The role of peptide loops of the Bordetella pertussis protein P.69 pertactin in antibody recognition

Abstract

Bordetella pertussis, the etiological agent of whooping cough, is re-emerging in several countries with a traditionally high vaccine uptake. In these B. pertussis strains, polymorphisms were found in several proteins, including P.69 pertactin (P.69 Prn). P.69 Prn, an adhesin, contains two variable regions which are composed of repeats, one of which flanks the receptor binding site. Antibody titers against P.69 Prn correlate with protection and P.69 Prn is one of the components of acellular pertussis vaccines. Nevertheless, little is known about the structure and location of P.69 Prn epitopes. We used a three pronged approach to identify discontinuous epitopes that are recognized by mouse monoclonal antibodies, i.e. site-directed mutagenesis, deletion mapping and competition assays. Site-directed mutagenesis was focused on regions of P.69 Prn predicted to form loops according to the crystal structure. In this report we describe the location of several discontinuous epitopes that are also recognized by human antibodies. Our results reveal an important role of the N-terminus in immune recognition. We provide data for an indirect role of loops in immune evasion by masking of epitopes. We propose that the repeat regions have evolved to allow rapid antigenic variation to deflect the immune response from the functional domain of P.69 Prn. The results presented here provide a better understanding of the structure and function of variable loops and their role in the persistence of pathogens in immunologically primed populations.

Introduction

Bordetella pertussis, a small gram-negative bacterium, is the causative agent of whooping cough or pertussis. Although vaccination against pertussis has been highly successful in reducing morbidity and mortality, it has remained one of the 10 most common causes of death from infectious diseases worldwide [1]. During the past 10 years there has been a resurgence of pertussis in countries with a high vaccine uptake [1], [2], [3], [4], [5], [6]. Several explanations have been suggested for the re-emergence of pertussis in these countries including increased awareness, improved diagnosis of the disease, waning immunity in both adolescents and adults and the adaptation of the B. pertussis population [2]. Pathogen adaptation has probably played an important role in the resurgence of pertussis in the Netherlands [2], [7]. Analysis of clinical isolates collected in the last 50 years revealed antigenic divergence between vaccine strains and circulating strains in both Europe, the United States, Japan and Australia [4], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. Polymorphisms were found in at least two proteins implicated in protective immunity: P.69 pertactin (P.69 Prn) and pertussis toxin (Ptx) [2], [18].

Several observations indicate an important role of P.69 Prn in protective immunity. Antibody levels to P.69 Prn have been shown to correlate with clinical protection [19], [20]. Acellular vaccines (ACV's) containing Ptx, filamentous hemagglutinin (FHA) and P.69 Prn were more effective compared to ACV's containing Ptx and FHA only [21], [22], [23]. Passive and active immunization studies in mice and pigs have shown that antibodies against P.69 Prn confer protective immunity [24], [25]. Anti-P.69 Prn antibodies, but not anti-Ptx, anti-fimbriae, or anti-filamentous hemagglutinin antibodies, were found to be crucial for B. pertussis phagocytosis [26]. Further, P.69 Prn variants induce type-specific antibodies [27] and, finally, the efficacy of the Dutch whole cell vaccine was also shown to be affected by variation of P.69 Prn in a mouse model [24].

P.69 Prn, belongs to the family of so-called autotransporter proteins [28], [29], [30] which undergo autoproteolytic processing [29]. P.69 Prn is processed from a 93 kDa large precursor to a 69 kDa (P.69) and 22 kDa (P.30) protein [31]. The unprocessed polypeptide is directed via a signal peptide to the secretory machinery in the inner membrane where the signal peptide is cleaved. Subsequently, the polypeptide is directed towards the outer membrane where P.30 forms a pore through which the 69-kDa protein is transported. After secretion via the autotransporter domain, proteolytic activities shape the 69-kDa protein to its final 60.37 or 58.34 kDa form [32]. These final forms (referred to as P.69 Prn), stay noncovalently bound to the bacterial cell surface and are used in most ACVs [22].

The X-ray crystal structure of P.69 pertactin has been determined to a resolution of 2.5 Å. The protein fold consists of a 16-stranded parallel β-helix with a V-shaped cross-section. The structure appears as a helix from which several loops protrude, one of which contains the sequence motif associated with the biological activity of the protein; adherence to host tissues [33].

P.69 Prn is polymorphic, and 13 variants (P.69 Prn1–P.69 Prn13) have been identified so far [34]. Variation is mainly limited to two regions, designated region 1 and 2, which are comprised of Gly-Gly-X-X-Pro (r1 repeat) and Pro-Gln-Pro (r2 repeat) repeats, respectively. Most variation is found in region 1 which is located proximal to the N-terminus and flanks the Arg-Gly-Asp (RGD) motif, implicated in ligand–receptor interactions in eukaryotes. It has been shown that the RGD motif is involved in P.69 Prn-mediated attachment of B. pertussis to mammalian cells [35], [36].

Although a number of studies in both animals and humans have indicated that P.69 Prn can elicit protective antibodies [19], [20], [24], [37], information about the location of epitopes to which these antibodies are directed is limited. Previously, we described the location of several linear epitopes on P.69 Prn of both human serum antibodies and mouse mAbs [38]. The aim of this study is to define the location of discontinuous epitopes on P.69 Prn, recognized by human antibodies. This would allow us to gain more insight into the role that P.69 Prn plays in immunity and immune evasion.