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Infection and Immunity, November 1999, p. 6119-6129, Vol. 67, No. 11
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification of Regions of the Chromosome of Neisseria
meningitidis and Neisseria gonorrhoeae Which Are
Specific to the Pathogenic Neisseria Species
Agnes
Perrin,
Xavier
Nassif,* and
Colin
Tinsley
Laboratoire de Microbiologie, INSERM U411,
Faculté de Médecine Necker-Enfants Malades, 75015 Paris, France
Received 7 May 1999/Returned for modification 21 June 1999/Accepted 26 July 1999
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ABSTRACT |
Neisseria meningitidis and Neisseria
gonorrhoeae give rise to dramatically different diseases. Their
interactions with the host, however, do share common characteristics:
they are both human pathogens which do not survive in the environment
and which colonize and invade mucosa at their port of entry. It is
therefore likely that they have common properties that might not be
found in nonpathogenic bacteria belonging to the same genetically
related group, such as Neisseria lactamica. Their common
properties may be determined by chromosomal regions found only in
the pathogenic Neisseria species. To address this issue, we
used a previously described technique (C. R. Tinsley and X. Nassif, Proc. Natl. Acad. Sci. USA 93:11109-11114, 1996) to identify
sequences of DNA specific for pathogenic neisseriae and not found in
N. lactamica. Sequences present in N. lactamica
were physically subtracted from the N. meningitidis Z2491
sequence and also from the N. gonorrhoeae FA1090
sequence. The clones obtained from each subtraction were tested by
Southern blotting for their reactivity with the three species, and only
those which reacted with both N. meningitidis and N. gonorrhoeae (i.e., not specific to either one of
the pathogens) were further investigated. In a first step, these clones
were mapped onto the chromosomes of both N. meningitidis and N. gonorrhoeae. The majority of the
clones were arranged in clusters extending up to 10 kb,
suggesting the presence of chromosomal regions common to N. meningitidis and N. gonorrhoeae which distinguish
these pathogens from the commensal N. lactamica. The
sequences surrounding these clones were determined from the N. meningitidis genome-sequencing project. Several clones
corresponded to previously described factors required for colonization
and survival at the port of entry, such as immunoglobulin A protease
and PilC. Others were homologous to virulence-associated proteins in
other bacteria, demonstrating that the subtractive clones are
capable of pinpointing chromosomal regions shared by N. meningitidis and N. gonorrhoeae which are involved in common aspects of the host interaction of both pathogens.
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INTRODUCTION |
Neisseria
meningitidis and Neisseria gonorrhoeae are two human
pathogens which belong to the same genospecies. Furthermore, phylogenetic analyses by rRNA similarities and DNA-DNA
hybridizations have placed N. meningitidis, N. gonorrhoeae, N. lactamica, and N. cinerea in
a subgroup with particularly close interspecies relatedness (19,
27, 39). Although these bacteria are closely related, they
express very different pathogenicities. N. lactamica and
N. cinerea are nonpathogenic. N. meningitidis
colonizes the nasopharynx, from where it may spread into the
bloodstream before crossing the blood-brain barrier to induce
meningitis. N. gonorrhoeae colonizes and invades the
epithelium of the genitourinary tract and may cause a localized
inflammatory process or an ascending infection leading to salpingitis.
However, even though N. meningitidis and N. gonorrhoeae give rise to two very different diseases, they both
have to colonize and cross an epithelium at their port of entry. This
is consistent with the fact that in addition to having specific
virulence factors, they have common virulence attributes such as pili,
immunoglobulin A (IgA) proteases, and class 5 outer membrane proteins.
However other as yet unidentified proteins, some of which are specific
for the pathogenic Neisseria species and are not found in
N. lactamica, are most probably involved in this common step
of interaction of these bacterial pathogens with their host.
While differences in pathogenic potential may theoretically result from
differential expression or subtly differing proteins, the situation is
more generally found to involve the possession of pathogen-specific
sequences. Attributes of bacterial virulence are often grouped in
islands and frequently are passed horizontally between more or less
closely related species (22). Representational difference
analysis (33, 44) provides a quick means of cloning DNA
corresponding to such species-specific sequences, by direct physical
subtraction of the chromosomal DNA of a closely related, avirulent
strain from the chromosomal DNA of the pathogen. Thus large islands of
DNA which may encode N. meningitidis-specific virulence
factors which are not present in N. gonorrhoeae have recently been identified. To identify the chromosomal regions that are
common to pathogenic Neisseria species and are responsible for the colonization and survival at the port of entry, we first subtracted from N. meningitidis those sequences which were
also present in the commensal N. lactamica and then
performed a similar experiment subtracting the N. lactamica sequences from the chromosome of N. gonorrhoeae. The results of these experiments confirmed that both
pathogens have common sequences which are absent from the nonpathogenic
N. lactamica and identify putative virulence factors
involved in survival and dissemination from the port of entry.
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MATERIALS AND METHODS |
Strains, plasmids, and growth conditions.
N.
meningitidis Z2491 and N. gonorrhoeae FA1090 were
chosen as reference pathogenic Neisseria strains; both are
in the process of being sequenced, and both also have many of their
important genetic markers positioned on published macrorestriction maps (10, 11). Two strains of N. lactamica, 8064 and
9764, from this laboratory were used to provide DNA for subtraction.
Other strains came from the collection of X. Nassif.
Neisseria strains were grown on GCB (Difco) agar plates,
containing the Kellogg supplements and ferric nitrate (26),
for 14 to 16 h at 37°C in a humid atmosphere containing 5%
CO2.
Molecular genetic techniques.
Routine molecular biological
techniques were carried out as recommended (3, 41). DNA
sequences were determined by using an ABI-Prism 370 automated sequencer
with the Big Dye primer-sequencing kit. Southern blotting was performed
as previously described (7, 44) but omitting the bovine
serum albumin from the hybridization buffer. DNA fragments were
labelled for Southern hybridizations by random-primed incorporation of
[
-32P]dCTP. Chromosomal DNA extraction was
performed on cells grown in broth or scraped from agar plates. Bacteria
from one 7-cm plate or from 10 ml of broth were suspended in 1 ml of 10 mM Tris-HCl (pH 8.0)-10 mM EDTA-100 mM NaCl containing 2 µg of
RNase A. After addition of 50 µl of 20% sodium dodecyl sulfate and
incubation at 65°C for 30 min, the mixtures were digested for 2 h at 37°C with proteinase K (100 µg). The solutions were then
extracted once with an equal volume of phenol (pH 8), twice with
phenol-choroform-isopentanol (25:24:1), and once with
chloroform-isopentanol (24:1). The solution was overlaid with an equal
volume of ethanol and cooled to 0°C, and the DNA was spooled from the
interface by mixing with a glass Pasteur pipette. The fibrous DNA was
washed in 70% ethanol, partially dried, and then redissolved in TE
buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA).
Chromosomal DNA was quantified by UV spectrophotometry. For
quantification of fragmented DNA, preparations were diluted into TE
buffer containing 1 µg of ethidium bromide per ml and appropriate dilutions were performed to produce 20-µl samples containing 1, 1/3,
1/10, 1/30, and 1/100 µl of the DNA preparation. In parallel, solutions of 200, 100, 50, 20, 10, 5, and 0 ng of standard DNA per 20 µl (HindIII digest of lambda phage) were prepared in
the same solution. Drops were placed on a sheet of polyethylene film, illuminated with UV light, and photographed. The concentration of the
DNA preparation was measured against the scale of luminosities of the
lambda DNA standards.
Representational difference analysis.
Clones of DNA
fragments present in the genome of N. meningitidis and/or
that of N. gonorrhoeae but absent from N. lactamica were prepared essentially as described previously
(44) (Fig. 1). Six banks were
created, three for N. gonorrhoeae and three for N. meningitidis. Briefly, 20 µg of DNA from N. gonorrhoeae or N. meningitidis was cleaved with
MboI, MspI, or Tsp509I, precipitated with ethanol-sodium acetate, and ligated with 5 nmol of the appropriate oligonucleotide adapter pair (RBam12 and RBam24, RCla12 and RCla24, or
REco12 and REco24 [Table 1]) for
18 h at 11°C. The mixture was gel purified on 2%
low-melting-point agarose (taking fragments above 200 bp) to remove
unincorporated primers, phenol purified, precipitated, and redissolved
in TE buffer. This procedure results in DNA fragments whose two 5' ends
are covalently linked to the 24-base adapter. To prepare the
subtracting DNA, chromosomes of two strains of N. lactamica
were sheared by repeated passage through a hypodermic needle to give
fragments ranging from about 3 to 10 kb. The DNA was repurified by
phenol extraction, precipitated, and redissolved in TE buffer. Equal
quantities of the two were mixed to make the subtracting DNA.
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FIG. 1.
Procedure for representational difference analysis.
Sequences specific to the pathogen are represented in grey; those in
common with N. lactamica are hatched. DNA from N. meningitidis or from N. gonorrhoeae was digested with
frequently cutting restriction endonucleases and ligated to adapter
pairs such that only the 5' end of each DNA stand was covalently linked
to the 24-bp adapter. On denaturing, mixing, and reannealing, only the
(pathogen-specific) sequences with an adapter covalently linked were
able to rehybridize with their complementary sequence. The fragments of
randomly sheared N. lactamica chromosome are generally over
10 times as long as the restriction fragments from the pathogen and not
only sequester all pathogen fragments having homologies in N. lactamica but also, in the large majority of cases, prevent the
polymerase from synthesizing the complement of the adapter during the
filling-in procedure. Hence, these common fragments are effectively
prevented from being amplified, and only the pathogen-specific
fragments possessing an adapter at each end can be exponentially
amplified.
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The first subtractive hybridization was performed with 40 µg of
N. lactamica subtracting DNA and 200 ng (
MboI or
MspI digested)
or 400 ng (
Tsp509I digested) of
R-adapter-linked pathogen DNA
fragments. The DNA was mixed, ethanol
precipitated, and redissolved
in 8µl of EE buffer [10 mM
N-(2-hydroxyethyl)piperazine-
N'-(3-propanesulfonic
acid), 1 mM EDTA (pH 8.0)]. The liquid was overlaid with 30 µl
of
mineral oil, denatured at 100°C for 2 min, and then placed
at 55°C.
After the addition of 2 µl of 5 M NaCl, the mixture was
left to
hybridize at 55°C for 48
h.
The reaction mixture was then diluted 10-fold with preheated EE
buffer-NaCl and immediately placed on ice. A portion of the
subtraction
mixture (10 µl) was diluted into 400 µl of PCR mix
(10 mM Tris-HCl
[pH 9.0], 50 mM KCl, 1.5 mM MgCl
2, 0.1% Triton
X-100,
0.125 mM each deoxynucleoside triphosphate, 100 U of
Taq polymerase per ml) to fill in the ends corresponding to the 24-base
adapter. The reaction mixtures were diluted a further 10-fold,
and PCR
amplifications were performed on 400 µl of the dilutions.
After
denaturation for 5 min at 94°C and addition of the appropriate
24-base oligonucleotide, the mixtures were amplified by PCR (30
cycles
of 1 min at 70°C, 3 min at 72°C, and 1 min at 94°C, followed
by 1 cycle of 1 min at 94°C and 10 min at 72°C [Perkin-Elmer GeneAmp
9600 thermal cycler]). The amplified meningococcal DNA was separated
by agarose gel electrophoresis from the primers and
high-molecular-weight
subtracting
DNA.
The first adapters (R) were cleaved from the PCR products with the
appropriate restriction enzymes, and the second-round adapters
were
ligated (2 µg of subtractive fragments and 2 nmol of adapters
[JBam12 and JBam24, JCla12 and JCla24, or JEco12 and JEco24; Table
1]
in a volume of 50 µl). The ligated fragments were gel purified
and
phenol
extracted.
The second-round subtractive hybridization was performed with 25 ng of
DNA from the pathogens (first-round products, cleaved
and religated to
the J adapters) and 40 µg of DNA from
N. lactamica.
Fragments amplified from the second round were cleaved with the
appropriate enzyme, gel purified, and cloned into pBluescript
(Stratagene) cleaved with the appropriate enzyme (
BamHI for
the
MboI fragments,
ClaI for the
MspI
fragments, and
EcoRI for the
Tsp509I fragments).
The recombinant plasmids were maintained in
Escherichia coli
DH5
. Subsequent manipulations all used the PCR
product corresponding
to the inserted DNA, amplified between primers
flanking the polycloning
site of
pBluescript.
Cloned DNA fragments were tested first by Southern blotting for their
reactivity with
N. meningitidis and/or
N. gonorrhoeae and absence of reactivity with either of the strains
of
N. lactamica;
this also permitted the elimination of
obvious duplicate clones.
Sequences were compared against other
subtractive clones and against
public-domain databases by using the
BLAST algorithm (National
Center for Biotechnology Information,
Bethesda, Md.) (
2). The
locations of the genes on the
published macrorestriction maps
of
N. meningitidis Z2491 and
of
N. gonorrhoeae FA1090 were determined
as described
previously (
44). The sequences were also used to
extract the
sequence of the chromosomal DNA surrounding the subtractive
clones from
the databases of the Z2491 genome sequencing project
(
46a)
and FA1090 (
44a). From this data, open reading frames
(ORFs)
were predicted by using the programs MacVector (Oxford
Molecular Group,
Oxford, United Kingdom) and CodonUse (Conrad
Halling, Monsanto Corp.).
These were also compared to sequences
in public-domain databases by
using
BLAST.
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RESULTS AND DISCUSSION |
Production of libraries of clones specific to the pathogenic
species.
In a first experiment, three banks of N. meningitidis-specific clones were prepared by subtracting the
chromosome of N. lactamica from meningococcal
chromosomal DNA, cleaved with three restriction enzymes.
Meningococcal DNA from strain Z2491 was cleaved with MboI (GATC, compatible with BamHI),
MspI (CCGG, compatible with ClaI) and
Tsp509I (AATT, compatible with EcoRI) and
subjected to two rounds of subtraction by using DNA mixed from two
strains of N. lactamica. The use of two strains of
N. lactamica ensured that clones isolated were not taken as
being N. meningitidis specific due to their absence from one
particular strain of N. lactamica. The N. meningitidis-specific fragments were cloned into
pBluescript. PCR products corresponding to the inserts, were
radiolabelled and used in an initial screening by Southern blotting
against chromosomal DNA from the meningococcus Z2491, the gonococcus
FA1090, and the two strains of N. lactamica used for
subtraction, each cleaved with ClaI. Of 237 clones initially
isolated, 41 showed a double specificity for N. gonorrhoeae and N. meningitidis and no reactivity with
N. lactamica. These were chosen for further study.
Pathogen-specific DNA sequences should be equally attainable by the
subtraction of
N. lactamica DNA from gonococcal DNA. To
test
the completeness of the bank obtained by subtraction of
N. lactamica from
N. meningitidis and to increase the
representativity
of the subtractive clones, another three banks were
produced as
above, but this time subtracting the DNA of the two strains
of
N. lactamica from
N. gonorrhoeae FA1090 DNA.
Again, 20 of 83 clones
showing reactivity with both
N. meningitidis and
N. gonorrhoeae were
kept.
Clones derived from the subtraction involving meningococcal
MboI fragments were designated Bm001, Bm002, etc.;
those involving
the
MspI fragments were named Cm001,
etc., and those involving
the
Tsp509I fragments were
named Em001, etc.; the letters B, C,
and E refer to the
corresponding
BamHI,
ClaI, and
EcoRI
sites
used for their cloning, respectively, and the letter m refers
to
the originating species
N. meningitidis. Clones derived from
N. gonorrhoeae were designated Bg001, Cg001, Eg001, etc. The
positions
of the 61 clones which were retained were determined in
relation
to the published macrorestriction maps of
N. gonorrhoeae FA1090
(
10) and
N. meningitidis
Z2491 (
11) by probing Southern blots
of chromosomal DNA
cleaved with infrequently cutting restriction
enzymes and subsequent
comparison of the reactive bands with their
published maps. In
addition, the subtractive clones were sequenced,
and, following BLAST
searches of the partially sequenced chromosomes
of
N. gonorrhoeae FA1090 and
N. meningitidis Z2491, the
corresponding
contigs were extracted from the genome sequence data
of
N. meningitidis Z2491 and analyzed to permit a tentative
mapping of the subtractive
clones on a smaller scale, relative to one
another and to other
defined genes. Figures
2 and
3
show the positions of the clones
on the chromosome of
N. meningitidis Z2491 and
N. gonorrhoeae FA1090. In
addition, in some cases the sequences surrounding these
contigs were
annotated and are shown in Fig.
4.
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FIG. 2.
Position of the pathogen-specific clones on the
chromosomal map of N. meningitidis Z2491. Clones were mapped
by Southern blotting and by comparison with the published partial
genome sequence. Those derived from the N. gonorrhoeae-minus-N. lactamica subtraction are shown on
the left (G-L libraries), and those from the N. meningitidis-minus-N. lactamica subtraction are
shown on the right (M-L libraries). Clones from the two libraries
derived from the same pathogen-specific region are marked with the same
shading. Some clones were present in multiple copies (generally
insertion sequences), and these are mapped only where they coincide
with another identified locus.
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FIG. 3.
Comparison of the positions of the pathogen-specific
clones on the chromosomes of N. gonorrhoeae FA1090 and
N. meningitidis Z2491. The relative positions follow the
lines of dislocation between the two chromosomes as previously
described (11), with certain exceptions.
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FIG. 4.
Genetic arrangement of the regions surrounding
pathogen-specific clones. Genes are shown as arrows, yellow for those
with homologies to proteins in the databases and grey for ORFs without
significant homology. Transposases are shown in red, and Correia
sequences (marked C) are shown in blue. The positions of the
subtractive clones are shown as orange bars below the bar representing
the genes. Regions previously discovered as being N. meningitidis specific are shown in green. A scale (in kilobases)
is shown above the sequences. (A) The pathogen-specific clones flank a
region of low G+C content (46%) containing several ORFs with no
homologies to previously described genes. Homologies of surrounding
ORFs, at the amino acid level, are as follows: 1, SubI, E. coli; 2a, 2b, 2c, transposase, IS1106, N. meningitidis; 3, ORF B, IS150, E. coli; 4, integrase, phage  R73; 5, transposase IS1106, N. meningitidis; 6, transposase, Synechocystis sp.
(accession no. BAA10234); 7, (3' end) HI0270, H. influenzae;
8, (5' end) GlcD, Synechocystis sp. (3' end) and GlpC,
Helicobacter pylori. (B) The pathogen-specific clones
correspond to a region of particularly low G+C content (42%),
containing ORFs with no homologies. Homologies of surrounding ORFs, at
the amino acid level, are as follows: 1, NuoF, Rickettsia
prowazekii; 2, NuoE, R. prowazekii; 3, NuoD, R. prowazekii; 4, NuoC, Rhodobacter capsulatus; 5, NuoB,
Rickettsia prowazekii; 6, NuoA, Sinorhizobium
meliloti; 7, transposase, IS1016, H. influenzae; 8, UvrD, E. coli; 9, HI1731, H. influenzae; 10, LamB homolog, H. influenzae; 11, BraB
homolog, H. influenzae; 12; MTH939, Methanobacterium
thermoautotrophicum; 13, transposase IS4351, N. meningitidis; 14, GlnE, E. coli; 15: PyrD,
Salmonella typhimurium. (C) Homologies are as follows: 1, transposase IS4351, N. meningitidis; 2, ORF 288, Coxiella burnetii; 3, ORF 1244, Sphingomonas
aromaticivorans; 4, YaeC, E. coli; 5, YaeE, E. coli; 6, ABC transporter (accession no. P30750), E. coli; 7, SLT70 transglycosylase (accession no. S56616), E. coli; 8, ribosomal protein S21, Burkholderia
pseudomallei; 9, LporfX, Legionella pneumophila; 10, RegG, N. gonorrhoeae; 11, RegF, N. gonorrhoeae;
12, CadD, Staphylococcus aureus; 13, ribosomal protein L31,
Haemophilus ducreyi; 14, putative acetyltransferase
(accession no. CAA90593), Schizosaccharomyces pombe; 15, ResA, Bacillus subtilis; 16, YbaW, E. coli; 17, VacJ, Rickettsia prowazekii; 18, YrbC, E. coli;
19, HI1085, H. influenzae; 20, HI1086, H. influenzae; 21, HI1087, H. influenzae; 22, AldA,
E. coli; 23, SsaI, Pasteurella haemolytica; 24, PabB, Helicobacter pylori; 25, OmpU, N. meningitidis; 26, HpuA, N. gonorrhoeae; 27, HpuB,
N. meningitidis; 28, GroEL, N. gonorrhoeae; 29, GroES, N. gonorrhoeae; 30, transposase, IS1016,
H. influenzae; 31, HI0736, H. influenzae; 32, LysA, Pseudomonas aeruginosa; 33, CyaY, E. coli;
34, HI1643, H. influenzae; 35, HI0931, H. influenzae; 36, YgaG, E. coli; 37, PolA, H. influenzae; 38, transposase IS1106, N. meningitidis; 39, Hap, H. influenzae; 40, ThdF,
E. coli. (D) The subtractive clones flank the previously
discovered N. meningitidis-specific region 2. Homologies are
as follows: 1, SecB, E. coli; 2, RecG H. influenzae; 3, ArgC, Synechocystis sp.; 4, CvaA,
plasmid ColV, E. coli; 5, CvaB, plasmid ColV, E. coli; 6, HI0276, H. influenzae; 7, YkvJ, Bacillus
subtilis; 8, HI1190, H. influenzae; 9, HI1189, H. influenzae; 10, YcfO, 11, HI1586, H. influenzae; 12, MucD/HtrA serine protease homolog, Pseudomonas; 13, (5' end)
HI0489, H. influenzae, (3' end) Nth endonuclease III,
H. influenzae; 14, GluP, Brucella abortus; 15, NhaC, Bacillus firmus; 16, (5' end) YbeY, E. coli, (3' end) YbeX, E. coli; 17, HemC,
Pseudomonas aeruginosa. (E) Homologies are as follows: 1, aq_1853, hypothetical protein, Aquifex aeolicus; 2, C09_orf404, Mycoplasma pneumoniae; 3, GepB,
Dichelobacter nodosus; 4, GlrA, Actinobacillus
actinomycetemcomitans; 5, RelA, Vibrio sp.; 6, putative
transposase (accession no. AAD10186), Streptococcus
pneumoniae; 7, TolC, E. coli; 8, HlyD, E. coli; 9, ORF C7, Ralstonia solanacearum/RstA1, CTX
phage, Vibrio cholerae; 10, ORF1, TspB, N. meningitidis; 11, TspB, N. meningitidis; 12, MdaB,
H. influenzae; 13, PntB, H. influenzae; 14, PntA,
E. coli.
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It is noticeable that these clones are not scattered throughout the
chromosomes of
N. gonorrhoeae and
N. meningitidis
but
are clustered only in some regions (Fig.
2). Furthermore, comparing
the maps of
N. gonorrhoeae and
N. meningitidis
(Fig.
3), it is
seen that in general the relative positions of the
clones on the
two chromosomes follow the lines linking the
previously mapped
markers (
11), whose positions have
presumably changed following
the chromosomal rearrangements which have
occurred since the divergence
of these two species. Of particular
interest is the group of clones
Em085, Cm024, and Em029, mapping near
argJ and
regF at 0.45 Mb
and separated
one from another by about 25 kb in
N. meningitidis.
In the
gonococcus, clone Em029 and
regF map around 0.45 Mb
whereas
the other clones map at about 1.65 Mb, indicating that a
large-scale
genetic rearrangement has occurred to separate these genes
in
N. gonorrhoeae or to bring them together in
N. meningitidis.
It should be pointed out that the clones cluster in the same regions of
the chromosome, whether they have been obtained by
subtracting the
N. lactamica genome from either
N. meningitidis or
N. gonorrhoeae, thus suggesting the completeness of the
bank
and validating the hypothesis that these clones designate regions
which are specific for pathogenic
neisseriae.
Functional classification of ORFs corresponding to the N. meningitidis- and N. gonorrhoeae-specific
clones.
To get some insight into the function of these regions
specific for pathogenic Neisseria species, the homologies at
the protein levels of the ORFs corresponding to the resulting
subtractive clones were noted after a BLAST search of the gene and
protein databases. The results are summarized in Table
2,
where the various homologies are divided into groups based on the
functionality of the homologous proteins.
(i) Sequences having homologies to known virulence factors.
A
few clones were located in sequences containing genes whose function
has been established as playing a role in the colonization and survival
of the port of entry, such as the IgA protease Iga (23, 29),
and the pilus-associated adhesion molecule PilC (40). The
fact that these genes are N. meningitidis and N. gonorrhoeae restricted confirms the original hypothesis that these
regions may encode virulence factors which are important in the first step of pathogenesis, i.e., the colonization of the epithelium and
survival at the port of entry. Furthermore, it suggests that the other,
as yet uninvestigated potential virulence factors (Table 2) which have
been identified on the basis of homology could be involved in common
steps of the disease.
(ii) Sequences related to DNA modifications and rearrangements,
insertion sequences, and viral recombinases.
The sequences related
to DNA modifications and rearrangements, insertion sequences, and viral
recombinases include methyltransferases DcmH and
HgiDIIM, transposases from IS1106 of
N. meningitidis, IS18 of
Acinetobacter, and IS150-like of N. gonorrhoeae, Synechocystis, and Aeromonas
salmonicida, the Correia sequences from N. meningitidis and N. gonorrhoeae, and proteins from phages Cf1c of
Xanthomonas campestris and CTX of Vibrio
cholerae. Hence a relatively large number of sequences identified
were related to DNA modifications, insertion sequences or transposons,
and phages. In the absence of further evidence, they may be taken to be
clonal in their distribution between the species, reflecting the closer
relationship between the gonococcus and the meningococcus rather than
genetic differences maintained by natural selection.
(iii) Sequences with homologies to proteins involved in metabolic
pathways or transporters.
The fact that metabolic genes may be
specific for pathogenic Neisseria species could be related
to the specific environment they both have to encounter. The outer
membrane porin PorA (5) belongs to this category. PorA is
found only in N. meningitidis, and the gene was initially
thought to be N. meningitidis specific; however, in N. gonorrhoeae the porA gene is not expressed, being a
pseudogene (15).
(iv) Sequences with weak homologies and homologies to
hypothetical proteins typically derived from genome-sequencing
projects.
The significance of sequences with weak homologies and
homologies to hypothetical proteins remains to be investigated.
Genetic arrangement of the pathogen-specific regions.
The
origin of the pathogenic Neisseria sequences is another
important question. In several bacterial species, which contain more or
less virulent variants (for example, E. coli,
Helicobacter pylori, Salmonella typhimurium, and
Yersinia enterocolitica), genes specifying the attributes of
increased pathogenic potential are clustered in so-called pathogenicity
islands (PAIs) (22). PAIs are usually large (50 to 200 kb),
often having a G+C content different from that of the host chromosome.
None of the regions had the characteristics typical of PAIs, of
bacteriophages, or of compound transposons, structures which are
associated with the introduction into bacterial chromosomes of foreign
DNA coding for virulence factors. Several of the regions were, however,
of particularly low G+C content (Fig. 4) and were associated with transposase and integrase genes, suggesting that at some time in the
genetic history of the species, the regions were the result of
recombinational events with DNA from other species. For example, the
region containing Cm016, Em024, and Cg004 at 1.17 Mb (Fig. 4A) contains
a region with a particularly low G+C content (46%, compared with 52%
for the chromosome in general) with no homologies to genes in the
databases, surrounded by ORFs with homologies to sequences encoding
transposases and a phage integrase, and may well represent DNA, as yet
unknown, acquired from another organism. A similar situation is seen
with the region corresponding to clones Cm020 and Eg024 (Fig. 4B).
The region between the clones Em085 and Cm024 and the
regF
gene is the site of one of the large chromosomal translocations
discovered by Dempsey et al. (
11). The surrounding region
(Fig.
4C) contains several copies of the Correia sequence, singly or
in
pairs, and these sequences are likely to be important in
intrachromosomal
rearrangements, as has been suggested previously
(
28).
Another striking feature of these regions is the association of many of
the clones with the previously described
N. meningitidis-specific
regions (
44). This suggests
that previously discovered
N. meningitidis-specific
islands,
at least in regions 2 and 7 (Fig.
4D and E), have inserted
into
preexisting pathogen-specific sequences. Together, these
data suggest
that these
N. meningitidis and
N. gonorrhoeae
regions
correspond to islands of pathogen-specific DNA, as was seen to
be the case in the
N. meningitidis-
N.
gonorrhoeae subtraction.
Conclusion.
Our data demonstrate that even though N. meningitidis and N. gonorrhoeae display very different
pathogeneses, they have regions of their chromosomes in common which
are not found in the nonpathogenic N. lactamica and which
are probably involved in common aspects of their life cycle, i.e.,
colonization and survival at the port of entry. The subtractive
technique has enabled us to identify novel candidate genes and regions
involved in these common steps. A further understanding of these steps
will require systematic mutagenesis of the genes located in these
regions. The postgenomic era has begun for many bacterial pathogens;
our data have confirmed that the technique of genomic subtraction has
the potential to pinpoint regions of chromosome that are most likely to
be involved in the differential virulence of bacterial pathogens. This
technique has therefore the potential to identify from among the
thousands of ORFs brought to light by genome sequencing a number of
potential targets for new therapies and vaccine production.
| |
ACKNOWLEDGMENTS |
This work was supported by the INSERM, the Université Paris
V René Descartes and the Fondation pour la Recherche
Médicale.
Thanks are due to N. meningitidis and N. gonorrhoeae sequencing teams at the Sanger Centre and the
University of Oklahoma, who made their sequences publicly available
throughout the progress of the genome projects.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: INSERM U411, 156 Rue de Vaugirard, 75015 Paris, France. Phone: 33 140615678. Fax: 33 140615592. E-mail: nassif{at}necker.fr.
Editor:
E. I. Tuomanen
| |
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Abstract
Introduction
