Bactericidal Effect of Cecropin A Fused Endolysin on Drug-Resistant Gram-Negative Pathogens

Abstract

The rapid spread of superbugs leads to the escalation of infectious diseases, which threatens public health. Endolysins derived from bacteriophages are spotlighted as promising alternative antibiotics against multi-drug resistant bacteria. In this study, we isolated and characterized the novel Salmonella typhimurium phage PBST08. Bioinformatics analysis of the PBST08 genome revealed putative endolysin ST01 with a lysozyme-like domain. Since the lytic activity of the purified ST01 was minor, probably owing to the outer membrane, which blocks accessibility to peptidoglycan, antimicrobial peptide cecropin A (CecA) was fused to the N-terminus of ST01 to disrupt the outer membrane. The resulting CecA::ST01 has been shown to have increased bactericidal activity against gram-negative pathogens including Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Enterobacter cloacae and the most affected target was A. baumannii. In the presence of 0.25 μM CecA::ST01, A. baumannii ATCC 17978 strain was completely killed and CCARM 12026 strain was wiped out by 0.5 μM CecA::ST01, which is a clinical isolate of A. baumannii and resistant to multiple drugs including carbapenem. Moreover, the larvae of Galleria mellonella could be rescued up to 58% or 49% by the administration of CecA::ST01 upon infection by A. baumannii 17978 or CCARM 12026 strain. Finally, the antibacterial activity of CecA::ST01 was verified using 31 strains of five gram-negative pathogens by evaluation of minimal inhibitory concentration. Thus, the results indicate that a fusion of antimicrobial peptide to endolysin can enhance antibacterial activity and the spectrum of endolysin where multi-drug resistant gram-negative pathogens can be efficiently controlled.

Keywords: Endolysin; antimicrobial peptide; bacteriophage; gram-negative pathogens; multiple-drug resistance.

Fig. 1. Identification of novel phage endolysin ST01.

(A) Cladogram of ST01 with 18 proteins using the Constraint-based Multiple Alignment Tool (COBALT). (B) Multiple alignment analysis of amino acid sequence of ST01 with Salmonella phage vB_SenS_ER21 lysozyme, Salmonella virus VSe101 lysin, and Salmonella phage vB_SenS_ER22 lysozyme. The alignment was performed using ClustalW. Black boxes represent 100% sequence identity. (C) The domain structure of ST01 was predicted by the InterPro tool. The predicted lysozyme-like domain is located between amino acid 34 and 140 of ST01.

Fig. 2. Lytic activity of ST01 against S. typhimurium.

(A) The peptidoglycan-degrading activity of ST01 was determined by plate lysis assay using a plate containing an autoclaved culture of Salmonella typhimurium ATCC 14028. SoluBL21 carrying pET21a (Empty) or pAS008 was grown in LB containing ampicillin until culture reached the midexponential phase and expression of ST01 was induced with the addition of 0 mM (-) or 1 mM IPTG (+). After 6 h culture at 37°C, 1 μl of each culture was spotted on the plate and air dried. The plates were incubated at 37°C overnight. The clear zone represents the lytic activity of ST01. (B) The antimicrobial activity of ST01 against S. typhimurium ATCC 14028 was tested by CFU reduction assay. Exponentially grown bacterial cells were adjusted as 1 × 10⁶ CFU in 20 mM Tris-HCl pH 7.5 and treated with 0, 0.2, and 2 μM of purified ST01 at 37°C for 2 h. The surviving bacterial cells were counted by plating on an LB plate. (C) The antimicrobial activity of ST01 was tested against P. aeruginosa PA01, A.baumannii ATCC 17978, K. pneumonia KCTC 2208, E. coli ATCC 8739, E. aerogenes F276, E. cloacae ATCC 13047 by CFU reduction assay. The bacterial cells were prepared as described above and treated with 0, 0.125, 0.25, 0.5, 1 μM of ST01. The experiments were repeated at least three times and data are presented as mean ± SD. Significance is shown as *p < 0.0392; **p < 0.0074; n.s. = not significant.

Fig. 3. Evaluation of lytic activity of CecA::ST01 against A. baumannii in vitro and in vivo.

(A). The antibacterial activity of CecA::ST01 was determined using S. typhimurium ATCC 14028 and A. baumannii ATCC 17978. Each bacterial suspension in 20 mM Tris-HCl pH 7.5 was treated with 0, 0.125, 0.25, 0.5, and 1 μM of CecA::ST01. Significance is indicated as **P<0.006; *P<0.0106. Data are presented as mean ± SD (n = 3) (B). Survival rates of A. baumannii ATCC 17978 infected larvae of G. mellonella treated with no (mock) or 5 μM of CecA::ST01. The larvae were infected by 2 × 10⁶ CFU of A. baumannii ATCC 17978. For endolysin treatment, 5 μM of CecA::ST01 was mixed with bacterial suspension immediately before injection. The survival of larvae was monitored by the time the larvae were kept at 30°C for 72 h (n = 10 per group). The experiment was repeated three times. Significance is shown as ****p < 0.0001. (C) The lytic activity of CecA::ST01 was determined using the clinical isolate of A. baumannii, CCARM 12026 by CFU reduction assay. Significance is indicated as *p < 0.0201. Data are presented as mean ± SD (n = 3) (D). The larvae of G. mellonella was infected by 2 × 10⁶ CFU of A. baumannii CCARM 12026. For endolysin treatment, 5 μM of CecA::ST01 was mixed with bacterial suspension immediately before injection. As a control, the same amount of 1×PBS was used (mock). The survival of larvae was monitored by the time the larvae were kept at 37°C for 72 h (n = 10 per group). The experiment was repeated three times. Significance is shown as ***p < 0.0009.