Identification of Membrane-Bound Lytic Murein Transglycosylase A (MltA) as a Growth Factor for Francisella novicida in a Silkworm Infection Model.

Takemasa Nakamura,Masahisa Watarai,Takashi Shimizu,Shib Shankar Saha,Shoma Okazaki,Akihiko Uda,Fumiya Inagaki,Kenta Watanabe

doi:10.3389/fcimb.2020.581864

Abstract

Francisella tularensis, the causative agent of tularemia, is transmitted by arthropod vectors within mammalian hosts. The detailed mechanisms contributing to growth and survival of Francisella within arthropod remain poorly understood. To identify novel factors supporting growth and survival of Francisella within arthropods, a transposon mutant library of F. tularensis subsp. novicida (F. novicida) was screened using an F. novicida–silkworm infection model. Among 750 transposon mutants screened, the mltA-encoding membrane-bound lytic murein transglycosylase A (MltA) was identified as a novel growth factor of F. novicida in silkworms. Silkworms infection with an mltA deletion mutant (ΔmltA) resulted in a reduction in the number of bacteria and prolonged survival. The ΔmltA strain exhibited limited intracellular growth and cytotoxicity in BmN4 silkworm ovary cells. Moreover, the ΔmltA strain induced higher expression of the antimicrobial peptide in silkworms compared to the wild-type strain. These results suggest that F. novicida MltA contributes to the survival of F. novicida in silkworms via immune suppression-related mechanisms. Intracellular growth of the ΔmltA strain was also reduced in human monocyte THP-1 cells. These results also suggest the contribution of MltA to pathogenicity in humans and utility of the F. novicida–silkworm infection model to explore Francisella infection.

Highlights

Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia (Ellis et al, 2002)
Among the 26 mutants, we identified 8 mutants that permitted more than 80% survival; these were identified as strains with minimal pathogenicity in the silkworm infection model (Figure 1)
We focused on the characteristics and pathogenicity associated with mltA (FTN_1286), the gene encoding hypothetical membrane-bound lytic transglycosylase MltA

Summary

Introduction

Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia (Ellis et al, 2002). It shows high infectivity via aerosol transmission; as few as 10 F. tularensis bacterial cells can cause disease in humans (McLendon et al, 2006). F. tularensis has been identified in a wide range of organisms, including mammals, birds, amphibians, and invertebrates (Keim et al, 2007), the disease tularemia predominantly occurs in rodents, voles, hares, rabbits, and humans (Carvalho et al, 2014). The detailed mechanisms of the growth and survival of F. tularensis in arthropod vectors remain poorly understood

Methods

Results

Conclusion