A Novel Bacteriophage Lysin-Human Defensin Fusion Protein Is Effective in Treatment of Clostridioides difficile Infection in Mice.

Zhong Peng,Xingmin Sun,Jianfeng Cai,Mussie Gide,Shaohui Wang,Hiran Malinda Lamabadu Warnakulasuriya Patabendige,Chunhui Li,Duolong Zhu

doi:10.3389/fmicb.2018.03234

Abstract

Clostridioides difficile is the leading cause of worldwide antibiotics-associated diarrhea. In this study, we report the construction and evaluation of a novel bacteriophage lysin-human defensin fusion protein targeting C. difficile. The fusion protein, designated LHD, is composed of two parts connected by a 3-repeating unit linker “(GGGGS)3”: the catalytic domain of a lysin protein from a C. difficile bacteriophage phiC2 (LCD), and the functional domain of a human defensin protein HD5. Lytic assays showed that LHD protein had a potent lytic activity against different types of clinical C. difficile strains, including the epidemic 027, 078, 012, and 087 strains. The minimum inhibitory concentration (MIC) of LHD was 0.78 μg/ml, which was lower than the MIC of the protein LCD (1.56 μg/ml), and the MICs of metronidazole (4 μg/ml) and vancomycin (4 μg/ml). In addition, the LHD protein could lyse C. different strains in different pHs (6.0, 7.0, and 8.0). Evaluation of LHD potency in vivo using mouse model of C. difficile infection (CDI) showed that administration of the LHD protein (twice daily for 7 days) was effective in mitigating the symptoms and reducing the death from CDI. Treatment with LHD also significantly decreased the number of C. difficile spores and the toxin level in feces from the infected mice. Our data suggest that this novel lysin-human defensin fusion protein has a potential on CDI control.

Highlights

Clostridium difficile, reclassified as C. difficile (Lawson et al, 2016), is a Gram-positive, sporeforming, anaerobic, and toxin-producing nosocomial pathogen
The novel bacteriophage lysin-human defensin fusion protein LHD was designed by linking the catalytic domain (LCD, 179aa) of a lysin protein from phage phiC2 (NCBI reference sequence NC_009231) and human alpha-defensin 5 (HD5, designated HD in this paper) (Furci et al, 2015) with a 3-repeating unit linker (“GGGGS”)3 (Figure 1)
Phage therapy is proposed to be suited for C. difficile infection (CDI) treatment, the technical difficulties of working with anaerobes limits the research in this area (Hargreaves and Clokie, 2014)

Summary

Introduction

Clostridium difficile, reclassified as C. difficile (Lawson et al, 2016), is a Gram-positive, sporeforming, anaerobic, and toxin-producing nosocomial pathogen. In Europe, CDI was associated with considerable short or long term disability, 8382 deaths per year (Cassini et al, 2016), and an annual economic burden of €3 billion euro (Reigadas Ramirez and Bouza, 2018) Oral antibiotics such as metronidazole, vancomycin, and fidaxomicin is still recommended treatment for CDI (Debast et al, 2014; McDonald et al, 2018). C. difficile isolates with significantly reduced susceptibility, and even resistance to these recommended antibiotics have been frequently identified and reported (Spigaglia, 2016; Peng et al, 2017a,b) In this regard, development of novel antibiotics and/or alternative treatment strategies for CDI receives increasing attentions nowadays. We report the generation of a novel fusion protein containing bacteriophage lysin and human defensin, which showed potent lytic activity in vitro, and was effective in treatment of CDI in mice

Methods

Results

Conclusion