Structural basis for fragmenting the exopolysaccharide of Acinetobacter baumannii by bacteriophage ΦAB6 tailspike protein

Abstract

With an increase in antibiotic-resistant strains, the nosocomial pathogen Acinetobacter baumannii has become a serious threat to global health. Glycoconjugate vaccines containing fragments of bacterial exopolysaccharide (EPS) are an emerging therapeutic to combat bacterial infection. Herein, we characterize the bacteriophage ΦAB6 tailspike protein (TSP), which specifically hydrolyzed the EPS of A. baumannii strain 54149 (Ab-54149). Ab-54149 EPS exhibited the same chemical structure as two antibiotic-resistant A. baumannii strains. The ΦAB6 TSP-digested products comprised oligosaccharides of two repeat units, typically with stoichiometric pseudaminic acid (Pse). The 1.48-1.89-Å resolution crystal structures of an N-terminally-truncated ΦAB6 TSP and its complexes with the semi-hydrolyzed products revealed a trimeric β-helix architecture that bears intersubunit carbohydrate-binding grooves, with some features unusual to the TSP family. The structures suggest that Pse in the substrate is an important recognition site for ΦAB6 TSP. A region in the carbohydrate-binding groove is identified as the determinant of product specificity. The structures also elucidated a retaining mechanism, for which the catalytic residues were verified by site-directed mutagenesis. Our findings provide a structural basis for engineering the enzyme to produce desired oligosaccharides, which is useful for the development of glycoconjugate vaccines against A. baumannii infection.

Functional Analysis and Antivirulence Properties of a New Depolymerase from a Myovirus That Infects Acinetobacter baumannii Capsule K45

“…Depolymerase degradation of extracted exopolysaccharides. Exopolysaccharides (EPS) were extracted from K30 strain NIPH 190, K45 strain NIPH 201, and K3 strain NIPH 501, using an adapted protocol (60). Briefly, A. baumannii strains were grown on 20 TSA plates supplemented with 0.5% glucose at 37°C for 5 days.…”

Ability of phages to infect Acinetobacter calcoaceticus‐Acinetobacter baumannii complex species through acquisition of different pectate lyase depolymerase domains

“…The genomes of P1, P2, B1, B3 and B5 have a length ranging from 40 598 to 41 608 bp, an average G + C content of 39.1–39.3%, which overlaps with the range of 38.6–39.3% found for the 31 ACB bacterial genomes included in this work. They encode 49–56 CDSs with high identities (> 85% average amino acid identity) to proteins of other A. baumannii podophages, such as phages Abp1 (NC_021316), phiAB1 (NC_028675), PD‐AB9 (NC_028679), PD‐6A3 (NC_028684), Fri1 (NC_028848), phiAB6 (NC_031086), AS11 (KY268296), AS12 (KY268295), WCHABP5 (KY888680), IME200 (NC_028987), SH‐Ab 15519 (KY082667), Petty (NC_023570) and Acibel007 (NC_025457) (Chang et al ., ; Huang et al ., ; Mumm et al ., ; Merabishvili et al ., ; Lee et al ., ; Popova et al ., ).…”

“…The non-sequenced phage B2 recognizes capsule K44 and is, therefore, expected to encode another non-homologous depolymerase. Conversely, phages with homologous depolymerases infect bacteria with similar K types; for example, depolymerases of B1 and B5 recognize K9, B3, phiAB6 and WCHAABP5 recognize K2 (Lee et al, 2017;Popova et al, 2017), Fri1 and AS11 recognize K19 (Kenyon et al, 2016) and IME200 and SH-Ab-155519 recognize the same strain of unknown K type. It is interesting to note that these depolymerases are so specific that they are unable to recognize K types extremely close phylogenetically (see Fig.…”

“…SH-Ab 15519 (KY082667), Petty (NC_023570) and Acibel007 (NC_025457) (Chang et al, 2011;Huang et al, 2013;Mumm et al, 2013;Merabishvili et al, 2014;Lee et al, 2017;Popova et al, 2017).…”

“…The activity of the P1 depolymerase was also tested against A. pittii strains CEB-P1 (P1 host) and NIPH 290 (P2 host) and A. baumannii strains NIPH 528 (B1 and B5 hosts) and NIPH 2061 (B3 host exopolysaccharides. The method applied has been previously described (MIGL, 2012;Lee et al, 2017). Briefly, CEB-P1 exopolysaccharides were extracted from 5-day-old cultures plates at 378C, by adding 2.5 ml of 0.9% (wt/vol) NaCl per plate and by harvesting the cells.…”