Nicole Guiso

Global Population Structure and Evolution of Bordetella pertussis and Their Relationship with Vaccination

ABSTRACT Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines. IMPORTANCE Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape. Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape.

Deciphering the ancient and complex evolutionary history of human arylamine N-acetyltransferase genes.

The human N-acetyltransferase genes NAT1 and NAT2 encode two phase-II enzymes that metabolize various drugs and carcinogens. Functional variability at these genes has been associated with adverse drug reactions and cancer susceptibility. Mutations in NAT2 leading to the so-called slow-acetylation phenotype reach high frequencies worldwide, which questions the significance of altered acetylation in human adaptation. To investigate the role of population history and natural selection in shaping NATs variation, we characterized genetic diversity through the resequencing and genotyping of NAT1, NAT2, and the pseudogene NATP in a collection of 13 different populations with distinct ethnic backgrounds and demographic pasts. This combined study design allowed us to define a detailed map of linkage disequilibrium of the NATs region as well as to perform a number of sequence-based neutrality tests and the long-range haplotype (LRH) test. Our data revealed distinctive patterns of variability for the two genes: the reduced diversity observed at NAT1 is consistent with the action of purifying selection, whereas NAT2 functional variation contributes to high levels of diversity. In addition, the LRH test identified a particular NAT2 haplotype (NAT2*5B) under recent positive selection in western/central Eurasians. This haplotype harbors the mutation 341T-->C and encodes the slowest-acetylator NAT2 enzyme, suggesting a general selective advantage for the slow-acetylator phenotype. Interestingly, the NAT2*5B haplotype, which seems to have conferred a selective advantage during the past approximately 6,500 years, exhibits today the strongest association with susceptibility to bladder cancer and adverse drug reactions. On the whole, the patterns observed for NAT2 well illustrate how geographically and temporally fluctuating xenobiotic environments may have influenced not only our genome variability but also our present-day susceptibility to disease.

Analysis of Bordetella pertussis Populations in European Countries with Different Vaccination Policies

ABSTRACT Despite the widespread use of pertussis vaccines during the last decades, pertussis has remained an endemic disease with frequent epidemic outbreaks. Currently two types of vaccines are used: whole-cell vaccines (WCVs) and recently developed acellular vaccines (ACVs). The long-term aim of our studies is to assess the effect of different vaccination policies on the population structure of Bordetella pertussis and ultimately on the disease burden in Europe. In the present study, a total of 102 B. pertussis isolates from the period 1998 to 2001 from five European countries (Finland, Sweden, Germany, The Netherlands, and France) were characterized. The isolates were analyzed by typing based on variable number of tandem repeats (VNTR); by sequencing of polymorphic genes encoding the surface proteins pertussis toxin S1 and S3 subunits (ptxA and ptxC), pertactin (prn), and tracheal colonization factor (tcfA); and by fimbrial serotyping. The results reveal a relationship between geographic location and VNTR types, the frequency of the ptxC alleles, and serotypes. We have not observed a relationship between the strain characteristics we studied and vaccination programs. Our results provide a baseline which can be used to reveal changes in the B. pertussis population in Europe in the coming years.

Comparison of Serological and Real-Time PCR Assays To Diagnose Bordetella pertussis Infection in 2007

ABSTRACT Bacterial culture for diagnosing pertussis infection has high specificity but poor sensitivity and is slow. Highly sensitive real-time PCR assays and single-serum pertussis serology have been developed to overcome these limitations, but there are few data available on the relative sensitivities and specificities of such assays for pertussis diagnosis. Using data on 195 participants (≥7 years old) from an epidemiological study, we assessed the sensitivity, specificity, and performance (Youden index) for pertussis diagnosis of the pertussis toxin enzyme-linked immunosorbent assay (using single and paired serology) and of real-time PCR assays (using the IS481 and ptxA-Pr targets). All available diagnostic information (clinical and laboratory) was pooled to serve as the gold standard. Single serology was the most efficient diagnostic test (Youden index, 0.57 to 0.58), with relatively high sensitivity (>64%) and high specificity (>90%), independent of the cutoff level. IS481 PCR performance was superior to that of ptxA-Pr PCR, and it was the second-most-efficient tool (Youden index, 0.30). Performing both ptxA-Pr and IS481 PCRs did not improve diagnostic performance. The greatest test efficiency (Youden index, 0.69 to 0.74) was achieved when single-serum serology was used in combination with IS481 or ptxA-Pr PCR or paired serology. Combining single serology with one PCR or paired serology increased the sensitivity with an associated limited decrease in specificity. The most specific tests for diagnosis of pertussis were single serology and ptxA-Pr PCR, and the most sensitive diagnostic tool was the combination of IS481 PCR with single serology.

Antigenic polymorphism of the lipopolysaccharides from human and animal isolates of Bordetella bronchiseptica

Six monoclonal antibodies (mAbs) against lipopolysaccharides (LPS) from Bordetella pertussis (P1P3, 60.5), B. parapertussis (PP2, PP6, PPB) and B. bronchiseptica (BRg1) were used to examine the presence of antigenic determinants of LPS on B. bronchiseptica cells. Forty-eight clinical isolates of this Gram-negative bacterium (4 canine, 3 equine, 6 porcine, 4 rabbit and 31 human) were examined. Significant cross-reactivities with the heterologous anti-pertussis and anti-parapertussis mAbs were observed. The isolates also exhibited marked antigenic polymorphism. The 48 isolates could be classified in six immunogroups. Purified LPS preparations extracted from some isolates were analysed by ELISA, thin-layer chromatography, and tricine-SDS-PAGE. The results show that four main types of antigenic polymorphism of B. bronchiseptica LPSs exist: (a) heterogeneity of the core, (b) presence or absence of O-chains, (c) differences in the hinge region between O-chain and core, and (d) differences in interactions of LPS with other cell-surface constituents. Smooth-type LPS molecules, detectable with mAb PP6, were more frequently observed in animal isolates (94%) than in human isolates (52%). Reverse frequencies were found with mAb 60.5 (48% of human isolates, 18% of animal isolates), which is unable to react with long-chain LPSs. This observation could be due to the general absence of some lectin-like receptor, specific to the O-chain, on human bronchoalveolar tissues.

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

ABSTRACT The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b− cells. The nonhemolytic AC+ Hly− bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC+ Hly− mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b− cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>107 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.

Photo Quiz: Multiple Skin Lesions on a 9-Year-Old Boy Returning from Mali

A 9-year-old boy presented to the pediatric emergency service for multiple skin lesions of the upper and lower limbs ([Fig. 1A][1] and [B][1]). These lesions had been present for 1 month. The patient had no prior medical history and had been correctly vaccinated. He lives in mainland France but had