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Journal of Clinical Microbiology, September 2006, p. 3397-3400, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.00247-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Differential In Vitro Expression of the brkA Gene in Bordetella pertussis and Bordetella parapertussis Clinical Isolates

Paola Stefanelli,1 Maurizio Sanguinetti,2 Cecilia Fazio,1 Brunella Posteraro,2 Giovanni Fadda,2 and Paola Mastrantonio1*

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy,1 Institute of Microbiology, Università Cattolica del Sacro Cuore, Rome, Italy2

Received 3 February 2006/ Returned for modification 13 March 2006/ Accepted 17 July 2006


   ABSTRACT
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In this study, we set up a real-time reverse transcriptase PCR assay to measure the relative amounts of brkA transcripts in 50 Bordetella isolates. The results suggested that brkA expression is strain dependent and its level may play a role in determining the serum resistance or susceptibility phenotype. Pertussis immunocompetent sera were unable to kill Bordetella parapertussis via complement deposition.


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BrkA (bordetella resistance killing antigen) is considered an important Bordetella pertussis virulence factor due to its ability to mediate serum resistance (1). The BrkA-encoding gene is also present in the two Bordetella species strictly related to B. pertussis, specifically B. parapertussis and B. bronchiseptica (5). In particular, B. parapertussis, as well as B. pertussis, may cause episodes of whooping cough. Several studies have been performed that demonstrated a cross-immunity between the two species (2), but it has also been hypothesized that, since both infect humans, they have developed mechanisms to evade any cross-immunity (6, 11).

There are, however, several conflicting reports on the ability of B. pertussis to survive complement killing and also on the different abilities of vaccinated subjects to develop a measurable bactericidal immune response (13-15).

To measure brkA mRNA expression in clinical strains belonging to Bordetella spp., 37 B. pertussis strains and 13 B. parapertussis strains collected from infants with whooping cough, and one reference strain, B. pertussis 338, were tested using a quantitative reverse transcriptase PCR (RT-PCR) assay.

Real-time RT-PCR. For each strain, 5 ml of Stainer Scholte (SS) broth suspension with a concentration of about 109 bacteria per ml (optical density at 600 nm of 0.5), harvested after a single passage from charcoal agar (CA) plates at different time points during the growth, was used to extract total RNA using the RNeasy Protect Mini kit, following the producer's instructions (QIAGEN, Hilden, Germany). Expression of the brkA and recA genes was quantitatively assessed with real-time RT-PCR using an i-Cycler iQ system (Bio-Rad Laboratories, Hercules, Calif.) (10). The recA housekeeping gene was used as a reference gene, since it is not under the BvgA/S regulon control (16). For each gene, a set of primer pairs and a Taqman probe was designed with Beacon Designer 2 (version 2.06) software (Premier Biosoft International, Palo Alto, Calif.) and was synthesized by MWG Biotech (Florence, Italy) (Table 1). Each reaction was run in quadruplicate, and the amplification efficiency was determined for all the genes (9). Since the PCRs worked with equal efficiencies, the relative mRNA expression levels of brkA were normalized for input RNA against the levels of recA gene transcripts. The relative mRNA expression levels of the brkA gene in each sample were calculated using the comparative cycle time method (8). In addition, to assess the validity of recA as a reference gene for comparative studies of gene expression, we performed an absolute quantification of recA transcripts. Thus, a standard curve was constructed by plotting serial dilutions of a cloned recA gene fragment (1010 to 104 copies/reaction) and used to quantify recA mRNA in samples of RNA extracted from B. pertussis 338 cells harvested at various time points (50 ng of total RNA for each sample). After the number of CFU for each sample was determined, the results were expressed as the number of recA transcripts per CFU. Similar numbers of recA transcript were found in the cells grown at the different time points (1.49 copies/CFU at 6 h, 1.62 copies/CFU at 20 h, 1.70 copies/CFU at 24 h, 1.65 copies/CFU at 32 h, 1.71 copies/CFU at 40 h, and 1.59 copies/CFU at 48 h). This finding demonstrates that recA is expressed at nearly identical levels in B. pertussis cells grown in vitro at different time points.


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  		TABLE 1. Primers and fluorescent probes used in real-time RT-PCR

 
Comparison between the categorical variables was performed by using one-way analysis of variance with a Dunnett correction posttest. A P value of less than 0.05 was considered statistically significant.

Serum killing assay. Twenty-seven immunoresponsive serum samples from children recovering from pertussis or vaccinated with a three-component acellular pertussis vaccine and 20 sera from infants with coughs due to B. parapertussis were used, after heat inactivation, in the serum bactericidal activity assay, performed following the procedures already described (12). Guinea pig Hartley serum (in-house preparation) was used as the source of complement.

Significant differences (P < 0.05) were found for 18 B. pertussis strains compared to B. pertussis 338, chosen as the reference strain for brkA expression after 20 h of growth. Of the 18 strains, 8 isolates showed lower brkA expression levels (ranging from 0.025 to 0.06) and 10 showed higher levels (ranging from 0.18 to 0.38) compared to the level in B. pertussis 338 (0.11). Significantly lower levels of expression were detected in 5 B. parapertussis strains (ranging from 0.045 to 0.075) of the 13 strains analyzed in relation to both the B. parapertussis 02726 reference strain and B. pertussis 338. The latter two showed the same expression level of the gene. The levels of brkA expression from the remaining isolates were not statistically different from that of the reference B. pertussis strain (P > 0.05) (data not shown).

The brkA expression profiles obtained during the whole growth phase separated Bordetella isolates into three groups: B. pertussis isolates with low levels of transcripts (Fig. 1A), B. pertussis isolates with high levels of transcripts (Fig. 1B), and B. parapertussis isolates (Fig. 1C). B. pertussis isolates with high levels of brkA expression at 20 h showed a greater expression of the gene all over the growth curve with a slight increase during the late growth phase (between 24 and 32 h) shown by some strains. Conversely, B. pertussis isolates with low levels of brkA transcripts at 20 h showed reduced expression throughout the whole growth period. B. parapertussis isolates produced the maximum amount of transcripts at 20 h.


Figure 1
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  		FIG. 1. Expression of the brkA gene at different time points during growth of representative Bordetella strains, as determined by real-time RT-PCR analysis. (A and B) Kinetics of brkA expression in representative B. pertussis isolates with low or high levels of transcript, respectively. (C) Kinetics of brkA expression in B. parapertussis strains. Each extract was tested in quadruplicate per each time point by RT-PCR. Error bars show standard deviations.

 
Twenty-seven immunocompetent sera (18 convalescent-phase sera and 9 postvaccination sera) were tested separately against the 18 B. pertussis strains with a significantly different level of brkA expression compared to the reference strain. Figure 2 shows the results expressed as a percentage of bacterial survival for one strain (14782), representative of the strains strongly expressing the gene, for one isolate (05168), representative of the 8 strains weakly expressing the gene, and for 1 B. parapertussis strain representative of the 13 strains tested. The percentages of B. pertussis 14782 survival were high, 98.4 ± 1.8 and 87 ± 4.9 using postvaccination or convalescent-phase sera, respectively. The other nine strains, expressing high levels of brkA expression, showed similar survival levels ranging from 74 to 100% (data not shown) and included those strains with a further brkA increase during the late growth phase. Similar high percentages of survival were observed for the bacteria with high levels of brkA expression after either 16 (70 to 85%) or 40 h of growth (100%) (data not shown).


Figure 2
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  		FIG. 2. Percentage of bacterial survival obtained in the serum killing assay after 20 h of bacterial growth, using 27 immunocompetent pertussis sera, tested individually (postvaccination sera [a bars] and convalescent-phase sera [b bars]). The b^ bar refers to 20 convalescent-phase sera obtained from infants recovering from B. parapertussis infection. The results are the means (numbers above the bars) and standard errors (error bars) for three separate experiments. Bp14782 and Bp05168, representative of B. pertussis with high and low levels of brkA transcripts, respectively; Bpp02726, B. parapertussis reference strain; Bp338, B. pertussis reference strain 338. Asterisks indicate statistically significant differences compared to the value for B. pertussis reference strain 338 (P < 0.05).

 
In contrast, B. pertussis 05168 was much more susceptible to the same postvaccination or convalescent-phase sera with 44% ± 12% and 28% ± 6.7% survival, respectively. The other strains with low levels of brkA expression showed similar percentages of colonies surviving. For B. parapertussis 02726 and for the other B. parapertussis strains, including the five strains with low levels of brkA expression, the percentage of survival was high with all pertussis competent sera. However, when the strains were tested against 20 different immune-competent sera against B. parapertussis, more than 96% ± 1.9% of bacteria were killed (Fig. 2, b^ bar).

Although other authors have shown that there is substantial B. pertussis strain variation in serum sensitivity due to brkA expression (4), this is the first study in which several B. pertussis and B. parapertussis clinical isolates were examined by real-time RT-PCR to quantify the relative amounts of brkA transcripts at different time points during bacterial growth. The results point out differences among B. pertussis strains in terms of the level of expression of the brkA gene. Moreover, the observed differences in serum sensitivity of the strains correlate well with the different levels of the gene expressed. This may suggest that the ability to promote complement deposition is dependent not only on serum competence, in terms of specific antibodies, but also on the different levels of brkA expressed by the bacteria. The results of the bactericidal assay using immunocompetent sera collected after vaccination or disease agree with those already reported by other authors: in particular, acellular postvaccination sera are scarcely efficient in killing bacteria (15).

Furthermore, the results obtained in this work confirm the lack of cross-reaction in the bactericidal activity between B. pertussis and B. parapertussis, even if the relative levels of brkA transcripts were similar. It is known that the two species are really distinct in terms of surface-exposed proteins and a different set of prominent surface molecules may prevent effective immune cross-protection (7). An example is the O antigen, present in B. parapertussis but absent in B. pertussis, and known to inhibit complement deposition (3).

Further investigation will be necessary to elucidate the molecular mechanisms involved in the expression of the brkA gene at different levels and to assess the correlation with levels of protein expression in B. pertussis and B. parapertussis clinical isolates.


   ACKNOWLEDGMENTS
 
This study was partly supported by ISS-NIH Scientific Cooperation Agreement (N5303).

We thank Tonino Sofia for editing the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, V. le Regina Elena 299, 00161 Rome, Italy. Phone: 390649902335. Fax: 390649387112. E-mail: pmastran{at}iss.it. Back


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Journal of Clinical Microbiology, September 2006, p. 3397-3400, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.00247-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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