Identification of Polysialic Acid and Chondroitin-like Polysaccharides of Moraxella bovis Strains Associated with Infectious Bovine Keratoconjunctivitis

Abstract

Moraxella bovis is a major etiologic agent for infectious bovine keratoconjunctivitis (IBK), commonly known as bovine pink eye. IBK has been a major economic burden to the cattle and dairy industries due to its economic and welfare impacts on affected cattle herds. Antimicrobial treatment of acute IBK infections is often challenging. Vaccine formulations widely used in industry have poor efficacy for the prevention of IBK. Capsular polysaccharides of some bacterial pathogens are important epidemiological markers and are successfully used in vaccines for humans. Currently, there are limited data demonstrating the presence of capsular polysaccharides in M. bovis. In this study, we show by genomic analysis that a broad selection of M. bovis strains obtained from the eyes of cattle harbor a gene cluster for expressing capsular polysaccharides. The isolates potentially express either a chondroitin-like polysaccharide or an α(2-8) polysialic acid. We isolated a polysaccharide from cultures of a well-studied model strain for IBK, the Epp63 strain, structurally identical to capsule α(2-8) polysialic acid of the human pathogens Escherichia coli K1 and Neisseria meningitidis Group B. The gene cluster in M. bovis Epp63 encodes a polysialyltransferase similar to other bacterial polysialyltransferases. Other M. bovis strains analyzed in this study possess a gene homologous to that of bacterial chondroitin synthase. We isolated a capsular polysaccharide from M. bovis genotypes 1 and 2 that has the repeat unit identical to nonsulfated chondroitin. These findings provide a tool for the study of M. bovis IBK pathogenesis that could lead to approaches for better control of the disease.

Keywords: Moraxella bovis; bacterial capsule; chondroitin; infectious bovine keratoconjunctivitis; polysaccharide structure; polysialic acid.

1

E. coli K1, E. coli K92, and M. bovis Epp63 gene clusters encoding polysialic acid capsule.

2

Partial bacterial polysialyltransferases sequence alignment includes the polysialyltransferase sequence of Mannheimia haemolytica (GenBank #QEB64866.1­(”noncrystal”)), M. haemolytica polysialyltransferase crystal structure (chain A, accession #5WC6, (“crystal”)), M. bovis Epp63 (GenBank # WP_112741710.1), Moraxella nonliquefaciens (GenBank # WP_239258942.1), E. coli K92 (GenBank# AAA24215.1), E. coli K1 (GenBank# AAA24213.1), N. meningitidis serogroup B (GenBank#QXZ29499), and N. meningitidis serogroup C GenBank# CAM09375.1. The intervening nonhomologous sequence unique to M. bovis Epp63 and/or M. nonliquefaciens is highlighted in the red box.

3

Detection of polysialic acid in M. bovis Epp63 cultures by immunodiffusion. Polysialic acid capsule was detected in M. bovis cultures by using an agar gel immunodiffusion assay. Several colonies of M. bovis cultured on solid media were suspended in PBS and added to one of 6 peripheral wells in the agar and diffused against meningococcal B equine antiserum in the center well for 1 h at room temperature and then at 4 °C. Wells were examined for precipitate line formation after 24 h. Negative and positive control wells contained PBS only and a 0.15 mg/mL solution of purified E. coli K1 capsular polysaccharide, respectively.

4

Overlaid ¹H, ¹³C-HSQC (black) and LR-HSQMBC (red) of the purified polysaccharide from M. bovis Epp63 cultures. The spectra were collected at 25 °C, pH 7, on a 16 mg/mL M. bovis Epp63 capsular polysaccharide sample. Resonance assignments, in Arabic numerals, are consistent with those reported for α(2–8)-linked 5-N-acetylneuraminic acid polymer (α(2–8) polysialic acid or pSia, inset). The dotted line along the C2 frequency is included to serve as a guide for all ¹H atoms that are long-range scalar-coupled to C2. The red square highlights the H8,C2 correlation detected in the LR-HSQMBC experiment, confirming the α(2–8) glycosidic linkage.

5

Comparison of the activity of purified M. bovis Epp63 polysialyltransferase with N. meningitidis Group C polysialyltransferase. M. bovis Epp63 and N. meningitidis Group C polysialyltransferases (PST) were assayed for polymerase activity by HPLC ion exchange chromatography using the fluorescent acceptor substrate GD₃FCHASE. (A) N. meningitidis PST and (B) M. bovis PST were incubated with CMP-N-acetylneuraminic acid for 30 min prior to chromatography. Similarly, the PST enzymes were incubated with CMP-N-glycolylneuraminic acid (C) N. meningitidis and (D) M. bovis for 20 h due to the slow reaction rate with this acceptor.

6

E. coli and M. bovis gene clusters encoding chondroitin capsules.

7

Multiple alignment of bacterial chondroitin synthase sequences. Labels in the alignment show GenBank accession numbers for the sequences. The alignment contains the chondroitin synthase sequence from the crystallized E. coli chondroitin synthase (K4CP). Active site residues are outlined in red. Sheet and helix motifs of the crystal structure are represented with yellow and black blocks, respectively. Unmodeled residues in the crystal structure are represented with orange blocks.

8

Digestion of M. bovis polysaccharides with chondroitinase. Polysaccharide was purified from M. bovis strains 57868, USMARC 58166, and M. bovoculi strain USMARC 57922. Polysaccharide from each strain was digested for 1 h at 37 °C with either hyaluronidase, chondroitinase AC, or heparinase III. The resulting reaction mixtures were subjected to electrophoresis in agarose gel for 2 h and stained overnight with Stains-All to visualize polysaccharides.

9

¹³C 1D NMR spectra of purified polysaccharides of M. bovis genotypes 1 (top panel) and 2 (bottom panel). Resonance assignments are shown in Arabic numerals for atom assignment and Roman numerals for residue assignment.