Limits...
Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis.

Primon-Barros M, Rigo GV, Frasson AP, Santos Od, Smiderle L, Almeida S, Macedo AJ, Tasca T - Mem. Inst. Oswaldo Cruz (2015)

Bottom Line: During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite.This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling.Under bovine serum limitation conditions, no significant differences were observed.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Pesquisa em Parasitologia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil.

ABSTRACT
Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.

Show MeSH

Related in: MedlinePlus

effect of different iron sources treatment, ferrous sulphate (FS),haemoglobin (HB) and haemin (HM), on Trichomonas vaginalisadenosine deaminase activity. No significant alteration in enzyme activity wasobserved. Bars represent the mean ± standard deviation of at least threeexperiments, each in triplicate, evaluated by analysis of variance followed byTukey's test (p ≤ 0.05). TV-LACH1 and TV-LACH2: fresh clinical isolates frommale patients; TV-LACM1 and TV-LACM2: fresh clinical isolates from femalepatients.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4660616&req=5

f02: effect of different iron sources treatment, ferrous sulphate (FS),haemoglobin (HB) and haemin (HM), on Trichomonas vaginalisadenosine deaminase activity. No significant alteration in enzyme activity wasobserved. Bars represent the mean ± standard deviation of at least threeexperiments, each in triplicate, evaluated by analysis of variance followed byTukey's test (p ≤ 0.05). TV-LACH1 and TV-LACH2: fresh clinical isolates frommale patients; TV-LACM1 and TV-LACM2: fresh clinical isolates from femalepatients.

Mentions: The effect of the different iron treatments on adenosine deamination in T.vaginalis was then evaluated. Cellular integrity was assessed by motilityand viability of the trophozoites by trypan blue exclusion before and after allenzymatic assays. Trophozoite integrity was not affected by treatments with iron. TheATCC isolates that had been cultured long-term and the fresh clinical isolates fromfemale or male patients had a decrease in ADA activity after treatment with 2,2-DP(Fig. 1). Decreased enzymatic activity wasobserved in ATCC 30236 (29%), ATCC 30238 (67.8%), TV-LACM1 (41.8%), TV-LACM2 (27.7%),TV-LACH1 (47.6%) and TV-LACH2 (68.1%). However, trophozoites treated for 24 h with FS,HB and HM did not exhibit significant differences in ADA activity compared with thecontrols, which indicates that the activity was re-established after treatment with theiron chelator (Fig. 2). To assess whether thedecrease in ADA activity that followed 2,2-DP treatment was due to an effect on ADA geneexpression inT. vaginalis, qPCR experiments were performed. The geneexpression of two ADA sequences obtained from the T. vaginalisgenomewas evaluated: TVAG_430670 (ada 125) and TVAG_416510 (ada231), both with 733 aa. A comparison of the gene expression in the controltrophozoites and that of the T. vaginalistreated with the iron chelator2,2-DP was performed by evaluating the Ct and the baseline obtained during theexponential phase of the qRT-PCR technique and normalising them to the α-tubulinhousekeeping gene by calculating the enzyme/α-tubulin ratio. The results indicated that2,2-DP treatment increased both TVAG_430670 (ada 125) and, to a greaterextent, TVAG_416510 (ada 231) transcript levels in T.vaginalisisolates (Fig. 3).TVAG_430670 (ada 125) and TVAG_416510 (ada 231) genestranscript levels were increased 1.5 and 2.41 times in the ATCC 30236 isolate, 1.37 and1.50 times in ATCC30238, 1.36 and 3.3 times in TV-LACM1, 5.65 and 12.81 times inTV-LACM2, 7.41 and 7.78 times in TV-LACH1 and 6.19 and 13.83 times in TV-LACH2 (Fig. 3), respectively. To confirm the previousfindings, another iron chelator, ferrozine, was tested. Treatment with ferrozine led tothe same profile found after 2,2-DP treatment: an 81.7% decrease was observed in theenzymatic activity of the ATCC 30236 isolate. However, for the isolates ATCC 30238,TV-LACM1, TV-LACM2, TV-LACH1 and TV-LACH2, the activity decreased by 51.8%, 72.2%,80.7%, 65.82% and 66%, respectively (Fig. 4).Additionally, an increase in the transcript levels of TVAG_430670 (ada125) and TVAG_416510 (ada 231) genes were observed in ATCC30236 (1.64 and 1.52 times, respectively), ATCC 30238 (1.34 and 1.76 times,respectively), TV-LACM1 (3.58 and 11.79 times, respectively), TV-LACM2 (1.71 and 6.68times, respectively), TV-LACH1 (1.50 and 2.62 times, respectively) and TV-LACH2 (2.62and 11.63 times, respectively) (Fig. 5).


Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis.

Primon-Barros M, Rigo GV, Frasson AP, Santos Od, Smiderle L, Almeida S, Macedo AJ, Tasca T - Mem. Inst. Oswaldo Cruz (2015)

effect of different iron sources treatment, ferrous sulphate (FS),haemoglobin (HB) and haemin (HM), on Trichomonas vaginalisadenosine deaminase activity. No significant alteration in enzyme activity wasobserved. Bars represent the mean ± standard deviation of at least threeexperiments, each in triplicate, evaluated by analysis of variance followed byTukey's test (p ≤ 0.05). TV-LACH1 and TV-LACH2: fresh clinical isolates frommale patients; TV-LACM1 and TV-LACM2: fresh clinical isolates from femalepatients.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660616&req=5

f02: effect of different iron sources treatment, ferrous sulphate (FS),haemoglobin (HB) and haemin (HM), on Trichomonas vaginalisadenosine deaminase activity. No significant alteration in enzyme activity wasobserved. Bars represent the mean ± standard deviation of at least threeexperiments, each in triplicate, evaluated by analysis of variance followed byTukey's test (p ≤ 0.05). TV-LACH1 and TV-LACH2: fresh clinical isolates frommale patients; TV-LACM1 and TV-LACM2: fresh clinical isolates from femalepatients.
Mentions: The effect of the different iron treatments on adenosine deamination in T.vaginalis was then evaluated. Cellular integrity was assessed by motilityand viability of the trophozoites by trypan blue exclusion before and after allenzymatic assays. Trophozoite integrity was not affected by treatments with iron. TheATCC isolates that had been cultured long-term and the fresh clinical isolates fromfemale or male patients had a decrease in ADA activity after treatment with 2,2-DP(Fig. 1). Decreased enzymatic activity wasobserved in ATCC 30236 (29%), ATCC 30238 (67.8%), TV-LACM1 (41.8%), TV-LACM2 (27.7%),TV-LACH1 (47.6%) and TV-LACH2 (68.1%). However, trophozoites treated for 24 h with FS,HB and HM did not exhibit significant differences in ADA activity compared with thecontrols, which indicates that the activity was re-established after treatment with theiron chelator (Fig. 2). To assess whether thedecrease in ADA activity that followed 2,2-DP treatment was due to an effect on ADA geneexpression inT. vaginalis, qPCR experiments were performed. The geneexpression of two ADA sequences obtained from the T. vaginalisgenomewas evaluated: TVAG_430670 (ada 125) and TVAG_416510 (ada231), both with 733 aa. A comparison of the gene expression in the controltrophozoites and that of the T. vaginalistreated with the iron chelator2,2-DP was performed by evaluating the Ct and the baseline obtained during theexponential phase of the qRT-PCR technique and normalising them to the α-tubulinhousekeeping gene by calculating the enzyme/α-tubulin ratio. The results indicated that2,2-DP treatment increased both TVAG_430670 (ada 125) and, to a greaterextent, TVAG_416510 (ada 231) transcript levels in T.vaginalisisolates (Fig. 3).TVAG_430670 (ada 125) and TVAG_416510 (ada 231) genestranscript levels were increased 1.5 and 2.41 times in the ATCC 30236 isolate, 1.37 and1.50 times in ATCC30238, 1.36 and 3.3 times in TV-LACM1, 5.65 and 12.81 times inTV-LACM2, 7.41 and 7.78 times in TV-LACH1 and 6.19 and 13.83 times in TV-LACH2 (Fig. 3), respectively. To confirm the previousfindings, another iron chelator, ferrozine, was tested. Treatment with ferrozine led tothe same profile found after 2,2-DP treatment: an 81.7% decrease was observed in theenzymatic activity of the ATCC 30236 isolate. However, for the isolates ATCC 30238,TV-LACM1, TV-LACM2, TV-LACH1 and TV-LACH2, the activity decreased by 51.8%, 72.2%,80.7%, 65.82% and 66%, respectively (Fig. 4).Additionally, an increase in the transcript levels of TVAG_430670 (ada125) and TVAG_416510 (ada 231) genes were observed in ATCC30236 (1.64 and 1.52 times, respectively), ATCC 30238 (1.34 and 1.76 times,respectively), TV-LACM1 (3.58 and 11.79 times, respectively), TV-LACM2 (1.71 and 6.68times, respectively), TV-LACH1 (1.50 and 2.62 times, respectively) and TV-LACH2 (2.62and 11.63 times, respectively) (Fig. 5).

Bottom Line: During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite.This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling.Under bovine serum limitation conditions, no significant differences were observed.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Pesquisa em Parasitologia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil.

ABSTRACT
Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.

Show MeSH
Related in: MedlinePlus