Characterization of a biofilm-forming Shigella flexneri phenotype due to deficiency in Hep biosynthesis.

Dan Xu,Yongping Shao,Bing Zhang,Chongbing Liao,Wei Zhang

doi:10.7717/peerj.2178

Abstract

Deficiency in biosynthesis of inner core of lipopolysaccharide (LPS) rendered a characteristic biofilm-forming phenotype in E. coli. The pathological implications of this new phenotype in Shigella flexneri, a highly contagious enteric Gram-negative bacteria that is closely related to E. coli, were investigated in this study. The ΔrfaC (also referred as waaC) mutant, with incomplete inner core of LPS due to deficiency in Hep biosynthesis, was characteristic of strong biofilm formation ability and exhibited much more pronounced adhesiveness and invasiveness to human epithelial cells than the parental strain and other LPS mutants, which also showed distinct pattern of F-actin recruitment. Failure to cause keratoconjunctivitis and colonize in the intestine in guinea pigs revealed that the fitness gain on host adhesion resulted from biofilm formation is not sufficient to offset the loss of fitness on survivability caused by LPS deletion. Our study suggests a clear positive relationship between increased surface hydrophobicity and adhesiveness of Shigella flexneri, which should be put into consideration of virulence of Shigella, especially when therapeutic strategy targeting the core oligosaccharide (OS) is considered an alternative to deal with bacterial antibiotics-resistance.

Highlights

Shigella flexneri is a highly contagious facultative intracellular pathogen causing acute inflammatory enteritis in human
While the invasion process and immune evasion of S. flexneri have been extensively studied, the early adhesion process has not been adequately understood since Shigella flexneri lacks general adhesion machinery and exhibits relatively poor adhesiveness compared with other pathogenic enterobacteria
To generate Shigella LPS mutants of different chain length, we knocked out wzy, waaL and rfaC individually using the lambda red system (Fig. S1)

Summary

Introduction

Shigella flexneri is a highly contagious facultative intracellular pathogen causing acute inflammatory enteritis in human. LPS is a glycolipid located in the outer membrane of Gram-negative bacteria. It is composed of three covalently linked domains: lipid A, which is embedded in the outer membrane; the oligosaccharide core including inner and outer parts; and repeats of the O-polysaccharide or O-antigen, which cover the bacterial surface. As an essential pathogenic component, LPS of gram-negative bacteria triggers strong immune responses, which are directly related to the adverse clinical outcomes (Alexander & Rietschel, 2001; Wang & Quinn, 2010). In contrast to the highly variable and antigenic O-antigen portion, the core oligosaccharide (OS), especially the inner (lipid A-proximal) core, composed of two 3-deoxy-Dmanno-oct-2-ulosonic acids (Kdo) and three L-glycero-D-mannoheptose (Hep), called HepI, HepII, and HepIII, is conserved across E. coli, Shigella and Salmonella and possesses limited structural variation. Inhibition of Kdo biosynthesis is usually lethal to bacteria, a defect in Hep biosynthesis results in a viable bacterial cell with a characteristic ‘‘deep rough’’ phenotype (Grizot et al, 2006; Klena et al, 2005)

Methods

Results

Conclusion