Characterization of a glycoside hydrolase endolysin from Acinetobacter baumannii phage AbTZA1 with high antibacterial potency and novel structural features

Abstract

The classification of Acinetobacter baumannii by WHO as 'priority 1' antibiotic-resistant pathogen underlines the urgent need for novel antimicrobial agents towards this pathogen. In this work, screening of the A. baumannii phage AbTZA1 genome allowed the identification of a putative endolysin (AbLys1, EC3.2.1.17) that belongs to the glycoside hydrolase family 24 (GH24). The sequence of AbLys1 was cloned, expressed in E. coli and purified. The lytic activity and specificity of AbLys1 were evaluated against a range of Gram-positive and Gram-negative human pathogens. AbLys1 was found to display a high selectivity towards A. baumannii. Kinetic analysis was carried out to characterize the dependence of its lytic activity on pH. The enzyme shows its maximal activity at pH values 7-8. The structure of AbLys1 was determined by X-ray crystallography to 1.82 Å resolution. The overall structure revealed two helical domains: a small, antenna-like, N-terminal domain and a larger C-terminal domain with six α-helices and a β-hairpin. Both the antenna-like and β-hairpin regions contain short sequences (AMseq1 and AMseq2) with predicted antimicrobial activity. Engineering studies revealed a key role of AMseq1 and AMseq2 on the enzyme's lytic activity towards A. baumannii cells but not towards purified peptidoglycan. This suggests that both sequences affect the destabilization of the outer membrane, thus providing access of the catalytic domain to the peptidoglycan. In addition, the deletion of AMseq1 enhanced the enzyme stability, whereas the deletion of AMseq2 diminished it. The results suggest that AbLys1 is a promising new enzybiotic with efficient lytic and antimicrobial activity.

Structural and functional features of a broad-spectrum prophage-encoded enzybiotic from Enterococcus faecium

“…A melting temperature (T m ) of 49.6 ± 0.2 °C was determined. The measured T m for Ef Ami1 falls within the expected range for a mesophilic enzyme and is close to that reported for other endolysins 15 , 17 . However, it is significantly lower to that reported by Żebrowska et al 43 for the endolysin from the thermophilic bacteriophage TP-84 (77.6 °C).…”

“…It has been proposed that certain endolysins possess specific regions in their structure that display antimicrobial activity, acting as surfactant-like peptides or providing initial point of association with the bacterial membrane 17 , 56 . Prediction of regions with antimicrobial activity in Ef Ami1 sequence was achieved using the AMPA algorithm 57 .…”

“…The thermal stability of Ef Ami1was measured using DSF on an Applied Biosystems ® real-time PCR StepOne™ instrument, as described by Premetis et al 17 . SYPRO™ Orange dye was used for monitoring the thermal denaturation of the enzyme.…”

“…Antimicrobial activity assays using disc diffusion method were carried out as described by Premetis et al 17 . Four bacteria strains were used: E. faecium, E. faecalis, S. aureus and A. baumannii .…”

Characterization and Engineering Studies of a New Endolysin from the Propionibacterium acnes Bacteriophage PAC1 for the Development of a Broad-Spectrum Artilysin with Altered Specificity

“…The continuous increase in the emergence of multidrug-resistant (MDR) bacteria is estimated to raise the number of deaths to 10 million annually by 2050 [1,2]. The exploitation of endolysins is a promising approach for the treatment of infections caused by MDR bacteria [3][4][5][6][7]. Endolysins are hydrolytic enzymes encoded by bacteriophages and degrade the peptidoglycan (PG) layer of the host bacterial cell at the end of the lytic replication cycle, causing disruption of the cell's integrity.…”