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1.  Pyruvate:ferredoxin oxidoreductase and thioredoxin reductase are involved in 5-nitroimidazole activation while flavin metabolism is linked to 5-nitroimidazole resistance in Giardia lamblia 
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 (MTRr) 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 MTRr 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 MTRr Giardia. However, reduction of flavins is suppressed in highly MTRr 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 MTRr 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.
doi:10.1093/jac/dkr192
PMCID: PMC3133484  PMID: 21602576
metronidazole; ronidazole; tinidazole; Blastocystis; NADPH oxidase
2.  Impaired Parasite Attachment as Fitness Cost of Metronidazole Resistance in Giardia lamblia▿ 
Antimicrobial Agents and Chemotherapy  2011;55(10):4643-4651.
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 (Mzr) G. lamblia has rarely been isolated from patients. To understand why clinical Mzr isolates are rare, we questioned whether Mz resistance entails fitness costs to the parasite. Our studies employed several newly generated and established isogenic Mzr 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 Mzr cell lines could not establish infection, while two Mzr 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 Mzr 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 Mzr isolates of Giardia. However, the data also caution that some forms of Mz resistance do not markedly interfere with in vivo infectivity.
doi:10.1128/AAC.00384-11
PMCID: PMC3186953  PMID: 21825286
3.  Microsatellite polymorphism in the sexually transmitted human pathogen Trichomonas vaginalis indicates a genetically diverse parasite 
Given the growing appreciation of serious health sequelae from widespread Trichomonas vaginalis infection, new tools are needed to study the parasite's genetic diversity. To this end we have identified and characterized a panel of 21 microsatellites and six single-copy genes from the T. vaginalis genome, using seven laboratory strains of diverse origin. We have (1) adapted our microsatellite typing method to incorporate affordable fluorescent labeling, (2) determined that the microsatellite loci remain stable in parasites continuously cultured up to 17 months, and (3) evaluated microsatellite marker coverage of the six chromosomes that comprise the T. vaginalis genome using fluorescent in situ hybridization (FISH). We have used the markers to show that T. vaginalis is a genetically diverse parasite in a population of commonly used laboratory strains. In addition, we have used phylogenetic methods to infer evolutionary relationships from our markers in order to validate their utility in future population analyses. Our panel is the first series of robust polymorphic genetic markers for T. vaginalis that can be used to classify and monitor lab strains, as well as provide a means to measure the genetic diversity and population structure of extant and future T. vaginalis isolates.
doi:10.1016/j.molbiopara.2010.08.006
PMCID: PMC2974001  PMID: 20813140
Population genetics; Trichomonas vaginalis; sexually transmitted infection; microsatellite; FISH; genetic markers
4.  A new-generation 5-nitroimidazole can induce highly metronidazole-resistant Giardia lamblia in vitro 
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 Giardia lamblia 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 ID90 values (concentration of drug at which 10% of control parasite ATP levels are detected) for MTR of >200 μM, whilst lines induced to be highly resistant to MTR in vitro have maximum ID90 values around 100 μM (MTR-susceptible isolates typically have an ID90 of 5–12.8 μM). 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.
doi:10.1016/j.ijantimicag.2010.03.004
PMCID: PMC3103471  PMID: 20456926
Pyruvate:ferredoxin oxidoreductase; Tinidazole; Ronidazole; 5-Nitroimidazole; Cross-resistance
5.  Mutations in the Thumb Allow Human Immunodeficiency Virus Type 1 Reverse Transcriptase To Be Cleaved by Protease in Virions ▿  
Journal of Virology  2009;83(23):12336-12344.
Although human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has been extensively studied, there are still significant questions about the effects of mutations on the maturation and stability of RT. We show here that a significant fraction (>80%) of the single point mutations we generated in the thumb subdomain of HIV-1 (RT) affect the stability of RT in virions. Fragments of the unstable mutant RTs can be detected in Western blots of virion proteins; however, the degree of degradation varies. The titers of the mutants whose virions contain degraded RTs are reduced. Some, but not all, of the unstable RT thumb subdomain mutants we analyzed have a temperature-sensitive phenotype. A preliminary survey of mutations in other subdomains of RT shows that some of these mutations also destabilize RT. The stability of the RT mutants is enhanced by the addition of a protease inhibitor, suggesting that the viral protease plays an important role in the degradation of the mutant RTs. These results confirm and extend earlier reports of mutations that affect the stability of RT in virions. The data suggest that the stability of a mutant RT in virions could be a major factor in determining the virus titer and, by extension, viral fitness, which could affect whether a mutation in RT is acceptable to the virus.
doi:10.1128/JVI.00676-09
PMCID: PMC2786724  PMID: 19759158
6.  5-Nitroimidazole Drugs Effective against Metronidazole-Resistant Trichomonas vaginalis and Giardia duodenalis 
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.
doi:10.1128/AAC.50.1.344-347.2006
PMCID: PMC1346785  PMID: 16377707
7.  Overlooked and underserved in Harlem: a population-based survey of adults with asthma. 
Environmental Health Perspectives  2002;110(Suppl 2):217-220.
The prevalence of asthma has increased over the past two decades; if this trend persists over the next two decades, the number of individuals with asthma in the United States will double by 2020, affecting 29 million Americans. Many of these individuals will be adults. Recent community-based participatory research in Harlem has focused on children with asthma, but little is known about the prevalence and burden of asthma among adults. We conducted a population-based probability sample of Central Harlem adults 18-65 years of age from 1992 to 1994. Asthma was one of three ambulatory care-sensitive conditions surveyed. We used an additional set of questions regarding asthma management and burden for those respondents who reported they had asthma. The prevalence of self-reported asthma was 14% in this population-based sample of Central Harlem adults. Respondents with asthma reported remarkably high rates of emergency department (ED) visits for asthma, but women were more likely than men to report two or more ED visits in the year prior to interview (38% vs. 18%). Women with asthma were also more likely than men with asthma to report activity restrictions because of asthma (61% vs. 26%). The burden of asthma among adults in Central Harlem is considerable. We urgently need comprehensive health approaches to address the high prevalence of health risks related to multiple chronic diseases, notably smoking and obesity. Key priorities are to determine which community education, prevention, and promotion programs are most effective and will best serve Harlem adults.
PMCID: PMC1241166  PMID: 11929731
8.  Orally Administered Giardia duodenalis Extracts Enhance an Antigen-Specific Antibody Response 
Infection and Immunity  2001;69(10):6503-6510.
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.
doi:10.1128/IAI.69.10.6503-6510.2001
PMCID: PMC98786  PMID: 11553595
9.  The N-Terminal Domain of IκBα Masks the Nuclear Localization Signal(s) of p50 and c-Rel Homodimers 
Molecular and Cellular Biology  1998;18(5):2640-2649.
Members of the Rel/NF-κB family of transcription factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding, dimerization, and binding to inhibitor. At the C-terminal end of the RHD, each protein has a nuclear localization signal (NLS). The crystal structures of the p50 and RelA family members show that the RHD consists of two regions: an N-terminal section which contains some of the DNA contacts and a C-terminal section which contains the remaining DNA contacts and controls dimerization. In unstimulated cells, the homo- or heterodimeric Rel/NF-κB proteins are cytoplasmic by virtue of binding to an inhibitor protein (IκB) which somehow masks the NLS of each member of the dimer. The IκB proteins consist of an ankyrin-repeat-containing domain that is required for binding to dimers and N- and C-terminal domains that are dispensable for binding to most dimers. In this study, we examined the interaction between IκBα and Rel family homodimers by mutational analysis. We show that (i) the dimerization regions of p50, RelA, and c-Rel are sufficient for binding to IκBα, (ii) the NLSs of RelA and c-Rel are not required for binding to IκBα but do stabilize the interaction, (iii) the NLS of p50 is required for binding to IκBα, (iv) only certain residues within the p50 NLS are required for binding, and (v) in a p50-IκBα complex or a c-Rel-IκBα complex, the N terminus of IκBα either directly or indirectly masks one or both of the dimer NLSs.
PMCID: PMC110643  PMID: 9566883

Results 1-9 (9)