Elucidation of the cell surface lipooligosaccharide structure of Moraxella bovoculi and its influence on the growth and biological activity of the microorganism

Abstract

Moraxella bovoculi is a Gram-negative microorganism that has shown potential to aide in the pathogenesis of infectious bovine keratoconjunctivitis (IBK) or ‘pink-eye’ in cattle. An ocular disease, IBK has shown to cause a significant economic loss to the cattle and dairy industry with reported losses of AUD 21 million annually to the beef industry in Australia.1 Infected animals show symptoms of keratitis, conjunctivitis, corneal ulceration and in severe cases, IBK can lead to permanent blindness.2 Currently, the only known cause for IBK is the Gram-negative bacterium Moraxella bovis. However, M. bovoculi is a recently isolated bacterium from calves which could also play a role in the pathogenesis of IBK with reports suggesting it may play a role in host colonisation.2-4 Currently, no investigations on the lipooligosaccharide (LOS) structure of M. bovoculi have been completed which is a major virulence factor found in Gram-negative bacteria.3,5,6 This following study aimed to further understand the biological role of LOS in M. bovoculi as well as compare it to the previously studied Moraxella species, M. bovis and M. catarrhalis. The first aim investigated the growth profile and biological activity of Moraxella bovoculi. The second aim investigated the extraction and purification of the LOS of M. bovoculi leading to the eventual analysis of the oligosaccharide structure using NMR spectroscopy. An investigation into the biological role of LOS in M. bovoculi examined the growth and biological activity. The growth characteristics of M. bovoculi showed that the bacterium reached its stationary phase at 15 hours. This information was exploited to optimise the yield of viable bacteria for future isolation and purification of LOS. Endotoxin activity was quantified using a Limulus Amebocyte Lysate (LAL) assay. The endotoxin activity of M. bovoculi is identical to M. bovis Epp63 Lgt2Δ strains but smaller than Mb25, L183/2 and Epp63 M. bovis strains as well as the wild-type M. catarrhalis O35E strain. An Auto-aggregation assay that measured sedimentation rates in liquid media were found to be similar to M. catarrhalis 035E strain. The percentage of adherent bacterial cells to HeLa and Chang cells was examined and revealed significantly decreased percentages towards all strains except the mutant M. bovis Epp63 Lgt2Δ and M. catarrhalis O35EkdtA strains. Susceptibility to the hydrophobic agents Tween 20 and Triton X-100 was analysed using a disk diffusion assay at a concentration of 5%. M. bovoculi is less sensitive to Tween-20 when compared to all M. bovis strains but is more sensitive when compared to M. catarrhalis O35E. M. bovoculi is less sensitive to Triton X-100 when compared to M. bovis Mb25, and M. catarrhalis O35EkdtA strains but is more sensitive when compared to M. bovis L183/2, Epp63Lgt2Δ and M. catarrhalis O35E. Susceptibility to a selection of nine antibiotic agents was also examined using a disk diffusion assay. M. bovoculi saw a significantly higher susceptibility to chloramphenicol when compared to M. bovis Epp63 strain. Conversely, M. bovoculi saw a significantly lower susceptibility to nalidixic acid when compared to M. bovis L183/2 strain. M. bovoculi presented a significantly higher susceptibility to novobiocin and polymyxin B when compared to M. bovis Mb25, L183/2, Epp63 and the mutant Epp63 Lgt2Δ strains. However, M. bovoculi saw a significantly lower susceptibility to rifampicin when compared to M. bovis Mb25 and Epp63 Lgt2Δ strains. Lastly, M. bovoculi saw a significantly higher susceptibility to vancomycin when compared to M. bovis Mb25, Epp63 and Epp63 Lgt2Δ but is not when compared to M. bovis L183/2. It was also analysed here that β-lactamase activity was observed in M. bovoculi when comparing Ampicillin and Penicillin G to the β-lactamase inhibitor, Amoxicillin/Clavulanic Acid presenting with a higher susceptibility. Lastly, a bactericidal assay using bovine serum was conducted using bacterial cell counts. No difference is observed between the concentrations 0% and 25% for M. bovoculi. The LOS of M. bovoculi was extracted using three protocols sequentially to identify which protocol would acquire the highest and purest yield to use for NMR analysis. These protocols included phenol/chloroform/petroleum ether method, the phenol/EDTA/TEA method and the hot phenol/water method in that order. It was determined that the hot phenol/water method gave the highest yield of LOS material once it was liberated of nucleic acid and protein contamination using DNase, RNase and Proteinase K. An SDS-PAGE confirmed that M. bovoculi was devoid of an O-antigen due to absence of higher molecular weight banding patterns confirming it to be a LOS structure. LOS was subjected to mild acid hydrolysis to cleave the Lipid A from the Oligosaccharide. The oligosaccharide was then purified further using size exclusion gel chromatography that determined the oligosaccharide to be high in molecular weight as it travelled down the Bio-gel P4 rapidly. The pure oligosaccharide sample was examined using 1H NMR spectroscopy for purity. Pure oligosaccharide samples were analysed using an Avance Bruker 600 MHz spectrophotometer using 1D and 2D NMR experiments. Spectral assignment of M. bovoculi determined that the oligosaccharide structure contains six sugar residues in addition to the Kdo residue. A central trifunctional α-glucopyranose residue is determined to be linked to the O-5 of the Kdo. The α-glucopyranose is linked to two β-glucopyranose residues at the four and six position carbons. The α-glucopyranose is also linked to a β-galactopyranose at the three-position carbon which extends to a α-galactopyranose from its six-position carbon. The α-galactopyranose extends further to another α-galactopyranose residue at the four-position carbon. The oligosaccharide of M. bovoculi had unusual features such as it is devoid of heptose residues as previously seen in species like M. bovis and M. catarrhalis. However, the structure did not contain any N-Acetylglucosamine residues unlike serotypes A and C of M. catarrhalis, and N-Acetylgalactosamine resides unlike M. bovis Epp63 and Mb25 strains.7,8