The Specific Capsule Depolymerase of Phage PMK34 Sensitizes Acinetobacter baumannii to Serum Killing

Abstract

The rising antimicrobial resistance is particularly alarming for Acinetobacter baumannii, calling for the discovery and evaluation of alternatives to treat A. baumannii infections. Some bacteriophages produce a structural protein that depolymerizes capsular exopolysaccharide. Such purified depolymerases are considered as novel antivirulence compounds. We identified and characterized a depolymerase (DpoMK34) from Acinetobacter phage vB_AbaP_PMK34 active against the clinical isolate A. baumannii MK34. In silico analysis reveals a modular protein displaying a conserved N-terminal domain for anchoring to the phage tail, and variable central and C-terminal domains for enzymatic activity and specificity. AlphaFold-Multimer predicts a trimeric protein adopting an elongated structure due to a long α-helix, an enzymatic β-helix domain and a hypervariable 4 amino acid hotspot in the most ultimate loop of the C-terminal domain. In contrast to the tail fiber of phage T3, this hypervariable hotspot appears unrelated with the primary receptor. The functional characterization of DpoMK34 revealed a mesophilic enzyme active up to 50 °C across a wide pH range (4 to 11) and specific for the capsule of A. baumannii MK34. Enzymatic degradation of the A. baumannii MK34 capsule causes a significant drop in phage adsorption from 95% to 9% after 5 min. Although lacking intrinsic antibacterial activity, DpoMK34 renders A. baumannii MK34 fully susceptible to serum killing in a serum concentration dependent manner. Unlike phage PMK34, DpoMK34 does not easily select for resistant mutants either against PMK34 or itself. In sum, DpoMK34 is a potential antivirulence compound that can be included in a depolymerase cocktail to control difficult to treat A. baumannii infections.

Characterisation and sequencing of the novel phage Abp95, which is effective against multi-genotypes of carbapenem-resistant Acinetobacter baumannii

“…It is reported that depolymerase has shown good effects in the treatment of bacterial infections, for it can improve the sensitivity of antibiotics 54 and enhance the sensitivity of the complement system to serum killing 55 . The combination of depolymerase and antibiotics can effectively destroy the biofilm 56 . Not only that, depolymerase also shows certain potential in vaccine research and development 57 .…”

A perfect fit: Bacteriophage receptor-binding proteins for diagnostic and therapeutic applications

“…Similarly, TSPs have been developed as antivirulence agents against Acinetobacter baumannii, another CPS 'specialist' and significant cause of hospitalborne infections worldwide [61]. For example, the anticapsule TSP from A. baumannii phage PMK34 was used to remove the CPS barrier of A. baumannii cells in vitro and resensitize the pathogen to serum killing, enabling a greater than 5-log reduction of A. baumannii in human serum [62]. Finally, an array of capsule (K1, K5, and K30)-specific TSPs, for example, K1F (Figure 2i), have been used to resensitize E. coli to serum killing, however, with varying efficacy when tested in a mouse thigh infection model that was linked to the stability of the different recombinant TSPs [43].…”

“…The last few years has also seen incredible advancement in artificial intelligence-based modeling tools, such as AlphaFold [42], which are revolutionizing the field of structural biology by providing high-confidence models of proteins and their complexes within relatively short timeframes. It is therefore of no surprise that these tools are already being used to explore the structures and functions of interesting RBPs (Figure 1; Table 2) [62,[68][69][70]. Nevertheless, despite these breakthroughs in protein modeling, combined with a growing number of RBP and phage tail structures solved empirically using X-ray crystallography and (cryo-)electron microscopy (Table 2), there remain a number of 'blind spots' in our understanding.…”

Genome-wide phage susceptibility analysis in Acinetobacter baumannii reveals capsule modulation strategies that determine phage infectivity

“…These costs likely emerge due to the A . baumannii capsule frequently serving as a requisite receptor for initiating phage infection [ 20 – 23 ].…”