Department of Microbiology, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7758, USA1
Author for correspondence: John F. Alderete. Tel: +1 210 567 3940. Fax: +1 210 567 6612. e-mail: ALDERETE{at}UTHSCSA.EDU
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: colonization, fibronectin, laminin, pathogenesis, Trichomonas vaginalis
Abbreviations: BM, basement membrane; ECM, extracellular matrix; FN, fibronectin; LM, laminin; TLCK, N-p-tosyl-L-lysine chloromethyl ketone; VECs, vaginal epithelial cells
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The vaginal wall consists of a stratified squamous outer epithelium and the underlying connective tissue. The hormones of the menstrual cycle, particularly oestrogen and progesterone, control the growth and differentiation of epithelial cells and ultimately lead to the terminal differentiation and exfoliation of vaginal epithelial cells (VECs). Maintaining and supporting the epithelium is a network of macromolecules comprising the extracellular matrix (ECM) and the basement membrane (BM), such as fibronectin (FN) and laminin (LM).
As with other STD-causing pathogens, persistence within the urogenital tract by T. vaginalis would predictably require specific binding of the parasite to host structures. As such, these organisms adhere to VECs via surface adhesins (Alderete & Garza, 1985 , 1988
; Alderete et al., 1988
; Arroyo & Alderete, 1989
; Arroyo et al., 1992
, 1995
). However, exfoliation of VECs from the vaginal epithelium in addition to trichomonal cysteine-proteinase-mediated cytotoxicity (Alderete & Pearlman, 1984
) point towards parasites possibly residing at sites below the epithelial surface. This possibility may help explain the non-self-limiting nature of trichomonosis.
This report supports the existence of another mechanism by which T. vaginalis colonizes host tissues during trichomonosis. We hypothesized that persistence of parasites in the vagina may be due to the interaction of T. vaginalis with FN and LM. Early studies by us demonstrated the interaction of plasma proteins, including FN, with the T. vaginalis surface (Peterson & Alderete, 1982 , 1984a
). In addition, parasite attachment to LM has been reported (Casta e Silva Filho et al., 1988
; Benchimol et al., 1990
). That LM does not reside on the surface of VECs (unpublished observations) suggests that specific parasite binding to FN may contribute to host parasitism. In this report, we demonstrate that T. vaginalis interacts with both FN and LM and that these associations are mediated by different mechanisms and surface sites from the previously identified adhesins (Arroyo et al., 1992
). The relevance of these findings with regard to pathogenesis of trichomonosis is discussed.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ECM components and coating of cover-slips.
FN was purified from human plasma, obtained from the UTHSCSA hospital blood bank, by gelatin-Sepharose affinity chromatography according to the manufacturers instructions (Pharmacia Biotech). Briefly, human plasma was diluted 1:1 (v/v) in PBS prior to chromatography. Purified FN, at concentrations of 0·51 mg ml-1, was dialysed for 48 h with three changes of PBS. Protein concentration was determined using bicinchoninic acid (BCA; Pierce Chemical). Entactin-free mouse LM was purchased from Collaborative Research (Becton Dickenson Labs). Cover-slips (12 mm diam.; Bellco Glass) were coated with 1 µg FN or LM by spreading a 100 µl volume of the protein solution in PBS over the entire surface, as before (Peterson et al., 1983 ; Thomas et al., 1985
). Cover-slips were then air-dried overnight at room temperature and washed by immersion in PBS before placing individual cover-slips in 24-well plates. The uniform coating of cover-slips with each protein was confirmed by indirect immunofluorescence using specific antisera to each protein.
Parasite-binding assay.
Parasites at 105 ml-1 were grown in normal medium containing 510 mCi [3H]thymidine ml-1 (185 MBq; Dupont, NEN Research Products). Efficient radiolabelling of parasites and the extent of radiolabel for each density of parasites used (specific activity) was monitored throughout, as before (Alderete & Pearlman, 1984 ; Alderete & Garza, 1985
, 1988
; Arroyo et al., 1992
).
Trichomonads used in all binding assays were from the mid-exponential phase of growth (~20 h) at 106 ml-1 (Peterson & Alderete, 1982 ). Except for the experiment presented in Fig. 2
, radiolabelled parasites were then centrifuged and suspended in the same volume of medium without radioisotope for an additional 2 h at 37 °C before use in binding assays. Trichomonads were then harvested and washed twice in minimal binding (MB) buffer (120 mM NaCl, 1·3 mM KCl, 0·9 mM NaH2PO4, 5·5 mM glucose and 26 mM NaHCO3, pH 5·0) by centrifuging at 670 g for 5 min. Parasites were enumerated using a Neubauer haemocytometer (Alderete & Pearlman, 1984
; Alderete & Garza, 1985
; Peterson & Alderete, 1982
). Different numbers of organisms from 5x105 to 5x106 parasites in 1 ml MB buffer supplemented with 400 µM N
-p-tosyl-l-lysine chloromethyl ketone (TLCK; Sigma) were added to individual wells containing the protein-coated cover-slips. These were incubated for 30 min at 37 °C or as stated for each experiment. Cover-slips were washed by immersing several times in 37 °C pre-warmed MB buffer. Bound radioactivity remaining on the cover-slips was measured by scintillation spectroscopy. The number of c.p.m. on cover-slips reflected the number of parasites bound to FN or LM and was confirmed by light microscopy and enumeration of organisms in individual fields.
|
Periodate and trypsin treatment of live trichomonads.
Washed, pelleted radiolabelled parasites (2x107) were suspended in 10 ml 1 mM periodate prepared in PBS, as before (Alderete & Garza, 1985 ), under conditions that do not adversely affect trichomonal motility and viability. Trichomonads were then incubated on ice for 30 min before washing twice in PBS prior to suspending in MB buffer for use in the binding assay, as described above. In a competition assay, the MB buffer was supplemented with 250 mM maltose, glucose, sucrose or N-acetylgalactosamine (Alderete & Garza, 1985
; Warton & Honigberg, 1980
, 1983
). Trichomonads were suspended in the modified MB immediately prior to incubation with coated cover-slips at 37 °C for 30 min. MB buffer without sugars was used as a control.
Prior to protease treatment, 2x107 washed radiolabelled organisms from batch culture were incubated for an additional 2 h in fresh TYM-serum. Parasites were pelleted by centrifugation at 670 g, suspended in 10 ml MB buffer containing 30 mg trypsin (Sigma) and incubated for 30 min at 37 °C. The reaction was then terminated by the addition of equal amounts of trypsin inhibitor (Sigma). The cells were washed and suspended in MB buffer at the appropriate density for binding assays. In addition, two aliquots of trypsinized cells were placed in TYM-serum medium for an additional 2 h with and without 10 nM cycloheximide, a protein synthesis inhibitor (Alderete & Garza, 1985 ). Parasites were monitored throughout to ensure that experimental conditions did not affect viability and motility.
Immunofluorescence.
FN- and LM-coated cover-slips were placed in individual wells of a 24-well plate with 200 µl PBS containing 5 µl of either rabbit anti-FN or anti-LM serum, respectively. Normal rabbit serum (NRS) served as a negative control on cover-slips handled similarly. Cover-slips were incubated on a shaker for 30 min at 4 °C before being washed in cold PBS. Then, 250 µl cold PBS containing 5 µl goat anti-rabbit antibody conjugated with fluorescein isothiocyanate (Sigma) was added. Cover-slips were incubated in the dark for 30 min at 4 °C followed by washing with cold PBS and placing onto glass slides for observation by epifluorescence.
Antibody pretreatment of trichomonads.
IgG antibody was purified from prebleed NRS and anti-adhesin sera, reported and used previously by us (Arroyo et al., 1992 ). Parasites were suspended in MB buffer at 2x106 ml-1 in the presence of 400 µM TLCK to inhibit cysteine proteinases known to degrade immunoglobulins (Provenzano & Alderete, 1995
). One microgram, 5 µg and 50 µg of each of the four anti-adhesin IgGs were pooled prior to mixing with live trichomonads. As a control, 50 µg NRS IgG was used. Trichomonads were pretreated with IgG for 10 min at 37 °C prior to addition of the parasiteantibody mixture to wells containing coated cover-slips.
Electrophoresis and immunoblotting.
Total protein preparations of parasites, grown either in high- or low-iron TYM-serum medium, were prepared as described before (Lehker & Alderete, 1992 ). Briefly, 2x107 cells were washed and suspended in 1 ml cold PBS-TCA (10%, v/v), and total proteins were precipitated overnight at 4 °C. The protein pellet was collected by microcentrifugation at 4 °C and washed three times with cold PBS. Proteins were dissolved by suspension and boiling in electrophoresis dissolving buffer (Laemmli, 1970
). Standard SDS-PAGE was performed using 4% stacking and 7% separating acrylamide gels (Laemmli, 1970
). The proteins and molecular size markers (Amersham) were visualized by staining of gels with Coomassie brilliant blue R (Laemmli, 1970
).
To visualize FN, proteins from supernatants or cell pellets were blotted onto nitrocellulose after SDS-PAGE using a standard protocol (Towbin et al., 1979 ; Lehker & Alderete, 1992
). Membranes were incubated in TNT (10 mM Tris/HCl, pH 8·0; 150 mM NaCl; 0·05% Tween 20) buffer with 5% non-fat milk as a blocking agent. Nitrocellulose blots were then incubated overnight at 4 °C in goat anti-FN IgG (1:400), followed by washing in TNT buffer, and incubated for 1 h at room temperature in rabbit anti-goat secondary antibody conjugated to alkaline phosphatase (1:4000) in TNT buffer with 3% milk. Blots were finally washed three times in TNT for development (Sambrook et al., 1989
).
Statistical analysis.
Unless otherwise stated, each experiment was performed at least three times, and each representative experiment included quadruplicate samples. Except where noted differently, percentage binding of parasites to substrate on cover-slips is c.p.m. bound divided by total c.p.m added per well and multiplied by 100. As all the data have a normal distribution, the Students t test was performed where indicated.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
During our optimization of the binding assay and since addition of TLCK to the interaction buffer affected binding to FN only (Fig. 1b, c
), we tested whether trichomonads degraded FN present on the cover-slips. To this end, cover-slips coated with 30 µg FN were incubated with parasites as described in Methods. As shown in Fig. 3(a)
, supernatants after interaction of cover-slips with trichomonads contained soluble FN that was degraded in a time-dependent manner. We also examined for FN degradation under the optimal experimental conditions employed for measuring binding to this substrate (Methods). As seen in Fig. 3(b)
, supernatants contained degraded FN released from the coated cover-slips, and this degradation was inhibited by TLCK. Analysis by immunoblot of total proteins from washed parasites recovered from supernatants detected FN bound to trichomonads (data not shown), possibly leading to the decrease in numbers bound to cover-slips as shown in Fig. 2
. These results show the ability of T. vaginalis organisms to actively release and degrade immobilized FN substrate. Although this aspect of parasitesubstrate interactions was not examined for LM, it is likely that similar events also occur, as we have shown previously that trichomonads degrade BM (Provenzano & Alderete, 1995
), of which LM is a component. Collectively, the data suggest that a combination of modifications occurring on trichomonads and target substrate affects binding levels of parasites at extended periods.
|
|
T. vaginalis interaction with FN and LM is distinct from cytoadherence
Because synthesis of adhesins and levels of cytoadherence are up-regulated by iron (Arroyo et al., 1993 ; Lehker et al., 1991
), we further compared levels of binding of high- versus low-iron-grown parasites onto FN- or LM-coated cover-slips. As a control, the iron status of trichomonads was confirmed by electrophoretic analysis of differentially expressed proteins (Peterson & Alderete, 1984b
; Lehker & Alderete, 1992
). Binding to FN or LM was unaffected by the iron status of the organism (data not shown). Not unexpectedly, incubation of parasites with a mixture of antibodies to the T. vaginalis adhesins (Arroyo et al., 1992
, 1995
) had no effect on levels of organisms bound to FN-coated cover-slips. These results strongly indicate that the mode of T. vaginalis association with ECM proteins is distinct from cytoadherence.
All T. vaginalis isolates tested interact with the immobilized substrates
Finally, we felt it important to verify that other fresh clinical isolates of T. vaginalis bound comparably to FN and LM on cover-slips. Fig. 5 illustrates the results of several binding assays for isolate T016 and four additional trichomonal isolates. Data represent the mean of four separate experiments, and although binding for each isolate between each of the four experiments varied, statistical analysis shows that levels of binding to either FN or LM were not significantly different between isolates (Students t test, P>>0·05, n=4).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There are many reports in the literature on the specific nature of associations between microbial pathogens and ECM/BM membrane components (Westerlund & Korhonen, 1993 ; Patti et al., 1994
; Patti & Hook, 1994
). Candida albicans, an opportunistic yeast, attaches to ECM and BM molecules, and it is presumed that the interaction with immobilized FN and other ECM molecules enhances the establishment and maintenance of infection at sites outside its normal niche (Calderone & Scheld, 1987
; Klotz, 1994
). Other mucosal pathogens, such as the streptococci, adhere to immobilized FN in a time- and dose-dependent manner (Kuusela et al., 1985
; van der Flier et al., 1995
). It may not be surprising, therefore, that T. vaginalis, a mucosal pathogen of the vaginal tract, utilizes components of the ECM and BM, such as FN and LM, to establish colonization and cause persistent infection.
In this report, we demonstrate and characterize the nature of T. vaginalis binding to immobilized FN and LM. Trichomonad surface proteins appear to be involved in both FN and LM associations, as evidenced by the effect of protease treatment on live organisms (Fig. 4). This was supported by the ability to regenerate binding to FN upon placement of trypsinized cells back into culture. The absence of similar regeneration for the trichomonad association with LM may not be surprising, as the data indicate a role for surface proteins as well as complex carbohydrate structures on T. vaginalis in this interaction. It is important to note, however, that, while a protein moiety is involved in FN binding, the structure may still be in the form of a glycoprotein. Molecular characterization of these structures awaits future experimentation.
The adhesin proteins mediating cytoadherence of T. vaginalis to VECs (Arroyo et al., 1992 ) were not involved in recognition and binding to FN and LM. Also, iron, an important modulator of expression of the adhesin genes (Lehker et al., 1991
), played no role in parasite associations with either FN- or LM-coated cover-slips. These results suggest that variations in the concentration of lactoferrin, a known in vivo iron source for the parasite (Peterson & Alderete, 1984b
; Lehker & Alderete, 1992
), would not affect the ability of T. vaginalis to colonize host tissues. We have described the morphological transformation that occurs upon cytoadherence to VECs of T. vaginalis organisms (Arroyo et al., 1993
). A change from the culture ellipsoid form to an amoeboid morphology was also seen on the immobilized FN and LM surfaces within the 30 min time point. These observations indicate the existence of signals similar to the ones observed during cytoadherence.
Under our experimental conditions, the binding assays did not allow all parasites to associate with the protein-coated cover-slips. Experiments were performed in which organisms that remained unbound after a 20 min incubation with immobilized FN were placed onto new FN-coated surfaces. In this manner, all trichomonads were found to attach to FN. These data suggest that the observed variations (Fig. 5) are probably due to the constraints within the assay system. It would also appear that trichomonads are capable of simultaneously adhering to VECs and attaching to ECM and BM regardless of the iron status at the site of infection.
We show that immobilized FN can be degraded by the parasite cysteine proteinases (Fig. 3). These results are consistent with earlier reports showing the ability of trichomonad cysteine proteinases, produced in vivo (Alderete et al., 1991
), to degrade FN and ECM components as determined by substrate gel analyses (Provenzano & Alderete, 1995
). Degradation of FN at the site of infection may be significant. Studies with mammalian cells have shown that particular FN fragments can elicit various responses, such as changes in morphology (Chen & Culp, 1998
), attachment or detachment of cells from surfaces (Saiki et al., 1991
; Fukai et al., 1997
), and even apoptosis (Fukai et al., 1998
). It is conceivable that, during infection, parasite degradation of ECM components could lead to unforeseen aspects of cytopathology (Draper et al., 1995
) and/or host cell and parasite responses.
These data further highlight the complexity of this hostparasite relationship. These studies will ultimately help in understanding the persistence of parasites in an environment that undergoes dramatic changes over the course of the menstrual cycle. Furthermore, this report emphasizes the need to continue the characterization of the mechanisms and molecules involved in the T. vaginalis interaction with host cells and tissue components, including FN and LM, to fully clarify the non-self-limiting nature of infection.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alderete, J. F. & Garza, G. E. (1988). Identification and properties of Trichomonas vaginalis proteins involved in cytoadherence. Infect Immun 56, 28-33.[Medline]
Alderete, J. F. & Pearlman, E. (1984). Pathogenic Trichomonas vaginalis cytotoxicity to cell culture monolayers. Br J Vener Dis 60, 99-105.[Medline]
Alderete, J. F., Deme, P., Gombo
ova, A., Valent, M., Fabu
ová, M., Jano
ká, A.,
tefanovic, J. & Arroyo, R. (1988). Specific parasitism of purified vaginal epithelial cells by Trichomonas vaginalis. Infect Immun 56, 2558-2562.[Medline]
Alderete, J. F., Newton, E., Dennis, C. & Neale, K. A. (1991). The vagina of women infected with Trichomonas vaginalis has numerous proteinases and antibody to trichomonad proteinases. Genitourin Med 674, 469-474.
Arroyo, R. & Alderete, J. F. (1989). Trichomonas vaginalis surface proteinase activity is necessary for parasite adherence to epithelial cells. Infect Immun 57, 2991-2997.[Medline]
Arroyo, R., Engbring, J. A. & Alderete, J. F. (1992). Molecular basis of host epithelial cell recognition by Trichomonas vaginalis. Mol Microbiol 6, 853-862.[Medline]
Arroyo, R., Gonzalez-Robles, A., Martinez-Palomo, A. & Alderete, J. F. (1993). Signaling of Trichomonas vaginalis for amoeboid transformation and adhesin synthesis follows cytoadherence. Mol Microbiol 7, 299-309.[Medline]
Arroyo, R., Engbring, J. A., Nguyen, J., Musatovova, O., Lopez, O., Lauriano, C. & Alderete, J. F. (1995). Characterization of cDNAs encoding proteins involved in Trichomonas vaginalis cytoadherence. Arch Med Res 26, 361-369.[Medline]
Benchimol, M., Batista, C. & de Souza, W. (1990). Fibronectin- and laminin-mediated endocytic activity in the parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus. J Submicrosc Cytol Pathol 22, 39-45.[Medline]
Calderone, R. A. & Scheld, W. M. (1987). Role of fibronectin in the pathogenesis of candidal infections. Rev Infect Dis 9 (suppl. 4), S400403.
Casta e Silva Filho, F., de Souza, W. & Lopes, J. D. (1988). Presence of laminin-binding proteins in trichomonads and their role in adhesion. Proc Natl Acad Sci USA 85, 8042-8046.[Abstract]
Chen, W. & Culp, L. A. (1998). Adhesion of fibronectins EDb domain induces tyrosine phosphorylation of focal adhesion proteins in Balb/c 3T3 cells. Clin Exp Metastasis 16, 30-42.[Medline]
Cotch, M. F., Pastorek, J. G.II, Nugent, R. P., Yerg, D. E., Martin, D. H. & Eschenbach, D. A. (1991). Demographic and behavioral predictors of Trichomonas vaginalis infection among pregnant women. Obstet Gynecol 78, 1087-1092.[Abstract]
Diamond, L. S. (1957). The establishment of various Trichomonas of animals and man in axenic cultures. J Parasitol 43, 488-490.
Draper, D., McGregor, J., Hall, J., Jones, W., Beutz, M., Heine, R. P. & Porreco, R. (1995). Elevated protease activities in human amnion and chorion correlate with preterm premature rupture of membranes. Am J Obstet Gynecol 173, 1506-1512.[Medline]
van der Flier, M., Chhun, N., Wizemann, T. M., Min, J., McCarthy, J. B. & Tuomanen, E. I. (1995). Adherence of Streptococcus pneumoniae to immobilized fibronectin. Infect Immun 63, 4317-4322.[Abstract]
Fukai, F., Hasebe, S., Ueki, M., Mutoh, M., Ohgi, C., Takahashi, H., Takeda, K. & Katayama, T. (1997). Identification of the anti-adhesive site buried within the heparin-binding domain of fibronectin. J Biochem 121, 189-192.[Abstract]
Fukai, F., Mashimo, M., Akiyama, K., Goto, T., Tanuma, S. & Katayama, T. (1998). Modulation of apoptotic cell death by extracellular matrix proteins and fibronectin-derived antiadhesive peptides. Exp Cell Res 242, 92-99.[Medline]
Hardy, P. H., Hardy, J. B., Nell, E. E., Graham, D. A., Spence, M. R. & Rosenbaum, R. C. (1984). Prevalence of six sexually transmitted disease agents among pregnant inner-city adolescents and pregnancy outcome. Lancet ii, 333-337.
Klotz, S. A. (1994). Plasma and extracellular matrix proteins mediate in the fate of Candida albicans in the human host. Med Hypotheses 42, 328-334.[Medline]
Krieger, J. N., Ravdin, J. I. & Rein, M. F. (1985). Contact-dependent cytopathogenic mechanisms of Trichomonas vaginalis. Infect Immun 50, 778-786.[Medline]
Krieger, J. N., Wolner-Hanssen, P., Stevens, C. & Holmes, K. K. (1990). Characteristics of Trichomonas vaginalis isolates from women with and without colpitis macularis. J Infect Dis 161, 307-311.[Medline]
Kuusela, P., Vartio, T., Vuento, M. & Myrhe, E. B. (1985). Attachment of staphylococci and streptococci on fibronectin, fibronectin fragments, and fibrinogen bound to solid phase. Infect Immun 50, 77-81.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680-685.
Laga, M. A., Manoka, A., Kivuvu, M. & 10 other authors (1993). Non-ulcerative sexually transmitted diseases as risk factors for HIV-1 transmission in women: results from a cohort study. AIDS 7, 95102.[Medline]
Lehker, M. W. & Alderete, J. F. (1992). Iron regulates growth of Trichomonas vaginalis and the expression of immunogenic trichomonad proteins. Mol Microbiol 6, 123-132.[Medline]
Lehker, M. W., Arroyo, R. & Alderete, J. F. (1991). The regulation by iron of the synthesis of adhesins and cytoadherence levels in the protozoan Trichomonas vaginalis. J Exp Med 174, 311-318.[Abstract]
Lockwood, B. C., North, M. J. & Coombs, G. H. (1988). The release of hydrolases from Trichomonas vaginalis and Tritrichomonas foetus. Mol Biochem Parasitol 30, 135-142.[Medline]
Minkoff, H., Grunebaum, A. N., Schwarz, R. H., Feldman, J., Cummings, M., Crobleholme, W., Clark, L., Pringle, G. & McCormack, W. M. (1984). Risk factors for prematurity and premature rupture of membranes: a prospective study of the vaginal flora in pregnancy. Am J Obstet Gynecol 150, 965-972.[Medline]
Neale, K. A. & Alderete, J. F. (1990). Analysis of the proteinases of representative Trichomonas vaginalis isolates. Infect Immun 58, 157-162.[Medline]
Patti, J. M. & Hook, M. (1994). Microbial adhesins recognizing extracellular matrix macromolecules. Curr Opin Cell Biol 6, 752-758.[Medline]
Patti, J. M., Allen, B. L., McGavin, M. J. & Hook, M. (1994). MSCRAMM-mediated adherence of microorganisms to host tissues. Annu Rev Microbiol 48, 585-617.[Medline]
Peterson, K. M. & Alderete, J. F. (1982). Host plasma proteins on the surface of pathogenic Trichomonas vaginalis. Infect Immun 37, 755-762.[Medline]
Peterson, K. M. & Alderete, J. F. (1984a). Selective acquisition of plasma proteins by Trichomonas vaginalis and human lipoproteins as growth requirements for this species. Mol Biochem Parasitol 12, 37-48.[Medline]
Peterson, K. M. & Alderete, J. F. (1984b). Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors. J Exp Med 160, 398-410.[Abstract]
Peterson, K. M., Baseman, J. B. & Alderete, J. F. (1983). Treponema pallidum receptor binding proteins interact with fibronectin. J Exp Med 157, 1958-1970.[Abstract]
Provenzano, D. & Alderete, J. F. (1995). Analysis of human immunoglobulin-degrading cysteine proteinases of Trichomonas vaginalis. Infect Immun 63, 3388-3395.[Abstract]
Provenzano, D., Koshnan, A. & Alderete, J. F. (1997). Involvement of dsRNA virus in the protein composition and growth kinetics of host Trichomonas vaginalis. Arch Virol 142, 939-952.[Medline]
Read, J. S. & Klebanoff, M. A. (1993). Sexual intercourse during pregnancy and preterm delivery: effects of vaginal microorganisms. Am J Obstet Gynecol 129, 629-636.
Saiki, I., Makabe, T., Yoneda, J., Murata, J., Ishizaki, Y., Kimisuka, F., Kato, I. & Azuma, I. (1991). Inhibitory effects of fibronectin and recombinant polypeptides on the adhesion of metastatic melanoma cells to laminin. Jpn J Cancer Res 82, 1112-1119.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Soper, D. E., Bump, R. C. & Hurt, G. W. (1990). Bacterial vaginosis and trichomoniasis vaginitis are risk factors for cuff cellulitis after abdominal hysterectomy. Am J Obstet Gynecol 163, 1016-1023.[Medline]
Thomas, D. D., Baseman, J. B. & Alderete, J. F. (1985). Fibronectin mediates Treponema pallidum cytadherence through recognition of fibronectin cell-binding domain. J Exp Med 161, 514-525.[Abstract]
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350-4354.[Abstract]
Warton, A. & Honigberg, B. M. (1980). Lectin analysis of surface saccharides in two Trichomonas vaginalis strains differing in pathogenicity. J Protozool 27, 410-419.[Medline]
Warton, A. & Honigberg, B. M. (1983). Analysis of surface saccharides in Trichomonas vaginalis strains with various pathogenicity levels by fluorescein-conjugated plant lectins. Z Parasitenkd 69, 149-159.[Medline]
Wasserheit, J. N. (1992). Interrelationship between human immunodeficiency virus infection and other sexually transmitted diseases. Sex Transm Dis 19, 61-77.[Medline]
Westerlund, B. & Korhonen, T. K. (1993). Bacterial proteins binding to the mammalian extracellular matrix. Mol Microbiol 9, 687-694.[Medline]
Wolner-Hanssen, P., Krieger, J. N., Stevens, C. E., Kiviat, N. B., Koutsky, L., Crichlow, C., DeRouen, T., Hillier, S. & Holmes, K. K. (1989). Clinical manifestations of vaginal trichomoniasis. JAMA (J Am Med Assoc) 261, 571576.
World Health Organization (1995). An overview of selected curable sexually transmitted diseases. WHO Global Programme on AIDS Report.
Zhang, Z. & Begg, C. B. (1994). Is Trichomonas vaginalis a cause of cervical neoplasma? Results from a combined analysis of 24 studies. Int J Epidemiol 23, 682-690.[Abstract]
Received 11 February 1999;
revised 6 May 1999;
accepted 4 June 1999.