Multispecies activity screening of microcin J25 mutants yields antimicrobials with increased specificity toward pathogenic Salmonella species relative to human commensal Escherichia coli

Seth C Ritter, Mike L. Yang, Yiannis Kaznessis, Benjamin J Hackel

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Modern large-scale agricultural practices that incorporate high density farming with subtherapeutic antibiotic dosing are considered a major contributor to the rise of antibiotic-resistant bacterial infections of humans with species of Salmonella being a leading agriculture-based bacterial infection. Microcin J25, a potent and highly stable antimicrobial peptide active against Enterobacteriaceae, is a candidate antimicrobial against multiple Salmonella species. Emerging evidence supports the hypothesis that the composition of the microbiota of the gastrointestinal tract prevents a variety of diseases by preventing infectious agents from proliferating. Reducing clearance of off-target bacteria may decrease susceptibility to secondary infection. Of the Enterobacteriaceae susceptible to microcin J25, Escherichia coli are the most abundant within the human gut. To explore the modulation of specificity, a collection of 207 mutants encompassing 12 positions in both the ring and loop of microcin J25 was built and tested for activity against Salmonella and E. coli strains. As has been found previously, mutational tolerance of ring residues was lower than loop residues, with 22% and 51% of mutations, respectively, retaining activity toward at least one target within the target organism test panel. The multitarget screening elucidated increased mutational tolerance at position G2, G3, and G14 than previously identified in panels composed of single targets. Multiple mutations conferred differential response between the different targets. Examination of specificity differences between mutants found that 30% showed significant improvements to specificity toward any of the targets. Generation and testing of a combinatorial library designed from the point-mutant study revealed that microcin J25I13T reduces off-target activity toward commensal human-derived E. coli isolates by 81% relative to Salmonella enterica serovar Enteritidis. These in vitro specificity improvements are likely to improve in vivo treatment efficacy by reducing clearance of commensal bacteria in the gastrointestinal tract of hosts.

Original languageEnglish (US)
Pages (from-to)2394-2404
Number of pages11
JournalBiotechnology and bioengineering
Volume115
Issue number10
DOIs
StatePublished - Oct 2018

Bibliographical note

Funding Information:
National Institutes of Health, Grant/Award Numbers: GM111358, GM121777, T32GM008347; National Science Foundation, Grant/Award Number: CBET1412283

Funding Information:
This work was supported by grants from the National Institutes of Health (GM121777 and GM111358) and a grant from the National Science Foundation (CBET?1412283). S.C. Ritter was also supported by a Predoctoral National Institutes of Health Traineeship (T32GM008347). We thank Dr. Timothy Johnson and Dr. Michael Sadowsky of the University of Minnesota for their donations of pathogenic E. coli and Salmonella strains used in this study, and Dr. James Johnson of the Veterans Affairs Hospital of Minneapolis for his donation of pathogenic and commensal E. coli strains used in this study. We?also thank Dr. James Link of Princeton for providing the pJP3 vector. Support from the University of Minnesota Genomics Center, the Digital Technology Center, and the Biotechnology Institute is gratefully acknowledged.

Funding Information:
FIG U RE 5 Proteolytic and acid stability of MccJ25 and MccJ25I13T. (a) The supernatant of wild‐type MccJ25 (WT, open circles) and MccJ25I13T (I13T, filled circles) were incubated at 60°C for 10 min in the presence of varying concentrations of Proteinase K followed by Proteinase K inactivation by incubating at 98°C for 20 min. These processed supernatants were then deposited on SE‐seeded LB agar plates. After growth, the size of the zones of growth inhibition was measured to determine residual activity. (b) The supernatant of WT and I13T were incubated at 37°C for 30 min at pH 1.5 (via HCl, gray bars) followed by normalization to pH 7.0 (via NaOH). Residual activity was then determined using SE as an indicator strain. MccJ25: microcin J25, SE, serovar Enteritidis We thank Dr. Timothy Johnson and Dr. Michael Sadowsky of the University of Minnesota for their donations of pathogenic E. coli and Salmonella strains used in this study, and Dr. James Johnson of the Veterans Affairs Hospital of Minneapolis for his donation of pathogenic and commensal E. coli strains used in this study. We also thank Dr. James Link of Princeton for providing the pJP3 vector. Support from the University of Minnesota Genomics Center, the Digital Technology Center, and the Biotechnology Institute is gratefully acknowledged.

Funding Information:
This work was supported by grants from the National Institutes of Health (GM121777 and GM111358) and a grant from the National Science Foundation (CBET1412283). S.C. Ritter was also supported by a Predoctoral National Institutes of Health Traineeship (T32GM008347).

Publisher Copyright:
© 2018 Wiley Periodicals, Inc.

Keywords

  • antimicrobial peptide (AMPs)
  • combinatorial library
  • pathogenic Salmonella
  • protein engineering
  • specificity

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