Received 26 May 2000/Returned for modification 6 October
2000/Accepted 6 November 2000
We developed and evaluated a PCR-based-restriction
endonuclease analysis method to detect and analyze the tonB
gene of Haemophilus influenzae and Haemophilus
parainfluenzae from pediatric patients undergoing tonsillectomy
and adenoidectomy. Multiple sites from the same patient, including the
surface of adenoids and tonsils, as well as the core of tonsils,
were cultured on chocolate agar and identified using standard
procedures and the API NH Kit. A total of 55 H. influenzae isolates were recovered from different sites of 20 patients, and 32 H. parainfluenzae isolates were
recovered from various sites of 12 patients. DNA was extracted
from American Type Culture Collection strains and test
isolates by the PureGene kit. Two primers, G1 (21-mer)
and G2 (23-mer), were designed by us to amplify by
PCR the tonB gene that consists of an 813-bp fragment. A
nested PCR using primers T1 (23-mer) and T2 (24-mer) that flank an
internal sequence to the gene of the order of 257 bp and restriction
endonuclease digestion using XhoI and BglII were done to detect whether heterogeneity within the gene exists between the two species. Reverse transcription-PCR (RT-PCR) was finally
done to detect transcription of the gene in both
species. Our data have shown that the tonB gene was
detected in both species. It is known to encode a virulent
protein, TonB, in H. influenzae; however,
demonstration of its presence in H. parainfluenzae is novel. Nested-PCR and restriction endonuclease analysis have shown that
the tonB gene is apparently structurally the same
in both species, with possible differences that may exist in certain
H. parainfluenzae isolates. RT-PCR done on selected
numbers of H. influenzae and H. parainfluenzae have shown that the tonB gene was
transcribed in both species. This shows that the TonB protein, if
expressed, may play a different role in the virulence in H. parainfluenzae since it is not needed for heme or heme complexes uptake as with H. influenzae.
 |
INTRODUCTION |
Chronic tonsillitis represents
persistent inflammatory changes despite antibiotic therapy. Sore
throat and cervical lymphadenopathy may persist, and peritonsillar
abscesses, although rare, can complicate acute or recurrent
tonsillitis, thus increasing the morbidity of these patients and the
need for surgical management. Even though the symptoms are the same,
tonsillitis may be caused by either viruses or bacteria. Generally,
younger preschool children tend to have viral tonsillitis, caused
mainly by adenoviruses, influenza viruses, parainfluenza viruses,
enteroviruses, and herpes simplex viruses. Other children and adults
acquire bacterial infections. The most common bacterial agents are
beta-hemolytic group A streptococcus (20% of the cases),
Streptococcus pyogenes, and Haemophilus spp. (6).
In a preliminary pilot study conducted by us on the determination of
the most prevalent etiology of tonsillitis in a group of Lebanese
patients presenting to the otolaryngology and Head and Neck Surgery
department of the American University of Beirut Medical Center
(AUBMC) over a period of 1 year, Haemophilus
influenzae (40%) and Haemophilus parainfluenzae
(24%) were found to be the most prevalent etiologies. Association of
these two species was clinically established with the illness, since
they were isolated from culture plates either singly or along with
normal flora such as alpha-hemolytic Streptococcus or
Neisseria species.
H. influenzae and H. parainfluenzae are
facultatively anaerobic gram-negative coccobacilli (7).
H. influenzae is a human-specific pathogen that
must colonize the human mucosal surface to avoid extinction. Although
it is in a commensal relationship with its host, it is also found
in upper and lower respiratory infections in adults and
children, in whom H. influenzae remains a leading cause
of disease (11, 12, 13). H. parainfluenzae, on the other hand, is not human specific, and it
is better known as a commensal bacterium that is part of the normal
flora (2, 7). Both organisms have several virulence
factors, but an unconventional factor that is recently drawing the
attention of researchers is the TonB protein, an energy-transducing
transmembrane protein responsible for the transport of different
essential metabolites into various bacteria. TonB has proven to be a
potent virulence factor in H. influenzae
(5). It is responsible for the active intake into the
periplasm of iron-bound transferrin (4), heme, heme:hemopexin (1), heme-albumin, hemoglobin,
hemoglobin:naptoglobin (8), after each of these chemicals
binds to its proper outer cell surface receptor.
Unlike H. influenzae, H. parainfluenzae's requirement for porphyrin is not essential due
to the ability of the latter to synthesize it from delta amino
levulinic acid (ALA) (7). In addition, the fact that
H. parainfluenzae is unable to bind or acquire iron from transferrin or heme from hemoglobin-haptoglobin or heme-hemopexin contributes to its avirulence and commensalism (3, 9, 10, 14,
15). However, since H. parainfluenzae was found
to be the etiology of tonsillitis in our patients and since the TonB protein is considered a potent virulence factor in H. influenzae through the active uptake of iron, heme, and other
essential metabolites (1, 4, 5, 8), we sought to detect
and carry out a comparative analysis of the tonB gene
encoding the TonB protein in both H. influenzae and
H. parainfluenzae species by PCR amplification and
endonuclease restriction analysis. In addition, we sought to check by
reverse transcription-PCR (RT-PCR) the transcription of mRNA as a
preliminary indication for expression of the protein. To do this, we
designed primers that flank a 813-bp fragment which constitutes the
whole tonB gene, and we developed and evaluated a PCR
restriction endonuclease-RT-PCR-based method for the detection, analysis, and transcription of the gene in H. influenzae and H. parainfluenzae.
| |
MATERIALS AND METHODS |
Source and identification of the isolates.
Isolates were
collected over a period of 1 year from six different tonsillar sites of
32 patients suffering from tonsillitis and undergoing
adenotonsillectomy at the AUBMC. Specimens were taken aseptically from
the surface and core of the adenoids and similarly from the right and
left tonsils. The specimens were cultured on blood and on chocolate
agar plates and incubated overnight at 37°C. Chocolate agar plates
were incubated in the presence of 5% CO2. Isolates
recovered were identified by using standard procedures and the API NH
kit. A variety of bacteria were identified (unpublished data),
including 55 H. influenzae and 32 H. parainfluenzae isolates that were used in this study.
DNA extraction, PCR, and Nested PCR.
Total DNA was extracted
from Haemophilus isolates and American Type Culture
Collection (ATCC) strains (H. influenzae ATCC 49427 and
H. parainfluenzae ATCC 9796 strains) using the
Pure-Gene Kit (Gentra Systems, Inc.). PCR was done on all DNA extracts
to amplify the whole 813-bp gene using the G1
(5'-ATTATGCAAACAAAACGTTCG-3') and G2
(5'-GAAGAGTAAAACTAATTGCACAC-3') (Amersham Pharmacia Biotech, Freiburg, Germany) primers designed by us using the GenBank database. Nested PCR was done using T1 (5'-GCAAGCACAACAAGTGCAGCTAA-3')
and T2 (5'-GCCGCCTTATCTAAACTTTCATCG-3') on 813-bp
amplicons to amplify a 257-bp amplicon.
PCR amplifications were carried out in 100-µl reaction mixtures
consisting of 10 µl of DNA (2 ng/µl) and 90 µl of the
amplification mix, which contained the following components: 20 pmol
each of the G1 and G2 primers (for the amplification of the 813-bp
amplicon) or the T1 and T2 primers (for the nested PCR that amplifies a 250-bp amplicon), 0.5 mM MgCl2, 200 µm concentrations of
each deoxynucleoside triphosphate, 10 µl of PCR buffer (Amersham
Pharmacia Biotech), and 2.5 U of Taq DNA polymerase
(Amersham Pharmacia Biotech). PCR amplification was performed in a
minicycler (M.J. Research, Watertown, Mass.) for 34 cycles. Each cycle
consisted of 1 min at 95°C for denaturation, 1 min at 55°C for
annealing, and 1 min at 72°C for extension. A final extension for 10 min at 72°C was also done. Amplicons were detected by electrophoresis on a 1% agarose (Sigma, St. Louis, Mo.) gel in 1× Tris-borate-EDTA buffer at 100 V for 1 h. Gels were stained with ethidium bromide (1 mg/ml), observed under UV light, and photographed with type 667 Polaroid film. PCR controls included DNA from ATCC strains and a
reagent blank.
Restriction endonuclease analysis.
For digestion of the gene
(813-bp amplicon), 10 µl of PCR-amplified DNA was restricted with 10 U of the restriction endonucleases XhoI and BglII
in a total volume of 15 µl, according to the manufacturer's specification (Amersham Pharmacia Biotech). The restricted fragments were separated by electrophoresis on agarose gels (2.5% Nusieve agarose, 3:1; FMC Bioproducts, Rockland, Maine) at 60 V for 2 h.
Gels were stained with ethidium bromide, and fragments were visualized
under UV light and photographed as described above.
RT-PCR.
RT-PCR was done on RNA from two H. influenzae and six H. parainfluenzae isolates of
different patients to demonstrate possible transcription of the
tonB gene detected in H. parainfluenzae in comparison to H. influenzae. To that purpose, RNA was
extracted using the RNeasy Mini Kit (Qiagen) according to
manufacturer's specifications. cDNA strand was synthesized from RNA
using the Ready-To-Go Kit (Amersham Pharmacia Biotech) according to the manufacturer's specification. RT-PCR was done on cDNA-generated strand
using the G1 and G2 primers and the PCR conditions described above.
Control tubes of extracted RNA were subjected to PCR to rule out the
presence of DNA in the starting RNA samples. Amplicons were visualized
on an ethidium bromide-stained gel and photographed.
| |
RESULTS |
Our data show that all 55 H. influenzae and 32 H. parainfluenzae isolates, in addition to both
H. influenzae and H. parainfluenzae ATCC strains, amplified the 813-bp sequence (Fig.
1A). Nested PCR amplified the 257-bp
amplicon (Fig. 2) in all isolates with the exception of
four H. parainfluenzae isolates, indicating that these
have nucleotide sequence differences where the primers bind. Restriction analysis of the tonB gene with XhoI
showed a similar DNA pattern between ATCC strains and the tested
isolates (Fig. 3). The original 813-bp
sequence from all isolates of both species was digested by
XhoI into 200- and 600-bp fragments. However, the
BglII restriction enzyme cut 28 of 55 H. influenzae and 15 of 32 H. parainfluenzae
amplicons into two fragments (ca. 388 and 425 bp), and these are
similar to the expected values derived from the DNA sequence. A
total of 27 of 55 H. influenzae and 17 of 32 H. parainfluenzae strains, as well as both ATCC
strains, were not digested by this enzyme. The six-base recognition
sequence of XhoI cuts nucleotides of the tonB
gene corresponding to the 130(K), 131(D), and 132(L) amino acids of the
TonB protein, while the BglII recognition sequence cuts
nucleotides of the gene corresponding to the 190(T), 191(R), and 192(A)
amino acids of the protein (5). Based on digestion with
the two enzymes, two composite patterns, I and II, were generated
(Table 1). RT-PCR has shown that the tonB gene in selected H. influenzae and
H. parainfluenzae isolates was transcribed (Fig. 1B).
View larger version (78K):
[in this window]
[in a new window]
|
FIG. 1.
(A) PCR 813-bp amplicons of a representative sample of
H. influenzae, and H. parainfluenzae
test isolates. Lane 1, 100-bp ladder; lane 2, negative control; lanes 3 and 4, H. influenzae test isolates; lanes 5 to 10, H. parainfluenzae test isolates. (B) The corresponding
RT-PCR amplicons for the PCR amplicons in panel A. Lane 1, 100-bp
ladder; lane 2, positive control; lanes 3 and 4, H. influenzae test isolates; lanes 5 to 10, H. parainfluenzae test isolates.
|
|
View larger version (83K):
[in this window]
[in a new window]
|
FIG. 2.
Representative nested-PCR amplicons (257 bp) of the
total tonB gene from H. influenzae and
H. parainfluenzae ATCC strains and test isolates. Lane
1, 50-bp ladder; lane 2, negative control; lane 3, H. influenzae ATCC; lane 4, H. influenzae test
isolate; lane 5, H. parainfluenzae ATCC strain; lane 6, H. parainfluenzae test isolate.
|
|
View larger version (44K):
[in this window]
[in a new window]
|
FIG. 3.
Representative uncut and digested PCR amplicons of the
tonB gene from H. influenzae and
H. parainfluenzae ATCC strains and test isolates. Lane
1, 100-bp ladder; lane 2, negative control; lanes 3 to 8, H. influenzae amplicons; lane 3, ATCC strain uncut amplicon; lane 4, ATCC strain digested amplicon with XhoI; lane 5, ATCC strain
digested amplicon with BglII; lane 6, test isolate uncut
amplicon; lane 7, test isolate digested amplicon with XhoI;
lane 8, test isolate digested amplicon with BglII; lanes 9 to 14, H. parainfluenzae amplicons; lane 9, ATCC strain
uncut amplicon; lane 10, ATCC strain digested with XhoI;
lane 11, ATCC strain digested with BglII; lane 12, test
isolate uncut amplicon; lane 13, test isolate digested with
XhoI; lane 14, test isolate digested with
BglII.
|
|
| |
DISCUSSION |
Generated data has shown that the tonB gene is present
in all tested H. influenzae isolates, as well as in all
tested H. parainfluenzae isolates, a fact not reported
earlier for H. parainfluenzae. This observation
indicates that H. parainfluenzae has the potential to
exhibit virulence similar to that seen in H. influenzae. Nested-PCR analyses have shown minor heterogeneity at
the primer annealing site of four H. parainfluenzae
isolates, a fact that hindered the amplification of the internal
sequence of the tonB gene by nested PCR of these four
isolates. All other isolates of H. influenzae and
H. parainfluenzae amplified the internal sequence of
the gene by nested PCR.
Restriction endonuclease analysis, on the other hand, has shown two
composite patterns, I and II, using XhoI and
BglII in both H. influenzae and
H. parainfluenzae (Table 1), showing that the gene is
structurally the same in both species with minor nucleotide sequence
variations also observed in both species. This nucleotide variation is
reflected at the level of amino acids 190(T), 191(R), and 192(A)
downstream of the TonB protein (4, 5). This observed alteration does not seem to affect either survival or the function of
both species. The 130(K), 131(D), and 132(L) amino acids of the TonB
protein, on the other hand, are well conserved in both H. influenzae and H. parainfluenzae (4,
5), reflecting the functional importance of this conserved
region. Based on this molecular analysis, the tonB gene
seems to have a common nucleotide sequence in both species, with minor
variations. Being structurally the same, the tonB gene in
H. parainfluenzae may encode a protein that could have
the same functionality as that of H. influenzae; however, since iron is apparently not essential for the survival of
H. parainfluenzae, it is believed that it may play an
important role in enhancing H. parainfluenzae's
virulence if the species has an iron acquisition mechanism
(10). For that reason the role of the tonB gene
and TonB protein in H. parainfluenzae may be to allow
either the acquisition of iron bound to certain carriers not yet tested
or the acquisition of other essential metabolites. Both cases reflect
the virulence potential of the tonB gene.
The tonB gene is shown to be transcribed into mRNA in both
H. parainfluenzae and H. influenzae.
This indicates that expression of the gene is initiated in both
species. Since the tonB gene is transcribed in H. parainfluenzae and since the species is reported not to acquire
iron-bound transferrin nor heme:hemopexin or hemoglobin:haptoglobin (15), however, it may be assumed that the TonB protein, if
translated, may very well be involved in the acquisition of other
essential metabolites, as is the case in other bacteria
(5). It may constitute a virulence factor since its gene
is carried by the species genome and is detected in patients with
tonsillitis. On the other hand, it may be also assumed that the
translation of the TonB protein is blocked and thus the protein is not
expressed. Detection of the protein in H. parainfluenzae would clarify its role as a virulence factor. In
addition, generation of a tonB mutant strain of
H. parainfluenzae would enhance our understanding of
its role in the pathogenesis of the organism.
| 1.
|
Cope, L. D.,
R. Yogev,
U. Muller-Eberhard, and E. Hansen.
1995.
A gene cluster involved in the utilisation of both free heme and heme:hemopexin by Haemophilus influenzae type b.
J. Bacteriol.
177:2644-2653[Abstract/Free Full Text].
|
| 2.
|
Forsgren, J.,
A. Samuelson,
A. Ahlin,
J. Joasson,
B. R. Dagoo, and A. Lindberg.
1994.
Haemophilus influenzae resides and multiplies intracellularly in human adenoid tissue as demonstrated by in situ hybridization and bacterial viability essay.
Infect. Immun.
62:673-679[Abstract/Free Full Text].
|
| 3.
|
Hanson, D. J.,
S. E. Pelzel,
J. Latimer,
U. Muller-Eberhard, and E. J. Hansen.
1992.
Identification of a genetic locus of Haemophilus influenzae type b necessary for the binding and utilization of heme bound to human hemoexin.
Proc. Natl. Acad. Sci. USA
89:1973-1977[Abstract/Free Full Text].
|
| 4.
|
Jarosik, G. P.,
I. Maciver, and E. Hansen.
1995.
Utilization of transferin-bound iron by Haemophilus influenzae requires an intact tonB gene.
Infect. Immun.
63:710-713[Abstract].
|
| 5.
|
Jarosik, G. P.,
J. D. Sanders,
L. D. Cope,
U. Muller-Eberhard, and E. J. Hansen.
1994.
A functional tonB gene is required for both utilization of heme and virulence.
Infect. Immun.
62:2470-2477[Abstract/Free Full Text].
|
| 6.
|
Johnson, J. T., and V. L. Yu.
1997.
Infectious diseases and antimicrobial therapy of the ears, nose and throat.
The W. B. Saunders Company, Philadelphia, Pa.
|
| 7.
|
Lennette, E. H., et al.
1985.
Manual of Clinical Microbiology, 4th ed.
American Society for Microbiology, Washington, D.C.
|
| 8.
|
Maciver, I.,
J. L. Latimer,
H. H. Liem,
U. Muller-Eberhard,
Z. Hrkal, and E. J. Hansen.
1996.
Identification of an outer membrane protein involved in the utilization of hemoglobin-haptoglobin complexes by nontypeable Haemophilus influenzae.
Infect. Immun.
64:3703-3712[Abstract].
|
| 9.
|
Morton, D. J., and P. Williams.
1989.
Utilization of transferrin-bound iron by Haemophilus species of human and porcine origin.
FEMS Microbiol. Lett.
65:123-128[CrossRef].
|
| 10.
|
Morton, D. J., and P. Williams.
1990.
Siderophore-independent acquisition of transferrin-bound iron by Haemophilus influenzae type b.
Mol. Microbiol.
3:1979-1803.
|
| 11.
|
Moxon, E. R.
1990.
Haemophilus influenzae, p. 1722-1729.
In
G. L. Mandell, R. G. Douglas, Jr., and J. E. Bennett (ed.), Principles and practice of infectious diseases, 3rd ed. Churchill Livingstone, Inc., New York, N.Y.
|
| 12.
|
Turk, D. C.
1982.
Clinical importance of Haemophilus influenzae, p. 3-9.
In
S. H. Sell, and P. F. Wright (ed.), Haemophilus influenzae: epidemiology, immunology and prevention of disease. Elsevier/North-Holland Publishing Co., New York, N.Y.
|
| 13.
|
Turk, D. C.
1984.
The pathogenicity of Haemophilus influenzae.
J. Med. Microbiol.
18:1-16[Medline].
|
| 14.
|
Williams, P.,
D. J. Morton,
K. J. Towner,
P. Stevenson, and E. Griffiths.
1990.
Utilisation of enterobactin and other exogenous iron source by Haemophilus influenzae, Haemophilus parainfluenzae, and Haemophilus paraphrphilus.
J. Gen. Microbiol.
136:2343-2350[Medline].
|
| 15.
|
Wong, C. Y. J.,
R. Patel,
D. Kendall,
P. W. Whitey,
A. Smith,
J. Holland, and P. Williams.
1995.
Affinity, conservation, and surface exposure of hemopexin-binding proteins in Haemophilus influenzae.
Infect. Immun.
63:2327-2333[Abstract].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
