Seminar Report #3
Critique of article - Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions. Experimental Parasitology (107): 125 – 135.
Trichomonosis is a chronic sexually transmitted disease caused by infection of the urogenital tract by Trichomonas vaginalis, a flagellated parasitic protozoan possessing a high quantity of cysteine proteinases (CPs) (Hernandez-Gutierrez, 2004). Cysteine proteinases being enzymes responsible for the catalyzing the hydrolysis of various proteinswhich also contain the essential amino acid residue cysteine within their active site (Oliveira, 2003). Trichomonad CPs are thought to play a critical role in the pathogenicity of the parasite (Hernandez-Gutierrez, 2004).
Evidence substantiating this claim includes the proteolytic degradation of mucin by secreted trichomonad CPs which aides the parasite in circumventing the mucous layer covering epithelial cells at the site of infection (Lehker and Sweeney, 1999). T. vaginalis cysteine proteinases have also been proven to be critical in the parasite’s attachment to human epithelial cells as inhibition of CPs has resulted in decreased levels of cytoadherence and consequently diminished contact-dependent cytotoxicity (Arroyo and Alderete, 1995). Inhibition of trichomonad CPs also reduced the haemolytic activity of live T. vaginalis parasites (Dailey et al., 1990).
T. vaginalis is also thought to escape the host defense mechanism by degrading the immunoglobulins which are released as a humoral response to infection, and also by supplying nutrients to parasites by degradation of hemoglobin (Min et al., 1998) which also has a cytotoxic
effect on the host cell. Resistance to the lysis of T. vaginalis through activation of the alternative complement pathway through CP degradation of the trichomonal cell surface protein C3 also adds to the parasite’s pathogenicity (Alderete et al., 1995). Degradation of the integral spectrin
protein found within the target cell membrane skeleton by the nonsecreted CP 30- kDa proteinase (Fiori et al., 1997), leads to the disruption of the cytoskeleton of red blood cells.
In order to further devolve the role CPs in the pathogenesis of T. vaginalis, the
researchers focused their investigation on characterizing a trichomonad 39-kDa proteinase, CP39, an active surface proteinase secreted during vaginal trichomonosis infection (Hernandez-Gutierrez, 2004).
To highlight the presence of 39-kDa proteinase in the vaginal secretions of a patient with symptomatic trichomonosis, zymograms, which are strips or bands of electrophoretic medium used to quantitate enzymes or isoenzymes by electrophoresis separation, were used to assess proteinase activity of 39-kDa proteinase in the supernatant from vaginal washes (VW) taken from an infected patient (Hernandez-Gutierrez, 2004). The possibility that the CPs present in vaginal secretions may have been shed during infection was demonstrated by using in vitro secretion assay which showed that both 65- and 39-kDa proteinases could be secreted by viable and metabolically active trichomonads under in vitro controlled conditions (Hernandez-Gutierrez, 2004). The researchers sought to confirm that the 39-kDa proteinase was secreted during active trichomonosis by analyzing the VWs obtained from infected patients. Digestion observed on gelatin gels was used to determine proteolytic activity. All of the assayed VWs which were culture-positive exhibited proteolytic activity between the molecular weight range of 116- to 21.5-kDa, none of the culture negative VWs exhibited the same proteolytic activity. This
suggested that CP39 along with other trichomonad proteinases is likely released in varying amounts in the vagina of patients during infection (Hernandez-Gutierrez, 2004).
Next the researchers attempted to determine whether or not CP39 proteinase was able to bind to the surface of different human urogenital cell types. Standard cell-binding assay showed that CP39 was able to bind to Hela epithelial cells, vaginal epithelial cells (VECs) and human prostatic cancer DU-145 cell lines but not colon cancer (CaCo) cells, suggesting a tissue specific binding mechanism for the parasite CP39 (Hernandez-Gutierrez, 2004). To determine whether CP39 was present in different T. vaginalis isolates 1-dimensional gelatin-gel electrophoresis of
six different isolates were analyzed. Proteolytic bands ranging from 21.5- to 116-kDa with different intensities were observed in the parasite extracts of all the isolates assayed. The zymorgams showed CP39 to be present with varying intensity in all isolates (Hernandez-Gutierrez, 2004). To confirm the cysteine proteinase nature of CP39, proteolytic activity of CP39 was shown to be completely eliminated by TLCK and E-64, known proteinase inhibitors (Hernandez-Gutierrez, 2004).
To establish whether CP39 was active at pH and temperatures found in the vagina during infection, the stability of the CP39 activity at pH 4.5, the vaginal pH of healthy women, was assessed over an extensive range of temperatures. CP39 was shown to be active between 4 and 50 ?C suggesting that CP39 activity might be stable during T. vaginalis infection (Hernandez-
In further analyzing the role of CP39 in the pathogenicity T. vaginalis the ability of the
proteinase to degrade various crucial cellular proteins including immunoglobins which are involved in the cells defensive response, was assessed by analyzing its proteolytic activity on substrate gel electrophoresis. The proteins Coll I, III, IV, V, human Fn, Hb, IgA, and IgG were
all degraded by CP39 as substrates (Hernandez-Gutierrez, 2004). To more specifically determine the ability of CP39 present in vaginal secretions to degrade secretory IgAs, the degradation of IgA by the proteinases present on two CP39-containing VWs from culture-positive patients was evaluated. IgA-containing gels were degraded by the 39-kDa-proteinase band present in both VWs tested (Hernandez-Gutierrez, 2004).
Towards the end of the investigation to determine the location of CP39 in the parasitic membrane and cytoplasm was analyzed utilizing an anti-CP39 mouse antibody and indirect immunofluorescence assays. The results of these analyses indeed showed that CP39 was indeed another surface proteinase as mentioned earlier (Hernandez-Gutierrez, 2004).
Overall, the presence of CP39 in vaginal secretions in infected patients, coupled with the cell binding, stability and proteolytic properties of the proteinase reported in this study continue to suggest as previous studies have done that CPs play a critical role in the infection of T.
vaginalis and are active during trichomonosis.
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