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Metronidazole and other 5-nitroimidazoles (5-NI) are among the most effective antimicrobials available against many important anaerobic pathogens, but evolving resistance is threatening their long-term clinical utility. The common 5-NIs were developed decades ago, yet little 5-NI drug development has since taken place, leaving the true potential of this important drug class unexplored. Here we report on a unique approach to the modular synthesis of diversified 5-NIs for broad exploration of their antimicrobial potential. Many of the more than 650 synthesized compounds, carrying structurally diverse functional groups, have vastly improved activity against a range of microbes, including the pathogenic protozoa Giardia lamblia and Trichomonas vaginalis, and the bacterial pathogens Helicobacter pylori, Clostridium difficile, and Bacteroides fragilis. Furthermore, they can overcome different forms of drug resistance, and are active and nontoxic in animal infection models. These findings provide impetus to the development of structurally diverse, next-generation 5-NI drugs as agents in the antimicrobial armamentarium, thus ensuring their future viability as primary therapeutic agents against many clinically important infections.

Metronidazole (MTR) is frequently used for the treatment of Blastocystis infections, but with variable effectiveness, and often with treatment failures as a possible result of drug resistance. We have developed two Blastocystis MTR-resistant (MTR(R)) subtype 4 WR1 lines (WR1-M4 and WR1-M5), with variable susceptibility to a panel of anti-protozoal agents including various 5-nitroimidazoles, nitazoxanide and furazolidone. WR1-M4 and WR1-M5 were developed and assessed over an 18-month period and displayed persistent MTR resistance, being more than 2.5-fold less susceptible to MTR than the parent isolate. The MTR(R) lines grew with a similar g time to WR1, but were morphologically less consistent with a mixture of size. All Blastocystis isolates and the MTR(R) lines were most susceptible to the 5-nitroimidazole drug ronidazole. WR1-M5 was apparently cross-resistant to satranidazole and furazolidone, and WR1-M4 was cross-resistant to nitazoxanide. These MTR(R) lines now provide a valuable tool for the continued assessment of the efficacy and mechanism of action of new and established drugs against a range of Blastocystis sp. subtypes, in order to identify a universally effective drug and to facilitate understanding of the mechanisms of drug action and resistance in Blastocystis.

Infections with the diarrheagenic protozoan pathogen Giardia lamblia are most commonly treated with metronidazole (Mz). Treatment failures with Mz occur in 10 to 20% of cases and Mz resistance develops in the laboratory, yet clinically, Mz-resistant (Mz(r)) G. lamblia has rarely been isolated from patients. To understand why clinical Mz(r) isolates are rare, we questioned whether Mz resistance entails fitness costs to the parasite. Our studies employed several newly generated and established isogenic Mz(r) cell lines with stable, high-level resistance to Mz and significant cross-resistance to tinidazole, nitazoxanide, and furazolidone. Oral infection of suckling mice revealed that three of five Mz(r) cell lines could not establish infection, while two Mz(r) cell lines infected pups, albeit with reduced efficiencies. Failure to colonize resulted from a diminished capacity of the parasite to attach to the intestinal mucosa in vivo and to epithelial cells and plastic surfaces in vitro. The attachment defect was related to impaired glucose metabolism, since the noninfectious Mz(r) lines consumed less glucose, and glucose promoted ATP-independent parasite attachment in the parental lines. Thus, resistance of Giardia to Mz is accompanied by a glucose metabolism-related attachment defect that can interfere with colonization of the host. Because glucose-metabolizing pathways are important for activation of the prodrug Mz, it follows that a fitness trade-off exists between diminished Mz activation and reduced infectivity, which may explain the observed paucity of clinical Mz(r) isolates of Giardia. However, the data also caution that some forms of Mz resistance do not markedly interfere with in vivo infectivity.

Objectives: The mechanism of action of, and resistance to, metronidazole in the anaerobic (or micro-aerotolerant) protozoan parasite Giardia lamblia has long been associated with the reduction of ferredoxin (Fd) by the enzyme pyruvate:ferredoxin oxidoreductase (PFOR) and the subsequent activation of metronidazole by Fd to toxic radical species. Resistance to metronidazole has been associated with down-regulation of PFOR and Fd. The aim of this study was to determine whether the PFOR/Fd couple is the only pathway involved in metronidazole activation in Giardia., Methods: PFOR and Fd activities were measured in extracts of highly metronidazole-resistant (MTR(r)) lines and activities of recombinant G. lamblia thioredoxin reductase (GlTrxR) and NADPH oxidase were assessed for their involvement in metronidazole activation and resistance., Results: We demonstrated that several lines of highly MTR(r) G. lamblia have fully functional PFOR and Fd indicating that PFOR/Fd-independent mechanisms are involved in metronidazole activation and resistance in these cells. Flavin-dependent GlTrxR, like TrxR of other anaerobic protozoa, reduces 5-nitroimidazole compounds including metronidazole, although expression of TrxR is not decreased in MTR(r) Giardia. However, reduction of flavins is suppressed in highly MTR(r) cells, as evidenced by as much as an 80% decrease in NADPH oxidase flavin mononucleotide reduction activity. This suppression is consistent with generalized impaired flavin metabolism in highly MTR(r) Trichomonas vaginalis., Conclusions: These data add to the mounting evidence against the dogma that PFOR/Fd is the only couple with a low enough redox potential to reduce metronidazole in anaerobes and point to the multi-factorial nature of metronidazole resistance.

We barcoded 25 in vitro isolates (representing 92 samples) of Giardia duodenalis from humans and other animals, which have been assembled by the Upcroft team at the Queensland Institute of Medical Research over a period of almost three decades. We used mutation scanning-coupled sequencing of loci in the triosephosphate isomerase, glutamate dehydrogenase and β-giardin genes, combined with phylogenetic analysis, to genetically characterise them. Specifically, the isolates (n514) of G. duodenalis from humans from Australia (AD113; BRIS/83/HEPU/106; BRIS/87/HEPU/713; BRIS/89/HEPU/1003; BRIS/92/HEPU/1541; BRIS/92/HEPU/1590; BRIS/92/HEPU/2443; BRIS/93/HEPU/1706), Malaysia (KL/92/IMR/1106) and Afghanistan (WB), a cat from Australia (BAC2), a sheep from Canada (OAS1) and a sulphur-crested cockatoo from Australia (BRIS/95/HEPU/2041) represented assemblage A (sub-assemblage AI-1, AI-2 or AII-2); isolates (n510) from humans from Australia (BRIS/91/HEPU/1279; BRIS/92/HEPU/2342; BRIS/92/HEPU/2348; BRIS/93/HEPU/1638; BRIS/93/HEPU/1653; BRIS/93/HEPU/1705; BRIS/93/HEPU/1718; BRIS/93/HEPU/1727), Papua New Guinea (BRIS/92/HEPU/1487) and Canada (H7) represented assemblage B (sub-assemblage BIV) and an isolate from cattle from Australia (BRIS/92/HEPU/1709) had a match to assemblage E. Isolate BRIS/90/HEPU/1229 from a human from Australia was shown to represent a mixed population of assemblages A and B. These barcoded isolates (including stocks and derived lines) now allow direct comparisons of experimental data among laboratories and represent a massive resource for transcriptomic, proteomic, metabolic and functional genomic studies using advanced molecular technologies.

Two genotypes, assemblages A and B, of the pathogenic gut protozoan parasite Giardia lamblia infect humans. Symptoms of infection range from asymptomatic to chronic diarrhea. Giardia chromosomes have long been characterized but not until the publication of the first Giardia genome sequence was chromosome mapping work, commenced nearly two decades ago, completed. Initial mapping studies identified and ordered Not I chromosome segments (summating to 1.8 Mb) of the estimated 2 Mb chromosome 3. The resulting map was confirmed with the release of the Giardia genome sequence and this revitalized mapping. The result is that 93% of the WB isolate genome sequence has now been assigned to one of five major chromosomes, and community access to these data has been made available through GiardiaDB, the database for Giardia genomes., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

This study investigates the susceptibility of a clinically metronidazole (Mz)-resistant isolate of Trichomonas vaginalis to alternative anti-trichomonal compounds. The microaerobic minimal inhibitory concentration (MIC) of the 5-nitroimidazole (NI) drug, Mz, against a typical Mz-susceptible isolate of T. vaginalis is around 3.2 microM Mz while the clinically, highly Mz-resistant isolate has an MIC of 50-100 microM. This isolate was cross-resistant to other members of the 5-NI family of compounds including tinidazole and other experimental compounds and maintained resistance under anaerobic conditions. In addition, this isolate was cross-resistant to the 5-nitrothiazole compound nitazoxanide and the 5-nitrofuran derivative, furazolidone. Adenosine analogues toyocamycin and 2-fluoro-2'-deoxyadenosine with no nitro group were also less effective against the clinically Mz-resistant isolate than a Mz-susceptible one. Three other isolates which were determined to be Mz-resistant soon after isolation lost resistance in the long term. One other isolate has maintained some level of permanent Mz resistance (MIC of 25 microM). A multi-drug resistance mechanism may be involved in these clinically Mz-resistant isolates.

The 5-nitroimidazole (NI) compound C17, with a side chain carrying a remote phenyl group in the 2-position of the imidazole ring, is at least 14-fold more active against the gut protozoan parasite Giardialamblia than the 5-NI drug metronidazole (MTR), with a side chain in the 1-position of the imidazole ring, which is the primary drug for the treatment of giardiasis. Over 10 months, lines resistant to C17 were induced in vitro and were at least 12-fold more resistant to C17 than the parent strains. However, these lines had ID(90) values (concentration of drug at which 10% of control parasite ATP levels are detected) for MTR of >200 microM, whilst lines induced to be highly resistant to MTR in vitro have maximum ID(90) values around 100 microM (MTR-susceptible isolates typically have an ID(90) of 5-12.8 microM). The mechanism of MTR activation in Giardia apparently involves reduction to toxic radicals by the activity of pyruvate:ferredoxin oxidoreductase (PFOR) and the electron acceptor ferredoxin. MTR-resistant Giardia have decreased PFOR activity, which is consistent with decreased activation of MTR in these lines, but C17-resistant lines have normal levels of PFOR. Therefore, an alternative mechanism of resistance in Giardia must account for these super-MTR-resistant cells., ((c) 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

The gut protozoan parasite, Giardia lamblia (Assemblage A), has 5 major chromosomes, 1 of which is 2 Mb, as determined from gel separations of whole chromosomes. We originally published a physical map of this chromosome and, now, using the sequence data from 46 chromosome-specific probes, have produced a sequence map of the 2 Mb chromosome. Comparison of the probe sequences with the Giardia genome database (http://GiardiaDB.org) has identified 4 scaffolds (CH991771, CH991780, CH991782, and CH991767) belonging to the 2 Mb, Assemblage A, chromosome. Because of the density of probe sequences, we have been able to predict the orientation of the scaffolds and have identified erroneous inclusions in scaffold CH991767. Exclusion of erroneously included sequences resulted in a 1.96 Mb chromosome sequence. This study brings together experimental data and the GiardiaDB data to compile the sequence of a whole chromosome.

Trichomonas vaginalis is the most common sexually transmitted protozoan in the world and its resistance to metronidazole is increasing. The purpose of this study was to demonstrate that clinical metronidazole resistance in T. vaginalis does not occur via the same mechanism as laboratory-induced metronidazole resistance--that is, via hydrogenosome down sizing. Ultrathin sections of this parasite were examined using transmission electron microscopy and the size and area of the cell and hydrogenosomes were compared between drug-resistant laboratory lines and clinically resistant isolates. Clinical metronidazole-resistant T. vaginalis had similar-sized hydrogenosomes as a metronidazole-sensitive isolate. Inducing metronidazole resistance in both of these isolates caused down sizing of hydrogenosomes. Inducing toyocamycin resistance did not cause any ultrastructural changes to the cell or to the hydrogenosome. No correlation between hydrogenosome number and the drug-resistant status of T. vaginalis isolates and lines was observed. This report demonstrates that clinical metronidazole resistance is not associated with down-sized hydrogenosomes, thus indicating that an alternative resistance mechanism is used by T. vaginalis.

A 58-year-old Caucasian woman presented with a 14-month history of persistent trichomoniasis not responsive to numerous courses of metronidazole and tinidazole. Vaginal secretion samples were obtained for sensitivity testing and treatment recommendations. In vitro susceptibility testing revealed the patient's Trichomonas vaginalis isolate was highly resistant to both metronidazole and tinidazole. She was treated with topical furazolidone and experienced a complete symptomatic cure and culture remained negative 35 days post furazolidone treatment.

Background: The prevalence of the sexually transmissible protozoan parasite Trichomonas vaginalis in the highlands of Papua New Guinea (PNG) has been reported to be as high as 46% and although not previously studied in Papua New Guinea, clinical resistance against metronidazole (Mz), the drug most commonly used to treat trichomoniasis, is well documented worldwide. This study was primarily aimed at assessing resistance to Mz in T. vaginalis strains from the Goroka region., Methods: Consenting patients presenting at the Goroka Base Hospital Sexually Transmitted Diseases (STD) Clinic and local women were asked to provide two vaginal swabs: one for culturing of the parasite; and one for polymerase chain reaction detection of T. vaginalis, Chlamydia trachomatis and Neisseria gonorrhoeae. T. vaginalis isolates were assayed for Mz susceptibility and a selection was genotyped., Results: The prevalence of T. vaginalis was determined to be 32.9% by culture and polymerase chain reaction of swabs among 82 local women and patients from the STD clinic. An unexpectedly high level of in vitro Mz resistance was determined with 17.4% of isolates displaying unexpectedly high resistance to Mz. The ability to identify isolates of T. vaginalis by genotyping was confirmed and the results revealed a more homogeneous T. vaginalis population in Papua New Guinea compared with isolates from elsewhere., Conclusion: T. vaginalis is highly prevalent in the Goroka region and in vitro Mz resistance data suggest that clinical resistance may become an issue.

Infections with the diarrheagenic pathogen, Giardia lamblia, are commonly treated with the 5-nitroimidazole (5-NI) metronidazole (Mz), and yet treatment failures and Mz resistance occur. Using a panel of new 2-ethenyl and 2-ethanyl 5-NI derivatives, we found that compounds with a saturated bridge between the 5-NI core and a pendant ring system exhibited only modestly increased antigiardial activity and could not overcome Mz resistance. By contrast, olefins with a conjugated bridge connecting the core and a substituted phenyl or heterocyclic ring showed greatly increased antigiardial activity without toxicity, and several overcame Mz resistance and were more effective than Mz in a murine giardiasis model. Determination of the half-wave potential of the initial one-electron transfer by cyclic voltammetry revealed that easier redox activation correlated with greater antigiardial activity and capacity to overcome Mz resistance. These studies show the potential of combining systematic synthetic approaches with biological and electrochemical evaluations in developing improved 5-NI drugs.

The genome of the gut protozoan parasite Giardia duodenalis (assemblage A) has been sequenced and compiled as contigs and scaffolds (GiardiaDB- http://GiardiaDB.org ), but specific chromosome location of all scaffolds is unknown. To determine which scaffolds belong to the 3-Mb chromosome, a library of probes specific for this chromosome was constructed. The probes were hybridised to NotI-cleaved whole chromosomes, and the combined size of different NotI segments identified by the probes was 2,225 kb indicating the probes were well distributed along the 3-Mb chromosome. Six scaffolds (CH991814, CH991779, CH991793, CH991763, CH991764, and CH991761) were identified as belonging to the 3-Mb chromosome, and these scaffolds were ordered and oriented according to scaffold features including I-PpoI sites and hybridisation pattern. However, the combined size of scaffolds was more than 4 Mb. Approximately, 1 Mb of scaffold CH991763 carrying previously identified sequences specific for the 1.5-Mb chromosome(s) including subtelomeric sequence was reassigned, and several other anomalies were addressed such that the final size of the apparently 3-Mb chromosome is estimated to be 2,885 kb. This work addresses erroneous computer-based assignment of a number of contigs and emphasises the need for alternative and confirmatory methods of scaffold construction.

Objectives: Two of the major diagnostic methods for Trichomonas vaginalis, wet mount and culture, rely on the continued viability of the organism. Methods to increase the viability of T. vaginalis in urine are needed., Goal: The goal of this study was to develop a method that increases the time of viability of T. vaginalis in urine., Study Design: Urine samples were inoculated with trichomonads, held at either room temperature or 37 degrees C, and processed through a column and frit, which was then placed in either a tube of culture medium containing antibiotics or a TV InPouch., Results: The column and polyethylene frit system was found to increase the duration of viability for T. vaginalis from urine specimens at least 6-fold., Conclusion: This novel method, which uses a column and frit system to increase the duration of viability of the organism, has the potential to increase the sensitivity of diagnostic tests.

We describe the genome sequence of the protist Trichomonas vaginalis, a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the approximately 160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.

Antiretroviral protease inhibitors were assessed in vitro for their activity against Giardia duodenalis and Trichomonas vaginalis. Kaletra (a co-formulation of ritonavir and lopinavir) was the most effective overall, with 50% effective drug concentrations (EC(50)) of 1.1-2.7 microM (ritonavir concentration) against G. duodenalis and 6.8-8 microM against metronidazole-sensitive and clinically metronidazole-resistant T. vaginalis. Minimal inhibitory concentrations were 2-2.5 microM and 10-50 microM for G. duodenalis and T. vaginalis, respectively. Within the range of human plasma concentrations for ritonavir, only G. duodenalis was inhibited. Lopinavir alone was less inhibitory than ritonavir but was associated with a blockage in cytokinesis of G. duodenalis trophozoites. Saquinavir was not effective. These findings are significant considering the association between human immunodeficiency virus and T. vaginalis, and between G. duodenalis and homosexual behaviour.

A genotyping method has been developed to distinguish each Trichomonas vaginalis isolate and has provided the first genome mapping studies of this protist with an estimated 180Mb genome. The technique was developed using high molecular weight DNA prepared from five laboratory isolates from Australia and USA and 20 clinical isolates from South Africa. Inhibition of the notorious T. vaginalis endogenous nucleases by addition of potent inhibitors was essential to the success of this study. Chromosomal DNA larger than 2.2Mb was macrorestricted to a minimum segment size of approximately 50kb, separated by pulsed field gel electrophoresis and hybridised with a variety of gene probes. Each isolate generated a unique pattern that was distinguished by each of the probes. Four single copy gene probes (fd, hmp35, ibp39 and pfoD) were identified but probes which identified several bands (pfoB and alpha-scs) per isolate were most informative for genotyping. The pyruvate:ferredoxin oxidoreductase B gene probe identified two to seven copies of pfoB (or its closely related homologue pfoA) per genome in different isolates and is an obvious candidate probe to identify epidemiological linkage between infections by this genotyping method. Cleavage of the genomes into smaller fragments failed to distinguish isolates from diverse locations indicating the proximal regions of genes are conserved.

Metronidazole (Mz)-resistant Giardia and Trichomonas were inhibited by 1 of 30 new 5-nitroimidazole drugs. Another five drugs were effective against some but not all of the Mz-resistant parasites. This study provides the incentive for the continued design of 5-nitroimidazole drugs to bypass cross-resistance among established 5-nitromidazole antiparasitic drugs.

Giardia duodenalis has linear chromosomes capped with typical eukaryotic repeats [(TAGGG)n], subtelomeric rRNA genes, and telomere gene units. The absence of two closely associated NotI sites in the large-subunit rRNA gene was used as an indicator in hybridizations of one- and two-dimensional NotI-cleaved Giardia chromosome separations that some chromosomes carry only rearranged and, by deduction, nonfunctional rRNA genes.

The flagellated protozoa Giardia duodenalis is the most commonly detected parasite in the intestinal tract of humans. Infections with the parasite result in diarrhoeal disease in humans and animals, with infants at risk from failure-to-thrive syndrome. The incidence of giardiasis worldwide may be as high as 1000 million cases. Current recommended treatments include the nitroheterocyclic drugs tinidazole, metronidazole and furazolidone, the substituted acridine, quinacrine, and the benzimidazole, albendazole. Paromomycin is also used in some situations, and nitazoxanide is proving to be useful. However, treatment failures have been reported with all of the common antigiardial agents, and drug resistance to all available drugs has been demonstrated in the laboratory. In addition, clinical resistance has been reported, including cases where patients failed both metronidazole and albendazole treatments. The identification of new antigiardial drugs is an important consideration for the future, but maintaining the usefulness of the existing drugs is the most cost-effective measure to ensure the continued availability of antigiardial drugs.

Trichomoniasis is the most common, sexually transmitted infection. It is caused by the flagellated protozoan parasite Trichomonas vaginalis. Symptoms include vaginitis and infections have been associated with preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDS and 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, with some cases remaining unresolved. The mechanism of metronidazole resistance in T. vaginalis from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasing metronidazole pressure. In the latter situation, hydrogenosomal function which is involved in activation of the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal of drug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintain their resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded as a 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. vaginalis appears to be on the increase and improved surveillance of treatment failures is urged.

This study examines the ability of Giardia duodenalis trophozoites, isolated from a wild bird, to colonize the intestinal tracts of companion animals (kittens) and domestic ruminants (lambs). Trophozoites colonized the intestinal tracts of intraduodenally inoculated animals as demonstrated by increasing parasite burdens within the duodenum and jejunum and by fecal passage of cysts within 4 days post-inoculation. The pathogenesis of the trophozoites was further investigated in kittens. In these animals, infection significantly reduced jejunal brush border microvillous length and density, which resulted in a loss of overall epithelial brush border surface area. This injury was associated with the production of diarrhea in four of five infected kittens. These findings indicate that some bird species may carry G. duodenalis that represent a possible health threat to companion animals and livestock. Our results describe the first successful colonization of avian-derived G. duodenalis trophozoites in the small intestines of domestic kittens and lambs.

Primary vaccine strategies against group A streptococci (GAS) have focused on the M protein--the target of opsonic antibodies important for protective immunity. We have previously reported protection of mice against GAS infection following parenteral delivery of a multi-epitope vaccine construct, referred to as a heteropolymer. This current report has assessed mucosal (intranasal (i.n.) and oral) delivery of the heteropolymer in mice with regard to the induction and specificity of mucosal and systemic antibody responses, and compared this to parenteral delivery. GAS-specific IgA responses were detected in saliva and gut upon i.n. and oral delivery of the heteropolymer co-administered with cholera toxin B subunit, respectively. High titre serum IgG responses were elicited to the heteropolymer following all routes of delivery when administered with adjuvant. Moreover, as with parenteral delivery, serum IgG antibodies were detected to the individual heteropolymer peptides following i.n. but not oral delivery. These data support the potential of the i.n. route in the mucosal delivery of a GAS vaccine.

Two series of benzimidazole derivatives were sythesised. The first one was based on 5,6-dinitrobenzimidazole, the second one comprises 2-thioalkyl- and thioaryl-substituted modified benzimidazoles. Antibacterial and antiprotozoal activity of the newly obtained compounds was studied. Some thioalkyl derivatives showed remarkable activity against nosocomial strains of Stenotrophomonas malthophilia, and an activity comparable to that of metronidazole against gram-positive and gram-negative bacteria. Of the tested compounds, 5,6-dichloro-2-(4-nitrobenzylthio)-benzimidazole showed the most distinct antiprotozoal activity.

Entamoeba histolytica is the pathogenic species of Entamoeba that causes amoebic dysentery and other invasive disease. The morphologically similar species, E. dispar, is non-pathogenic and accounts for about 90% of the previously estimated 500 million E. histolytica infections world-wide. Because of the recent redefinition of E. histolytica and E. dispar, and the limited number of drugs available to treat amoebiasis, a new approach to treatment of individuals carrying these parasites is necessary. A meeting of eminent scientists has recently agreed that on no account should prophylaxis against amoebiasis be given, and no treatment without symptoms should be administered. The expense of treating asymptomatic individuals, both monetary and at the risk of over-use of precious drugs, does not appear to be justified. It would seem wise that we preserve currently effective anti-amoebic drugs and avoid the development of drug-resistant E. histolytica.

We have identified novel adjuvant activity in specific cytosol fractions from trophozoites of Giardia isolate BRIS/95/HEPU/2041 (J. A. Upcroft, P. A. McDonnell, and P. Upcroft, Parasitol. Today, 14:281-284, 1998). Adjuvant activity was demonstrated in the systemic and mucosal compartments when Giardia extract was coadministered orally with antigen to mice. Enhanced antigen-specific serum antibody responses were demonstrated by enzyme-linked immunosorbent assay to be comparable to those generated by the "gold standard," mucosal adjuvant cholera toxin. A source of adjuvant activity was localized to the cytosolic component of the parasite. Fractionation of the cytosol produced fraction pools, some of which, when coadministered with antigen, stimulated an enhanced antigen-specific serum response. The toxic component of conventional mucosal adjuvants is associated with adjuvant activity; therefore, in a similar way, the toxin-like attributes of BRIS/95/HEPU/2041 may be responsible for its adjuvanticity. Complete characterization of the adjuvant is under way.

A simple technique for routine, reproducible global surveillance of the drug susceptibility status of the anaerobic protozoa Trichomonas, Entamoeba, and Giardia is described. Data collected using this technique can be readily compared among different laboratories and with previously reported data. The technique employs a commercially available sachet and bag system to generate a low-oxygen environment and log(2) drug dilutions in microtiter plates, which can be monitored without aerobic exposure, to assay drug-resistant laboratory lines and clinically resistant isolates. MICs (after 2 days) of 3.2 and 25 microM indicated metronidazole-sensitive and highly clinically resistant isolates of T. vaginalis in anaerobic assays, respectively. The aerobic MICs were 25 and >200 microM. MICs (1 day) of 12.5 to 25 microM were found for axenic lines of E. histolytica, and MICs for G. duodenalis (3 days) ranged from 6.3 microM for metronidazole-sensitive isolates to 50 microM for laboratory metronidazole-resistant lines. This technique should encourage more extensive monitoring of drug resistance in these organisms.

The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract, fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent, and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined. Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated using laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.

Numerous studies have demonstrated various strain differences between Giardia isolates, but little is known about the immunology and pathogenesis of infections. This study aimed to compare host responses to strains of Giardi duodenalis differing in levels of virulence and pathogenicity and, by doing so, elucidate the mechanisms via which pathogenic strains establish infections. Marked differences were found in the infection dynamics, histopathological responses and serum antibody responses of neonatal mice infected with either G. duodenalis strain BRIS/83/HEPU/106 (isolated from a human) or BRIS/95/HEPU/2041 (isolated from a sulphur-crested cockatoo, Cacatua galerita). Infections with the bird strain were more intense (6.7-times greater) and persisted longer (by 14days) than infections with the human strain. The bird strain was more pathogenic and caused greater pathophysiological alteration to the gut mucosa, including increased villous atrophy, hyperplasia of goblet cells and vacuolated epithelial cells. Mice infected with the bird strain produced less serum anti-Giardia IgA and IgM, but more total (non-specific) serum IgA than those infected with the human strain of Giardia. This suggests that avian G. duodenalis strains are infective for mammalian hosts and may contribute to zoonotic infections. Furthermore, infection of mice with BRIS/95/HEPU/2041 serves as a good experimental model to provide further insight into the mechanisms via which G. duodenalis causes disease.

In anaerobes, decarboxylation of pyruvate is executed by the enzyme pyruvate:ferredoxin oxidoreductase, which donates electrons to ferredoxin. The pyruvate:ferredoxin oxidoreductase and its homologues utilise many alternative substrates in bacterial anaerobes. The pyruvate:ferredoxin oxidoreductase from anaerobic protozoa, such as Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica have retained this diversity in usage of alternative keto acids for energy production utilising a wide variety of substrates. In addition to this flexibility, both T. vaginalis and G. duodenalis have alternative enzymes that are active in metronidazole-resistant parasites and that do not necessarily involve donation of electrons to characterized ferredoxins. Giardia duodenalis has two oxoacid oxidoreductases, including pyruvate:ferredoxin oxidoreductase and T. vaginalis has at least three. These alternative oxoacid oxidoreductases apparently do not share homology with the characterized pyruvate:ferredoxin oxidoreductase in either organism. Independently, both G. duodenalis and T. vaginalis have retained alternative oxoacid oxidoreductase activities that are clearly important for the survival of these parasitic protists.

We have induced high levels of resistance to metronidazole (1 mM or 170 microg ml(-1)) in two different strains of Trichomonas vaginalis (BRIS/92/STDL/F1623 and BRIS/92/STDL/B7708) and have used one strain to identify two alternative T. vaginalis 2-keto acid oxidoreductases (KOR) both of which are distinct from the already characterised pyruvate:ferredoxin oxidoreductase (PFOR). Unlike the characterised PFOR which is severely down-regulated in metronidazole-resistant parasites, both of the alternative KORs are fully active in metronidazole-resistant T. vaginalis. The first, KORI, localized in all membrane fractions but predominantly in the hydrogenosome fraction, is soluble in Triton X-100 and the second, KOR2, is extractable in 1 M acetate from membrane fractions of metronidazole-resistant parasites. PFOR and both KORI and KOR2 use a broad range of 2-keto acids as substrates (pyruvate, alpha-ketobutyrate, alpha-ketomalonate), including the deaminated forms of aromatic amino acids (indolepyruvate and phenylpyruvate). However, unlike PFOR neither KORI or KOR2 was able to use oz-ketoglutarate. Deaminated forms of branched chain amino acids (alpha-ketoisovalerate) were not substrates for T. vaginalis KORs. Since KOR I and KOR2 do not apparently donate electrons to ferredoxin, and are not down-regulated in metronidazole-resistant parasites, we propose that KORI and KOR2 provide metronidazole-resistant parasites with an alternative energy production pathway(s) which circumvents metronidazole activation.

The efficacies of 12 5-nitroimidazole compounds and 1 previously described lactam-substituted nitroimidazole with antiparasitic activity, synthesized via SRN1 and subsequent reactions, were assayed against the protozoan parasites Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica. Two metronidazole-sensitive lines and two metronidazole-resistant lines of Giardia and one line each of metronidazole-sensitive and -resistant Trichomonas were tested. All except one of the compounds were as effective or more effective than metronidazole against Giardia and Trichomonas, but none was as effective overall as the previously described 2-lactam-substituted 5-nitroimidazole. None of the compounds was markedly more effective than metronidazole against Entamoeba. Significant cross-resistance between most of the drugs tested and metronidazole was evident among metronidazole-resistant lines of Giardia and Trichomonas. However, some drugs were lethal to metronidazole-resistant Giardia and had minimum lethal concentrations similar to that of metronidazole for drug-susceptible parasites. This study emphasizes the potential in developing new nitroimidazole drugs which are more effective than metronidazole and which may prove to be useful clinical alternatives to metronidazole.

Early in 1995, a sulphur-crested cockatoo captured in the wild died along with several other cage mates, apparently of an overwhelming, acute infection of Giardia. Trophozoites isolated from the dead bird and established in traditional Giardia axenic medium were infective to mice and established chronic infections associated with weight gain impairment. Genetically and morphologically, the Giardia isolated from the bird belonged to the duodenalis group. Here, Jacqui Upcroft, Ann McDonnell and Peter Upcroft present data on pathogenic avian Giardia with he potential to contaminate watersheds and discuss the implications.

A gene encoding an unusually small dynein heavy chain homologue, hDYHH, was cloned from the genome of a free-living diplomonad, Hexamita inflata (Hi). The open reading frame (ORF) of hDYHH is 867bp and encodes a polypeptide of 289 amino acids (aa), hDYHH. hDYHH is homologous to the region around the third P-loop ATP-binding site of several dynein heavy chain polypeptides that are around 4000aa. Northern blot analysis showed that hDYHH is expressed in vivo and that the mRNA length (approximately 1.8kb) is consistent with the gene length (1.67kb). Southern blot analysis indicated that there are hDYHH homologues within the Hi genome, possibly including a longer dynein heavy chain gene. An hDYHH homologue was also identified in Hexamita pusilla (Hp). hDYHH is the first full-length protein-encoding gene cloned from Hexamita.

The protozoan parasite, Giardia duodenalis, shares many metabolic and genetic attributes of the bacteria, including fermentative energy metabolism which relies heavily on pyrophosphate rather than adenosine triphosphate and as a result contains two typically bacterial glycolytic enzymes which are pyrophosphate dependent. Pyruvate decarboxylation and subsequent electron transport to as yet unidentified anaerobic electron acceptors relies on a eubacterial-like pyruvate:ferredoxin oxidoreductase and an archaebacterial/eubacterial-like ferredoxin. The presence of another 2-ketoacid oxidoreductase (with a preference for alpha-ketobutyrate) and multiple ferredoxins in Giardia is also a trait shared with the anaerobic bacteria. Giardia pyruvate:ferredoxin oxidoreductase is distinct from the pyruvate dehydrogenase multienzyme complex invariably found in mitochondria. This is consistent with a lack of mitochondria, citric acid cycle, oxidative phosphorylation and glutathione in Giardia. Giardia duodenalis actively consumes oxygen and yet lacks the conventional mechanisms of oxidative stress management, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. In their place Giardia contains a prokaryotic H2O-producing NADH oxidase, a membrane-associated NADH peroxidase, a broad-range prokaryotic thioredoxin reductase-like disulphide reductase and the low molecular weight thiols, cysteine, thioglycolate, sulphite and coenzyme A. NADH oxidase is a major component of the electron transport pathway of Giardia which, in conjunction with disulphide reductase, protects oxygen-labile proteins such as ferredoxin and pyruvate:ferredoxin oxidoreductase against oxidative stress by maintaining a reduced intracellular environment. As the terminal oxidase, NADH oxidase provides a means of removing excess H+, thereby enabling continued pyruvate decarboxylation and the resultant production of acetate and adenosine triphosphate. A further example of the bacterial-like metabolism of Giardia is the utilisation of the amino acid arginine as an energy source. Giardia contain the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, but not in other eukaryotes apart from trichomonads and Chlamydomonas reinhardtii. The pathway includes substrate level phosphorylation and is sufficiently active to make a major contribution to adenosine triphosphate production. Two enzymes of the pathway, arginine deiminase and carbamate kinase, are rare in eukaryotes and do not occur in higher animals. Arginine is transported into the trophozoite via a bacterial-like arginine:ornithine antiport. Together these metabolic pathways in Giardia provide a wide range of potential drug targets for future consideration.

The cytidine triphosphate synthetase genes from three diverse strains of Giardia duodenalis have been sequenced and found to vary significantly from one another. The isolates were chosen as representatives of three demes as determined by several criteria including divergence in the rDNA repeat unit. Inserts in the genes and protein are conserved in length but are the most divergent regions among the three sequences examined. Variation in the rest of the gene occurs primarily in the third base position resulting in many silent mutations. One of the isolates (1709) was found to contain two genes with high sequence homology.

An axenic culture of Giardia was established from a sample of infected intestine obtained following autopsy of a sulphur-crested cockatoo (Cacatua galerita). The cockatoo recently captured in the wild and with good muscle tone died along with several other cage mates, apparently of an overwhelming, acute infection of Giardia. Trophozoites which established in the traditional, axenic Giardia medium (TYI-S-33 with supplementary bile) were morphologically identical to G. duodenalis. When outbred Quackenbush Swiss neonatal mice were infected with trophozoites a chronic infection was established and parasites were still present at 38 days post-inoculation. Weight gain by infected mice was reduced by 20%, thus mimicking failure-to-thrive syndrome in children, and maximum parasite load was more than 3-fold higher in comparison with other G. duodenalis strains. Analysis of the electrophoretic karyotype, rDNA and hybridization studies together with Giemsa- and trichrome-stained samples, and scanning electron microscopy indicated that the bird-derived Giardia belonged to the duodenalis group. This is the first report of infection of mammals with Giardia isolated from a bird. These data may have potentially serious implications for contamination of watersheds and establishment of zoonotic infections.

Giardia duodenalis is the best-characterized example of the most ancient eukaryotes, which are primitively amitochondrial and anaerobic. The surface of Giardia is coated with cysteine-rich proteins. One family of these proteins, CRP136, varies among isolates and upon environmental stress. A repeat region within the CRP136 family is interchangeable by a cassette-like mechanism, generating further diversity in repeat size, copy number, and sequence. Flanking the 5' region of the CRP136 family is a novel protein kinase gene and an ankyrin homolog, creating a conserved unit. A short spacer separates the ankyrin gene from the variable, tandem array of rDNA gene units at a common breakpoint within the large subunit gene, which is followed by the (TAGGG)n telomeric sequence. Transcriptional up-regulation of the CRP136 family is accompanied by a switch in mRNA length and promoter, of de novo expression, and suggests that CRP136 mRNA induction is under the control of a telomerically regulated position effect, which evolved very early in the eukaryotic lineage.

We describe the purification and characterisation of a thioredoxin reductase-like disulphide reductase from the ancient protozoan parasite, Giardia duodenalis. This dimeric flavoprotein contains 1 mol FAD per subunit and had an apparent subunit molecular mass of 35 kDa. The purified enzyme catalysed the NADPH-dependent (Km = 8 microM) reduction of 5,5'-dithio-bis(2-nitrobenzoic acid) to thionitrobenzoate and was unable to utilise NADH as an electron donor. The sulphydryl-active compounds, N-ethylmaleimide, sodium arsenite and Zn2+ ions, strongly inhibited the enzyme suggesting that a thiol component forms part of the active site. Purified enzyme was able to utilise a variety of substrates, including cystine and oxidised glutathione, which suggests that it is a broad-range disulphide reductase, probably accounting for the majority of thiol cycling activity in this organism. While the G. duodenalis enzyme does not require an intermediate electron transport protein, analogous to thioredoxin, for activity, we have identified a candidate carrier protein which enhances DTNB turnover six fold, therefore implying that Giardia contains a thioredoxin-like system. Physical, enzymatic and spectral properties of the G. duodenalis disulphide reductase are also consistent with it being a member of the thioredoxin reductase-class of disulphide reductases. Furthermore, the internal amino acid sequence of a tryptic peptide generated from the purified protein was highly homologous with thioredoxin reductases from other sources. This is the first report of a disulphide reductase to be purified from the anaerobic protozoa and explains the so called "glutathione-induced thiol-reductase activity' previously observed in G. duodenalis.

Two protein kinase (PK) genes, gPK1 and gPK2, were cloned from the genome of the ancient protozoan parasite, Giardia duodenalis (Gd). Both gPK genes and their products are highly homologous (85% and 77% identical, respectively). gPK1 and gPK2 contain all the motifs characteristic of PK, but they are not highly homologous to other PK and therefore belong to a novel PK gene family. Northern blot analysis showed that the gPK genes are expressed in vivo. Southern blot analysis indicated that there are other homologous PK genes in the Gd genome. gPK1 and gPK2 are the first full-length PK genes cloned from this primitive eukaryote. The unique amino acid (aa) sequences of gPK1 and gPK2 suggest that they are involved in unique biological functions in Gd.

We describe the purification of a H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. The enzyme is a monomeric flavoprotein containing flavin adenine dinucleotide in a 1:1 molar ratio with the polypeptide. The NADH oxidase has an apparent molecular mass of 46 kDa and was homogenous as determined by denaturing gel electrophoresis and N-terminal amino acid sequencing. NADPH could substitute for NADH as an electron donor with a K(m) value of 4.2 microM for NADH and 16 microM for NADPH (pH 7.8 at room temperature). With oxygen as the primary electron acceptor under aerobic conditions, the pure enzyme did not produce O.-2 nor H2O2 as stoichiometric products of oxygen reduction, implicating H2O as the end product and obviating the need for superoxide dismutase. The ability to utilise oxygen explains the apparent respiration of the amitochondrial fermentative metabolism of Giardia. Mercurials, flavoantagonists and heavy metals (Cu2+ and Zn2+) inhibited this activity. Under anaerobic conditions the enzyme catalysed electron transfer at lower efficiencies to other electron acceptors including nitroblue tetrazolium, potassium ferricyanide, FAD and FMN, using either NADH or NADPH as electron donors. NADPH, however, was a more efficient electron donor. Cytochrome c was not reduced under any assay conditions used. The enzyme reduced the nitrofuran drugs, furazolidone (an antigiardial) and nitrofurantoin, to their toxic radical forms as determined by EPR. Metronidazole, a nitroimidazole, was not reduced. Pure NADH oxidase did not demonstrate ferredoxin:NAD(P)1 oxidoreductase activity since it could not accept electrons from reduced ferredoxin to regenerate NAD(P)H. The G. duodenalis NADH oxidase may, therefore, function as a terminal oxidase, similar to the mitochondrial cytochrome oxidase, and in the maintenance of an optimum intracellular redox ratio. This report of a flavoenzyme from Giardia places Giardia close to the anaerobic bacteria in evolutionary terms.

The major 2-oxoacid oxidoreductase (2-OR), pyruvate:ferredoxin oxidoreductase (PFOR) from Giardia duodenalis has been purified to apparent homogeneity. A second 2-OR with a preference for alpha-ketobutyrate as substrate was identified and was removed from PFOR containing fractions during purification. Only PFOR and the second 2-OR were identified in gels of crude Giardia extracts assayed for 2-OR activity. The native form of PFOR which is membrane associated, is a homodimer of 138 kDa subunits. Pyruvate is the preferred substrate: alpha-ketobutyrate and oxaloacetate, but not phenyl-pyruvate or alpha-ketoglutarate, are decarboxylated. PFOR from Giardia is more stable than PFOR from most other organisms and purified PFOR can be stored without deterioration at -70 degrees C. Purified PFOR donates electrons to Giardia ferredoxin (Fd I) with concomitant reduction of metronidazole. However, two other Giardia ferredoxins did not accept electrons from PFOR. Consistent with the involvement of PFOR in metronidazole activation, the activity of pyruvate dependent 2-OR activity was decreased in all metronidazole-resistant lines tested but not in furazolidone-resistant lines. The presence of three different ferredoxins and two 2-ORs in Giardia suggests that a number of different electron transport pathways operate in this organism providing unusual metabolic flexibility for a eukaryote.