Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

Trichomoniasis is the most common, sexually transmitted infection.It is caused by the flagellated protozoan parasite Trichomonas vagina/is. Symptoms include vaginitis and infections have been associatedwith preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDSand cervical cancer. Trichomoniasis has the highest prevalence and incidence of any sexually transmitted infection. The 5-nitroimidazole drugs, of which metronidazole is the most prescribed, are the only approved,effective drugs to treat trichomoniasis. Resistance against metronidazole is frequently reported and cross-resistance among the family of 5-nitroimidazole drugs is common, leaving no alternative for treatment, withsome cases remaining unresolved. The mechanism of metronidazole resistance in T. Vagina/is from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasingmet ronidazole pressure. In the latter situation, hydrog enosomal function which is involved in activationof the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal ofdrug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintaintheir resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded asa laboratory induced phenomenon, it clearly has been demonstrated in clinical isolates. Pursuit of both approaches will allow dissection of the underlying mechanisms. Many alternative drugs and treatments have been tested in vivo in cases of refractory trichomoniasis, as well as in vitro with some successes including the broad spectrum anti-parasitic drug nitazoxanide. Drug resistance incidence in T. Vagina/is appears to be on the increase and improved surveillance of treatment failures is urged.     

阴道毛滴虫多克隆抗体的制备及Fd基因的原核表达

目的 将阴道毛滴虫铁氧还蛋白（ferredoxin,Fd）基因在大肠埃希菌中诱导表达。方法制备阴道毛滴虫可溶性抗原,多点注射免疫家兔,获得的血清用ELISA测定其抗体效价。将原核表达重组质粒pET3C-Fd转化入大肠埃希菌BL21（DE3）感受态细胞中,异丙基-β-D-硫代半乳糖苷（IPTG）诱导蛋白质表达。结果制备出抗阴道毛滴虫多克隆抗体,抗体效价在1：8000以上,用于免疫印迹实验。经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹（Western blot）分析,重组质粒在大肠埃希菌中表达出Fd。结论在大肠埃希菌中表达出了Fd。     

阴道毛滴虫铁氧还蛋白与甲硝唑抵抗的研究进展

氢化酶体是阴道毛滴虫重要的代谢器官,该器官内存在的铁氧还蛋白不仅是虫体代谢过程中主要的电子传递介体,而且也在甲硝唑的激活中起关键作用。近年来阴道毛滴虫的甲硝唑抵抗株在临床和实验室都有报道,实验研究发现活化药物的铁氧还蛋白减少或缺失,因此对铁氧还蛋白与甲硝唑抵抗的相关性研究越来越受到医学及药学界的重视。本文总结近年来该领域的研究成果及发展动态,以期对滴虫药物抵抗的发生机制以及滴虫病防治的研究提供有价值的资料。     

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.     

Location of the cell-binding domain of CP65, a 65kDa cysteine proteinase involved in Trichomonas vaginalis cytotoxicity

The cysteine proteinase (CP) of 65 kDa, CP65, binds to the surface of HeLa cells and is involved in Trichomonas vaginalis cellular damage. To identify and locate the CP65 cellular-binding domain, we enriched the CP65 protein band by ammonium sulfate fractionation and ion-exchange chromatography and the N-terminal sequence was obtained. A 618 bp gene fragment was obtained by PCR using genomic DNA as template and primers derived from the N-terminal sequence of CP65 and the Asn papain-catalytic conserved region. This gene fragment encodes for 206 amino acid (aa) residues corresponding to the N-terminal region of a mature CP with 67–76% identity to the reported trichomonad cathepsin-L-like CPs. This gene fragment was expressed in a bacterial system for antibody production and functional analysis. Antibodies against the native trichomonad CP65 recognized the recombinant protein, referred to as rCP65, confirming its relationship with the CP65 gene. The rCP65 protein was bound to the surface of HeLa cells and competed with the native CP65 for binding. Antibodies to the rCP65 (-rCP65) reacted with the trichomonad CP65 located on the parasite surface, and inhibited trichomonal cytotoxicity in a concentration-dependent manner. These data strongly suggest that this gene fragment encodes for the putative cell-binding domain (CBD) of CP65 located at its N-terminal region.     

Taurine is a weak scavenger of peroxynitrite and does not attenuate sodium nitroprusside toxicity to cells in culture

Summary. Many studies have suggested an antioxidant role for taurine, but few studies have directly measured its free radical scavenging activity. The aim of the present study was to directly determine the action of taurine and taurine analogs to inhibit peroxynitrite-mediated oxidation of dihydrorhodamine 123 (DHR) to rhodamine. Taurine was also tested to determine if it could attenuate the toxicity of sodium nitroprusside (SNP) to neuronal cultures. Taurine at concentrations above 30 mM had a modest ability to inhibit peroxynitrite formation derived from SIN-1. Hypotaurine could inhibit peroxynitrite formation from both SIN-1 (↓75%) and SNP (↓50%) at 10 mM. Other taurine analogs (homotaurine, β-alanine & isethionic acid) slightly potentiated DHR oxidation by SIN-1. Short-term (1-hour) treatment of PC12 cultures with either SNP (1–2 mM) or taurine (20–40 mM) appeared to induce cellular proliferation. In contrast, 24-hour treatment with SNP (1 mM) induced cell death. Combination treatments with taurine and SNP appeared to interact in an additive fashion for both cell proliferation and neurotoxic actions. It appears unlikely that taurine is a major endogenous scavenger of peroxynitrite. Received May 9, 2000 Accepted June 13, 2000     

A short-term,simple method for detection of N-nitrosocompounds produced from sodium nitrite and morpholine in stomach

Syrian hamsters in the 11th or 12th day of pregnancy were given sodium nitrite and morpholine simultaneously by stomach tube. The embryonic cells were cultured for 72 h in normal MEM medium plus 10% fetal calf serum and then transferred into medium containing 8-azaguanine. After cultivation in the selection medium, number of 8 azaguanine-resistant colonies was scored. As the results, this oral concurrent transplacental application of sodium nitrite and morpholine can cause 8 azaguanine-resistant mutants on the cultured embryonic cells from mothers that received these chemicals. Nitrosomorpholine was only detected in stomach of animals treated with sodium nitrite and morpholine.     

A natural fusion of flavodiiron,rubredoxin, and rubredoxin oxidoreductase domains is a self-sufficient water-forming oxidase of Trichomonas vaginalis

Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H₂O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H₂O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD⁺ while simultaneously removing O₂ from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX–like enzyme and instead harbors three paralogous genes (FDPF1–3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a “stand-alone” FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.     

The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases

The pyrophosphate-dependent phosphofructokinase (PP_i-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5′ half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PP_i-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PP_i-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PP_i-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the α- and β-subunits of plant PP_i-PFKs. The third group (``X') containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group (``Y') comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PP_i-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers. Received: 5 December 1997 / Accepted: 18 March 1998