Department of Microbiology and Immunology, Northwestern University Medical School, 303 E. Chicago Ave, Searle 6-467, Chicago, IL 60611, USA1
Author for correspondence: H. Steven Seifert. Tel: +1 312 503 9788. Fax: +1 312 503 1339. e-mail: h-seifert{at}northwestern.edu
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ABSTRACT |
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Keywords: Neisseria gonorrhoeae, human epithelial cells, fluorescent labelling, flow cytometry
Abbreviations: CFDA-SE, 5- and 6-carboxyfluorescein diacetate, succinimidyl ester; FACS, fluorescence-activated cell sorter
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INTRODUCTION |
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The pilin protein is the major component of the gonococcal type IV pilus. Antigenic variation of this protein has been shown to affect pilus-mediated adherence to human cells in vitro (Jonsson et al., 1994 ; Long et al., 1998
; Rudel et al., 1992
). The PilC protein is a phase-variable, minor pilus component that also plays a role in adherence to epithelial cells (Jonsson et al., 1991
; Rudel et al., 1992
). PilC has been localized to the tip of the pilus (Rudel et al., 1995
) and to the bacterial cell surface (Rahman et al., 1997
). Piliated gonococcal pilC mutants show decreased adherence to epithelial cells (Nassif & So, 1995
; Rudel et al., 1992
), and purified PilC protein binds to epithelial cells in vitro (Rudel et al., 1995
) and blocks adherence of N. gonorrhoeae and Neisseria meningitidis (Scheuerpflug et al., 1999
). Both pilin and PilC have a role in pilus-mediated adherence to epithelial cells.
An epithelial cell-surface receptor for gonococcal pili is human membrane cofactor protein (MCP, CD46) as identified by Källström et al. (1997) . Transfected Chinese hamster ovary cells expressing CD46 bind piliated but not non-piliated gonococci, and this adherence can be blocked with recombinant CD46 protein and
-CD46 antibodies (Källström et al., 1997
). In addition, purified neisserial pili trigger a transient increase in internal calcium levels in target epithelial cells, and the calcium flux is blocked by
-CD46 antibodies (Källström et al., 1998
), suggesting that interaction of pili with CD46 results in signal transduction. Human CD46 is a widely distributed complement regulator whose main function is to protect host cells from autologous complement attack by acting as a cofactor for proteolytic inactivation of C3b and C4b by factor I (Liszewski et al., 1991
; Seya et al., 1986
). CD46 has been found on every human cell and tissue type examined except for erythrocytes (Seya et al., 1990
), and it is expressed at very high levels on most epithelial cell lines (Johnstone et al., 1993
; McNearney et al., 1989
). Due to alternative splicing, four major isoforms of CD46 are produced: BC1, BC2, C1 and C2 (Post et al., 1991
). Splicing occurs within a Ser-Thr-Pro (STP)-rich region, which is surface-exposed, resulting in the presence or absence of the B portion. A second splicing event determines which of two alternative cytoplasmic tails is expressed. All four of the major CD46 isoforms can be expressed on one cell, and predominance of certain isoforms on specific tissues has been noted (Russell et al., 1992
). In addition to being the gonococcal pilus receptor, CD46 is also a receptor for measles virus (Dorig et al., 1993
), human herpes virus-6 (HHV-6) (Santoro et al., 1999
) and M protein of group A Streptococcus pyogenes (Okada et al., 1995
). Also, a significant negative correlation between CD46 expression and Helicobacter pylori infection was observed on biopsies of human gastric mucosa (Sasaki et al., 1998
). Therefore, CD46 appears to be a highly conserved protein that is important to several microbial pathogens.
In this study, we developed a fluorescence-activated cell sorter (FACS)-based assay for measuring gonococcal adherence. Using this assay we observed different levels of pilus-mediated adherence to different pertinent human epithelial cell lines. Surprisingly, we found that pilus-mediated adherence has an inverse correlation to the level of surface-exposed CD46 expression on these cell lines. CD46 isoform expression was determined for each of these epithelial cell lines, but the level of isoform expression did not correlate with levels of pilus-mediated adherence.
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METHODS |
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Cell lines and growth conditions.
The human epithelial cell lines ME180 endocervical (ATCC HTB 33), Chang conjunctival (ATCC CCL 20.2), Hec-1B endometrial (ATCC HTB 113) and PC-3 prostate (ATCC CRL 1435), and CHOK-1 Chinese hamster ovary cells (ATCC CCL 61) were maintained in RPMI 1640 (Gibco-BRL) supplemented with 5% fetal bovine serum (Gibco-BRL), penicillin (100 U ml-1)+streptomycin (100 µg ml-1) (Gibco-BRL) and amphotericin B solution (2·5 µg ml-1; Biologs) at 37 °C in 5% CO2. All experiments were carried out free from antibiotics and amphotericin.
Adherence assays.
The standard c.f.u. adherence assay was performed essentially as described by Long et al. (1998) . For the FACS-based adherence assay, 2x105 epithelial cells per well were plated in a 24-well plate 24 h prior to incubation with gonococci. Eighteen hours prior to labelling, gonococci were heavily streaked onto GCB plates and incubated at 37 °C in 5% CO2. Gonococci were swabbed up from the plates with Dacron swabs and resuspended in liquid GCB without Kellogg supplements. Bacteria were rinsed and resuspended in incomplete PBS (Gibco-BRL) to an OD550 of 0·9. Bacteria were labelled with 5- and 6-carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE; Molecular Probes) as described by Logan et al. (1998)
. Excess dye was washed off, and the labelled bacteria were resuspended in RPMI 1640. Labelled bacteria were incubated with the epithelial cell monolayer at 37 °C in 5% CO2 for 12 h to avoid internalization of the bacteria. Monolayers were washed three times with incomplete PBS to remove non-adherent gonococci and then incubated with 1% saponin in incomplete PBS for 10 min at 37 °C in 5% CO2 to disrupt the monolayer. Cells plus adherent, labelled gonococci were fixed with 1% formaldehyde in incomplete PBS. Flow cytometry (see below) was used to measure the level of fluorescence emitted by each epithelial cell, which corresponded to the relative number of adherent, fluorescing gonococci.
Analysis of CD46 levels.
Epithelial cells were plated 24 h before harvest at a concentration of 2x105 cells per well in a 24-well plate. At the time of harvest cells were approximately 80% confluent. However, no significant difference in CD46 expression was observed when cells were between 30 and 100% confluent. For flow cytometric analysis, cells were directly stained by incubating with FITC-conjugated -CD46 mAb N24 (PharMingen) or indirectly stained by incubating with
-CD46 mAb GB24 (J. Atkinson, Washington University, St Louis, MO, USA) and FITC-conjugated polyclonal antibodies against mouse IgG (PharMingen). Flow cytometry (see below) was used to measure the level of fluorescence.
Flow cytometry.
Flow cytometry was carried out on a FacsSort (Becton Dickinson) instrument with CellQuest software. Instrument settings were adjusted so that fluorescence of cells with either no bacteria or no antibody fell within the first decade of the logarithmic scale on which the emission is displayed. The mean fluorescence intensity of negative controls ranged from 2·0 to 4·0. For the adherence assays, the percentage of cells bound to bacteria was determined by enumerating cells fluorescing with intensity exceeding the first decade of the four-decade logarithmic scale of fluorescence intensity. The relative number of fluorescing bacteria or antibody bound per cell was expressed as the geometric mean of the histogram plotting level of fluorescence versus the number of events (epithelial cells) measured. Plots show at least 10000 events.
Fluorescence microscopy.
Adherence assays were performed as described above except that epithelial cells were grown on coverslips (Sarstedt) in 24-well plates. After the final wash, coverslips were mounted onto slides and sealed with nailpolish. An Olympus IX inverted fluorescence microscope with a Photometrix cooled charge-coupled device (CCD) camera (CH350/LCCD) driven by DeltaVision software from Applied Precision was used to detect fluorescently labelled gonococci bound to epithelial cells. Approximately twenty optical sections were taken through the depth of the cell, and DeltaVision software (softWoRx) was used to deconvolve these images.
Determination of CD46 isoform expression.
Reverse transcription was carried out as part of the Cells-to-cDNA kit (Ambion). The 3'-UT MCP-specific antisense primer (Post et al., 1991 ) was used to produce CD46-specific cDNA from each human cell line. CHOK-1 cells were used as a negative control, and no human CD46 was detected. PCR was used to amplify the CD46 gene from the cDNA obtained for each cell line. Primers to constant portions of the gene were used for the amplification: 3'-UT MCP (Post et al., 1991
) and 5'-MCP SCR4 (5'-GGTCAAATGTCGATTTCCAGTAGTCG-3'). Amplification products were separated on 2% Metaphor agarose (FMC Bioproducts) gels to distinguish between CD46 isoforms based upon size. As a control, the four major isoforms were amplified from plasmids containing each isoform (provided by J. Atkinson) using the conditions described above. Gels were photographed with a digital camera (AlphaImager 2000; Alpha Inotech) and the percentage of each isoform in each cell line was determined by densitometry using ImageQuant software (Molecular Dynamics). The percentage of each isoform was normalized to the total surface-exposed CD46 for each cell line by multiplying the mean fluorescence of total CD46 by the fraction (percentage) of isoform expression measured above. Results are plotted as relative fluorescence of each isoform in each epithelial cell line.
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RESULTS |
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DISCUSSION |
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Pilus-mediated adherence is a crucial step in the gonococcal infection process. Previous work by Källström et al. (1998) identified the abundant transmembrane glycoprotein CD46 as the pilus receptor. Our results demonstrate that pilus-mediated adherence varied significantly between human epithelial cell lines, in particular between ME180 endocervical cells and Chang conjunctival cells. Surprisingly, the levels of surface-exposed CD46 had an inverse correlation to the level of pilus-mediated adherence. In fact, the Chang cells had a threefold higher level of surface-exposed CD46 compared to ME180 cells. Since the antibody used to measure CD46 levels recognized all isoforms of CD46, these experiments did not address whether differential isoform expression could account for the adherence pattern observed. Recent results reported by Källström et al. (2001)
showed no difference in adherence of gonococcal strain MS11 to ME180 and Chang cells, whereas we have observed a significant difference in adherence to these cell lines using a piliated variant of strain MS11. It is important to note that different piliated variants of MS11 were used by each of our groups in gonococcal adherence assays, and piliation state is known to affect adherence (Long et al., 1998
). However, pilin variants of strain FA1090 had similar relative levels of adherence to the cell lines, indicating that piliation state does not affect differences in adherence between cell lines. A previous study using the ME180, Hec1B, PC3 and Chang epithelial cell lines showed that piliated variants of MS11 can vary in adherence to ME180, Hec1B and Chang cells, with Chang cells having the least number of gonococci bound in all cases (Rudel et al., 1992
). Another discrepancy between the observations of Källström et al. (2001)
and our results is that the former saw no significant difference in CD46 levels between ME180 and Chang cells using a polyclonal antibody to CD46, while we observed a large difference in expression using two independent mAbs to CD46. Both our group and Källström et al. (2001)
measured CD46 levels in a similar manner; however, specificity of the polyclonal anti-CD46 antibody versus the mAbs used in this study may contribute to the differences seen. Other factors, such as growth conditions and maintenance of the cell lines, may alter CD46 expression and explain the differences observed.
Examination of CD46 isoform levels in the epithelial cell lines used in this work revealed that each cell line had a distinct pattern of expression as measured by RT-PCR. Our RT-PCR results differ from those of Källström et al. (2001) for the ME180, Hec1B and Chang cell lines. Perhaps the cell lines alter their CD46 isoform expression. However, our results for the ME180 cell line match results previously reported by Wang et al. (2000)
for percentage isoform expression, indicating that our cell line and method of mRNA detection are consistent with those of other investigators. Prior reports have shown that more gonococci adhere to the BC isoforms than to the C isoforms when expressed on Chinese hamster ovary cells (Källström et al., 1998
). Our results indicate that the human cell line with the highest degree of adherence (ME180) predominately expresses the BC2 isoform. Also, cell lines, such as HEC-1B, with lower adherence levels express similar amounts of the BC1 and BC2 isoforms as ME180. In addition, we normalized the total surface-exposed CD46 to the percentage isoform expression for each cell line. The actual number of BC1 and BC2 isoforms possibly expressed on the surface of Chang cells is greater than on ME180 cells. If CD46 acts as a classic receptor for the pilus, then the number of piliated bacteria bound to a cell would correlate with the number of surface-exposed CD46 molecules. Our results indicate that no distinct correlation can be derived between gonococcal adherence and surface-exposed CD46 levels or CD46 isoform expression. Based upon our observations, we propose that CD46 does not act as a classic receptor in pilus-mediated adherence.
If CD46 does not act as a classic receptor for the gonococcal pilus, then what role does CD46 play in pilus-mediated adherence? Källström et al. (1998) have demonstrated signal transduction via CD46 stimulated with purified gonococcal pili. Signal transduction was measured as a calcium flux and was blocked by
-CD46 antibodies. CD46 has also been implicated in signalling events during measles virus infection (Karp et al., 1996
). Perhaps differences in CD46-mediated signal transduction in each cell line alter gonococcal adherence levels. Cell signalling may lead to stabilization and/or promotion of gonococcal attachment to epithelial cells. Another possibility is that differential adherence of piliated gonococci to these cell lines is independent of CD46. PilusCD46 interactions may be involved in an initial, transient attachment of gonococci, but other bacterialhost interactions may be required for the stabilization of this attachment. Differences in expression of host receptors for other gonococcal molecules may explain the differences in adherence levels that we observed. For example, expression of asialoglycoproteins, which interact with gonococcal lipooligosaccharide (Porat et al., 1995
), and CD66 molecules and heparan-sulfate-containing proteoglycans, which interact with Opa proteins (Chen et al., 1995
; Virji et al., 1996a
, b
), may affect adherence to various cell lines. Our results suggest that the precise role of CD46 in gonococcal adherence still remains to be determined.
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ACKNOWLEDGEMENTS |
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Received 20 December 2000;
revised 19 April 2001;
accepted 26 April 2001.