Date Published: September 14, 2015
Publisher: Public Library of Science
Author(s): Joseph Gault, Mathias Ferber, Silke Machata, Anne-Flore Imhaus, Christian Malosse, Arthur Charles-Orszag, Corinne Millien, Guillaume Bouvier, Benjamin Bardiaux, Gérard Péhau-Arnaudet, Kelly Klinge, Isabelle Podglajen, Marie Cécile Ploy, H. Steven Seifert, Michael Nilges, Julia Chamot-Rooke, Guillaume Duménil, Tomoko Kubori.
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.
Members of the Neisseria genus are Gram-negative proteobacteria that include several commensals such as N. sicca, N. lactamica or N. elongata and two human pathogens, N. gonorrheae and N. meningitidis. Both of these are highly adapted for interaction with humans, their unique host. N. gonorrheae colonizes the human urogenital tract and is responsible for a sexually transmitted infection characterized by a massive inflammatory response and purulent discharge. Neisseria meningitis is responsible for devastating sepsis and meningitis . N. meningitidis proliferates on the surface of epithelial cells lining the nasopharynx in approximately 5 to 30% of the total human population. Pathogenesis is initiated when bacteria access the bloodstream from the throat, survive and multiply in the blood. Systemic infection and perturbation of vascular function lead to sepsis, the most severe form of the disease associated with organ dysfunction, limb necrosis and death in certain cases. N. meningitidis can also cross the blood-brain barrier and access the cerebrospinal fluid, leading to meningitis.
Our results show that, unlike in class I pilins, a large portion of the pilus surface is coated with sugars in class II pilins. Over the years pilin glycosylation of class I pilins has been studied from 3 different Neisseria meningitidis strains demonstrating a single conserved glycosylation site at Ser63 (Table 3). Strain C311#3 displays a Gal(β1–4)Gal(α1–3)2,4-DATDH , strain 8013 one GATDH residue  and NIID280 a DATDH residue . In addition, N. gonorrhoeae strain N400 presents a hexose residue linked to a DATDH on its class I pilin also at Ser63 . In this single study using top-down mass spectrometry, we describe for the first time the glycosylation pattern of 5 different strains expressing class II pilins including the FAM18 reference strain. Pilins from all of these 5 strains display 3 to 5 glycosylation sites (Table 3). Independently of the serogroup, clonal complex, geographic site or temporal period of isolation of the strains (Table 2), class I pilins show a single site of glycosylation while class II pilins have multiple sites of glycosylation.