Previous Article | Next Article 
Infection and Immunity, February 2000, p. 906-911, Vol. 68, No. 2
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Effects of the Immunoglobulin A1 Protease on Neisseria
gonorrhoeae Trafficking across Polarized T84 Epithelial
Monolayers
Sylvia
Hopper,*
Brandi
Vasquez,
Alex
Merz,
Susan
Clary,
J. Scott
Wilbur, and
Magdalene
So
Department of Molecular Microbiology and
Immunology, Oregon Health Sciences University, Portland, Oregon
97201-3098
Received 13 September 1999/Returned for modification 23 September
1999/Accepted 29 October 1999
 |
ABSTRACT |
We previously demonstrated that the Neisseria IgA1
protease cleaves LAMP1 (lysosome-associated membrane protein 1), a
major integral membrane glycoprotein of lysosomes, thereby accelerating its degradation rate in infected A431 human epidermoid carcinoma cells
and resulting in the alteration of lysosomes in these cells. In this
study, we determined whether the IgA1 protease also affects the
trafficking of Neisseria gonorrhoeae across polarized T84 epithelial monolayers. We report that N. gonorrhoeae
infection of T84 monolayers, grown on a solid substrate or polarized on semiporous membranes, also results in IgA1 protease-mediated reduction of LAMP1. We demonstrate that iga mutants in two genetic
backgrounds exited polarized T84 monolayers in fewer numbers than the
corresponding wild-type strains. Finally, we present evidence that
these mutants have a statistically significant and reproducible defect
in their ability to traverse T84 monolayers. These results add to
our previous data by showing that the IgA1 protease alters lysosomal
content in polarized as well as unpolarized cells and
by demonstrating a role for the protease in the traversal of epithelial
barriers by N. gonorrhoeae.
| |
TEXT |
Neisseria gonorrhoeae
(i.e., the gonococcus [GC]) causes gonorrhea in humans, its only
host, and gains entry into the body via the mucosal surfaces. There is
no animal model for GC disease. Studies on the molecular requirements
of GC interactions with the epithelium rely on human fallopian tube
organ cultures (hFTOC), immortalized human epithelial cells grown on
solid substrates, and the human challenge model of urethral infection.
Such studies reveal that GC attach to and invade nonciliated cells of
the mucosal epithelium through the coupling of several bacterial
adhesins (type IV pili, PilC, and certain Opa variants) with their
cognate host cell receptors (CD46, CD66, and heparan sulfate
proteoglycans), which are present in a large number of human tissues
(3, 4, 7, 8, 11-13, 17, 18, 21-24, 27, 30, 35, 40-44,
47; J. G. Cannon, D. Johannsen, J. D. Hobbs, N. Hoffman, J. A. F. Dempsey, D. Johnston, H. Koyman, and
M. S. Cohen, presented at the Eleventh Int. Pathog.
Neisseria Conf., 1998).
The trafficking of GC within the epithelial cell was initially examined
using hFTOC (20, 29). However, the nature of the system
limits the type and scale of trafficking studies that can be performed.
Experimentation on Neisseria (GC and Neisseria
meningitidis [MC]) transepithelial trafficking has more recently
relied on polarized T84 human colorectal epithelial cell monolayers
(32, 33, 36, 37, 46). Similar to cells in native epithelia, T84 cells have the capacity to polarize and form impermeable barriers with high electrical resistance when grown on semiporous membranes (10, 26). This latter attribute permits the detection of
small changes in barrier integrity. T84 cells are derived from human colorectal epithelia and thus are a model system for a site that is
infected by GC in vivo. CD66 receptors have been demonstrated on the
apical membranes of polarized T84 monolayers, and Opa-mediated binding
of these receptors allowed rapid traversal of nonpiliated GC across the
barrier (46).
Neisserial infections of polarized T84 monolayers share key features
with infections of human organ cultures. GC invade, traverse, and exit
hFTOC and T84 monolayers in identical time courses (28, 32).
Very similar results were reported in studies comparing MC transcytosis
in T84 and infected human nasopharyngeal organ cultures (32, 36,
39). Furthermore, the process of transepithelial trafficking does
not disrupt monolayer barrier functions (32, 36, 46). The
trafficking of GC across polarized T84 monolayers is influenced by a
number of factors. Piliation modulates the speed of transepithelial
trafficking in a manner that is independent of its role in attachment
(32). Certain Opa variants also influence transcytosis.
Nonpiliated (P
) strains traverse T84 monolayers very
quickly, provided they express Opa variants that bind CD66
(46).
The pathogenic neisseriae, like a number of other mucosal pathogens,
secrete immunoglobulin A1 (IgA1) protease, an enzyme that cleaves the
hinge of human IgA1 (hIgA1) (34, 38). Numerous functions
have been ascribed to the IgA1 protease, but its role in pathogenesis
remains enigmatic. The protease has been proposed to promote bacterial
colonization through cleavage of hIgA1 on the mucosal surface. IgA1
protease activity, hIgA1 cleavage fragments (2), and
anti-IgA1 protease antibodies (16) have been found in the
cervical mucus of infected women. A recent human challenge study showed
that an iga (IgA1 protease gene) mutant was not impaired in
its ability to initiate an infection in the human male urethra (19).
The IgA1 protease also cleaves LAMP1 (15, 25), a major
integral membrane glycoprotein of lysosomes with an hIgA1-like hinge in
its luminal domain (5). Proteolysis accelerates the LAMP1
degradation rate (25) and results in multiple
alterations in the lysosomes of infected cells (1). An
iga mutant is defective in intracellular growth, compared to
the wild-type (WT) parent strain (25), and this phenotype is
likely to be due to the inability of the mutant to cleave LAMP1 and
alter lysosomes.
Studies on the effects of the IgA1 protease on host cell lysosomes were
performed using the A431 human epidermoid carcinoma cell line
grown on a solid support and the GC clinical isolate GCM740 and its
isogenic iga derivative, GCM740
4 (38).
To further our understanding of the virulence function(s) of the IgA1
protease, we sought to determine whether the protease affects GC
interactions with T84 monolayers, including transepithelial
trafficking. We report that infection of T84 cells, either grown on a
solid substrate or polarized on semiporous membranes, by GC strain MS11
variant A (MS11A) also resulted in reduced steady-state levels of
LAMP1. In contrast, T84 cells infected with MS11A500, an isogenic
iga mutant of MS11A, had near-normal LAMP1 levels. We show
that this iga mutant exited polarized T84 monolayers in
significantly fewer numbers than the WT strain. Finally, we demonstrate
that two iga mutants have a reproducible and statistically
significant defect in their traversal across polarized T84 monolayers.
Construction and characterization of an iga
mutant in strain MS11A.
In previous studies, we used GC
strain GCM7404, a P+ Opa clinical
isolate with a well-defined mutation in iga, the type
2 IgA1 protease gene (38). The iga mutant and
GCM740, its WT parent, have identical outer membrane protein
profiles and adhered to and invaded cells equally well (25).
We wished to perform further studies on IgA1 protease interactions with
T84 cells using an iga mutant in the MS11A background
(14), as this laboratory strain has been used extensively
for genetic studies and studies on cell adhesion, invasion, and
transcytosis. Both strains are type 2 IgA1 protease producers
(25).
To construct an iga null mutant in MS11A, the 4.2-kb
HindIII fragment within the iga gene (codon
140 to beyond the stop codon) was deleted and the 1.6-kb
HindIII fragment containing the kanamycin and bleomycin
resistance cassette from pKIXX (Pharmacia) was inserted at this site.
This construct, pKaniga2 (pBR322 background), contains ~2.7 kb of 5' iga sequence, the first 139 codons, and
~2.5 kb of 3' downstream sequence. The WT iga gene in
strain MS11A was replaced with the mutated iga gene in
pKaniga2 by transformation and allelic exchange. In the
resulting mutant, MS11A500, >95% of the iga coding
sequence has been exchanged for the kanamycin and bleomycin resistance
cassette, while the 5' upstream and 3' downstream flanking sequences
are intact (data not shown).
MS11A500 was tested for IgA1 protease activity. hIgA1 (4 µg;
Calbiochem) was incubated with the supernatant (5.5 µl) from
a 7.5-h
culture of MS11A or MS11A500 grown in supplemented GCB
broth as
described previously (
45). As a control, hIgA1 was
incubated
with purified recombinant
Neisseria IgA1 protease (0.1
µg;
Boehringer) or sterile medium. The proteins were separated
by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
stained
with Coomassie blue. Results show that hIgA1 was cleaved
by the MS11A
supernatant and by purified IgA1 protease, but not
by GCB medium or by
the MS11A500 supernatant (Fig.
1).
MS11A500
therefore has no detectable IgA1 protease activity.
View larger version (82K):
[in this window]
[in a new window]
|
FIG. 1.
IgA1 protease activities of WT MS11A and its isogenic
iga mutant MS11A500. Human IgA1 was incubated for 4 h
at 37°C in growth medium (lane 1) or with purified recombinant
Neisseria type 2 IgA1 protease (lane 2), the supernatant
from a broth culture of MS11A (lane 3), or the supernatant from a broth
culture of MS11A500 (lane 4), as described in the text. The reactions
were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and stained with Coomassie blue. The arrow indicates
the position of the IgA1 cleavage products of the hIgA1 heavy chain.
The arrowhead indicates the position of the full-length hIgA1 heavy
chain.
|
|
Next, MS11A500 was assessed for its ability to adhere to cells.
T84 cells from the American Type Culture Collection were
propagated
as described previously (
10) and infected with
MS11A500 or the
WT parent MS11A strain at a multiplicity of infection
(MOI) of
10. At 2 and 4 h postinfection, the adhesion index of the
infecting
strains was determined as described before (
45).
WT MS11A adhered
to T84 (Table
1) and
A431 (
32) cells comparably. MS11A500 adhered
slightly better
to T84 cells than MS11A (Table
1), although the
difference is
statistically significant only for the 2-h time
point (
P < 0.05, two-tailed
t test for unpaired samples). MS11A500
also adhered slightly better to A431 human epithelial cells early
after
infection (data not shown).
Attempts to compare the invasion indices of MS11A and MS11A500 using
T84 cells were unsuccessful and yielded variable data.
In these
experiments, a significant number of extracellular bacteria
survived
gentamicin treatment, even at an antibiotic concentration
of 400 µg/ml. The inability of gentamicin to kill all extracellular
bacteria
in T84 cultures may be due to the fact that T84 cells,
like many
epithelial cells (
48), secrete large amounts of heavily
glycosylated mucins to the luminal (apical) surface. This mucous
layer
is likely to reduce access of gentamicin to adherent bacteria.
The
invasiveness of MS11A500 was therefore assessed using the
A431 human
epithelial cell line as described previously (
25).
In these
experiments, MS11A500 and MS11A invaded A431 cells equally
well (Table
1), indicating that the null mutation in the MS11A
iga gene
does not affect the invasiveness of the
strain.
LAMP1 levels in infected T84 cells.
Previous studies from our
laboratory indicated that Neisseria infection of A431 cells
resulted in reduced steady-state levels of LAMP1 (25). To
determine whether T84 cells respond similarly to GC infection, LAMP1
levels were quantitated in GC-infected T84 cultures as described for
A431 cells (25). T84 monolayers were infected with MS11A for
8 h at an MOI of 50, and total cell proteins from these cultures
were immunoblotted with monoclonal antibodies against LAMP1 (H4A3) and
-tubulin (E7) (Developmental Studies Hybridoma Bank, University of
Iowa [6]). The blots were developed using anti-mouse
alkaline phosphatase antibodies (Boehringer) by a colorimetric reaction
(Boehringer), and the signals from the blots were scanned and
quantitated using the NIH Image version 1.61 program. -Tubulin
levels served as our internal control (1, 25). Results
indicate that LAMP1 levels in cultures infected with WT MS11A were
noticeably reduced compared to those in uninfected cells (Fig.
2A, compare lanes 1 and 2 with lanes 5 and 6). Normalization of LAMP1 signals to their respective internal
-tubulin signals revealed a 71% decrease in LAMP1 signals in
MS11A-infected cultures. LAMP1 levels were higher in cultures infected
with lower MOIs (S. Hopper, P. Ayala, and M. So, unpublished data), and
the 71% reduction in steady-state LAMP1 levels calculated in this
study is very similar to the value reported for A431 cultures infected
with WT Neisseria at lower MOIs for longer periods of time
(1, 25). Thus, the degree of LAMP1 reduction is dependent in
part on the exposure of infected cells to the IgA1 protease.
View larger version (49K):
[in this window]
[in a new window]
|
FIG. 2.
LAMP1 levels in infected T84 cultures grown on a solid
substrate (A) or polarized on semipermeable membranes (B). (A) Lanes 1 and 2, uninfected cultures; lanes 3 and 4, cultures infected with
iga MS11A500; lanes 5 and 6, cultures infected with WT
MS11A. (B) Lanes 1 and 2, uninfected monolayers; lanes 3 and 4, monolayers infected with WT MS11A.
|
|
To determine whether LAMP1 reduction is due to the IgA1 protease,
MS11A500 was used to infect T84 cells under the same conditions
as
described above, and LAMP1 levels in these cultures were quantitated
by
immunoblotting. As shown in Fig.
2A, LAMP1 levels in
MS11A500-infected
cultures (lanes 3 and 4) were nearly identical to
those in uninfected
cultures (lanes 1 and 2). This is consistent with
previous observations
that A431 cells infected with the
iga
mutant GCM740
4 have nearly
normal LAMP1 levels
(
25).
LAMP1 levels were next quantitated in polarized T84 monolayers. T84
cultures were maintained and polarized as described previously
(
32) and infected with MS11A for 17 h at an MOI of 10. Total
proteins from infected and uninfected monolayers were quantitated
by immunoblotting as described before (
25). LAMP1 levels in
infected polarized T84 monolayers were also reduced (Fig.
2B,
compare
lanes 1 and 2 with lanes 3 and 4). In this representative
example,
there was a 61% decrease in
LAMP1.
Double immunofluorescence confocal microscopy of infected polarized T84
monolayers supported these immunoblot results. Polarized
T84 monolayers
were infected with MS11A at an MOI of 1 and processed
for microscopy
after 12 h as described before (Fig.
3) (
31,
33). The monolayer was
infected at a low MOI to allow comparison
of LAMP1 levels in adjacent
infected and uninfected cells. At
this time, P
+
Opa
MS11A organisms were mostly observed in the apical
region of
the cell. The panels in Fig.
3 represent successive
~1-µm-thick
optical cross sections of one such monolayer, beginning
at the
apical plasma membrane (A) and ending near the basal membrane
(I). These micrographs illustrate that cells infected with bacteria
(red) have few LAMP1 signals (green) throughout their entire length
and
demonstrate the extent of LAMP1 reduction in individual infected
T84
cells. Taken together, these results demonstrate that GC infection
of
T84 cells, grown on solid supports and polarized on microporous
membranes, also results in reduced LAMP1 levels and that this
reduction
is due to the bacterial IgA1 protease.
View larger version (95K):
[in this window]
[in a new window]
|
FIG. 3.
Double immunofluorescence laser scanning confocal
microscopy of GC-infected T84 monolayers. Polarized T84 monolayers were
infected with MS11A and fixed and stained for bacteria (red) and LAMP1
(green) using polyclonal anti-total GC protein antibody 8547 (33) and monoclonal anti-LAMP1 antibody H4A3 and secondary
BODIPY anti-mouse and Texas red anti-rabbit antibodies (Molecular
Probes). Each panel represents an ~1-µm-thick optical cross section
of an infected monolayer, starting at the apical membrane (A) and
ending near the basal membrane (I). The arrows mark an infected region
of the monolayer. Images were acquired with a Leica (L900) confocal
laser scanning microscope and were compiled and processed using Adobe
Photoshop version 4 software.
|
|
Transcytosis of WT MS11A and MS11A500 across polarized T84
monolayers.
The influence of the IgA1 protease on GC transcytosis
was next examined. T84 monolayers were polarized as described before (32) and infected with MS11A or MS11A500 at an MOI of 10 (12 filters per strain). Between 22 and 34 h postinfection, the basal medium of each infected filter was plated every 2 h on GCB agar for determination of total bacterial counts (Fig.
4). In complete infection medium, the
doubling time of the two GC strains is approximately 35 min (data not
shown). The bacterial counts in the basal well are therefore not an
accurate indication of the actual numbers of bacteria that have exited
the monolayers between two time points. However, since the
extracellular growth rates of these two GC strains are identical (data
not shown), the relative bacterial counts should be the same, allowing
a comparison of the WT and iga strains. In such an
experiment, the number of exocytosed bacteria cannot be normalized to
the total number of infecting bacteria, as to quantitate the latter
would require lysing of the monolayers for determination of
cell-associated bacterial counts.
View larger version (11K):
[in this window]
[in a new window]
|
FIG. 4.
Means and standard deviations of MS11A and MS11A500
exiting polarized T84 monolayers. Polarized T84 monolayers were
infected with MS11A (black bars) or its isogenic iga mutant
MS11A500 (white bars) (12 filters per strain). At various times, the
basal medium from each infected filter was plated for determination of
total bacterial counts. The values are the averages of all 12 filters.
*, P < 0.05, two-tailed t test for
unpaired samples.
|
|
At 24 h postinfection, no bacteria were recovered from the basal
well of any infected filter; at 26 and 28 h, only a small
number
of MS11A and MS11A500 organisms had crossed their monolayers.
At 30, 32, and 34 h, sizeable numbers of MS11A were recovered
from the
basal wells. In contrast, significantly fewer MS11A500
organisms were
recovered. For instance, at the 34-h time point,
there were 4.8 times
as many MS11A organisms in the basal well
as MS11A500 organisms
(
P < 0.05, two-tailed
t test for unpaired
samples). At the last time point, the electrical resistance of
all
infected monolayers was high, indicating that infection did
not
noticeably affect barrier integrity. An experiment comparing
GCM740 and
GCM740
4 yielded similar results (data not shown).
Thus, fewer
iga mutants than WT parental strains exited polarized
T84
monolayers.
Finally, the transcytosis times of WT and
iga mutants were
examined. The apical wells of polarized monolayers were inoculated
with
WT bacteria (GCM740 or MS11A) or their isogenic
iga mutants
(GCM740
4 or MS11A500). At each time point, each infected filter
was
transferred to a new well containing sterile medium in another
microtiter plate, and this new plate and the plate of basal medium
from
the previous incubation period were incubated further. The
presence of
bacteria in the basal medium from the previous time
point was
determined by the turbidity and the yellow color of
the medium after
overnight incubation. The basal medium was scored
as either positive or
negative for bacteria. A positive score
for a particular basal medium
was taken as an indication that
by that time point bacteria had crossed
the filters. For each
bacterial strain, the number of wells yielding
bacteria in the
basal medium was expressed as the percentage of the
total number
of wells infected. In all, 67 experiments were performed,
using
a total of 3,180 polarized T84 filters; 786 filters were infected
with MS11A, 845 filters were infected with MS11A500, 772 filters
were
infected with GCM740, and 777 filters were infected with
GCM740
4.
Results are shown in Fig.
5. For each
strain, each time
point represents an average of the sum of filters
from all experiments.
The electrical resistance of all monolayers was
determined before
and after each experiment. The measurements were
consistently
high for all monolayers up to the 36-h time point (data
not shown).
Thus, the bacteria were not destroying the cellular tight
junctions
and entering the basal medium through intercellular spaces or
damaged cells.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 5.
Transcytosis times of WT GC strains and their isogenic
iga mutants across polarized T84 monolayers. At defined
intervals after inoculation, the basal medium was removed and incubated
further to determine the presence of viable bacteria. At each time
point, the number of infected filters yielding bacteria in the basal
medium is expressed as a percentage of the total number of filters
infected. The least-likelihood-ratio test of each pair of strains
yielded a P value of <0.005 in both cases.
|
|
Results indicate that in both the GCM740 and MS11A genetic backgrounds
the
iga mutants were 3 to 5 h slower in crossing the
monolayers than the WT strains at earlier stages of infection.
The
differences in transcytosis times between WT and
iga mutants
are statistically significant: the least-likelihood-ratio test
yielded
a
P value of < 0.005 for each pair of strains assayed.
In this experiment, bacteria were detected in the basal medium
as early
as 10 h postinfection. In contrast, results from the
previous
experiment indicate that bacteria did not enter the basal
medium until
26 h postinfection. The discrepancy in these two
results probably
lies in the fact that in this experiment the
presence of one bacterium
in the basal well will result in a positive
score for that filter. This
method is therefore more sensitive
than the preceding experiment (Fig.
4), which requires plating
a portion of each basal well for
determination of bacterial
counts.
In the present study, we have demonstrated that GC infection of T84
cells, either grown on solid substrates or polarized on
semiporous
membranes, results in reduced steady-state levels of
LAMP1 and that
this reduction is due to the IgA1 protease (Fig.
2). We have also
provided evidence that the MS11A500
iga mutant
exited
polarized T84 monolayers in fewer numbers than its parental
WT MS11A
strain (Fig.
4). This defect cannot be due to a reduced
adhesion or
invasion ability, as MS11A500 and MS11A invaded epithelial
cells equally well and, in fact, MS11A500 was
slightly more adherent
than MS11A at 2 h postinfection
(Table
1). It was reported previously
that the
iga mutant is
defective in its ability to replicate within
A431 cells
(
25). This traversal phenotype of the MS11A500
iga mutant may reflect a similar growth defect in T84 cells,
although
the hypothesis cannot be confirmed: intracellular growth
assays
cannot be performed in T84 cells due to the intrinsic
limitations
of the cell line. Alternately, the traversal phenotype may
be
due to an undefined trafficking
defect.
We have also provided evidence that in the earlier stages of bacterial
transcytosis (up to 36 h postinfection),
iga mutants
in
the GCM740 and MS11A genetic backgrounds were 3 to 5 h slower
than
the WT strain in crossing polarized T84 monolayers (
P < 0.005
for each pair of strains [Fig.
5]). These results strongly
support
the notion that the IgA1 protease influences GC transcytosis
across
polarized T84 monolayers. The partial transcytosis defect of
these
iga mutants may be related to the defect in
intracellular replication
observed for the GCM740
iga mutant
in A431 cells (
25), although
we cannot offer an explanation
for how fewer intracellular bacteria
might result in a
slower-trafficking phenotype. Alternatively,
the slow-transcytosis
phenotype of the
iga mutants may be due
to the defect in
IgA1 protease-mediated processing of bacterial
(
38) and/or
host cell membrane
proteins.
That an
iga mutant is affected in its intracellular
trafficking is not at variance with the results from a previous study
which concluded that an
iga mutant and its isogenic WT
parent
behaved identically in infected hFTOC (
9). The nature
of the
hFTOC assay precluded the detailed examination of bacterial
transcytosis
and exocytosis. Nor can our results be compared to those
from
a recent study which demonstrated that expression of the
CD66-binding
Opa variants by GC and
Escherichia coli
recombinants resulted
in rapid bacterial transcytosis across polarized
T84 monolayers
(
46). That study compared P
Opa
with P
Opa52-expressing MS11, while all
our strains were P
+ Opa
. A P
+
Opa
MS11 strain in that study did not cross polarized T84
monolayers
within the time frame of the experiment (24 h), in contrast
to
the results from this and other studies (
32; S. Hopper and M.
So, unpublished data; S. Clary and M. So, unpublished
data), which
indicate that P
+ Opa
MS11A
transcytoses T84 monolayers at a predictable time course
of 36 to
48 h. We believe that the discrepancy in these results
is likely
to be due to the pilin variants expressed by these strains
(S. D. Gray-Owens, personal
communication).
Recent studies indicated that an
iga mutant of GC strain
FA1090 was able to colonize the urethra in male volunteers and cause
gonococcal urethritis (
19). Thus, the IgA1 protease is not
strictly
required for establishing a urethral infection. In relating
these
observations to our findings, it must be borne in mind that the
human challenge model, although powerful in many respects, has
practical limitations that do not permit a full examination of
the role
of the IgA1 protease in gonococcal disease. As stated
by the authors of
reference
19, the challenge model does not
detect
slight differences in bacterial infectivity. Our findings
that the IgA1
protease has a mild effect on GC traversal across
T84 monolayers would
likely be missed in the challenge study.
For ethical reasons, the human
challenge study permits examination
only of early stages of an
infection and thus cannot address issues
pertinent to later stages of a
gonorrhea infection, such as persistence.
GC is able to colonize and
infect a number of sites besides the
urethra, all of which have
distinguishing characteristics. It
also causes disease in women. If the
IgA1 protease plays a role
in the infectivity of bacteria for these
other sites, this would
be missed by the challenge
model.
In summary, our studies strongly suggest that the IgA1 protease plays a
role in GC transepithelial trafficking. The ability
to detect such
subtle defects illustrates the power and sensitivity
of the T84 system
for such studies. It will be interesting to
determine the exact
molecular basis for these trafficking defects
and the significance of
these findings to gonococcal
disease.
| |
ACKNOWLEDGMENTS |
This work was supported in part by NIH grant RO1 AI32493
awarded to M. So.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology and Immunology, L220, Oregon Health Sciences
University, 3181 SW Sam Jackson Park Rd., Portland, OR 97201-3098. Phone: (503) 494-6840. Fax: (503) 494-6862. E-mail:
hoppers{at}ohsu.edu.
Editor:
E. I. Tuomanen
| |
REFERENCES |
| 1.
|
Ayala, P.,
L. Lin,
S. Hopper,
M. Fukuda, and M. So.
1998.
Infection of epithelial cells by pathogenic neisseriae reduces the levels of multiple lysosomal constituents.
Infect. Immun.
66:5001-5007[Abstract/Free Full Text].
|
| 2.
|
Blake, M.,
K. K. Holmes, and J. Swanson.
1979.
Studies on gonococcus infection. XVII. IgA1-cleaving protease in vaginal washings from women with gonorrhea.
J. Infect. Dis.
139:89-92[Medline].
|
| 3.
|
Bos, M. P.,
F. Grunert, and R. J. Belland.
1997.
Differential recognition of members of the carcinoembryonic antigen family by Opa variants of Neisseria gonorrhoeae.
Infect. Immun.
65:2353-2361[Abstract].
|
| 4.
|
Buchanan, T. M.,
W. A. Pearce,
G. K. Schoolnik, and R. J. Arko.
1977.
Protection against infection with Neisseria gonorrhoeae by immunization with outer membrane protein complex and purified pili.
J. Infect. Dis.
136(Suppl.):S132-S137.
|
| 5.
|
Carlsson, S. R.,
J. Roth,
F. Piller, and M. Fukuda.
1988.
Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp1 and h-lamp-2.
J. Biol. Chem.
263:18911-18919[Abstract/Free Full Text].
|
| 6.
|
Chen, J. W.,
G. L. Chen,
M. P. D'Souza,
T. L. Murphy, and J. T. August.
1986.
Lysosomal membrane glycoproteins: properties of LAMP-1 and LAMP-2.
Biol. Soc. Symp.
51:97-112.
|
| 7.
|
Chen, T., and E. C. Gotschlich.
1996.
CGM1a antigen of neutrophils, a receptor of gonococcal opacity proteins.
Proc. Natl. Acad. Sci. USA
93:14851-14856[Abstract/Free Full Text].
|
| 8.
|
Chen, T.,
F. Grunert,
A. Medina-Marino, and E. C. Gotschlich.
1997.
Several carcinoembryonic antigens (CD66) serve as receptors for gonococcal opacity proteins.
J. Exp. Med.
185:1557-1564[Abstract/Free Full Text].
|
| 9.
|
Cooper, M. D.,
Z. A. McGee,
M. H. Mulks,
J. M. Kooney, and T. L. Hindman.
1984.
Attachment to and invasion of human fallopian tube mucosa by an IgA1 protease-deficient mutant of Neisseria gonorrhoeae and its wild type parent.
J. Infect. Dis.
150:737-744[Medline].
|
| 10.
|
Dharmsathaphorn, K., and J. L. Madara.
1990.
Established intestinal cell lines as model systems for electrolyte transport studies.
Methods Enzymol.
192:354-389[Medline].
|
| 11.
|
Grassme, H. U.,
R. M. Ireland, and J. P. van Putten.
1996.
Gonococcal opacity protein promotes bacterial entry-associated rearrangements of the epithelial cell actin cytoskeleton.
Infect. Immun.
64:1621-1630[Abstract].
|
| 12.
|
Gray-Owen, S. D.,
C. Dehio,
A. Haude,
F. Grunert, and T. F. Meyer.
1997.
CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes.
EMBO J.
16:3435-3445[CrossRef][Medline].
|
| 13.
|
Gray-Owen, S. D.,
D. R. Lorenzen,
A. Haude,
T. F. Meyer, and C. Dehio.
1997.
Differential Opa specificities for CD66 receptors influence tissue interactions and cellular response to Neisseria gonorrhoeae.
Mol. Microbiol.
26:971-980[CrossRef][Medline].
|
| 14.
|
Hagblom, P.,
E. Segal,
E. Billyard, and M. So.
1985.
Intragenic recombination leads to pilus antigenic variation in Neisseria gonorrhoeae.
Nature
315:156-158[CrossRef][Medline].
|
| 15.
|
Hauck, C. R., and T. F. Meyer.
1997.
The lysosomal/phagosomal membrane protein h-lamp-1 is a target of the IgA1 protease of Neisseria gonorrhoeae.
FEBS Lett.
405:86-90[CrossRef][Medline].
|
| 16.
|
Hedges, S. R.,
M. S. Mayo,
L. Kallman,
J. Mestecky,
E. W. Hook III, and M. W. Russell.
1998.
Evaluation of immunoglobulin A1 (IgA1) protease and IgA1 protease-inhibitory activity in human female genital infection with Neisseria gonorrhoeae.
Infect. Immun.
66:5826-5832[Abstract/Free Full Text].
|
| 17.
|
James, J. F., and J. Swanson.
1978.
Studies on gonococcus infection. XIII. Occurrence of color/opacity colonial variants in clinical cultures.
Infect. Immun.
19:332-340[Abstract/Free Full Text].
|
| 18.
|
Jerse, A. E.,
M. S. Cohen,
P. M. Drown,
L. G. Whicker,
S. F. Isbey,
H. S. Seifert, and J. G. Cannon.
1994.
Multiple gonococcal opacity proteins are expressed during experimental urethral infection in the male.
J. Exp. Med.
179:911-920[Abstract/Free Full Text].
|
| 19.
|
Johannsen, D. B.,
D. M. Johnston,
H. O. Koymen,
M. S. Cohen, and J. G. Cannon.
1999.
A Neisseria gonorrhoeae immunoglobulin A1 protease mutant is infectious in the human challenge model of urethral infection.
Infect. Immun.
67:3009-3013[Abstract/Free Full Text].
|
| 20.
|
Johnson, A. P.,
D. Taylor-Robinson, and Z. A. McGee.
1977.
Species specificity of attachment and damage to oviduct mucosa by Neisseria gonorrhoeae.
Infect. Immun.
18:833-839[Abstract/Free Full Text].
|
| 21.
|
Kallstrom, H.,
M. K. Liszewski,
J. P. Atkinson, and A. B. Jonsson.
1997.
Membrane cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic Neisseria.
Mol. Microbiol.
25:639-647[CrossRef][Medline].
|
| 22.
|
Kellogg, D. S., Jr.,
I. R. Cohen,
L. C. Norins,
A. L. Schroeter, and G. Reising.
1968.
Neisseria gonorrhoeae. II. Colonial variation and pathogenicity during 35 months in vitro.
J. Bacteriol.
96:596-605[Abstract/Free Full Text].
|
| 23.
|
Kellogg, D. S., Jr.,
W. L. Peacock, Jr.,
W. E. Deacon,
L. Brown, and C. I. Pirkle.
1963.
Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation.
J. Bacteriol.
85:1274-1279[Abstract/Free Full Text].
|
| 24.
|
Kupsch, E. M.,
B. Knepper,
T. Kuroki,
I. Heuer, and T. F. Meyer.
1993.
Variable opacity (Opa) outer membrane proteins account for the cell tropisms displayed by Neisseria gonorrhoeae for human leukocytes and epithelial cells.
EMBO J.
12:641-650[Medline].
|
| 25.
|
Lin, L.,
P. Ayala,
J. Larson,
M. Mulks,
M. Fukuda,
S. R. Carlsson,
C. Enns, and M. So.
1997.
The Neisseria type 2 IgA1 protease cleaves LAMP1 and promotes survival of bacteria within epithelial cells.
Mol. Microbiol.
24:1083-1094[CrossRef][Medline].
|
| 26.
|
Madara, J. L.,
J. Stafford,
K. Dharmsathaphorn, and S. Carlson.
1987.
Structural analysis of a human intestinal epithelial cell line.
Gastroenterology
92:1133-1145[Medline].
|
| 27.
|
Makino, S.,
J. P. van Putten, and T. F. Meyer.
1991.
Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells.
EMBO J.
10:1307-1315[Medline].
|
| 28.
|
McGee, Z.,
D. Stephens,
L. Hoffman,
W. Schlech III, and R. Horn.
1983.
Mechanisms of mucosal invasion by pathogenic Neisseria.
Rev. Infect. Dis.
5(Suppl. 4, Sept.-Oct.):S708-S714.
|
| 29.
|
McGee, Z. A., and M. L. Woods, Jr.
1987.
Use of organ cultures in microbiological research.
Annu. Rev. Microbiol.
41:291-300[CrossRef][Medline].
|
| 30.
|
McGee, Z. A.,
A. P. Johnson, and D. Taylor-Robinson.
1981.
Pathogenic mechanisms of Neisseria gonorrhoeae: observations on damage to human fallopian tubes in organ culture by gonococci of colony type 1 or type 4.
J. Infect. Dis.
143:413-422[Medline].
|
| 31.
|
Merz, A. J.,
C. A. Enns, and M. So.
1999.
Type IV pili of pathogenic neisseriae elicit cortical plaque formation in epithelial cells.
Mol. Microbiol.
32:1316-1332[CrossRef][Medline].
|
| 32.
|
Merz, A. J.,
D. B. Rifenbery,
C. G. Arvidson, and M. So.
1996.
Traversal of a polarized epithelium by pathogenic Neisseriae: facilitation by type IV pili and maintenance of epithelial barrier function.
Mol. Med.
2:745-754[Medline].
|
| 33.
|
Merz, A. J., and M. So.
1997.
Attachment of piliated, Opa and Opc gonococci and meningococci to epithelial cells elicits cortical actin rearrangements and clustering of tyrosine-phosphorylated proteins.
Infect. Immun.
65:4341-4349[Abstract].
|
| 34.
|
Plaut, A. G.,
J. V. Gilbert,
M. S. Artenstein, and J. D. Capra.
1975.
Neisseria gonorrhoeae and Neisseria meningitidis: extracellular enzyme cleaves human immunoglobulin A.
Science
190:1103-1105[Abstract/Free Full Text].
|
| 35.
|
Prall, F.,
P. Nollau,
M. Neumaier,
H. D. Haubeck,
Z. Drzeniek,
U. Helmchen,
T. Loning, and C. Wagener.
1996.
CD66a (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues.
J. Histochem. Cytochem.
44:35-41[Abstract].
|
| 36.
|
Pujol, C.,
E. Eugene,
L. de Saint Martin, and X. Nassif.
1997.
Interaction of Neisseria meningitidis with a polarized monolayer of epithelial cells.
Infect. Immun.
65:4836-4842[Abstract].
|
| 37.
|
Pujol, C.,
E. Eugene,
M. Marceau, and X. Nassif.
1999.
The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion.
Proc. Natl. Acad. Sci. USA
96:4017-4022[Abstract/Free Full Text].
|
| 38.
|
Shoberg, R. J., and M. H. Mulks.
1991.
Proteolysis of bacterial membrane proteins by Neisseria gonorrhoeae type 2 immunoglobulin A1 protease.
Infect. Immun.
59:2535-2541[Abstract/Free Full Text].
|
| 39.
|
Stephens, D. S., and M. M. Farley.
1991.
Pathogenic events during infection of the human nasopharynx with Neisseria meningitidis and Haemophilus influenzae.
Rev. Infect. Dis.
13:22-33[Medline].
|
| 40.
|
Swanson, J.
1973.
Studies on gonococcus infection. IV. Pili: their role in attachment of gonococci to tissue culture cells.
J. Exp. Med.
137:571-589[Abstract].
|
| 41.
|
van Putten, J. P.,
T. D. Duensing, and R. L. Cole.
1998.
Entry of OpaA+ gonococci into HEp-2 cells requires concerted action of glycosaminoglycans, fibronectin and integrin receptors.
Mol. Microbiol.
29:369-379[CrossRef][Medline].
|
| 42.
|
Virji, M., and J. S. Everson.
1981.
Comparative virulence of opacity variants of Neisseria gonorrhoeae strain P9.
Infect. Immun.
31:965-970[Abstract/Free Full Text].
|
| 43.
|
Virji, M.,
K. Makepeace,
D. J. Ferguson, and S. M. Watt.
1996.
Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic Neisseriae.
Mol. Microbiol.
22:941-950[CrossRef][Medline].
|
| 44.
|
Virji, M.,
S. M. Watt,
S. Barker,
K. Makepeace, and R. Doyonnas.
1996.
The N-domain of the human CD66a adhesion molecule is a target for Opa proteins of Neisseria meningitidis and Neisseria gonorrhoeae.
Mol. Microbiol.
22:929-939[CrossRef][Medline].
|
| 45.
|
Waldbeser, L. S.,
R. S. Ajioka,
A. J. Merz,
D. Puaoi,
L. Lin,
M. Thomas, and M. So.
1994.
The opaH locus of Neisseria gonorrhoeae MS11A is involved in epithelial cell invasion.
Mol. Microbiol.
13:919-928[CrossRef][Medline].
|
| 46.
|
Wang, J.,
S. D. Gray-Owen,
A. Knorre,
T. F. Meyer, and C. Dehio.
1998.
Opa binding to cellular CD66 receptors mediates the transcellular traversal of Neisseria gonorrhoeae across polarized T84 epithelial cell monolayers.
Mol. Microbiol.
30:657-671[CrossRef][Medline].
|
| 47.
|
Weel, G. F. L.,
C. T. P. Hopman, and J. P. M. Van Putten.
1991.
In situ expression and localization of Neisseria gonorrhoeae opacity proteins in infected epithelial cells: apparent role of Opa proteins in cellular invasion.
J. Exp. Med.
173:1395-1405[Abstract/Free Full Text].
|
| 48.
|
Zhang, K.,
D. Baeckstrom,
H. Brevinge, and G. C. Hansson.
1997.
Comparison of sialyl-Lewis a carrying CD43 and MUC1 mucins secreted from a colon carcinoma cell line for E-selectin binding and inhibition of leukocyte adhesion.
Tumor Biol.
18:175-187.
|
Infection and Immunity, February 2000, p. 906-911, Vol. 68, No. 2
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Duncan, J. A., Gao, X., Huang, M. T.-H., O'Connor, B. P., Thomas, C. E., Willingham, S. B., Bergstralh, D. T., Jarvis, G. A., Sparling, P. F., Ting, J. P.-Y.
(2009). Neisseria gonorrhoeae Activates the Proteinase Cathepsin B to Mediate the Signaling Activities of the NLRP3 and ASC-Containing Inflammasome. J. Immunol.
182: 6460-6469
[Abstract]
[Full Text]
-
Sjolinder, H., Eriksson, J., Maudsdotter, L., Aro, H., Jonsson, A.-B.
(2008). Meningococcal Outer Membrane Protein NhhA Is Essential for Colonization and Disease by Preventing Phagocytosis and Complement Attack. Infect. Immun.
76: 5412-5420
[Abstract]
[Full Text]
-
Han, X., Kennan, R. M., Davies, J. K., Reddacliff, L. A., Dhungyel, O. P., Whittington, R. J., Turnbull, L., Whitchurch, C. B., Rood, J. I.
(2008). Twitching Motility Is Essential for Virulence in Dichelobacter nodosus. J. Bacteriol.
190: 3323-3335
[Abstract]
[Full Text]
-
Fernaays, M. M., Lesse, A. J., Cai, X., Murphy, T. F.
(2006). Characterization of igaB, a Second Immunoglobulin A1 Protease Gene in Nontypeable Haemophilus influenzae.. Infect. Immun.
74: 5860-5870
[Abstract]
[Full Text]
-
Mann, B., Orihuela, C., Antikainen, J., Gao, G., Sublett, J., Korhonen, T. K., Tuomanen, E.
(2006). Multifunctional Role of Choline Binding Protein G in Pneumococcal Pathogenesis. Infect. Immun.
74: 821-829
[Abstract]
[Full Text]
-
Henderson, I. R., Navarro-Garcia, F., Desvaux, M., Fernandez, R. C., Ala'Aldeen, D.
(2004). Type V Protein Secretion Pathway: the Autotransporter Story. Microbiol. Mol. Biol. Rev.
68: 692-744
[Abstract]
[Full Text]
-
Dietrich, G., Kurz, S., Hubner, C., Aepinus, C., Theiss, S., Guckenberger, M., Panzner, U., Weber, J., Frosch, M.
(2003). Transcriptome Analysis of Neisseria meningitidis during Infection. J. Bacteriol.
185: 155-164
[Abstract]
[Full Text]
-
Perrin, A., Bonacorsi, S., Carbonnelle, E., Talibi, D., Dessen, P., Nassif, X., Tinsley, C.
(2002). Comparative Genomics Identifies the Genetic Islands That Distinguish Neisseria meningitidis, the Agent of Cerebrospinal Meningitis, from Other Neisseria Species. Infect. Immun.
70: 7063-7072
[Abstract]
[Full Text]
-
Ayala, P., Vasquez, B., Wetzler, L., So, M.
(2002). Neisseria gonorrhoeae Porin P1.B Induces Endosome Exocytosis and a Redistribution of Lamp1 to the Plasma Membrane. Infect. Immun.
70: 5965-5971
[Abstract]
[Full Text]
-
Vitovski, S., Dunkin, K. T., Howard, A. J., Sayers, J. R.
(2002). Nontypeable Haemophilus influenzae in Carriage and Disease: A Difference in IgA1 Protease Activity Levels. JAMA
287: 1699-1705
[Abstract]
[Full Text]
-
Tsirpouchtsidis, A., Hurwitz, R., Brinkmann, V., Meyer, T. F., Haas, G.
(2002). Neisserial Immunoglobulin A1 Protease Induces Specific T-Cell Responses in Humans. Infect. Immun.
70: 335-344
[Abstract]
[Full Text]
-
Henderson, I. R., Nataro, J. P.
(2001). Virulence Functions of Autotransporter Proteins. Infect. Immun.
69: 1231-1243
[Full Text]
-
Hopper, S., Wilbur, J. S., Vasquez, B. L., Larson, J., Clary, S., Mehr, I. J., Seifert, H. S., So, M.
(2000). Isolation of Neisseria gonorrhoeae Mutants That Show Enhanced Trafficking across Polarized T84 Epithelial Monolayers. Infect. Immun.
68: 896-905
[Abstract]
[Full Text]
Top
Abstract
Text
