Pertactin Is Required forBordetellaSpecies To Resist Neutrophil-Mediated Clearance

Abstract

Pertactin (PRN) is an autotransporter protein produced by all members of the Bordetella bronchiseptica cluster, which includes B. pertussis, B. parapertussis, and B. bronchiseptica. It is a primary component of acellular pertussis vaccines, and anti-PRN antibody titers correlate with protection. In vitro studies have suggested that PRN functions as an adhesin and that an RGD motif located in the center of the passenger domain is important for this function. Two regions of PRN that contain sequence repeats (region 1 [R1] and R2) show polymorphisms among strains and have been implicated in vaccine-driven evolution. We investigated the role of PRN in pathogenesis using B. bronchiseptica and natural-host animal models. A ⌬prn mutant did not differ from wild-type B. bronchiseptica in its ability to adhere to epithelial and macrophage-like cells in vitro or to establish respiratory infection in rats but was cleared much faster than wild-type bacteria in a mouse lung inflammation model. Unlike wild-type B. bronchiseptica, the ⌬prn mutant was unable to cause a lethal infection in SCID-Bg mice, but, like wild-type bacteria, it was lethal for neutropenic mice. These results suggest that PRN plays a critical role in allowing Bordetella to resist neutrophil-mediated clearance. Mutants producing PRN proteins in which the RGD motif was replaced with RGE or in which R1 and R2 were deleted were indistinguishable from wild-type bacteria in all assays, suggesting that these sequences do not contribute to PRN function.Bordetella pertussis and Bordetella bronchiseptica cause respiratory infections in mammals. These two closely related bacterial subspecies display a high level of biochemical and genomic similarity and produce a similar set of virulence factors that are regulated at the transcriptional level by a highly conserved two-component signaling system called BvgAS (8,46,50,65). However, they differ in symptomatic manifestations and host ranges: B. pertussis infects only humans and causes the acute disease whooping cough, while B. bronchiseptica has been isolated from nearly all mammals and typically causes chronic asymptomatic infections (41, 52). Nevertheless, some crucial factors are functionally interchangeable, such as filamentous hemagglutinin (FHA) (30) and the BvgAS signaling system (40), suggesting that studying B. bronchiseptica can reveal insights into the function of B. pertussis factors and vice versa. B. bronchiseptica and its natural-host animal models are therefore valuable tools for exploring molecular mechanisms of Bordetella pathogenesis.Pertactin (PRN) is a putative Bordetella virulence factor belonging to the autotransporter (AT) family. It was discovered as a surface-localized immunogen of B. bronchiseptica in 1985 (49) and was found to have highly conserved homologs in B. pertussis and B. parapertussis shortly thereafter (5, 38). Many subsequent studies focused on the immunogenic and protective properties of PRN, leading to its inclusion in acellular pertussis vaccines (34, 48), and efficacy trials have...

A Novel Sensor Kinase Is Required for Bordetella bronchiseptica To Colonize the Lower Respiratory Tract

“…Inflammation has been documented by recruitment of macrophages and neutrophils via observation of stained lung sections of infected mice (26). In this model, wild-type B. bronchiseptica induces a significant inflammatory response and is able to persist in the lungs for several weeks (26,27,32,68). We wondered if plrS might contribute to the bacteria's ability to survive under these experimental conditions.…”

“…Plasmid pMD11 was transformed into E. coli SM10 pir, conjugated into RB50, and recombined into the chromosome; and cointegrates were selected on BG agar supplemented with 50 mM MgSO 4 as described previously (27). To create an in-frame deletion in BB0264, cointegrates were streaked on BG agar plates without added MgSO 4 to select for the excision of pMD11 (27), resulting in strain RB50⌬plrS. The chromosomal in-frame deletion in BB0264 was confirmed by PCR analysis.…”

“…To create an in-frame deletion in BB0264, we utilized a recently described allelic exchange system based on plasmid pSS4245 (27). A derivative of this plasmid, pMD11, was constructed as follows: primers 0264.380fwd (5Ј-GATTA AGCTTCTATTTCACCGCCGACCTGC-3Ј) and 0264.12rev (5Ј-GACCTCTA GAAAACATTCTCATGAGGGGTCCC-3Ј) were used to amplify a 0.4-kb DNA fragment corresponding to the 5Ј upstream region and containing the first four codons of BB0264.…”

“…DNA sequence analysis was used to confirm the correct sequence of all DNA fragments generated by PCR, as well as the junction of the in-frame deletion generated during the cloning step. Plasmid pMD11 was transformed into E. coli SM10 pir, conjugated into RB50, and recombined into the chromosome; and cointegrates were selected on BG agar supplemented with 50 mM MgSO 4 as described previously (27). To create an in-frame deletion in BB0264, cointegrates were streaked on BG agar plates without added MgSO 4 to select for the excision of pMD11 (27), resulting in strain RB50⌬plrS.…”

Pertussis Vaccine Effectiveness in the Setting of Pertactin-Deficient Pertussis

“…[16][17][18] Pertactin is an autotransporter potentially involved in bacterial adhesion to the respiratory tract and resistance to neutrophil-induced bacterial clearance. 19,20 It is also a component of all acellular pertussis vaccines currently administered in the United States. 21 Pertactin deficiency may have evolved in response to acellular vaccine-induced selection pressure, providing an advantage to the bacteria.…”