Previous Article | Next Article 
Infection and Immunity, January 1999, p. 187-192, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Fimbria-Mediated Enhanced Attachment of Nontypeable
Haemophilus influenzae to Respiratory Syncytial
Virus-Infected Respiratory Epithelial Cells
Zili
Jiang,1
Nobuo
Nagata,1,
Edgar
Molina,1,
Lauren O.
Bakaletz,2
Hal
Hawkins,3 and
Janak A.
Patel1,*
Departments of
Pediatrics1 and
Pathology,3 University of Texas Medical
Branch, Galveston, Texas, and
Otological Research Laboratories,
Department of Otolaryngology, Ohio State University, Columbus,
Ohio2
Received 19 August 1998/Returned for modification 17 September
1998/Accepted 8 October 1998
 |
ABSTRACT |
Respiratory syncytial virus (RSV) infection is known to predispose
children to otitis media and sinusitis due to bacteria such as
nontypeable Haemophilus influenzae (NTHI). In this study, we investigated the role of NTHI surface outer membrane protein P5-homologous fimbriae (P5-fimbriae) in attachment to RSV-exposed A549
epithelial cells. Analysis by fluorescence flow cytometry showed that a
live P5-fimbriated NTHI strain (NTHIF+) attached to a
higher proportion of RSV-exposed A549 cells than to control cells
(mean, 68% for RSV versus 29% for control; P = 0.008), while attachment of the P5-fimbriae-deficient isogenic mutant strain (NTHIF
) was significantly lower than in
control cells and rose only slightly following RSV exposure (mean, 17%
for RSV versus 10% for control, P = 0.229).
Attachment of NTHIF+ did not correlate with the amount of
RSV antigen expressed by A549 cells. Furthermore,
paraformaldehyde-fixed NTHIF+ also demonstrated an enhanced
binding to RSV-exposed cells. Observations by transmission electronic
microscopy showed that the mean number of bacteria attached per 100 RSV-exposed A549 cells was higher for NTHIF+ than
NTHIF (99 versus 18; P < 0.001). No
intracellular bacteria were identified. UV-irradiated conditioned
supernatants collected from RSV-infected A549 cultures (UV-cRSV) also
enhanced the attachment of NTHIF+ to A549, suggesting the
presence of a preformed soluble mediator(s) in UV-cRSV that enhances
the expression of receptors for P5-fimbriae on A549 cells. In summary,
RSV infection significantly enhances NTHI attachment to respiratory
epithelial cells. P5-fimbria is the critical appendage of NTHI that
participates in this attachment. In clinical settings, blocking of the
P5-fimbria-mediated attachment of NTHIF+ by passive or
active immunity may reduce the morbidity due to NTHI during RSV infection.
| |
INTRODUCTION |
Nontypeable Haemophilus
influenzae (NTHI) is a gram-negative, unencapsulated bacterium
that frequently colonizes the nasopharyngeal mucosa of healthy children
(14, 17). NTHI also accounts for a large proportion of
bacterial conjunctivitis, otitis media, sinusitis, bronchitis, and
pneumonia (22). Among the respiratory viruses, respiratory
syncytial virus (RSV) is the most common virus associated with the
development of otitis media due to NTHI in children (5, 6, 15,
29). Similar associations have been described for sinusitis and
pneumonia (18, 19, 32). Furthermore, we have previously
demonstrated in experimental animals that RSV infection enhances the
nasopharyngeal colonization with NTHI (23).
Recent studies show that a surface fimbria structure of NTHI, known as
outer membrane protein P5-homologous fimbria (P5-fimbria), is involved
in the attachment to mucus and respiratory epithelial cells (2, 3,
21). The P5-fimbriae are approximately 2.4 nm in diameter, are
long and flexible in appearance, and mediate attachment and not
hemagglutination (1, 2). Immune response against the
P5-fimbriae has been shown to reduce nasopharyngeal colonization as
well as occurrence of otitis media in experimental animals (3,
30). However, the role of P5-fimbriae in attachment of NTHI to
virus-infected airway epithelium has not been studied.
In the present study, we hypothesized that RSV infection enhances the
attachment of NTHI to respiratory epithelial cells and that the
enhancement is primarily mediated by the bacterial surface P5-fimbriae.
This hypothesis was evaluated in vitro in A549 cells (type II-like
alveolar epithelial cell carcinoma), as they have been used for studies
of RSV infection (24) as well as bacterial attachment
(13).
| |
MATERIALS AND METHODS |
Cell cultures.
A549 cells were obtained from the American
Type Culture Collection (Rockville, Md.) and grown in minimal essential
medium (MEM) containing 8% fetal bovine serum (Sigma, St. Louis, Mo.) and 2 mM L-glutamine. Maintenance MEM during RSV incubation
contained 1% fetal calf serum (FCS). The media did not contain any
antibiotics in order to allow study of live bacteria interactions.
RSV preparations.
The human Long strain of RSV (A2) was
grown in HEp-2 cells (human laryngeal epithelial carcinoma; American
Type Culture Collection) and purified by polyethylene glycol
precipitation followed by centrifugation on a 35 to 60% discontinuous
sucrose gradient (21). The titer of the purified RSV (pRSV)
pool was 109.25 PFU/ml. Monolayers of A549 cells grown in
24-well tissue culture plates were incubated with pRSV at a
multiplicity of infection (MOI) of 1 in MEM supplemented with 1% FCS
and then incubated at 37°C in 5% CO2. When the cells
exhibited extensive cytopathic effect at 48 h, the supernatant was
aspirated and centrifuged at 5,000 × g to remove large
cellular debris. This preparation is referred to as the conditioned RSV
(cRSV) pool. UV-inactivated conditioned supernatants from pRSV-infected
A549 cells (UV-cRSV) were prepared as previously described
(30). Control supernatants were prepared similarly without
RSV infection.
NTHI preparations.
P5-fimbriated NTHI strain 1128 (NTHIF+) was isolated from a child with otitis media and
was used to construct a nonfimbriated mutant (NTHIF) at
the Otological Research Laboratories, The Ohio State University, Columbus (1, 30). NTHI were grown on chocolate agar plates at 37°C in 5% CO2 overnight. Two to three colonies were
mixed in Haemophilus test medium (Becton Dickinson
Microbiology Systems, Cockeysville, Md.) at 37°C overnight. The next
day, 1-ml aliquots of bacteria suspensions were transferred to fresh
Haemophilus test medium and recultured under the same
conditions for about 4 h (mid-log phase). Bacteria were washed
with phosphate-buffered saline (PBS) and concentrated to approximately
2 × 109 CFU/ml as estimated by optical density and
confirmed by quantitative plate colony counts. For some experiments,
bacteria were also fixed by being incubated with 1% paraformaldehyde
for 15 min and washed three times with PBS. The nonviability of the
paraformaldehyde-fixed NTHI was confirmed by plate culture.
NTHI attachment assay by culture.
A549 cells were grown as
confluent monolayers in 24-well tissue culture plates. The cells were
then inoculated with pRSV (MOI = 1) and incubated for 24 h at
37°C in 5% CO2. The monolayers were then washed twice,
and 1 ml of Hanks' balanced salt solution (HBSS; Sigma) containing
NTHI was inoculated into each well (A549:NTHI = 1:0.5). After
incubation for 3 h at 37°C, the monolayers were washed gently
three times with prewarmed HBSS. A549 cells were then detached with
trypsin-EDTA (Sigma), washed twice with HBSS, and plated on chocolate
agar for quantitative colony counts.
NTHI attachment assay by flow cytometry.
Equivalent numbers
of NTHIF+ and NTHIF, as determined by optical
density and confirmed by quantitative plate colony count, were used to
compare bacterial attachment by flow cytometry. NTHI suspensions were
labeled with PKH-26, a red fluorescent dye (General Linker kit; Sigma).
In brief, approximately 2 × 109 CFU of NTHI was
suspended in 1 ml of diluent and mixed with 1 ml of PKH-26 linker
solution. The mixture was incubated at 25°C for 5 min. Equal volumes
of FCS were then added to stop the labeling reaction. Bacteria were
washed three times with PBS. Viability of the labeled bacteria was
tested by quantitative cultures. Preliminary studies showed that the
levels of PKH-26 labeling were comparable for NTHIF+ and
NTHIF.
Confluent monolayers of A549 cells were inoculated with pRSV or control
medium and incubated for 24 h at 37°C in 5% CO2.
The monolayers were then detached by incubation with 0.1 M EDTA (pH 7.5) for 10 min and washed twice with PBS. For some experiments, 5 × 105 cells were labeled with fluorescein
isothiocyanate-conjugated anti-RSV polyclonal antibody (Chemicon,
Temecula, Calif.) at 37°C in 5% CO2 for 1 h and
then washed three times with PBS. PKH-26-labeled bacteria were mixed
with RSV-exposed or control A549 cell suspensions at various ratios and
incubated at 37°C on an orbital shaker for 30 min. The cells were
washed three times by differential centrifugation to remove unattached
bacteria and then fixed in 1% paraformaldehyde prior to analysis by
two-color fluorescence flow cytometry (FACScan analyzer; Becton
Dickinson, San Jose, Calif.).
NTHI attachment assay by transmission electron microscopy.
A549 monolayers grown in 24-well tissue culture plates were inoculated
with pRSV (MOI = 1) or control medium for 24 h. NTHI suspensions were then added to the wells (A549:NTHI = 1:100) and incubated for 1 h. Unattached NTHI was removed by washing three times with prewarmed HBSS. The monolayers were then fixed with 2.5%
cacodylate-buffered glutaraldehyde and postfixed in 1% osmium tetroxide. The cells were detached by gentle scraping with a needle, dehydrated, and embedded by standard techniques. The cells were visualized under a transmission electron microscope by an examiner who
was unaware of the sample treatments. The sections were surveyed by
using a square grid, counting only those without holes or obstacles (en
face cuts excluded). Counts progressed back and forth across a section
until at least 100 cells had been counted. Only the cells with complete
nuclei were counted. Mitotic and degenerative cells were excluded. The
number of attached bacteria were then counted and were categorized as
tightly or loosely attached to the cell body or tightly attached to microvilli.
Statistics.
Nonparametric data were evaluated by
Mann-Whitney rank sum test, while parametric data were evaluated by
Student's t test. Analysis of proportions was evaluated by
z test. P values of <0.05 were considered
statistically significant.
| |
RESULTS |
Analysis of NTHI attachment by culture.
Initial NTHI
attachment studies were conducted by quantitative culture of bacteria
that remained attached to washed monolayers of A549 cells. The number
of NTHIF+ attached to RSV-exposed A549 cells was
significantly higher than for control A549 cells (Fig.
1). The culture technique, however, could
not exclude the possibility that the bacteria were loosely attached to
the cells merely by virtue of gravity or were attached to the inner
plastic surface of the well.
View larger version (11K):
[in this window]
[in a new window]
|
FIG. 1.
Effect of pRSV exposure (MOI = 1, 24 h) or
control medium on binding of NTHIF+ to A549 cell monolayers
(A549:NTHI = 1:0.5), as determined by the culture method. The
values are means of experiments performed in triplicate. Here and in
Fig. 3, 4, 7, and 8, error bars indicate standard deviations.
|
|
Role of NTHI P5-fimbriae in attachment.
We further evaluated
the role of P5-fimbriae in attachment by the fluorescence flow
cytometry method (Fig. 2). This method overcame some of the problems of the culture method since the NTHI-A549
coincubation was performed under rotational motion followed by vigorous
washing. The results showed that NTHIF+ attached to a
larger proportion of pRSV-exposed than control A549 cells (mean, 68%
for RSV versus 29% for control; P = 0.008) while the
attachment of NTHIF was only slightly higher for
pRSV-exposed cells than for control cells (mean, 17% for RSV versus
10% for control; P = 0.229) (Fig. 3). The relative density of NTHI attached
to both control and pRSV-exposed A549 cells, as determined by mean
fluorescence intensity, was also significantly higher for
NTHIF+ than NTHIF for both control and
RSV-exposed A549 cells (Fig. 4). Initial live RSV infection was required as exposure of A549 cells to
UV-irradiated pRSV did not result in any enhancement of NTHI attachment
(data not shown).
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 2.
Histographic representation of number of A549 cells
(y axis) expressing red fluorescence of PKH-26-labeled NTHI
(x axis), as determined by flow cytometry (A549:NTHI = 1:100). (A) Background fluorescence of pRSV-exposed (MOI = 1, 24 h) or control A549 cells; (B and C) fluorescence of
NTHIF+ and NTHIF attached to control or
pRSV-exposed A549 cells.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 3.
Percentage of pRSV-exposed (MOI = 1, 24 h) or
control A549 cells demonstrating attached NTHIF+ or
NTHIF (A549:NTHI = 1:100), as determined by flow
cytometry. The values are means of six experiments.
|
|
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 4.
Mean fluorescence intensity of PKH-26-labeled
NTHIF+ or NTHIF to 5 × 105
pRSV-exposed (MOI = 1, 24 h) or control A549 cells
(A549:NTHI = 1:100), as determined by flow cytometry. The values
are means of six experiments.
|
|
As with live NTHI
F+, paraformaldehyde-prefixed
NTHI
F+ attachment to pRSV-exposed A549 cells was higher
than that to control cells
(Fig.
5). The
slightly higher attachment for live NTHI than prefixed
NTHI was
directly related to the ongoing bacterial replication
of live NTHI
during the assay (data not shown). These results
suggested that
activation or modification of P5-fimbriae proteins
during contact with
A549 cells is not required for enhanced attachment.
View larger version (41K):
[in this window]
[in a new window]
|
FIG. 5.
Comparison of attachment of live or prefixed (1%
paraformaldehyde) NTHIF+ to pRSV-exposed (MOI = 1, 24 h) or control A549 cells (A549:NTHI = 1:100), as
determined by flow cytometry.
|
|
The attachment of NTHI was further evaluated by transmission electron
microscopy. The results showed that only a few NTHI
F+ and
NTHI
F were seen attached to the cell membrane of control
cells (Table
1). Following pRSV exposure,
however, >5-fold more NTHI
F+ than NTHI
F
attached to the cells, as evaluated by all parameters
tight or
loose
attachment to the cell body, and tight attachment to microvilli
(99 NTHI
F+ versus 18 NTHI
F per 100 A549 cells
[Table
1]). Nonetheless, the attachment of
NTHI
F was
slightly but significantly higher for RSV-exposed than control
cells
(3.5 NTHI
F+ versus 18 NTHI
F per 100 A549
cells), suggesting that adhesins other than P5-fimbriae
may also play a
role, albeit a very small one, in attachment.
A549 cells exhibited
electron-dense areas underlying attached
NTHI and microvilli
projections attaching to NTHI (Fig.
6).
No
intracellular NTHI was seen, suggesting that the enhanced
association
between NTHI and RSV-exposed A549 cells was purely at the
surface
membrane level.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Quantitative analysis of NTHI attachment to pRSV-exposed
or control A549 cells by transmission electron microscopy
|
|
View larger version (106K):
[in this window]
[in a new window]
|
FIG. 6.
Transmission electron micrographs of A549 cells
incubated with NTHIF+ (A549:NTHI = 1:100). (A) Control
cells (magnification, ×14,300), which do not show any attached NTHI.
(B) pRSV-exposed (MOI = 1, 24 h) cells (magnification,
×10,725); the arrow points to a microvillus projection adhering to
NTHI with an the electron-dense area underlying the attached NTHI.
|
|
Overall, the results of flow cytometry and electron microscopy studies
suggested that the P5-fimbriae are critical for NTHI
attachment to
control A549 cells. The increase in attachment to
pRSV-exposed cells is
also dependent on the P5-fimbriae.
Relationship between NTHI attachment and RSV antigen
expression.
The relationship between NTHI attachment and cellular
RSV antigen expression was analyzed to determine whether the bacterial attachment was directly influenced by viral replication within the
cell. At 24 and 48 h of pRSV exposure, studies by two-color flow
cytometry showed that while NTHIF+ attachment to A549 cells
increased over time, there was no statistically significant difference
in attachment to RSV antigen-positive or -negative cells (Fig.
7). Furthermore, studies by transmission electron microscopy revealed very few RSV particles in pRSV-exposed cells, with no clear association between the presence of
cell-associated virus and NTHI attachment.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 7.
Comparison of attachment of NTHIF+
(A549:NTHI = 1:100) to RSV antigen-positive ( )
or -negative ( ) A549 cells exposed to pRSV (MOI = 1, 24 h), as determined by two color flow cytometry. The values
are means of three experiments.
|
|
Role of soluble mediators in NTHI attachment.
As there was no
relationship between RSV antigen expression and NTHI attachment, we
next postulated that soluble mediators released by RSV-infected cells
could induce, in an autocrine or paracrine manner, the synthesis or
activation of NTHI-binding surface receptors on RSV-uninfected A549
cells. Conditioned supernatants from pRSV-exposed A549 cultures were
rendered noninfectious by UV irradiation and subsequently incubated
with fresh A549 cells (diluted 1:2 with fresh culture medium, 1 ml/well). After 24 h, NTHI attachment to suspended A549 cells was
analyzed by flow cytometry. The results showed that the effect of
UV-cRSV exposure on attachment of NTHIF+ and
NTHIF was comparable with that of live pRSV exposure
(Fig. 8).
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 8.
Percentages of pRSV-exposed (MOI = 1, 24 h)
and UV-cRSV-exposed (1:2 dilution, 24 h) A549 cells demonstrating
attached NTHIF+ or NTHIF (A549:NTHI = 1:100), as determined by flow cytometry. The values are means of four
experiments.
|
|
| |
DISCUSSION |
NTHI commonly exists in an asymptomatic nasopharyngeal
colonization state. Respiratory viral infection is the commonest factor associated with the spread of NTHI contiguously in the respiratory tract to produce disease. We have previously shown that RSV infection enhances NTHI nasopharyngeal colonization in experimental animals (23). Also, Raza et al. have shown that binding of H. influenzae type b and Neisseria meningitidis to
epithelial cells is enhanced by RSV infection in HEp-2 cells
(26). Several adhesins of NTHI have been described, but
their significance in the virus-enhanced disease state has not been
studied. In this respect, we report here that the specific NTHI
interaction with RSV-exposed respiratory epithelial cell in vitro
involves enhanced attachment of NTHI that is primarily mediated by its
P5-fimbriae.
In the present study, NTHIF bound less avidly to normal
as well as RSV-exposed A549 cells, suggesting that for the two NTHI strains used in the experiments, other adhesins of NTHI played a minor
role in attachment. The role of P5-fimbriae is further strengthened by
the previous observations that 100% of clinical strains are fimbriated
(2). Furthermore, in a chinchilla model of otitis media, the
P5-fimbriae have been shown to be important for adhesion and a proven
virulence factor (2, 3). The role of other NTHI adhesins in
clinical settings appears to be more variable. Approximately 32% of
strains express hemagglutinating pili, and nonpiliated strains adhere
just as well as piliated strains (13). Approximately 20 to
30% of NTHI isolates lack the HMW1/HMW2 family of adhesins
(32). Another adhesin, Hia, is universally absent from NTHI
that expresses HMW1 or HMW2 (32). Nonetheless, the roles of
these adhesins, singly or in combination, in virus-enhanced attachment
of NTHI need to be explored. Furthermore, our observations need to be
confirmed in vitro, using respiratory epithelial cells of diverse
origin, as well as in vivo.
We determined by transmission electron microscopy that the NTHI
interaction with epithelial cells was similar to that described by
Holmes and Bakaletz (16). A549 cells exhibited
electron-dense areas underlying the attached NTHI and microvilli
projections attaching to NTHI. The present study also showed that NTHI
was not internalized by A549 cells. In contrast, Gilsdorf et al.
(13) showed that H. influenzae type b is
internalized by A549 cells. However, only 4 to 5% of NTHI was
internalized, as determined by the gentamicin survivability test, and
no electron microscopic observations were made. Studies by St. Geme and
Falkow (31) demonstrated intracellular NTHI in Chang (human
conjunctival) epithelial cells by electron microscopy. Nonetheless, our
studies suggest that in A549 cells, the intracellular invasion is
probably an infrequent event and that it is not enhanced by RSV
infection. In vivo though, it is possible that NTHI exists in an
intracellular quiescent stage in respiratory tissues such as the
adenoids (10).
Since paraformaldehyde-fixed bacteria also adhered in enhanced numbers
to RSV-exposed cells, our results suggest that de novo bacterial
protein synthesis was not required for the P5-fimbriae to interact with
the cells. The modest increase in adherence of live compared with fixed
bacteria seen in our study, and possibly those seen in the studies of
St. Geme and Falkow (31), could be due to the ongoing
replication of live bacteria during the bacterium-epithelial cell
coincubation stage. On the other hand, initial live RSV infection of
epithelial cells was required to enhance the adhesiveness of the cells,
since incubation with UV-irradiated purified RSV did not result in
enhancement of NTHI attachment. The effect of live RSV infection was
probably mediated by a soluble mediator(s) produced by RSV-infected
cells. The identity of the bacterial attachment-enhancing mediator(s)
has not been determined, but potential candidates include RSV-induced
soluble cytokines by epithelial cells, such as interleukin-1
,
interleukin-1
, and tumor necrosis factor alpha (23).
These cytokines are known to activate several nuclear transcription
factors that regulate the synthesis of a variety of proteins.
Elahmer et al. have shown that RSV up-regulates the attachment of
N. meningitidis to HEp-2 cells via increased cellular
expression of CD14 (8). The nature of the NTHI
P5-fimbriae-binding receptor(s) expressed on the epithelial cells
exposed to RSV has not been studied. Previous studies have shown that
NTHI binds to sialylated oligosaccharides of normal epithelial cells.
NTHI also binds to oligosaccharide-containing mucin secreted by
epithelial cells (7, 9, 20). Specifically, Fakih et al. have
shown that NTHI fimbriae bind to sialylated gangliosides on HEp-2
epithelial cells (9). Whether RSV infection enhances the
P5-fimbria-mediated attachment of NTHI by increasing the expression of
gangliosides on epithelial cells needs further investigation. The
enhancement of attachment of bacteria by a variety of viruses may
be also explained by the fact that many bacterial species use
oligosaccharide receptors on epithelial cells and in mucin
(25).
In summary, the present study shows that RSV enhances attachment of
NTHI to epithelial cells which is largely mediated by NTHI
P5-fimbria-binding receptors. In clinical settings, blocking of
P5-fimbria-mediated attachment of NTHIF+ by passive or
active immunity may reduce the disease incidence and severity during
RSV infection.
| |
ACKNOWLEDGMENTS |
This work was supported by NIDCD grant DC-02129.
We thank Sandra Fuentes for secretarial assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Pediatric Infectious Diseases, Children's Hospital at the University
of Texas Medical Branch, 301 University Blvd., Galveston, TX
77555-0371. Phone: (409) 772-2798. Fax: (409) 747-1753. E-mail:
janak.patel{at}utmb.edu.
Present address: Tomakomai City General Hospital, Tomakomai, Japan.
Present address: Gateway Community Health Center, Laredo, Tex.
Editor:
P. E. Orndorff
| |
REFERENCES |
| 1.
|
Bakaletz, L. O.,
M. A. Ahmed,
I. J. Forney,
P. F. Kolatukudy, and D. J. Lim.
1992.
Cloning and sequence analysis of a pilin-like gene from an otitis media isolate of nontypable Haemophilus influenzae (strain # 1128).
J. Infect. Dis.
165:S201-S203.
|
| 2.
|
Bakaletz, L. O.,
B. M. Tallan,
T. Hoepf,
T. F. DeMaria,
H. G. Birck, and D. J. Lim.
1988.
Frequency of fimbriation of nontypeable Haemophilus influenzae and its ability to adhere to chinchilla and human respiratory epithelium.
Infect. Immun.
56:331-335[Abstract/Free Full Text].
|
| 3.
|
Bakaletz, L. O.,
B. M. Tallan,
W. J. Andrzejewski,
H. G. DeMaria, and D. J. Lim.
1989.
Immunological responsiveness of chinchillas to outer membrane and isolated fimbrial proteins on nontypeable Haemophilus influenzae.
Infect. Immun.
57:3226-3229[Abstract/Free Full Text].
|
| 4.
|
Brinton, C. C., Jr.,
M. J. Carter,
D. B. Derber,
S. Kar,
J. A. Kramarik, and S. W. Wood.
1989.
Design and development of pilus vaccines for Haemophilus influenzae diseases.
Pediatr. Infect. Dis. J.
8:S54-S61[Medline].
|
| 5.
|
Chonmaitree, A., and T. Heikkinen.
1997.
Role of viruses in middle-ear disease.
Ann. N.Y. Acad. Sci.
830:143-157[Medline].
|
| 6.
|
Chonmaitree, T.,
M. J. Owen,
J. A. Patel,
D. Hedgpeth,
D. Horlick, and V. M. Howie.
1992.
Effect of viral respiratory tract infection on outcome of acute otitis media.
J. Pediatr.
120:856-862[Medline].
|
| 7.
|
Davies, J.,
I. Carlstedt,
A. Nilsson,
A. Hakansson,
H. Sabharwal,
L. V. Alphen,
M. V. Ham, and C. Svanborg.
1995.
Binding of Haemophilus influenzae to purified mucins from the human respiratory tract.
Infect. Immun.
63:2485-2492[Abstract].
|
| 8.
|
Elahmer, O. R.,
N. W. Raza,
M. M. Ogilvie,
C. C. Blackwell,
D. M. Weir, and R. A. Elton.
1996.
The effect of respiratory syncytial virus infection on expression of cell surface antigens associated with binding of potentially pathogenic bacteria, p. 169-177.
In
I. Kahane, and I. Ofek (ed.), Toward anti-adhesion therapy for microbial diseases. Plenum Press, New York, N.Y.
|
| 9.
|
Fakih, M. G.,
T. F. Murphy,
M. A. Pattoli, and C. S. Berenson.
1997.
Specific binding of Haemophilus influenzae to minor gangliosides of human respiratory epithelial cells.
Infect. Immun.
65:1695-1700[Abstract].
|
| 10.
|
Farley, M. M.,
D. S. Stephens,
S. L. Kaplan, and E. O. Mason, Jr.
1990.
Pilus- and non-pilus-mediated interactions of Haemophilus influenzae type b with human erythrocytes and human nasopharyngeal mucosa.
J. Infect. Dis.
161:274-280[Medline].
|
| 11.
|
Ghafoor, A.,
N. K. Nomanl,
Z. Ishaq,
Z. Z. Sohail,
F. Anwar,
M. I. Burney,
W. Qureshi, and S. A. Ahmad.
1990.
Diagnoses of acute lower respiratory tract infections in children in Rawalpindi and Islamabad, Pakistan.
Rev. Infect. Dis.
12:S907-S914.
|
| 12.
|
Gilsdorf, J. R.,
K. W. McCrea, and C. F. Marrs.
1997.
Role of pili in Haemophilus influenzae adherence and colonization.
Infect. Immun.
65:2997-3002[Medline].
|
| 13.
|
Gilsdorf, J. R.,
M. Tucci, and C. F. Marrs.
1996.
Role of pili in Haemophilus influenzae adherence to, and internalization by, respiratory cells.
Pediatr. Res.
39:343-348[Medline].
|
| 14.
|
Harabuchi, Y.,
H. Faden,
N. Yamanaka,
L. Duffy,
J. Wolf,
D. Krystofik, and Tonawanda/Williamsville Pediatrics.
1994.
Nasopharyngeal colonozation with nontypeable Haemophilus influenzae and recurrent otitis media.
J. Infect. Dis.
170:862-866[Medline].
|
| 15.
|
Hietella, J.,
M. Uhari,
H. Tuokko, and M. Leionen.
1989.
Mixed bacterial and viral infections are common in children.
Pediatr. Infect. Dis. J.
8:683-686[Medline].
|
| 16.
|
Holmes, K. A., and L. O. Bakaletz.
1997.
Adherence of nontypable Haemophilus influenzae promotes reorganization of the actin cytoskeleton in human or chinchilla epithelial cells in vitro.
Microb. Pathog.
23:157-166[Medline].
|
| 17.
|
Ingvarsson, L.,
K. Lundgren, and J. Ursing.
1982.
The bacterial flora in nasopharynx in healthy children.
Acta Otolaryngol.
386:S94-S96.
|
| 18.
|
John, T. J.,
T. Cherian,
M. C. Steinhoff,
E. A. F. Simoes, and M. John.
1991.
Etiology of acute respiratory infections in children in tropical southern India.
Rev. Infect. Dis.
13:S463-S469.
|
| 19.
|
Korppi, M.,
M. Leinonen,
M. Koskela,
H. Makela, and K. Launiala.
1989.
Bacterial coinfection in children hospitalized with respiratory syncytial virus infections.
Pediatr. Infect. Dis. J.
8:687-692[Medline].
|
| 20.
|
Marieke Van Ham, S.,
L. Alphen,
F. R. Mool, and J. P. M. Putten.
1995.
Contribution of the major and minor subunits to fimbria-mediated adherence of Haemophilus influenzae to human epithelial cells and erythrocytes.
Infect. Immun.
63:4883-4889[Abstract].
|
| 21.
|
Miyamoto, N., and L. O. Bakaletz.
1996.
Selective attachment of non-typeable Haemophilus influenzae (NTHi) to mucus or epithelial cells in the chinchilla eustachian tube and middle ear.
Microb. Pathog.
21:343-356[Medline].
|
| 22.
|
Murphy, T. F., and M. A. Apicella.
1987.
Nontypable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection.
Rev. Infect. Dis.
9:1-15[Medline].
|
| 23.
|
Patel, J. A.,
H. Faden,
S. Sharma, and P. L. Ogra.
1992.
Effect of respiratory syncytial virus on attachment, colonization, and immunity of nontypable Haemophilus influenzae: implication in otitis media.
Int. J. Pediatr. Otorhinolaryngol.
23:15-23[Medline].
|
| 24.
|
Patel, J. A.,
M. Kunimoto,
R. Garofalo,
T. C. Sim,
O. Ruuskanen,
T. Chonmaitree,
P. L. Ogra, and F. Schmalstieg.
1995.
IL-1 mediates enhanced expression of ICAM-1 in pulmonary epithelial cells infected with respiratory syncytial virus.
Am. J. Respir. Cell Mol. Biol.
13:602-609[Abstract].
|
| 25.
|
Plotkowski, M. C.,
O. Bajolet-Laudinat, and E. Puchelle.
1993.
Cellular and molecular mechanisms of bacterial adhesion to respiratory mucosa.
Eur. Respir. J.
6:903-916[Abstract].
|
| 26.
|
Raza, M. W.,
M. M. Ogilvie,
C. C. Blackwell,
J. Stewart,
R. A. Elton, and D. M. Weir.
1993.
Effect of respiratory syncytial virus infection on binding of Neisseria meningitidis and Haemophilus influenzae type b to a human epithelial cell line (Hep-2).
Epidemiol. Infect.
110:339-347[Medline].
|
| 27.
|
Read, R. C.,
R. Wilson,
A. Rutman,
V. Lund,
H. C. Todd,
A. R. Brain,
P. K. Jeffery, and P. J. Cole.
1991.
Interaction of nontypable Haemophilus influenzae with human respiratory mucosa in vitro.
J. Infect. Dis.
163:549-558[Medline].
|
| 28.
|
Reddy, M. S.,
J. M. Bernstein,
T. F. Murphy, and H. S. Faden.
1996.
Binding between outer membrane proteins of nontypeable Haemophilus influenzae and human nasopharyneal mucin.
Infect. Immun.
64:1477-1479[Abstract].
|
| 29.
|
Ruuskanen, O., and T. Heikkinen.
1994.
Viral-bacterial interaction in acute otitis media.
Pediatr. Infect. Dis. J.
13:1047-1049[Medline].
|
| 30.
|
Sirakova, T.,
P. E. Kolattukudy,
D. Murwin,
J. Billy,
E. Leake,
D. Lim,
T. DeMaria, and L. Bakaletz.
1994.
Role of fimbriae expressed by nontypeable Haemophilus influenzae in pathogenesis of and protection against otitis media and relatedness of the fimbrin subunit to outer membrane protein A.
Infect. Immun.
62:2002-2020[Abstract/Free Full Text].
|
| 31.
|
St. Geme, J. W., III, and S. Falkow.
1990.
Haemophilus influenzae adheres to and enters cultured human epithelial cells.
Infect. Immun.
58:4036-4044[Abstract/Free Full Text].
|
| 32.
|
St. Geme, J. W., III,
V. V. Kumar,
D. Cutter, and S. J. Barenkamp.
1998.
Prevalence and distribution of the hmw and hia genes and the HMW and Hia adhesins among genetically diverse strains of nontypeable Haemophilus influenzae.
Infect. Immun.
66:364-368[Abstract/Free Full Text].
|
| 33.
|
Ueba, O.
1978.
Respiratory syncytial virus concentration and purification of the infectious virus.
Acta Med. Okayama
32:265-272.
|
Infection and Immunity, January 1999, p. 187-192, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Kisiela, D. I., Czuprynski, C. J.
(2009). Identification of Mannheimia haemolytica Adhesins Involved in Binding to Bovine Bronchial Epithelial Cells. Infect. Immun.
77: 446-455
[Abstract]
[Full Text]
-
Novotny, L. A., Partida-Sanchez, S., Munson, R. S. Jr., Bakaletz, L. O.
(2008). Differential Uptake and Processing of a Haemophilus influenzae P5-Derived Immunogen by Chinchilla Dendritic Cells. Infect. Immun.
76: 967-977
[Abstract]
[Full Text]
-
Bookwalter, J. E., Jurcisek, J. A., Gray-Owen, S. D., Fernandez, S., McGillivary, G., Bakaletz, L. O.
(2008). A Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Homologue Plays a Pivotal Role in Nontypeable Haemophilus influenzae Colonization of the Chinchilla Nasopharynx via the Outer Membrane Protein P5-Homologous Adhesin. Infect. Immun.
76: 48-55
[Abstract]
[Full Text]
-
Avadhanula, V., Rodriguez, C. A., DeVincenzo, J. P., Wang, Y., Webby, R. J., Ulett, G. C., Adderson, E. E.
(2006). Respiratory Viruses Augment the Adhesion of Bacterial Pathogens to Respiratory Epithelium in a Viral Species- and Cell Type-Dependent Manner. J. Virol.
80: 1629-1636
[Abstract]
[Full Text]
-
Harrison, A., Dyer, D. W., Gillaspy, A., Ray, W. C., Mungur, R., Carson, M. B., Zhong, H., Gipson, J., Gipson, M., Johnson, L. S., Lewis, L., Bakaletz, L. O., Munson, R. S. Jr.
(2005). Genomic Sequence of an Otitis Media Isolate of Nontypeable Haemophilus influenzae: Comparative Study with H. influenzae Serotype d, Strain KW20. J. Bacteriol.
187: 4627-4636
[Abstract]
[Full Text]
-
Swords, W. E., Moore, M. L., Godzicki, L., Bukofzer, G., Mitten, M. J., VonCannon, J.
(2004). Sialylation of Lipooligosaccharides Promotes Biofilm Formation by Nontypeable Haemophilus influenzae. Infect. Immun.
72: 106-113
[Abstract]
[Full Text]
-
Coleman, H. N., Daines, D. A., Jarisch, J., Smith, A. L.
(2003). Chemically Defined Media for Growth of Haemophilus influenzae Strains. J. Clin. Microbiol.
41: 4408-4410
[Abstract]
[Full Text]
-
Novotny, L. A., Bakaletz, L. O.
(2003). The Fourth Surface-Exposed Region of the Outer Membrane Protein P5-Homologous Adhesin of Nontypable Haemophilus influenzae Is an Immunodominant But Nonprotective Decoying Epitope. J. Immunol.
171: 1978-1983
[Abstract]
[Full Text]
-
Heikkinen, T., Chonmaitree, T.
(2003). Importance of Respiratory Viruses in Acute Otitis Media. Clin. Microbiol. Rev.
16: 230-241
[Abstract]
[Full Text]
-
Look, D. C., Walter, M. J., Williamson, M. R., Pang, L., You, Y., Sreshta, J. N., Johnson, J. E., Zander, D. S., Brody, S. L.
(2001). Effects of Paramyxoviral Infection on Airway Epithelial Cell Foxj1 Expression, Ciliogenesis, and Mucociliary Function. Am. J. Pathol.
159: 2055-2069
[Abstract]
[Full Text]
-
Novotny, L. A., Jurcisek, J. A., Pichichero, M. E., Bakaletz, L. O.
(2000). Epitope Mapping of the Outer Membrane Protein P5-Homologous Fimbrin Adhesin of Nontypeable Haemophilus influenzae. Infect. Immun.
68: 2119-2128
[Abstract]
[Full Text]
-
Bakaletz, L. O., Kennedy, B.-J., Novotny, L. A., Duquesne, G., Cohen, J., Lobet, Y.
(1999). Protection against Development of Otitis Media Induced by Nontypeable Haemophilus influenzae by Both Active and Passive Immunization in a Chinchilla Model of Virus-Bacterium Superinfection. Infect. Immun.
67: 2746-2762
[Abstract]
[Full Text]
Top
Abstract
Introduction
