The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis.

• Klíčová slova
• drug discovery, drug screening, parasite, protease inhibitor, proteasome, substrate specificity, trichomonas,
• MeSH
• inhibitory proteasomu farmakologie   chemie   MeSH
• katalytická doména * MeSH
• proteasomový endopeptidasový komplex * metabolismus   chemie   MeSH
• protozoální proteiny chemie   metabolismus   antagonisté a inhibitory   genetika   MeSH
• substrátová specifita MeSH
• Trichomonas vaginalis * enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inhibitory proteasomu MeSH
• proteasomový endopeptidasový komplex * MeSH
• protozoální proteiny MeSH

Tick-transmitted Babesia are a major global veterinary threat and an emerging risk to humans. Unlike their Plasmodium relatives, these erythrocyte-infecting Apicomplexa have been largely overlooked and lack specific treatment. Selective targeting of the Babesia proteasome holds promise for drug development. In this study, we screened a library of peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B for their activity against Babesia. Several of these compounds showed activity against both the asexual and sexual blood stages of Plasmodium falciparum. These compounds inactivate β5 proteasome subunit activity in the lysates of Babesia divergens and Babesia microti in the low nanomolar range. Several compounds were tested with the purified B. divergens proteasome and showed IC50 values comparable to carfilzomib, an approved anticancer proteasome inhibitor. They also inhibited B. divergens growth in bovine erythrocyte cultures with solid EC50 values, but importantly, they appeared less toxic to human cells than carfilzomib. These compounds therefore offer a wider therapeutic window and provide new insights into the development of small proteasome inhibitors as selective drugs for babesiosis.

• Publikační typ
• časopisecké články MeSH

The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.

• MeSH
• elektronová kryomikroskopie * MeSH
• inhibitory proteasomu * farmakologie   chemie   MeSH
• lidé MeSH
• molekulární modely MeSH
• proteasomový endopeptidasový komplex * metabolismus   MeSH
• protozoální proteiny metabolismus   antagonisté a inhibitory   genetika   chemie   MeSH
• rekombinantní proteiny * metabolismus   genetika   MeSH
• Trichomonas vaginalis * účinky léků   genetika   enzymologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inhibitory proteasomu * MeSH
• proteasomový endopeptidasový komplex * MeSH
• protozoální proteiny MeSH
• rekombinantní proteiny * MeSH

Iron, as an essential micronutrient, plays a crucial role in host-pathogen interactions. In order to limit the growth of the pathogen, a common strategy of innate immunity includes withdrawing available iron to interfere with the cellular processes of the microorganism. Against that, unicellular parasites have developed powerful strategies to scavenge iron, despite the effort of the host. Iron-sequestering compounds, such as the approved and potent chelator deferoxamine (DFO), are considered a viable option for therapeutic intervention. Since iron is heavily utilized in the mitochondrion, targeting iron chelators in this organelle could constitute an effective therapeutic strategy. This work presents mitochondrially targeted DFO, mitoDFO, as a candidate against a range of unicellular parasites with promising in vitro efficiency. Intracellular Leishmania infection can be cleared by this compound, and experimentation with Trypanosoma brucei 427 elucidates its possible mode of action. The compound not only affects iron homeostasis but also alters the physiochemical properties of the inner mitochondrial membrane, resulting in a loss of function. Furthermore, investigating the virulence factors of pathogenic yeasts confirms that mitoDFO is a viable candidate for therapeutic intervention against a wide spectrum of microbe-associated diseases.

• Klíčová slova
• chelation, iron, mitochondria, parasites, protists,
• MeSH
• antiinfekční látky * MeSH
• antiparazitární látky farmakologie   MeSH
• chelátory železa farmakologie   terapeutické užití   MeSH
• deferoxamin chemie   MeSH
• mitochondrie MeSH
• železo * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• antiinfekční látky * MeSH
• antiparazitární látky MeSH
• chelátory železa MeSH
• deferoxamin MeSH
• železo * MeSH

Babesia divergens is an emerging tick-borne pathogen considered as the principal causative agent of bovine babesiosis in Europe with a notable zoonotic risk to human health. Despite its increasing impact, considerable gaps persist in our understanding of the molecular interactions between this parasite and its hosts. In this study, we address the current limitation of functional genomic tools in B. divergens and introduce a stable transfection system specific to this parasite. We define the parameters for a drug selection system hdhfr-WR99210 and evaluate different transfection protocols for highly efficient generation of transgenic parasites expressing GFP. We proved that plasmid delivery into bovine erythrocytes prior to their infection is the most optimal transfection approach for B. divergens, providing novel evidence of Babesia parasites' ability to spontaneously uptake external DNA from erythrocytes cytoplasm. Furthermore, we validated the bidirectional and symmetrical activity of ef-tgtp promoter, enabling simultaneous expression of external genes. Lastly, we generated a B. divergens knockout line by targeting a 6-cys-e gene locus. The observed dispensability of this gene in intraerythrocytic parasite development makes it a suitable recipient locus for further transgenic application. The platform for genetic manipulations presented herein serves as the initial step towards developing advanced functional genomic tools enabling the discovery of B. divergens molecules involved in host-vector-pathogen interactions.

• Klíčová slova
• 6-cys-e gene knockout, Babesia divergens, GFP-expression, bidirectional promoter, erythrocytes pre-loading, gene targeting, transfection system,
• MeSH
• Babesia * genetika   MeSH
• babezióza * parazitologie   MeSH
• erytrocyty parazitologie   MeSH
• genový targeting MeSH
• lidé MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Proteasomes are essential for protein homeostasis in mammalian cells1-4 and in protozoan parasites such as Trichomonas vaginalis (Tv).5 Tv and other protozoan 20S proteasomes have been validated as druggable targets.6-8 However, in the case of Tv 20S proteasome (Tv20S), biochemical and structural studies were impeded by low yields and purity of the native proteasome. We successfully made recombinant Tv20S by expressing all seven α and seven β subunits together with the Ump-1 chaperone in insect cells. We isolated recombinant proteasome and showed that it was biochemically indistinguishable from the native enzyme. We confirmed that the recombinant Tv20S is inhibited by the natural product marizomib (MZB)9 and the recently developed peptide inhibitor carmaphycin-17 (CP-17)8,10. Specifically, MZB binds to the β1, β2 and β5 subunits, while CP-17 binds the β2 and β5 subunits. Next, we obtained cryo-EM structures of Tv20S in complex with these covalent inhibitors at 2.8Å resolution. The structures revealed the overall fold of the Tv20S and the binding mode of MZB and CP-17. Our work explains the low specificity of MZB and higher specificity of CP-17 towards Tv20S as compared to human proteasome and provides the platform for the development of Tv20S inhibitors for treatment of trichomoniasis.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH