Enhanced phagocytosis and complement-mediated killing of Mannheimia haemolytica serotype 1 following in-frame CMP-sialic acid synthetase (neuA) gene deletion

Authors: MENGHWAR, HARISH - Oak Ridge Institute For Science And Education (ORISE); Tatum, Fred; Briggs, Robert; Casas, Eduardo; Kaplan, Bryan; AZADI, PARASTOO - University Of Georgia; Dassanayake, Rohana

Submitted to: Microbiology Spectrum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/6/2023
Publication Date: 10/18/2023
Citation: Menghwar, H., Tatum, F.M., Briggs, R.E., Casas, E., Kaplan, B.S., Azadi, P., Dassanayake, R.P. 2023. Enhanced phagocytosis and complement-mediated killing of Mannheimia haemolytica serotype 1 following in-frame CMP-sialic acid synthetase (neuA) gene deletion. Microbiology Spectrum. Article e0294423. https://doi.org/10.1128/spectrum.02944-23.
DOI: https://doi.org/10.1128/spectrum.02944-23

Interpretive Summary: Sialic acids are nine-carbon amino sugars found in both prokaryotes and eukaryotes. Sialylation of the bacterial outer membrane (lipopolysaccharides, endotoxin) can camouflage bacteria from the host immunological responses. Although sialic acid is a known virulence factor in Histophilus somni and Pasteurella multocida, the significance of sialic acid in Mannheimia haemolytica virulence is currently unknown. Unlike H. somni and P. multocida, M. haemolytica does not have a sialic acid uptake mechanism. Therefore, we determined the role of sialic acid in M. haemolytica virulence by generating a sialic acid mutant. LPS from both wild-type and sialic acid mutant strains showed similar cytokine responses when incubated with cattle leukocytes suggesting sialylation of lipopolysaccharides does not reduce cytokine response. However, M. haemolytica sialic acid mutant strain was highly sensitive to phagocytic and complement-mediated killing compared to the wild-type M. haemolytica strain. These observations suggest that the sialylation of lipopolysaccharides can act as a stealth mechanism to protect M. haemolytica against phagocytic- and complement-mediated killing.

Technical Abstract: Mannheimia haemolytica is the most significant bacterial pathogen associated with the bovine respiratory disease complex. Although sialic acid is a known virulence factor in pathogenic members of the related Pasteurellaceae family, such as Histophilus somni and Pasteurella multocida, the significance of sialic acid in M. haemolytica virulence is currently unknown. Therefore, the role of sialic acid as a virulence determinant of M. haemolytica was determined by constructing in-frame neuA (CMP-N-acetylneuraminic acid (Neu5Ac/sialic) synthetase) gene deletion mutant. Both neuA mutant and wildtype strains showed similar growth rates in the growth curve assay. Chromatography analysis revealed a lack of sialic acid in neuA mutant lipopolysaccharides (LPS), but not the wildtype LPS. No differences in proinflammatory cytokine expressions (IL-1ß, IL-6 and IL-8) were observed when peripheral blood mononuclear cells were incubated with LPS from neuA mutant, or wild-type as determined by real-time qPCR and ELISA evaluation. Interestingly, three-to-four logs colony forming units reduction of neuA mutant, but not the wildtype, was detected in whole blood killing assay. Similar results were also observed in plasma and serum killing assays. In contrast, no-to-low killing was noticed when neuA mutant was incubated with heat-inactivated plasma or serum. Flow cytometry analyses showed higher percentages of neuA mutant phagocytosis by phagocytes, compared to wildtype, in the whole blood phagocytosis assay. Nevertheless, no difference in reactive oxygen species production in neutrophils or monocytes was detected for neuA mutant or wildtype. Taken together, these results suggest that sialylated LPS of M. haemolytica can reduce complement-mediated and phagocytic killing, thus camouflaging the bacterial surface from the host.