Metronidazole is the main treatment for T. vaginalis. Its derivative tinidazole is so similar that a strain resistant to one drug is oft resistant to the other. In the T. vaginalis hydrogenosome, these drugs are activated when ferredoxin transfers an electron to metronidazole's NO2 group and a lethal free radical forms. Sensitive parasites lacking hydrogenosomes (Giardia and Entamoeba) activate the drug in the cytosol.
Drug-resistant parasites were identified by culturing parasites from patients and measuring the mean lethal metronidazole concentration required for killing. Some strains were 10-50x more resistant to metronidazole. What is the basis of resistance? Are resistant parasites deficient in drug activation? Western blots, northern blots, complements, knockouts, microscopy and centrifugation were all used to answer these two questions.
|Western & Northern Blots||Western (protein) and northern (mRNA) blots showed that drug-resistant strains had low levels of the hydrogenosomal protein ferredoxin and the related protein PFO. Other underexpressed proteins included malic enzyme and hydrogenase.|
Ferredoxin was knocked out with NeoR plasmids. Restriction enzyme, northern and immunoblot analysis verified ablation of ferredoxin. Other genes were unaffected, except for hydrogenosomal PFO (↓↓) and cytosolic hydrogenase (↑↑).Trichomonas vaginalis GenomeThe Trichomonas vaginalis genome is ~160 Mb and haploid. It has over 26,000 genes, with 70% being repetitious. There are seven ferredoxin genes, varying 30-90% in identity to the one knocked out.
|Minimal Lethal Concentration||The minimal lethal concentration (MLC) of metronidazole was about the same for the parental strain and the ferredoxin knockout. Loss of ferredoxin alone therefore does not cause Trichomonas vaginalis cells to become resistant to metronidazole.|
|Ferredoxin Complementation||The metronidazole-resistant Trichomonas foetus strain KV1/MR100 was transformed with a ferredoxin plasmid. The minimal lethal concentration of un-transformed and transformed cells was then compared. Tranformation with ferredoxin significantly restored metronidazole sensitivity.|
|Hydrogenosome Morphology||Sensitive and resistant Trichomonas vaginalis strains underwent microscopy. Hydrogenosomes were equal in quantity across all strains, but were 3-4x smaller in resistant strains. Centrifugation found that resistant strains' hydrogenosomes were less dense and more homogeneous.|
|Interpretation||These results indicate that a loss of hydrogenosomal proteins is involved in drug resistance, but it is unclear if this is a cause or effect. Restoring ferredoxin to a resistant strain increases sensitivity; however, knocking out ferredoxin from a sensitive strain does not confer resistance.|
Drug resistant strains lack one or more hydrogenosomal proteins. Highly drug resistant strains lack multiple hydrogenosomal proteins, and their hydogenosomes are smaller, less dense and more homogenous (formed a narrow band upon centrifugation).
Ferredoxin complementation in a resistant strain confers sensitivity, but knocking out ferredoxin has no effect. Undetected ferredoxin genes may remain; or a ferredoxin- and PFO-independent pathway may exist.
Multi-Drug Resistant Protein
Multi-Drug Resistant Protein (aka MDR or P-glycoprotein) is a ~170 kDa glycoprotein. It is targeted to the plasma membrane, where it inserts twelve transmembrane domains and thereby forms a pore. MDR can bind and pump out numerous drugs, driving the reaction via ATP hydrolysis using its own ATPase domain. In resistant strains it is over-expressed.