Formation of the Francisella tularensis Biofilm is Affected by Cell Surface Glycosylation, Growth Medium, and a Glucan Exopolysaccharide

Anna E. Champion,Aloka B. Bandara,Kelly C. Freudenberger Catanzaro,Thomas J. Inzana

doi:10.1038/s41598-019-48697-x

Abstract

Biofilms are matrix-associated communities that enable bacteria to colonise environments unsuitable for free-living bacteria. The facultative intracellular pathogen Francisella tularensis can persist in water, amoebae, and arthropods, as well as within mammalian macrophages. F. tularensis Types A and B form poor biofilms, but F. tularensis mutants lacking lipopolysaccharide O-antigen, O-antigen capsule, and capsule-like complex formed up to 15-fold more biofilm than fully glycosylated cells. The Type B live vaccine strain was also 50% less capable of initiating surface attachment than mutants deficient in O-antigen and capsule-like complex. However, the growth medium of all strains tested also influenced the formation of biofilm, which contained a novel exopolysaccharide consisting of an amylose-like glucan. In addition, the surface polysaccharide composition of the bacterium affected the protein:DNA:polysaccharide composition of the biofilm matrix. In contrast, F. novicida attached to surfaces more efficiently and made a more robust biofilm than Type A or B strains, but loss of O-antigen or capsule-like complex did not significantly affect F. novicida biofilm formation. These results indicated that suppression of surface polysaccharides may promote biofilm formation by F. tularensis Types A and B. Whether biofilm formation enhances survival of F. tularensis in aquatic or other environmental niches has yet to be determined.

Highlights

Francisella tularensis is a gram-negative, facultative intracellular bacterium that can infect numerous mammals, arthropods and other eukaryotes, and is the etiologic agent of tularemia or “rabbit fever”[1,2,3]
There was no significant difference between the number of live vaccine strain (LVS) cells and LVS mutants (Table 1) deficient in O-antigen (Ag) (WbtIG191V) or capsule-like complex (CLC) (LVSΔ1423-22) that attached to polyvinyl 96-well plates (Fig. 1A), or the time required for attachment (1, 2, or 4 hours; data not shown)
Complementation of the O-Ag mutation or the CLC mutation in trans completely reversed the enhancement in biofilm formation (Fig. 1D)

Summary

Introduction

Francisella tularensis is a gram-negative, facultative intracellular bacterium that can infect numerous mammals, arthropods and other eukaryotes, and is the etiologic agent of tularemia or “rabbit fever”[1,2,3]. Holarctica generally do not form a substantial biofilm, they are capable of developing some biofilm on polystyrene 96-well plates[12] Despite their genetic similarity, the less virulent F. novicida strains differ from Types A and B (A/B) strains in their polysaccharide surface components. Whether the differences in surface glycosylation between Types A/B strains and F. novicida contribute to their differences in capability to form biofilm or development of the biofilm matrix has yet to be determined. Loss of the LPS O-Ag and/or loss of the capability to glycosylate CLC proteins resulted in enhanced biofilm formation sooner by Types A/B strains, but not F. novicida. We hypothesise that F. tularensis can phase vary its surface polysaccharides to successfully transition between a phenotype that forms a biofilm, which may enhance survival in the environment, and a planktonic phenotype that is a facultative intracellular pathogen in the mammalian host

Methods

Results

Conclusion