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Journal of Bacteriology, October 2000, p. 5902-5905, Vol. 182, No. 20
INSERM U447, Institut de Biologie de Lille,
Institut Pasteur de Lille, 59019 Lille Cedex, France
Received 1 May 2000/Accepted 24 July 2000
An in silico scan of the partially completed genome sequence of
Bordetella pertussis and analyses of transcriptional
fusions generated with a new integrational vector were used to identify new potential virulence genes. The genes encoding a putative
siderophore receptor, adhesins, and an autotransporter protein appeared
to be regulated in a manner similar to Bordetella virulence
genes by the global virulence regulator BvgAS. In contrast, the gene encoding a putative intimin-like protein appeared to be repressed under
conditions of virulence.
Bordetella pertussis is a
strictly human pathogen responsible for whooping cough, an acute
respiratory disease particularly severe in young children
(18). It produces a number of toxins and adhesins involved
in its pathogenicity. The coordinated expression of the virulence genes
is controlled by the global sensor and regulator BvgAS (1).
The genes under the positive control of BvgAS are called vag
(for virulence-activated gene) genes. In response to environmental
conditions, such as low temperature or the presence of
MgSO4 or nicotinic acid, B. pertussis undergoes a phenotypic modulation. The vag genes are downregulated,
while another set of genes called vrg (for
virulence-repressed gene) genes is upregulated (19).
We have scanned the B. pertussis sequences currently
available on the Sanger Center website
(www.sanger.ac.uk/Projects/B_pertussis) to identify new potential
virulence genes (Table 1). We have developed a new suicide vector, pFUS2, for rapid gene inactivation by
homologous recombination and generation of transcriptional fusions
between the interrupted genes and promoterless lacZ (Fig. 1). pFUS2 was derived from pQE30 (Qiagen,
Courtaboeuf, France) by (i) the replacement of the 960-bp
EcoRI-blunted BglI fragment containing the
tac promoter and the bla gene with a gentamicin resistance cassette, (ii) the insertion into the BamHI and
blunted EcoRI sites of the promoterless lacZ from
pUTminiTn5lacZ2 (5) contained on a 3-kb
BamHI-blunted HindIII fragment, (iii) the insertion of a PCR-amplified 760-bp fragment carrying the RP4 origin of
transfer from pJQ200mp18 (27) into the unique
XbaI site, and (iv) the insertion into the ClaI
and blunted BamHI sites of a PCR-amplified 910-bp fragment
containing the very 3' end of B. pertussis groES, the
intergenic groES-groEL region, and the 5' end of
groEL fused to the slightly truncated 5' end of lacZ.
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
New Virulence-Activated and Virulence-Repressed Genes Identified
by Systematic Gene Inactivation and Generation of Transcriptional
Fusions in Bordetella pertussis
ABSTRACT
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TABLE 1.
Characteristics of putative B. pertussis proteins
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FIG. 1.
Map of pFUS2. (a) Main features of the vector.
GmR, gentamicin resistance cassette; oriT, RP4 origin of
transfer, oriV, ColE1 origin of replication; to, a transcriptional
terminator to prevent transcription initiation from vector sequences.
(b) Sequence surrounding the multiple cloning site of pFUS2. The
portion of the nucleotide sequence corresponding to the 3' end of
groES, the intergenic groES-groEL region, and the
5' end of groEL is shown in bold. The 3' end of
groES and the 5' end of groEL have been
translated (in bold), as has the beginning of lacZ. SD
represents the putative ribosome binding site sequence of
groEL. 
-Gal, -galactosidase.
Gene inactivation with pFUS2 was first validated by targeting the known
Bvg-activated genes fhaB and ptx, coding for the
filamentous hemagglutinin (FHA) and pertussis toxin, respectively
(21), and one Bvg-repressed gene, vrg24
(19). Pairs of oligonucleotides were designed to amplify
350- to 550-bp internal fragments of the target genes by PCR. The
amplicons were cloned into pFUS2 so that translation of these genes
terminated at the groES stop codon, giving rise to
transcriptional fusions with lacZ. Gene inactivations were
performed in both B. pertussis Tohama I derivatives BPSM
(bvg+) (23) and BPLOW
(
bvg). BPLOW carries a chromosomal deletion of
bvgAS extending from the first EcoRI site
upstream of bvgA to the first EcoRI site within
bvgS. This strain was constructed by double homologous
recombination using pSS1129 as described previously (28).
Both ptx and fhaB fusions were expressed at high
levels in BPSM and were modulated by MgSO4 and nicotinic acid (Table 2). The expression of
ptx'-'lacZ was very low in BPLOW, and that of
fhaB'-'lacZ was undetectable. The expression of the
vrg24'-'lacZ fusion in BPSM increased 2.5-fold in the
presence of the modulators, and it was slightly higher in the
bvg background than in the bvg+
background. Altogether, these results confirm the usefulness of pFUS2
to identify vag genes as well as vrg genes.
Therefore, we used this vector to target genes encoding new potential
virulence factors (Table 1).
|
Adhesins. Two genes code for putative proteins homologous to FHA. One of them, called fhaL (for FHA-like, large), corresponds to the largest open reading frame (ORF) of the entire genome. The gene encoding the other FHA-like protein, named FhaS (for FHA-like, small), harbors two frameshifts, and its 5' region is not included in the current contig. Both genes appeared to be well expressed, albeit at much lower levels than fhaB (Table 2). Interestingly, both are regulated by Bvg and thus qualify as vag genes. The reasons for this apparent redundancy are not clear. FHA is of great importance to B. pertussis pathogenicity. Therefore, the bacterium may maintain backup gene copies. Alternatively, the poorly expressed copies might act as reservoirs for homologous recombination with the master gene to generate antigenic diversity, similar to the pilin genes in Neisseria (16). It is also conceivable that the three proteins play related but distinct functions, similar to the antigen 85 complex of Mycobacterium tuberculosis (2). Alternatively, the bacterium progressively sheds obsolete copies.
The gene encoding a putative signal peptide-bearing protein homologous to a salivary streptococcal adhesin (13) was identified and called adhS. This gene is part of an operon also including genes encoding a permease and an ATPase, and the corresponding protein shares limited similarity with periplasmic components of ATP-binding cassette transporters. It is therefore unclear whether it should be classified as an adhesin. This gene was expressed at a very low level and apparently was not modulated. A gene was identified coding for a protein whose closest homolog is the enteropathogenic Escherichia coli intimin, and it was therefore called bilA (for Bordetella intimin-like). However, this putative protein is somewhat longer than intimin, and it lacks the two disulfide-bonded cysteines shown to be essential for the binding activity of intimin (12). The expression of bilA was very high in thebvg
background and was dramatically upregulated by nicotinic acid and
MgSO4 in BPSM. These features indicate that bilA
is a vrg gene. The roles of the vrg genes in B. pertussis infection remain mysterious (1, 22).
Recent evidence suggests that these genes may be expressed upon entry into eukaryotic cells or at high bacterial cell densities
(25). The product of bilA has been recently
identified in Bordetella bronchiseptica and has been shown
to be involved in colonization in a rabbit model (K. E. Stockbauer, B. Fuchslocher, J. F. Miller, and P. A. Cotter,
Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. B-184, 2000). It
will be interesting to determine whether the protein is involved in
cytoskeleton rearrangements in the host cell, like intimin is
(6).
Capsule. The B. pertussis genome contains a complete operon for the biosynthesis of a polysaccharide capsule. Earlier literature reports have suggested that the bacterium is capsulated (20). A gene in the 5' region of this operon, named bexB (for Bordetella capsule export gene B) based on the usual nomenclature for other species, was targeted by pFUS2. No expression of bexB was detectable in either BPSM or BPLOW, but surprisingly a low but significant level of activity was observed in the presence of MgSO4. It is possible that the appropriate environmental conditions have not been met for optimal gene expression.
Enzymes. An ORF was found which encodes a putative signal peptide-bearing protein homologous to secreted DNases of several pathogens (3). We called this gene drnB (for DNase of Bordetella). Its expression was rather low and was not influenced by modulation.
The B. pertussis genome analysis revealed two ORFs encoding homologs of-cystathionase. These genes were named metC1
and metC2 based on the nomenclature for other species. The
product of metC1 is highly similar to Bordetella
avium osteotoxin, which metabolizes cystine into an
osteoblast-toxic molecule (14). metC1 was
expressed at a fairly high level but was not Bvg regulated. In
contrast, metC2 was upregulated twofold in the presence of MgSO4 or nicotinic acid, and its expression was slightly
higher in the bvg background than in the
bvg+ background, reminiscent of
vrg24. Therefore, metC2 might be a vrg
gene. The small amplitude of modulation may reflect the indirect effect
of modulating agents on vrg expression in B. pertussis. Whereas the transcription of vag genes is
directly activated by binding of the BvgA regulator to their promoter
regions, that of the vrg genes is indirectly regulated by a
Bvg-dependent repressor (24).
The sequence analysis also uncovered an ORF encoding a protein
homologous to the Legionella pneumophila legiolysin, which in fact is a dioxygenase (17, 30). After inactivation of the B. pertussis bllY (for Bordetella legiolysin)
gene, no strong modulation was observed. However, the -galactosidase
activity of that fusion in the bvg background was lower
than in the bvg+ background.
The B. pertussis chromosome contains several genes coding
for proteases other than housekeeping proteases. Two of them, called nrpB (for neutral protease of Bordetella) and
znpB (for Zn protease of Bordetella) in agreement
with the names given to homologs in other species, were selected for
inactivation by pFUS2. Both were expressed but were not regulated by Bvg.
Autotransporters. The genes for several autotransporters, in addition to BrkA, Tcf, pertactin, and Vag-8 (4, 8, 9, 11), were also identified in the genome of B. pertussis. Five of them (phg, aidB, sphB1 [for serine protease homolog of Bordetella], sphB2, and sphB3) were targeted with pFUS2. The expression levels of sphB2 and sphB3 were very low or undetectable, while phg, aidB, and sphB1 were better expressed. In addition, sphB1 was strongly activated by Bvg, making it a new vag gene.
Iron metabolism. A number of genes encoding potential siderophore and heme receptors were uncovered. In particular, an outer membrane protein of unknown function (26) was found to be homologous to TonB-dependent siderophore receptors of various bacterial species. Its gene is followed by a second gene, coding for a 56.7% identical protein. The two genes, called bfrD and bfrE (for Bordetella ferrisiderophore receptor), are separated by approximately 200 bp. Expression of bfrE was very low and did not appear to be Bvg regulated. In contrast, bfrD was expressed at a high level at a Bvg-dependent fashion, making it the first vag gene involved in iron acquisition in Bordetella. Interestingly, siderophore production is repressed by Bvg in certain B. bronchiseptica strains but not in B. pertussis (15). This divergent regulation may relate to considerable differences between the two species in the ability to survive outside of their hosts (1).
In conclusion, pFUS2 has allowed us to uncover several new Bvg-regulated genes. Undoubtedly, additional members of the Bvg regulon remain to be found. In fact, BvgAS appears to control various aspects of Bordetella physiology in addition to virulence, including the production of a cytochrome (7), a porin (10), and cell wall hydrolase(s) (29). Further characterization of the Bvg regulon will greatly benefit from the development of transcriptomic and proteomic tools.| |
ACKNOWLEDGMENTS |
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We thank K. Timmis for the kind gift of pUTminiTn5lacZ2 and Alain Baulard and Jean Dubuisson for critical reading of the manuscript. The technical help of E. Fort, E. Fontaine, and A. Levard is acknowledged.
F. J.-D. is Chargé de Recherche CNRS. This work was supported by INSERM, Institut Pasteur de Lille, Ministère de l'Education Nationale de la Recherche et de la Technologie, and Région Nord-Pas de Calais.
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FOOTNOTES |
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* Corresponding author. Mailing address: INSERM U447, Institut de Biologie de Lille, Institut Pasteur de Lille, 1 rue Calmette, 59019 Lille Cedex, France. Phone: 33 3 20 87 11 55. Fax: 33 3 20 87 11 58. E-mail: francoise.jacob{at}pasteur-lille.fr.
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Journal of Bacteriology, October 2000, p. 5902-5905, Vol. 182, No. 20
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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