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

is the most significant bacterial pathogen associated with the bovine respiratory disease complex. Although sialic acid is a known virulence factor in other members of Pasteurellaceae, such as and , the significance of sialic acid to the virulence of is currently unknown. Therefore, the role of sialic acid as a virulence determinant of was investigated by constructing an in-frame [CMP-N-acetylneuraminic acid (Neu5Ac/sialic) synthetase] mutant, which was shown by high-performance anion exchange chromatographic analysis (HPAEC) to be devoid of sialic acid on the lipopolysaccharide (LPS). Both the mutant and wild-type parent strains exhibited similar growth rates in the growth curve assay. Real-time qPCR and ELISA evaluation showed no differences in proinflammatory cytokine expressions (IL-1β, IL-6, and IL-8) between the mutant and parent strain when peripheral blood mononuclear cells were incubated with LPS. Interestingly, the mutant was three to four logs more sensitive to a whole-blood bacterial killing assay than the parent strain. Similar results were also observed in plasma and serum bacterial killing assays. Flow cytometry analyses showed higher uptake of mutant by phagocytes, compared to the parent strain, in the whole-blood phagocytosis assay; however, no difference in reactive oxygen species production in neutrophils or monocytes was detected for either strain. Taken together, these results indicate that sialylation of LPS plays a vital role in reducing complement-mediated and phagocytic killing. IMPORTANCE The Gram-negative coccobacillus is a natural inhabitant of the upper respiratory tract in ruminants and the most common bacterial agent involved in bovine respiratory disease complex development. Key virulence factors harbored by are leukotoxin, lipopolysaccharide, capsule, adhesins, and neuraminidase which are involved in evading innate and adaptive immune responses. In this study, we have shown that CMP-sialic acid synthetase () is necessary for the incorporation of sialic acid onto the membrane, and inactivation of results in increased phagocytosis and complement-mediated killing of thus demonstrating that sialylation contributes to the virulence of .