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The Role of Efflux Pumps in Schistosoma mansoni Praziquantel Resistant Phenotype.

Pinto-Almeida A, Mendes T, Armada A, Belo S, Carrilho E, Viveiros M, Afonso A - PLoS ONE (2015)

Bottom Line: Praziquantel (PZQ) is the drug of choice to treat this disease due to its high cure rates and no significant side effects.Low doses of Verapamil successfully reverted drug resistance.Our results might give an indication that a combination therapy with PZQ and natural or synthetic Pgp modulators can be an effective strategy for the treatment of confirmed cases of resistance to PZQ in S. mansoni.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Areas of Basic and Applied Biology, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal; Medical Parasitology Unit, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Lisbon, Portugal; Universidade de São Paulo, Instituto de Química de São Carlos, São Carlos, SP, Brazil.

ABSTRACT

Background: Schistosomiasis is a neglected disease caused by a trematode of the genus Schistosoma that is second only to malaria in public health significance in Africa, South America, and Asia. Praziquantel (PZQ) is the drug of choice to treat this disease due to its high cure rates and no significant side effects. However, in the last years increasingly cases of tolerance to PZQ have been reported, which has caused growing concerns regarding the emergency of resistance to this drug.

Methodology/principal findings: Here we describe the selection of a parasitic strain that has a stable resistance phenotype to PZQ. It has been reported that drug resistance in helminths might involve efflux pumps such as members of ATP-binding cassette transport proteins, including P-glycoprotein and multidrug resistance-associated protein families. Here we evaluate the role of efflux pumps in Schistosoma mansoni resistance to PZQ, by comparing the efflux pumps activity in susceptible and resistant strains. The evaluation of the efflux activity was performed by an ethidium bromide accumulation assay in presence and absence of Verapamil. The role of efflux pumps in resistance to PZQ was further investigated comparing the response of susceptible and resistant parasites in the absence and presence of different doses of Verapamil, in an ex vivo assay, and these results were further reinforced through the comparison of the expression levels of SmMDR2 RNA by RT-PCR.

Conclusions/significance: This work strongly suggests the involvement of Pgp-like transporters SMDR2 in Praziquantel drug resistance in S. mansoni. Low doses of Verapamil successfully reverted drug resistance. Our results might give an indication that a combination therapy with PZQ and natural or synthetic Pgp modulators can be an effective strategy for the treatment of confirmed cases of resistance to PZQ in S. mansoni.

No MeSH data available.


Related in: MedlinePlus

Relative expression level of SmMDR2 RNA.In males and females of PZQ-susceptible and PZQ-resistant parasite strains in the presence and absence of PZQ. White bars—RNA from adult worms without exposure to PZQ, and black bars—RNA from adult worms after exposure to PZQ. The n-fold changes were determined by quantitative RT-PCR using S. mansoni 18S (Sm18s) RNA of each group was used as a reference gene. Differences of the relative level of SmMDR2 between the groups was done using ANOVA, p < 0.05.
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pone.0140147.g012: Relative expression level of SmMDR2 RNA.In males and females of PZQ-susceptible and PZQ-resistant parasite strains in the presence and absence of PZQ. White bars—RNA from adult worms without exposure to PZQ, and black bars—RNA from adult worms after exposure to PZQ. The n-fold changes were determined by quantitative RT-PCR using S. mansoni 18S (Sm18s) RNA of each group was used as a reference gene. Differences of the relative level of SmMDR2 between the groups was done using ANOVA, p < 0.05.

Mentions: The relative expression levels of SmMDR2 gene were assessed using a quantitative RT-PCR method. Adult worms of each PZQ-strain were separated by sex and compared. Parasites were also compared in the presence and absence of 0.3 μM of PZQ for 3 hours. As shown in Fig 12, when comparing PZQ-susceptible males and females, before exposure to PZQ, females showed a relative increase in the expression level of SmMDR2 of approximately 4 times when compared to males (p < 0.05). When exposed to PZQ the expression level of SmMDR2 in susceptible males increased 17 times when compared to the expression level of the same gene in the absence of PZQ in females (p < 0.05). As expected PZQ-resistant males showed, in the absence of PZQ, an increase in the expression level of SmMDR2 of approximately 32 times when compared to PZQ-susceptible males (p < 0.05). Furthermore, after exposure to PZQ, SmMDR2 expression level of PZQ-resistant males was approximately 6 times higher than in the absence of PZQ (p < 0.05). Finally, PZQ-resistant females showed no significant change in SmMDR2 expression after exposure to PZQ (p > 0.05), and the expression was approximately 10 times lower than the PZQ-susceptible females (p < 0.05).


The Role of Efflux Pumps in Schistosoma mansoni Praziquantel Resistant Phenotype.

Pinto-Almeida A, Mendes T, Armada A, Belo S, Carrilho E, Viveiros M, Afonso A - PLoS ONE (2015)

Relative expression level of SmMDR2 RNA.In males and females of PZQ-susceptible and PZQ-resistant parasite strains in the presence and absence of PZQ. White bars—RNA from adult worms without exposure to PZQ, and black bars—RNA from adult worms after exposure to PZQ. The n-fold changes were determined by quantitative RT-PCR using S. mansoni 18S (Sm18s) RNA of each group was used as a reference gene. Differences of the relative level of SmMDR2 between the groups was done using ANOVA, p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4596880&req=5

pone.0140147.g012: Relative expression level of SmMDR2 RNA.In males and females of PZQ-susceptible and PZQ-resistant parasite strains in the presence and absence of PZQ. White bars—RNA from adult worms without exposure to PZQ, and black bars—RNA from adult worms after exposure to PZQ. The n-fold changes were determined by quantitative RT-PCR using S. mansoni 18S (Sm18s) RNA of each group was used as a reference gene. Differences of the relative level of SmMDR2 between the groups was done using ANOVA, p < 0.05.
Mentions: The relative expression levels of SmMDR2 gene were assessed using a quantitative RT-PCR method. Adult worms of each PZQ-strain were separated by sex and compared. Parasites were also compared in the presence and absence of 0.3 μM of PZQ for 3 hours. As shown in Fig 12, when comparing PZQ-susceptible males and females, before exposure to PZQ, females showed a relative increase in the expression level of SmMDR2 of approximately 4 times when compared to males (p < 0.05). When exposed to PZQ the expression level of SmMDR2 in susceptible males increased 17 times when compared to the expression level of the same gene in the absence of PZQ in females (p < 0.05). As expected PZQ-resistant males showed, in the absence of PZQ, an increase in the expression level of SmMDR2 of approximately 32 times when compared to PZQ-susceptible males (p < 0.05). Furthermore, after exposure to PZQ, SmMDR2 expression level of PZQ-resistant males was approximately 6 times higher than in the absence of PZQ (p < 0.05). Finally, PZQ-resistant females showed no significant change in SmMDR2 expression after exposure to PZQ (p > 0.05), and the expression was approximately 10 times lower than the PZQ-susceptible females (p < 0.05).

Bottom Line: Praziquantel (PZQ) is the drug of choice to treat this disease due to its high cure rates and no significant side effects.Low doses of Verapamil successfully reverted drug resistance.Our results might give an indication that a combination therapy with PZQ and natural or synthetic Pgp modulators can be an effective strategy for the treatment of confirmed cases of resistance to PZQ in S. mansoni.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Areas of Basic and Applied Biology, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal; Medical Parasitology Unit, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Lisbon, Portugal; Universidade de São Paulo, Instituto de Química de São Carlos, São Carlos, SP, Brazil.

ABSTRACT

Background: Schistosomiasis is a neglected disease caused by a trematode of the genus Schistosoma that is second only to malaria in public health significance in Africa, South America, and Asia. Praziquantel (PZQ) is the drug of choice to treat this disease due to its high cure rates and no significant side effects. However, in the last years increasingly cases of tolerance to PZQ have been reported, which has caused growing concerns regarding the emergency of resistance to this drug.

Methodology/principal findings: Here we describe the selection of a parasitic strain that has a stable resistance phenotype to PZQ. It has been reported that drug resistance in helminths might involve efflux pumps such as members of ATP-binding cassette transport proteins, including P-glycoprotein and multidrug resistance-associated protein families. Here we evaluate the role of efflux pumps in Schistosoma mansoni resistance to PZQ, by comparing the efflux pumps activity in susceptible and resistant strains. The evaluation of the efflux activity was performed by an ethidium bromide accumulation assay in presence and absence of Verapamil. The role of efflux pumps in resistance to PZQ was further investigated comparing the response of susceptible and resistant parasites in the absence and presence of different doses of Verapamil, in an ex vivo assay, and these results were further reinforced through the comparison of the expression levels of SmMDR2 RNA by RT-PCR.

Conclusions/significance: This work strongly suggests the involvement of Pgp-like transporters SMDR2 in Praziquantel drug resistance in S. mansoni. Low doses of Verapamil successfully reverted drug resistance. Our results might give an indication that a combination therapy with PZQ and natural or synthetic Pgp modulators can be an effective strategy for the treatment of confirmed cases of resistance to PZQ in S. mansoni.

No MeSH data available.


Related in: MedlinePlus