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J F Alderete

Publications and source records attributed to J F Alderete.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular analysis of the gene encoding the immunodominant phenotypically varying P270 protein of Trichomonas vaginalis.

Trichomonas vaginalisis a flagellated protozoan responsible for the most common non-viral sexually transmitted disease. The immunogen P270 was previously found to be up-regulated in expression and to undergo phenotypic variation between surface versus cytoplasmic localization in trichoImonads harbouring a dsRNA virus. In this report, we characterize the entire p270 open reading frame (ORF) and the unknown flanking 5;- and 3;-unique, non-repeat coding sequences of the gene in addition to untranslated regions. Consistent with an earlier report (Dailey & Alderete, 1991, Infect. Immun. 59: 2083-88), a significant portion of the gene consists of a tandemly repeated 333 bp element that contains the sequence coding for the epitope DREGRD detected by murine monoclonal antibody and antibody from the sera of patients. The non-repeat coding regions for the 5;- and 3;-ends were 69 nucleotides (23 amino acids) and 1183 nucleotides (395 amino acids), respectively. Sequencing of repeat elements showed them to be identical, affirming the highly-conserved nature of this element throughout the gene. The start codon was immediately preceded by the 12 nucleotide consensus sequence (TCATTTTTAATA) found in other trichomonad protein-coding genes. A very AT-rich, non-coding region was identified upstream of the p270 ORF. P270 appears to contain a leader sequence at the amino-terminus and transmembrane domain at the carboxy-terminus. No significant homology was found with any reported proteins at either the nucleotide or amino acid level.

Amino Acid Sequence↗
PubMed1998

Three genes encode distinct AP33 proteins involved in Trichomonas vaginalis cytoadherence.

Adherence to host cells is essential for the initiation and maintenance of infection by mucosal pathogens. The protozoan Trichomonas vaginalis colonizes the human urogenital tract via four surface proteins (AP65, AP51, AP33 and AP23). To characterize AP33 further, six cDNA clones were examined. Restriction mapping indicated that the six clones represented three similar genes. Southern analysis confirmed the existence of three single-copy AP33 genes and suggested a semi-conservative genomic arrangement between T. vaginalis isolates. Analysis of full-length sequences determined that each contained a 930bp open reading frame encoding a protein of approximately 33,000 Da. Sequence comparisons revealed a high degree of identity at both the DNA and the protein levels. N-terminal protein sequencing established the presence of leader peptides. Each of the three full-length recombinant proteins had a predicted pI of approximately 10, which was verified experimentally for the T. vaginalis AP33 adhesin. A database search revealed that AP33 had significant identity to the succinyl-CoA synthetase alpha-subunit of several different organisms and virtually 100% identity to the reported T. vaginalis subunit. Unlike commercially purchased enzyme, the recombinant proteins retained adhesive properties equal to the natural T. vaginalis AP33. The characteristics of the AP33 protein are similar to those of the other adhesins and emphasize a complex host-parasite relationship.

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PubMed1998

Characterization of Trichomonas vaginalis AP33 adhesin and cell surface interactive domains.

Adherence to host target cells is a critical step in establishing infection with the sexually transmitted pathogen Trichomonas vaginalis. Four parasite surface proteins mediating attachment to vaginal epithelial cells have been identified. One surface protein, termed AP33, was characterized further to identify domains interactive with previously generated antibodies and with host surface sites. N- and C-terminal deletion subclones were generated and tested for reactivity with both mAb and rabbit antiserum against AP33, and were also examined for their ability to bind to host cells. Surprisingly, the rabbit antiserum known to inhibit cytoadherence recognized an epitope(s) contained within 72 residues in the N-terminal half of the protein. However, the mAb epitope was immunoreactive with a 28-amino-acid region near the C-terminus. Subsequent mapping of this region with overlapping peptides identified a nine-amino-acid sequence reactive with the mAb. Equally surprising, two domains interactive with host cell surfaces were identified at distinct parts of AP33: one in the N-terminal half of the protein, and the other within 24 residues in the C-terminal third. Further analysis of the C-terminal binding domain revealed that a peptide representing this area could inhibit T. vaginalis cytoadherence by 40%.

Amino Acid Sequence↗
PubMed1997

Involvement of dsRNA virus in the protein composition and growth kinetics of host Trichomonas vaginalis.

Trichomonas vaginalis harbors a double-stranded (ds)-RNA virus, and the presence of virus is related to upregulated expression and phenotypic variation of a prominent immunogen (Khoshnan A, Alderete JF (1994) J Virol 68: 4035-4038). To further test the influence of virus on T. vaginalis, virus-infected (V+) isolates were compared to virus-free (V-), agar-cloned progeny trichomonads derived from the parental isolates for accumulation of total proteins and cysteine proteinases. Comparative high resolution two dimensional (2D)-SDS-PAGE was performed of trichomonads grown in a chemostat under identical conditions. At least 47 proteins were identified as specifically expressed by representative V+ isolate 347, and approximately 41 spots were specific to the corresponding V- progeny, showing an association between virus and the presence and absence of parasite proteins. Qualitatively and quantitatively dissimilar cysteine proteinase patterns were detected from numerous V+ isolates and the V- progeny. A 2D analysis for isolate 347 showed the appearance of unique proteinase activities for parental parasites and presence of at least one proteinase in the V- progeny. Finally, the V+ T. vaginalis isolate 347, but not the V- isolate 347 progeny nor other V+ isolates, underwent fluctuations in density during chemostat growth allowing for purification of virus particles from the V+ isolate 347 supernatants during decreased parasite density.

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PubMed1997

The vagina has reducing environment sufficient for activation of Trichomonas vaginalis cysteine proteinases.

BACKGROUND: Trichomonas vaginalis, a worldwide distributed sexually transmitted protozoan, is remarkable for synthesis of numerous, distinct cysteine proteinases, the significance of which is evidenced by the presence in vivo of soluble proteinases in secretions and antiproteinase antibody in serum of patients with trichomonosis. These proteinases purportedly play a role in host parasitism and immune evasion. OBJECTIVE: It is known that for cysteine proteinases to be functional, they must be activated by disulphide reducing reagents. Whether or not the host vaginal environment has the reducing environment essential for activation of the trichomonad cysteine proteinases is unknown. Our goal, therefore, was to determine whether or not vaginal secretions had sufficient reducing power to activate the trichomonad proteinases. METHODS: 48 vaginal washes (VWs) from patients were assayed for reducing equivalents and a score in dithiothreitol (DTT) reducing equivalents was assigned to each VW. Activation of trichomonad cysteine proteinases was then tested under the range of reducing equivalents detected from VWs. The possible protective effect of hydrogen peroxide, an oxidising agent produced by some Lactobacillus species, on proteinase activity was also determined. RESULTS: Nine of 48 VWs (18.7%) possessed < or = 10 microM DTT reducing equivalents, four VWs (8.3%) had from 20 microM DTT to 40 microM DTT reducing equivalents, and most (50%) were between 10 microM to 15 microM. Overall, the range in VWs was from approximately 10 microM to 40 microM reducing equivalents. Importantly, data suggest differential proteinase activation over this in vivo range of reducing level. Only two T vaginalis cysteine proteinase activities were stimulated at 2.5 microM DTT in contrast with all proteinase activities present at 40 microM DTT, albeit quantitatively diminished compared with the activity at 1 mM DTT, the concentration routinely used in vitro. Finally, hydrogen peroxide reversibly neutralised all trichomonad proteinases. CONCLUSIONS: These results show that the vagina of women has a reducing environment adequate for activation of trichomonad proteinases. The data underscore that the host environment plays a role in the host-parasite interrelation. Finally, hypotheses can now be formulated to help explain resistance and susceptibility to infection commonly reported among women and between men and women with trichomonosis.

Animals↗
PubMed1996

Molecular characterization of a third malic enzyme-like AP65 adhesin gene of Trichomonas vaginalis.

Adherence to the vaginal epithelium by the sexually transmitted parasite Trichomonas vaginalis is mediated by four trichomonad surface proteins (AP65, AP51, AP33 and AP23). We recently showed that the 65-kDa adhesin is a member of a multigene family comprised of two similar but distinct proteins, AP65-1 and AP65-2, encoded by the genes ap65-1 and ap65-2, respectively. An additional immuno-crossreactive clone, the 1.2 kb F11.1 cDNA, was isolated from a phagemid expression library and encoded a fusion protein of approximately 46,000 daltons (46 kDa) that bound to HeLa cell surfaces. A significant portion of the 5' end was missing which, using the 5'-RACE method, was obtained and combined with the F11.1 clone to give a full-length cDNA. The ap65-3 gene encoded for a protein of 567 amino acids with a molecular mass of 63.1 kDa. The gene showed 88% and 96% identity at the DNA level with ap65-1 and ap65-2, respectively. Restriction mapping confirmed that the three AP65 genes are different. Southern analysis revealed that the ap65-3 gene is present in the T. vaginalis genome in multiple copies. Experiments with agar clones of trichomonads showed that each gene of the multigene family is present in all parasites, and Northern analysis showed that ap65-3 is expressed and transcriptionally regulated by iron. The ap65-3 gene had a leader sequence and, as with ap65-1 and ap65-2, showed significant homology to malic enzyme. Finally, analysis of the 3'-untranslated regions revealed that the transcript of ap65-3 had a long poly (A) tail in comparison to ap65-1 and ap65-2. Even more intriguing, sequences were found that may relate to differential degradation of select AP65 transcripts, such as the sequence motifs AUUUA for ap65-1 mRNA and UUAUUUAU for the ap65-2 mRNA, which were not found for ap65-3.

Amino Acid Sequence↗
PubMed1995

Iron mediates Trichomonas vaginalis resistance to complement lysis.

Trichomonas vaginalis, a sexually transmitted disease agent in humans, is readily lysed by activation of the alternative complement pathway. The parasite became resistant following growth in medium supplemented by iron compared to parasites grown in medium depleted of iron, which were readily killed by complement. The resistance to complement was dependent on iron concentration while divalent cations other than iron were ineffective, showing specific regulation of this property by iron. Lactoferrin, but not transferrin, rendered low-iron-parasites resistant to complement lysis, reinforcing the in vivo modulation by a known source of iron for this parasite. Pretreatment of high-iron, complement-resistant parasites with proteinase inhibitors resulted in lysis by complement, indicating that resistance was likely due to proteinase degradation of C3 on the trichomonal surface.

Animals↗
PubMed1995

Cloning and molecular characterization of two genes encoding adhesion proteins involved in Trichomonas vaginalis cytoadherence.

Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagellate Trichomonas vaginalis. Four trichomonad surface proteins (AP65, AP51, AP33 and AP23) mediate cytoadherence. The cDNA encoding the AP65 adhesin was isolated from a phagemid cDNA expression library by screening with antiserum and monoclonal antibody (mAb) raised against the purified trichomonad AP65 protein. Two clones, F11.2 and F11.5, coded for immuno-crossreactive recombinant proteins that possessed functional properties equal to the T. vaginalis AP65 adhesin. Analysis of full-length sequences corresponding to the F11.2 and F11.5 cDNAs revealed that both contained 1701-base open reading frames (ORFs) that encoded proteins of 63 281 daltons and 83 087 daltons, respectively. Comparison of the full-length sequences showed 87% identity at the nucleotide level and 91% identity at the protein level. Restriction-enzyme mapping and Southern analysis reaffirmed the distinctness of the F11.2 and F11.5 cDNAs, indicating that two different AP65 genes (now called ap65-1 and ap65-2) are present in the T. vaginalis genome in at least two copies each. Northern analysis detected high levels of transcript of approximately 1.8 kb for both ap65-1 and ap65-2 genes in trichomonads grown only in high-iron medium, confirming the transcriptional regulation of adhesin synthesis by iron. Homology searches revealed significant similarity (38% amino acid identity and 54% nucleotide identity) to malic enzymes. However, purified malic enzyme and mAb to AP65 crossreactive with malic enzyme neither inhibited cytoadherence of T. vaginalis to host cells nor prevented binding of the trichomonad AP65 to HeLa cells in a ligand assay.

Amino Acid Sequence↗
PubMed1995

Analysis of human immunoglobulin-degrading cysteine proteinases of Trichomonas vaginalis.

Trichomonas vaginalis is a protozoan parasite that causes a widely distributed sexually transmitted disease (STD). Since immunoglobulin G (IgG) antibodies to specific trichomonad immunogens are found in serum and vaginal washes (VWs) from patients with trichomoniasis, a potential mechanism of immune evasion by this parasite might be the ability of T. vaginalis proteinases to degrade human immunoglobulins (Igs). Incubation of human IgG with lysates of T. vaginalis organisms resulted in time- and concentration-dependent degradation of the heavy chain. Secretory IgA was degraded similarly. Inhibitors of cysteine proteinases, when added to trichomonal lysates, abolished IgG and IgA degradation, while EDTA, a metalloproteinase inhibitor, did not. Substrate-gel electrophoresis with human IgG, IgM, or IgA copolymerized with acrylamide revealed several distinct cysteine proteinases in both lysates and culture supernatants from logarithmically growing parasites that degraded all classes of human antibodies. Trichomonal lysates and supernatants of numerous isolates tested all had Ig-degrading activity. Finally, proteolytic activity against IgG was detected in most (26 of 33; 78%) VWs from patients with trichomoniasis. In contrast, 18 of 28 (65%) VWs from women without trichomoniasis or from patients infected with other STDs had no detectable proteinases when tested in an identical manner. The other 10 of these 28 VWs (35%) had smaller amounts of detectable Ig-degrading proteinases. These differences in Ig-degrading proteinase activity between patients with and without trichomoniasis, regardless of coinfecting STDs, were statistically significant (P = 0.001). These results illustrate that T. vaginalis is capable of degrading human Igs.

Animals↗
PubMed1995

Characterization of double-stranded RNA satellites associated with the Trichomonas vaginalis virus.

Three small and distinct satellite double-stranded RNAs (dsRNAs) denoted s1, s1', and s2 were recently described for a Trichomonas vaginalis isolate harboring a dsRNA virus. Since characterization of these satellite dsRNAs might provide insight into the virus replication cycle and virus-host interactions, full-length cDNAs to s1 and s1' dsRNAs were synthesized and sequenced. s1 dsRNA has 688 bp, and s1' dsRNA has 616 bp. A 228-bp open reading frame that begins at nucleotide 37 was detected on a putative sense strand of s1. All satellite RNAs were found associated with RNA-dependent RNA polymerase (RDRP) activity that banded on CsCl gradients. Within carrier trichomonads, satellite RNAs synthesized single-stranded replicative intermediates. An in vitro assay was established to assess replication of satellite RNAs. Transcripts generated from s1 cDNA, for example, served as a template for the viral RDRP. These templates had a polarity similar to that of the replicative intermediate found in the satellite-harboring parasites. Importantly, the recognition of s1 RNA was shown to be specific, since unrelated RNAs did not serve as templates for RDRP under the same experimental conditions. The data indicate that the cDNA of s1 has a specific and essential sequence needed for recognition by the viral RDRP and for subsequent RNA synthesis. Both s1 and s1' have conserved domains, albeit of unproven function, but which may be required for replication.

Animals↗
PubMed1995

Characterization of cDNAs encoding adhesin proteins involved in trichomonas vaginalis cytoadherence.

Trichomonas vaginalis cytoadherence is mediated by four adhesins (AP65, AP51, AP33 and AP23). Adhesin gene expression was previously shown to be up-regulated by iron. Therefore, a cDNA library was constructed from mRNA of T. vaginalis grown in a high-iron medium. Ten cDNA clones (three for AP65, one for AP51, and six for AP33) were recognized with polyclonal antiserum or monoclonal antibody raised against these adhesins. No cross-hybridization among cDNAs of the three adhesins was observed in Southern analysis, confirming restriction mapping analysis of representative cDNAs. Southern analysis probed with representative cDNAs of the adhesins indicated multiple copies for each of the three adhesin genes. Northern analysis showed transcripts of 1.8 kilobases (kb), 1.4 kb, and 0.9 kb for AP65, AP51 and AP33, respectively. Consistent with adhesin expression, mRNAs for these adhesins were detected only when parasites were grown in high-iron conditions. Specific antibodies eluted from E. coli expressing recombinant proteins reacted only with the respective parasite adhesins. Recombinant proteins bind to fixed HeLa cells and competed with radiolabeled trichomonad adhesins for binding. Although proteinase activity is required for cytoadherence, recombinant proteins show no detectable proteinase activity. These data show that recombinant proteins from these clones exhibited characteristics of the trichomonad adhesins.

Animals↗
PubMed1995

Two Trichomonas vaginalis surface proteinases bind to host epithelial cells and are related to levels of cytoadherence and cytotoxicity.

Recent reports strongly suggest that cytoadherence and cytotoxicity by Trichomonas vaginalis require cysteine proteinase activity. Because of the large number of cysteine proteinases synthesized by T. vaginalis, a ligand assay was used to identify specific proteinases which may selectively target host cells. Two cysteine proteinases from trichomonal extracts with relative molecular masses (Mr) of 65,000 daltons (65-kDa) and 30-kDa were found to avidly bind to HeLa cell and vaginal epithelial cell surfaces. The two proteinases were distinguished by differential inhibition with leupeptin and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). Leupeptin pretreatment of live organisms inhibited the 30-kDa proteinase, which concomitantly reduced or eliminated cytoadherence. The T. vaginalis isolates with low levels of cytoadherence also had diminished or no detectable 30-kDa proteinase activity. On the other hand, TLCK pretreatment inhibited both the 30-kDa and 65-kDa proteinases, which resulted in decreased levels of cytoadherence and totally abolished contact-dependent cytotoxicity. Furthermore, isolates capable of attachment but with little or no cytotoxicity toward HeLa cells had no detectable host cell-bound 65-kDa proteinase. Finally, antiserum generated to each proteinase reacted by indirect immunofluorescence with live organisms, suggesting a surface location for both proteinases. This strategy and use of the ligand assay may permit for the deliniation of the role of two specific T. vaginalis surface proteinases in the properties of cytoadherence and cytotoxicity.

Animals↗
PubMed1994

Unique double-stranded RNAs associated with the Trichomonas vaginalis virus are synthesized by viral RNA-dependent RNA polymerase.

Most Trichomonas vaginalis isolates are carriers of the multisegmented double-stranded RNA (dsRNA) virus. In vitro polymerase assays were performed to demonstrate the RNA-dependent RNA polymerase (RDRP) activity of purified particles. Transcripts which comigrated with the dsRNAs of the virus were readily detected as synthesized products, indicating viral RDRP activity. In addition, smaller-sized dsRNA species, possibly two of approximately 700 bp (s1) and one of 500 bp (s2), were synthesized by purified virus particles of the CsCl gradient surrounding the virus peak. No cross-hybridization with either s1 or s2 and the dsRNA segments occurred, suggesting that s1 and s2 were synthesized from different templates. An RNase A protection assay revealed that the synthesized s1 and s2 polymerase products were double stranded. Furthermore, hybridization of products with strand-specific RNA of s1 generated from cDNA indicated that only one strand was synthesized in vitro. s1 and s2 were not visualized in ethidium bromide-stained agarose gels of dsRNA of infected trichomonads grown in batch cultures. However, dsRNA profiles of the same infected organisms cultivated under defined continuous-flow conditions contained readily detectable levels of s1 and s2, indicating that amplification of s1 and s2 occurred under specific environmental conditions. These newly discovered dsRNAs were not detected in all of the virus-carrying isolates. Finally, it is noteworthy that the s1 and s2 dsRNAs and the RDRP activity were not detected in trichomonal isolates without virus or in virus-negative progeny derived from virus-positive parental isolates. These data indicate the possibility of variations in the number of dsRNAs and/or of the presence of satellites in trichomonads infected with the multisegmented virus.

Animals↗
PubMed1994

Trichomonas vaginalis with a double-stranded RNA virus has upregulated levels of phenotypically variable immunogen mRNA.

Some isolates of Trichomonas vaginalis carry a double-stranded RNA virus (TVV) and undergo phenotypic variation between surface expression and cytoplasmic expression of a prominent immunogen (P270). Only trichomonads with TVV express P270 on the surface. Northern (RNA) blots using a specific cDNA encoding the repeat element of the phenotypically varying P270 immunogen as a probe showed accumulation of P270 transcript only in isolates with TVV (V+) in contrast to isolates without the virus (V-). To test further the influence of virus infection on P270 mRNA expression, V- progeny, derived from the parental V+ isolates, were tested. Trichomonads of V- progeny, like the fresh clinical V- isolates, also showed no accumulation of P270 mRNA. By immunoblotting with a monoclonal antibody to the key repeated epitope of P270, it was found that V- organisms had quantitatively less immunoreactive protein than the parental V+ isolates. Although V+ and V- isolates contained proteins immunoreactive with the monoclonal antibody to P270, it was necessary to test for the presence of the P270 gene among all the isolates. As expected, Southern blots demonstrated that V+ and V- trichomonads possessed the gene encoding P270. These data suggest that the double-stranded RNA virus of T. vaginalis is involved in the regulation of P270 mRNA accumulation.

Animals↗