Michael W. Lehker

Iron and contact with host cells induce expression of adhesins on surface of Trichomonas vaginalis

The proteins AP65, AP51, AP33 and AP23 synthesized by Trichomonas vaginalis organisms in high iron play a role in adherence. Multigene families encode enzymes of the hydrogenosome organelles, which have identity to adhesins. This fact raises questions regarding the compartmentalization of the proteins outside the organelle and about the interactions of adhesins with host cells. Data here demonstrate the presence of the proteins outside the organelle under high‐iron conditions. Fluorescence and immuno‐cytochemical experiments show that high‐iron‐grown organisms coexpressed adhesins on the surface and intracellularly in contrast with low‐iron parasites. Furthermore, the AP65 epitopes seen by rabbit anti‐AP65 serum that blocks adherence and detects surface proteins were identified, and a mAb reacting to those epitopes recognized the trichomonal surface. Two‐dimensional electrophoresis and immunoblot of adhesins from surface‐labelled parasites provided evidence that all members of the multigene family were co‐ordinately expressed and placed on the trichomonal surface. Similar two‐dimensional analysis of proteins from purified hydrogenosomes obtained from iodinated trichomonads confirmed the specific surface labelling of proteins. Contact of trichomonads with vaginal epithelial cells increased the amount of surface‐expressed adhesins. Moreover, we found a direct relationship between the levels of adherence and amount of adhesins bound to immortalized vaginal and ureter epithelial cells, further reinforcing specific associations. Finally, trichomonads of MR100, a drug‐resistant isolate absent in hydrogenosome proteins and adhesins, were non‐adherent. Overall, the results confirm an important role for iron and contact in the surface expression of adhesins of T. vaginalis organisms.

Biology of trichomonosis.

Trichomonas vaginalis is emerging as a major pathogen of men and women and is associated with serious health consequences. Advances in diagnosis and treatment are presented. The complexity of trichomonad pathogenesis is illustrated in the interaction of this parasite with human cells, tissues and the immune system. It is now becoming evident that the interaction of trichomonads with the host is frequently modulated by environmental signals. The molecular biology of trichomonads is still in its infancy, but analysis of genes, genomic structure and transcriptional mechanisms suggest that trichomonads combine both prokaryotic and eukaryotic features. Evidence for the ancient divergence of trichomonads from other eukaryotic lineages is discussed.

The mechanisms and molecules involved in cytoadherence and pathogenesis of Trichomonas vaginalis

Abstract All urogenital mucosal pathogens must overcome host factors and responses in order to colonize the mucosa and establish infection. These include the extensive mucus layer, nutrient-limiting conditions antibody responses and the constant fluid flow of the vagina. In this article, John Alderete, Michael Lehker and Rossana Arroyo review the recent work describing the specificity by which the protozoan. Trichomonas vaginalis , adheres to the vaginal epithelial cells via four surface proteins. They discuss three distinct signals that may be received by the parasite upon recognition and binding to these cells, illustrating the type of adaptive responses evolved in the establishment and maintenance of infection.

The complex fibronectin--Trichomonas vaginalis interactions and Trichomonosis.

Trichomonosis is the vaginitis caused by Trichomonas vaginalis. This sexually transmitted agent achieves successful host parasitism through various means including: (1). acquisition of nutrients through specific receptors; (2). recognition and binding to mucin followed by cytoadherence mediated by adhesins that resemble metabolic enzymes; (3). evasion of immune responses through (i). masking of organisms by host proteins, (ii). shedding of trichomonad proteins into the secretions and (iii). secretions of cysteine proteinases that degrade all immunoglobulin subclasses and complement; (4). alternating surface expression of at least two antigen repertoires; and (5) alternate and coordinate expression of virulence genes in response to host environmental factors. The fact that the parasite survives long term in the varying and adverse environment of the vagina attests to the highly evolved nature of this protist. An understanding of the non-self-limiting nature of this infection may come from recent findings illustrating the complexity of Trichomonas vaginalis-fibronectin (FN) interactions. The parasite readily attaches to surfaces with immobilized FN and binds to FN in a highly specific receptor-mediated fashion. The amount and affinity of bound FN by live organisms is influenced by concentrations in medium of both iron and calcium. De novo protein synthesis is required for optimal FN acquisition in the presence of calcium. Furthermore, the parasites bind with differing affinities to the N-terminal domain (NTD), the cell-binding domain (CBD) and the gelatin-binding domain (GBD) of FN. Iron modulates binding of NTD similar to that of FN. This minireview summarizes recent findings on the T. vaginalis-FN associations.

[33] Analysis for adhesins and specific cytoadhesion of Trichomonas vaginalis

Publisher Summary Trichomonas vaginalis is a protozoan parasite that is one of the most common clinically recognized sexually transmitted infectious agents. As with other mucosal pathogens, trichomonads must be capable of surmounting the mucous barrier and must cytoadhere to the squamous epithelial cells of the vaginal epithelium to overcome being expelled by the continuous fluid flow of the vagina. The chapter describes the assays developed and employed to establish the highly specific nature of T. vaginalis cytoadhesion and the identity of trichomonad surface proteins involved in this property. Prerequisite to the identification of important virulence factors, like adhesins, some degree of understanding is needed of the host factors of the vagina (or at the site of infection of a given microbial pathogen) that may provide environmental signals that regulate expression of the property being examined, in this case cytoadhesion and synthesis of adhesions. The cytoadhesion findings reinforce the idea that trichomonad surface proteins are mediators of specific host-cell attachment. The chapter also describes the ligand assay that has been developed by which specific microbial surface proteins involved in cytoadhesion are identified.

Resolution of six chromosomes of Trichomonas vaginalis and conservation of size and number among isolates.

The electrophoretic karyotype of Trichomonas vaginalis isolates was determined by contour-clamped homogeneous electric field electrophoresis. Six chromosomal bands ranging between 50 kbp and 6 Mbp were reliably resolved by our separation method. Trichomonad chromosomes fell into 3 distinct size classes. The 3 maxichromosomes were approximately 5,700, 4,700, and 3,500 kbp. Two intermediate-sized chromosomes were approximately 1,200 kbp and 1,100 kbp. A minichromosome was approximately 75 kbp. The same size and number of chromosomes were present in 15 T. vaginalis isolates obtained from different geographic regions, reinforcing the idea of a highly conserved karyotype among trichomonal isolates worldwide.

Molecular analysis of Trichomonas vaginalis surface protein repertoires

Important reasons necessitate interest and support for basic research on the sexually transmitted parasite, Trichomonas vaginalis (Table 6.1). Surprisingly, this protozoan remains one of the most poorly investigated infectious agents yet it is the most prominent parasite causing an illness in developed countries. In all world societies trichomoniasis causes an economic and emotional burden equal to other devastating pathogens of microbial etiology.

Tuning of Alternative Splicing - Switch From Proto- Oncogene to Tumor Suppressor

STAT5B, a specific member of the STAT family, is intimately associated with prostate tumor progression. While the full form of STAT5B is thought to promote tumor progression, a naturally occurring truncated isoform acts as a tumor suppressor. We previously demonstrated that truncated STAT5 is generated by insertion of an alternatively spliced exon and results in the introduction of an early termination codon. Present approaches targeting STAT proteins based on inhibition of functional domains of STATs, such as DNA-binding, cooperative binding (protein-protein interaction), dimerization and phosphorylation will halt the action of the entire gene, both the proto-oncogenic and tumor suppressor functions of Stat5B. In this report we develop a new approach aimed at inhibiting the expression of full-length STAT5B (a proto-oncogene) while simultaneously enhancing the expression of STAT5∆B (a tumor suppressor). We have demonstrated the feasibility of using steric-blocking splice-switching oligonucleotides (SSOs) with a complimentary sequence to the targeted exon-intron boundary to enhance alternative intron/exon retention (up to 10%). The functional effect of the intron/exon proportional tuning was validated by cell proliferation and clonogenic assays. The new scheme applies specific steric-blocking splice-switching oligonucleotides and opens an opportunity for anti-tumor treatment as well as for the alteration of functional abilities of other STAT proteins.

Identification of fibronectin as a receptor for bacterial cytoadherence

Publisher Summary This chapter discusses the identification of fibronectin as a receptor for bacterial cytoadherence. Studies on the associations between bacterial pathogens and certain host proteins likely resulted from serendipitous observations. Such was the case involving fibronectin and Staphylococcus aureus . The same could be said of perhaps many other examples of bacterial pathogens and fibronectin associations, and whether or not a bacterial pathogen bound a particular host protein was the result of a rather random screening of a library of commercially available proteins. The consequences to the host of being infected by organisms interacting with host serum proteins in general and extracellular matrix (ECM) proteins in particular are numerous, apart from the obvious feature of host parasitism through enhanced cytoadherence. For example, masking of the microbial surface might interfere with antigen presentation or prevent recognition of the foreign organism or antigens leading to an overall immune evasion strategy by the pathogen. Host proteins might impair further the more specialized functions of immune cells whose job is ligand recognition for phagocytosis. Autoimmune reactions might be envisioned, possibly the result of an altered conformation of the host protein and elicitation of autoreactive antibody, and contribute to disease pathogenesis.