Abstract
Public health is confronting the threat caused by antibiotic resistance and this means new antibacterial strategies must be developed urgently. Antimicrobial peptides (AMPs) have been considered as promising therapeutic candidates against infection in the post-antibiotic era. In this paper, we dismissed the significance of “Rana box” in the natural nigrocin-HL identified from skin secretion of Hylarana latouchii by comparing its activity with nigrocin-HLD without the motif. By substituting the “Rana box” sequence with an amidated phenylalanine residue, the natural peptide was modified into a shorter AMP nigrocin-HLM. Activities and toxicities of these two peptides in vitro and in vivo were compared. As a result, nigrocin-HLM not only displayed significantly increased potency against several representative microbes, but also high activity against the antibiotic-resistant methicillin-resistant S. aureus (MRSA, NCTC 12493 and ATCC43300 and several clinical isolates) as evidenced by markedly reduced minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC). More strikingly, nigrocin-HLM exhibited prominent inhibition against MRSA infection in a pneumonia mice model. In addition, the substitution attenuated the toxicity of nigrocin-HLM as evidenced by precipitously decreased hemolytic and cytotoxic activities in vitro, and acute toxicity to mice in vivo. Taken these results into consideration, nigrocin-HLM should be a promising therapeutic candidate for anti-infection. And in addition to dismiss an indispensable role of “Rana box” in maintaining antimicrobial activity of nigrocin-HL, our data provided an inspired strategy for peptide optimization.
Highlights
Antibiotic resistance has disseminated rapidly because of multifactorial reasons including indiscriminate use and some complex ancient antibiotic-resistance mechanisms of bacteria (Brown and Wright, 2016; Dickey et al, 2017)
The antibiotic resistance crisis caused by some developed bacteria, represented by MRSA, has outpaced the attempts to develop new antibiotics
We isolated and identified an Antimicrobial peptides (AMPs) named nigrocin-HL from skin secretion of the broad-folded frog, H. latouchii. This novel AMP bears a heptapeptide motif (CGLSGLC) named “Rana box,” which is shared throughout anuran AMPs including brevinins, gaegurins, esculentins, and nigrocins (Ponti et al, 1999; Kozic et al, 2015)
Summary
Antibiotic resistance has disseminated rapidly because of multifactorial reasons including indiscriminate use and some complex ancient antibiotic-resistance mechanisms of bacteria (Brown and Wright, 2016; Dickey et al, 2017). Nigrocin is a representative anuran AMP family, and after sequence alignment between nigrocin-HL and other members of nigrocin family reported previously, a highly conserved heptapeptide motif “CGLXGLC” named “Rana box” at C-terminus was confirmed in the nigrocin family (Park et al, 2001; Conlon et al, 2006; Li et al, 2007; Che et al, 2008; IwakoshiUkena et al, 2011b) It was not well determined if “Rana box” motif, as a widespread disulphide- bridged structure among anuran AMPs, was an indispensable domain for antimicrobial activity of these AMPs. Here, we describe the isolation and identification of a novel AMP named nigrocin-HL from the skin secretion of H. latouchii using “shotgun” molecular cloning and mass spectrometry. An analog of it named nigrocin-HLM was designed and synthesized by “Rana box”-targeted and C-terminus amidation, which possessed activity against antibiotic-resistant bacteria, but showed much less toxicity both in vitro and in vivo as well
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