Comparative structural and antigenic characterization of genetically distinct Flavobacterium psychrophilum O-polysaccharides

Authors: CISAR, JOHN - Retired Non ARS Employee; BUSH, ALLEN - University Of Maryland; Wiens, Gregory - Greg

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/25/2019
Publication Date: 5/8/2019
Citation: Cisar, J.O., Bush, A., Wiens, G.D. 2019. Comparative structural and antigenic characterization of genetically distinct Flavobacterium psychrophilum O-polysaccharides. Frontiers in Microbiology. 10:1041. https://doi.org/10.3389/fmicb.2019.01041.
DOI: https://doi.org/10.3389/fmicb.2019.01041

Interpretive Summary: Flavobacterium psychrophilum is a bacterial pathogen of rainbow trout that causes significant losses for trout farmers. Improved methods of control are needed but these efforts are hampered by a lack of understanding of how isolates vary from one another. Scientists have previously reported rabbit antibodies that distinguish particular strains; however, the precise genetic and biochemical differences were unresolved. In this manuscript, we report the analysis of the genome sequence of multiple F. psychrophilum isolates and identify the genes predicted to produce the O-polysaccharide, which is a heat-stable bacterial cell surface carbohydrate composed of three repeating sugars. We identified two strains that differ in only a single biosynthetic gene and purified the O-polysaccharide from both strains. Using chemical composition analysis and high-resolution nuclear magnetic resonance we determined that the two strains produce the same three sugars but they differ in a chemical linkage between two of the sugars. This difference has a profound effect on the binding of both rabbit and trout antibodies. In addition, we identify five other genes that vary between other isolates and correlate with changes in antibody binding likely by chemically modifying one of the three sugars. These results will allow the development of rapid typing assays to distinguish between strains and help us understand how O-polysaccharide variation correlates with strain virulence and host genetic resistance.

Technical Abstract: Little is known about the underlying basis of serotype specificity among strains of Flavobacterium psychrophilum, the agent of rainbow trout fry syndrome and bacterial cold-water disease. Different heat-stable O-antigens are displayed by this gram-negative pathogen, which points to structural variations in the O-polysaccharide (O-PS) moiety of cell surface lipopolysaccharide (LPS). A trisaccharide composed of L-Rha, L-FucNAc and D-Qui2NAc4NR, where R represents a dihydroxyhexanamido derivative, was previously identified as the repeating unit of Fp CSF259-93 O-PS. Interestingly, the O-PS gene cluster of this strain and that of Fp 950106-1/1, which belongs to a different O-serotype, are identical except for wzy, which encodes the putative polymerase that links trisaccharide repeats into O-PS chains. We have now found from results of glycosyl composition analysis and high-resolution nuclear magnetic resonance, that the only structural difference between O-PS from these strains is the linkage of D-Qui2NAc4NR to L-Rha, which is alpha1-2 for Fp CSF259-93 versus beta1-3 for Fp 950106-1/1. The corresponding difference in O-serotype specificity was established from the reactions of rabbit and trout anti-F. psychrophilum antibody with purified O-PS and LPS. Moreover, LPS-based differences in antigenicity were noted between strains with O-PS loci identical to those of Fp CSF259-93 or Fp 950106-1/1, except for the genes predicted to direct synthesis of different R-groups in Qui2NAc4NR. The findings provide a framework for defining the genetic basis of O-PS structure and antigenicity and suggest that the repertoire of F. psychrophilum O-serotypes extends beyond what is presently recognized from serological studies of this important fish pathogen.