannii is a capsular polysaccharide (CPS) composed of many oligosaccharide repeats (K units), which forms a thick protective layer around the bacterial cell. Due to polymorphism of the capsule gene locus (K locus, KL) CPS structures are highly diverse (by now more than 120 KL types have been identified). The CPSs are ligands of tailspike receptor proteins of bacteriophages specific to A. baumannii, which bind to and cleave the CPS prior to infection of bacterial cells. The aim of this work was elucidation of CPS structures, characterization of the K loci and mechanisms of the CPS cleavage with bacteriophage tailspike receptor proteins.
The СPS structures of 30 A. baumannii strains of various KL types were established by sugar analysis along with one- and two-dimensional NMR spectroscopy and selective cleavage by Smith degradation or solvolysis with trifluoroacetic acid. The CPSs were found to be built up of tri- to octa-saccharide K units containing common sugars (D-Glc, D-Gal, D-Man, D-GlcNAc, D-GalNAc), 6-deoxy hexoses (L-Rha, L-6dTal), uronic (GlcA) and amino uronic (GalNAcA) acids, derivatives of 6-deoxyamino hexoses (L-FucN, D-Fuc3N, D-QuiN4N) and 5,7-diamino-3,5,7,9-teradeoxynon-2-ulosonic acids (Pse and Leg). A number of A. baumannii CPSs are structurally and genetically related to each other. In some of them, the same K units are linked in different modes (by different linkages or between different monosaccharides). Others differ in the position of a glycosidic linkage between monosaccharides within K units or in the nature of an N-acyl group on an amino sugar. Accordingly, the K loci of strains with the related CPSs show the same organization and contain the same genes but those for polymerases (Wzy), the corresponding glycosyltransferases or acyltransferases are polymorphous. In addition, the CPSs may differ in the presence or absence of a side-chain glycosyl group or an O-acetyl group. The former is added to the K unit by an additional glycosyltransferase encoded in the K locus, whereas O-acetylation is ensured by an acyltransferase encoded by a gene located outside the K locus.
The CPSs were cleaved with recombinant bacteriophage tailspike receptor proteins that possess a polysaccharide-depolymerasing activity, and structures of derived oligosaccharides were established by NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. All depolymerases studied were found to cleave specifically the CPSs of A. baumannii by the hydrolytic mechanism to give monomers or/and oligomers of the K units. The proteins studied may be considered as potential therapeutic agents, and the reducing oligosaccharides obtained by the enzymatic cleavage of the CPSs as components of glycoconjugate vaccines against infections caused by A. baumannii.