Neisseria meningitidis Type IV Pili Composed of Sequence Invariable Pilins Are Masked by Multisite Glycosylation

Abstract

The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.

Structural Diversity in the Type IV Pili of Multidrug-resistant Acinetobacter

“…Because opsonization is mediated by the binding of host immune proteins (commonly antibodies), the latter studies support the notion that the purpose of C-terminal glycosylation could be to interfere with immune recognition in vivo. Similarly, Gault et al (25) recently showed that hypervirulent N. meningitidis strains with class II pilins are more heavily glycosylated than their class I counterparts.…”

“…If C-terminal glycosylation in Acinetobacter type IV pili reduces recognition by host immune proteins, one might expect that the pilins from strains lacking a C-terminal glycan would face greater pressure to diversify as has been shown in N. meningitidis (25). Using our alignment of 49 PilA sequences, we separated protein sequences into tfpOϪ and tfpOϩ groups and compared the variability of their surface-exposed residues.…”

“…Glycosylation is an additional source of variability in some type IV pilins; O-linked glycosylation has been observed in multiple strains of both Pseudomonas aeruginosa (20 -22) and Neisseria (23,24). Additional glycosylation sites have been found in class II strains of Neisseria meningitidis where they have been hypothesized to play a role in immune evasion (25).Among the many genera of Gram-negative bacteria that express type IV pili is Acinetobacter, a coccobacillus that is widely distributed in nature and can be isolated from the environment in the soil and in water as well as from a variety of mammalian hosts (26,27). Several species of Acinetobacter, chiefly Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter nosocomialis, and Acinetobacter pittii, are collectively referred to as the A. calcoaceticus-baumannii (Acb) 4 complex and constitute an increasingly common source of nosocomial infections (28 -30).…”

“…Glycosylation is an additional source of variability in some type IV pilins; O-linked glycosylation has been observed in multiple strains of both Pseudomonas aeruginosa (20 -22) and Neisseria (23,24). Additional glycosylation sites have been found in class II strains of Neisseria meningitidis where they have been hypothesized to play a role in immune evasion (25).…”

Abundant Lysine Methylation and N-Terminal Acetylation in Sulfolobus islandicus Revealed by Bottom-Up and Top-Down Proteomics

“…Top-down proteomics (15), which implies the analysis of complex protein samples without prior enzymatic digestion, emerged recently as a powerful method for unambiguous characterization of intact proteoforms (16) bearing multiple PTMs in various combinations (17,18). With the capabilities of the latest high-resolution tandem mass spectrometry systems, it is now possible to envision an ambitious goal: perform high-throughput discovery of proteoforms on a proteomewide scale (19), analyzing both qualitative (20) and quantitative (21,22) changes at the proteoform level.…”