Studies of sialic acid and sialidase in the oral spirochete, Treponema denticola.

摘要

The goal of my graduate research is to investigate the roles of sialic acid and sialidase (also known as neuraminidase) in the biology and pathogenicity of Treponema denticola, an oral spirochete that is strongly associated with human periodontitis. In the first part of this study (chapter 2), we identified and characterized a putative neuraminidase of T. denticola, TDE0471. Biochemical and genetic studies revealed that TDE0471 is a cell surface-exposed exo-neuraminidase that removes sialic acid from human serum proteins and that the released sialic acid can be utilized as nutrients for the spirochete growth. T. denticola is resistant to the serum killing. We found that a TDE0471-deficient mutant is more sensitive to the serum killing and the potential mechanism is that TDE0471 protects the spirochete from serum killing by preventing the deposition of membrane attack complexes on bacterial cell surfaces. Along with these observations, animal studies showed that the TDE0471-deficient mutant strain is less virulent than the wild-type strain and that T. denticola causes more severe tissue damage in complement-deficient mice. In this part of the study, we also identified several candidate proteins that might be affected by TDE0471 using proteomic analyses.;In the second part (chapter 3) of this study, we found that the TDE0471-deficient mutant forms more biofilms and is less motile compared to its parental wild-type strain. Further genetic and biochemical studies revealed that the flagellin proteins (FlaB1 and FlaB2) of T. denticola are glycosylated. Our preliminary results suggested that FlaB1 and FlaB 2 are also sialylated and that inactivation of TDE0471 affects the sialylation of FlaB1, which further impairs the cell motility.;In the last part (chapter 4) of this study, we identified a TRAP sialic acid transport system in T. denticola, which consists of three proteins: TDE1019, TDE1020, and TDE1021. Sequence alignment and structural modeling analyses revealed that TDE1020 shares a similar 3D structure to the sialic acid binding proteins from other bacteria and it contains a conserved sialic acid binding sites. Human serum contains high concentrations of sialic acid and bacterial pathogens often utilize specific transporters to uptake sialic acid from the serum. Genetic studies showed that deletion of TDE1020 impairs the spirochete to grow in the serum growth medium, suggesting that TDE1020 is probably involved in uptake and transport of sialic acid from the serum.;Based on these results, we conclude that T. denticola has evolved a complex system to acquire and utilize sialic acid, e.g., scavenging host sialic acid through its neuraminidase and specific TRAP transporter, and that sialic acid plays an important role in the biology and pathogenicity of T. denticola, e.g., nutrient acquisition, biofilm formation, motility, immune evasion, and virulence.