Biofilm formation is one of the main causes of increased antibiotic resistance in Acinetobacter baumannii infections. Bacteriophages and their derivatives, such as tail proteins with depolymerase activity, have shown considerable potential as antibacterial or antivirulence agents against bacterial infections. Here, we gained insights into the activity of a capsular polysaccharide (CPS) depolymerase, derived from the tailspike protein (TSP) of φAB6 phage, to degrade A. baumannii biofilm in vitro. Recombinant TSP showed enzymatic activity and was able to significantly inhibit biofilm formation and degrade formed biofilms; as low as 0.78 ng, the inhibition zone can still be formed on the bacterial lawn. Additionally, TSP inhibited the colonization of A. baumannii on the surface of Foley catheter sections, indicating that it can be used to prevent the adhesion of A. baumannii to medical device surfaces. Transmission and scanning electron microscopy demonstrated membrane leakage of bacterial cells treated with TSP, resulting in cell death. The therapeutic effect of TSP in zebrafish was also evaluated and the results showed that the survival rate was significantly improved (80%) compared with that of the untreated control group (10%). Altogether, we show that TSP derived from φAB6 is expected to become a new antibiotic against multi-drug resistant A. baumannii and a biocontrol agent that prevents the formation of biofilms on medical devices.

Motility plays an essential role in the host–parasite relationship of pathogenic bacteria, and is often associated with virulence. While many pathogenic bacteria use flagella for locomotion, Acinetobacter baumannii strains do not have flagella, but have other features that aid in their motility. To study the genes involved in motility, transposon mutagenesis was performed to construct A. baumannii mutant strains. Mutant strain MR14 was found to have reduced motility, compared to wild-type ATCC 17978. NCBI BLAST analysis revealed that the Tn10 transposon in the MR14 genome is integrated into the gene that encodes for carboxy-terminal processing protease (Ctp). Additionally, MR14 exhibits a mucoidy, sticky phenotype as the result of increased extracellular DNA (eDNA) caused by bacterial autolysis. Transmission and scanning electron microscopy revealed cytoplasmic content leaving the cell and multiple cell membrane depressions, respectively. MR14 showed higher sensitivity to environmental stressors. Mutation of the ctp gene reduced invasion and adhesion of A. baumannii to airway epithelial cells, potentially due to increased hydrophobicity. In the zebrafish model of infection, MR14 increased the survival rate by 40% compared to the wild-type. Taken together, the ctp gene in A. baumannii has a pivotal role in maintaining membrane integrity, adaptation to environmental stress, and controlling virulence.

Carboxy-terminal processing protease (Ctp) is a serine protease that controls multiple cellular processes through posttranslational modification of proteins. Acinetobacter baumannii ATCC 17978 ctp mutant, namely MR14, is known to cause cell wall defects and autolysis. The objective of this study was to investigate the role of ctp mutation–driven autolysis in regulating biofilms in A. baumannii and to evaluate the vesiculation caused by cell wall defects. We found that in A. baumannii, Ctp is localized in the cytoplasmic membrane, and loss of Ctp function enhances the biofilm-forming ability of A. baumannii. Quantification of the matrix components revealed that extracellular DNA (eDNA) and proteins were the chief constituents of MR14 biofilm, and the transmission electron microscopy further indicated the presence of numerous dead cells compared with ATCC 17978. The large number of MR14 dead cells is potentially the result of compromised outer membrane integrity, as demonstrated by its high sensitivity to sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). MR14 also exhibited the hypervesiculation phenotype, producing outer-membrane vesicles (OMVs) of large mean size. The MR14 OMVs were more cytotoxic toward A549 cells than ATCC 17978 OMVs. Our overall results indicate that A. baumanniictp negatively controls pathogenic traits through autolysis and OMV biogenesis.