Elsevier

Vaccine

Volume 27, Issue 4, 22 January 2009, Pages 542-548
Vaccine

Immunoproteomic analysis of Bordetella pertussis and identification of new immunogenic proteins

https://doi.org/10.1016/j.vaccine.2008.11.020Get rights and content

Abstract

Bordetella pertussis is the causative agent of highly communicable respiratory infection whooping cough (pertussis) which remains one of the world's leading causes of vaccine-preventable deaths. In the present study, total soluble proteins extracted from two B. pertussis strains, Tohama I and the local isolate Saadet were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the antisera obtained from the mice immunized with inactivated whole cells as well as those collected from the mice challenged intraperitoneally with live cells of each strain. Of a total of 25 immunogenic proteins identified, 21 were shown to be the novel antigens for B. pertussis.

Introduction

Whooping cough (pertussis) is a highly contagious, acute respiratory illness of humans that is caused by the gram-negative bacterium Bordetella pertussis. This bacterium is a strict human pathogen with no known animal or environmental reservoirs [1]. The introduction of mass vaccination of children in the mid-20th century decreased the incidence of pertussis; however, it is still one of the world's leading causes of vaccine-preventable deaths [2]. As reported by the World Health Organization (WHO), an estimated 50 million cases and 300,000 deaths occur every year; case-fatality rates in developing countries are estimated to be as high as 4% in infants (http://www.who.int/immunization_monitoring/diseases/pertussis_surveillance/en/, as of August 2008).

A new revolution in vaccine design has emerged from the use of post-genomic technologies [3], [4]. Much of information about immunogenic components can be derived from proteomics coupled to Western blotting, namely immunoproteomics which has been successfully applied for the discovery of antigens from various bacterial pathogens; examples including Helicobacter pylori[5], [6], Staphylococcus aureus[7], Bacillus anthracis[8], Shigella flexneri[9], Francisella tularensis[10], Corynebacterium diphtheriae[11], Streptococcus pyogenes[12], Chlamydia pneumoniae[13] and Neisseria meningitidis[14].

Sequencing of the genomes of B. pertussis, B. parapertussis and B. bronchiseptica[15] provided an essential database for applying proteomics approach to these organisms. First proteomics study on B. pertussis involved an analysis of differential protein expression under iron-limitation and iron-excess conditions [16]. Very recently, a comparative surfaceome analysis of the vaccine strain and the clinical strains of this pathogen was reported [17]. In the present study, we report the first immunoproteomic approach to B. pertussis Tohama I and Saadet strains which involved the identification of immunogenic proteins of their total soluble proteomes.

Section snippets

Bacterial strains and preparation of total soluble proteomes

The strains of B. pertussis used in this study were Tohama I and Saadet, the latter being a local strain. B. pertussis Tohama I and Saadet strains were grown on Cohen-Wheeler agar medium [18] for 48 h at 37 °C. For the preparation of whole cell lysates, the cells were collected and suspended in cold TE buffer and centrifuged at 4500 g for 10 min. The pellet was resuspended in 1 ml 0.04 M lysosyme in TE buffer and incubated at 37 °C for 35 min, followed by a centrifugation at 16,000 g for 10 min. The

Total soluble proteome

Total soluble proteomes of B. pertussis Tahoma I and Saadet strains were resolved by 2-DE. Three technical replicates of 2-DE gels for each of the three independent biological samples were run for each strain. 350 μg of extracted proteins were applied to the IPG strips. After being stained with colloidal CBB G250, over 600 spots could be detected on the gels within the pI range 3–10 (Fig. 1).

Identification of immunoreactive proteins

Western blotting of the 2-D gels using antisera designated as Th (sc), Sa (sc), Th (ip) and Sa (ip),

Discussion

To date, a number of virulence determinants of B. pertussis have been identified to be immunogenic, including filamentous hemagglutinin (FHA) and fimbriae (FIM) as two adhesins, pertactin (PRN), serum resistance protein BrkA and tracheal colonization factor (TcfA) as the autotransporters, three toxins, namely pertussis toxin (PT), adenylate cyclase (CyaA) and dermonecrotic toxin (DNT), and LPS, as extensively reviewed by Mattoo and Cherry [24]. Also, the heat shock protein 60 (Hsp60), which

Acknowledgements

This research was granted in part by TUBİTAK-TBAG (project no.107T444). We would like to acknowledge Dr. Dirk Albrecht for his helps in MALDI-TOF/MS analysis.

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