Flagellar Basal Body Structural Proteins FlhB, FliM, and FliY Are Required for Flagellar-Associated Protein Expression in Listeria monocytogenes.

Changyong Cheng,Xiao Han,Huifei Yu,Hang Wang,Weihuan Fang,Tiantian Ma,Lingli Jiang,Chang Cai,Fengdan Liu,Jing Sun,Yi Hang,Houhui Song,Zhongwei Chen,Yongchun Yang

doi:10.3389/fmicb.2018.00208

Abstract

Listeria monocytogenes is a food-associated bacterium that is responsible for food-related illnesses worldwide. In the L. monocytogenes EGD-e genome, FlhB, FliM, and FliY (encoded by lmo0679, lmo0699, and lmo0700, respectively) are annotated as putative flagella biosynthesis factors, but their functions remain unknown. To explore whether FlhB, FliM, and FliY are involved in Listeria flagella synthesis, we constructed flhB, fliM, fliY, and other flagellar-related gene deletion mutants using a homologous recombination strategy. Then, we analyzed the motility, flagella synthesis, and protein expression of these mutant strains. Motility and flagella synthesis were completely abolished in the absence of flhB, fliM, or fliY. These impaired phenotypes were fully restored in the complemented strains CΔflhB, CΔfliM, and CΔfliY. The transcriptional levels of flagellar-related genes, including flaA, fliM, fliY, lmo0695, lmo0698, fliI, and fliS, were downregulated markedly in the absence of flhB, fliM, or fliY. Deletion of flhB resulted in the complete abolishment of FlaA expression, while it decreased FliM and FliY expression. The expression of FlaA was abolished completely in the absence of fliM or fliY. No significant changes were found in the expression of FlhF and two flagella synthesis regulatory factors, MogR and GmaR. We demonstrate for the first time that FlhB, FliM, and FliY not only mediate Listeria motility, but also are involved in regulating flagella synthesis. This study provides novel insights that increase our understanding of the roles played by FlhB, FliM, and FliY in the flagellar type III secretion system in L. monocytogenes.

Highlights

The gram-positive bacterium Listeria monocytogenes is a ubiquitous intracellular pathogen, which has been implicated within the past decades as the causative organism in several outbreaks of foodborne disease
These results clearly show that the loss of flhB, fliM, and fliY abolished flagellar synthesis and motility in L. monocytogenes
The bacterial flagellum has previously been shown to play a critical role as an export apparatus in mediating extracellular secretion of non-flagella virulence factors and other important heterologous polypeptides (Haiko and Westerlund-Wikstrom, 2013)

Summary

Introduction

The gram-positive bacterium Listeria monocytogenes is a ubiquitous intracellular pathogen, which has been implicated within the past decades as the causative organism in several outbreaks of foodborne disease. In Gram-positive and Gram-negative bacteria, the flagellum is composed of three major parts, including the flagellar filament, the hook complex, and the basal body, which crosses the bacterial cell membrane, as well as a flagellar-associated cytoplasmic ring. There are dozens of flagellar-related proteins in L. monocytogenes, which serve as regulators, structural proteins, cofactors, an ATPase, and proteins that are required protein transport and modification These proteins play an important role in flagellar synthesis. At mammalian host physiologic temperature, 37◦C, most L. monocytogenes strains do not produce flagella and are non-motile (Peel et al, 1988; Shen and Higgins, 2006) This is due to MogR repression of flagellar gene transcription at 37◦C (Grundling et al, 2004). The flagellarmediated transport and secretion system in most Gram-negative pathogenic bacteria is a type III secretion system (T3SS)

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Conclusion