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 (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.

Sequence map of the 3-Mb Giardia duodenalis assemblage A chromosome.

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.

Susceptibility in vitro of clinically metronidazole-resistant Trichomonas vaginalis to nitazoxanide, toyocamycin, and 2-fluoro-2'-deoxyadenosine.

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 EBNA-3 gene family proteins disrupt the G2/M checkpoint.

The Epstein-Barr nuclear antigens (EBNA), EBNA-3, -4 and -6, have previously been shown to act as transcriptional regulators, however, this study identifies another function for these proteins, disruption of the G2/M checkpoint. Lymphoblastoid cell lines (LCLs) treated with a G2/M initiating drug azelaic bishydroxamine (ABHA) did not show a G2/M checkpoint response, but rather they display an increase in cell death, a characteristic of sensitivity to the cytotoxic effects of the drug. Cell cycle analysis demonstrated that the individual expression of EBNA-3, -4 or -6 are capable of disrupting the G2/M checkpoint response induced by ABHA resulting in increased toxicity, whereas EBNA-2, and -5 were not. EBNA-3 gene family protein expression also disrupted the G2/M checkpoint initiated in response to the genotoxin etoposide and the S phase inhibitor hydroxyurea. The G2 arrest in response to these drugs were sensitive to caffeine, suggesting that ATM/ATR signalling in these checkpoint responses may be blocked by the EBNA-3 family proteins. The function of EBNA-3, -4 and -6 proteins appears to be more complex than anticipated and these data suggest a role for these proteins in disrupting the host cell cycle machinery.

A histone deacetylase inhibitor, azelaic bishydroxamic acid, shows cytotoxicity on Epstein-Barr virus transformed B-cell lines: a potential therapy for posttransplant lymphoproliferative disease.

BACKGROUND: Posttransplant lymphoproliferative disease (PTLD), driven by the presence of Epstein-Barr virus (EBV), is becoming an increasingly important clinical problem after solid organ transplantation. The use of immunosuppressive therapy leads to the inhibition of the cytotoxic T cells that normally control the EBV latently infected B cells. The prognosis for many patients with PTLD is poor, and the optimal treatment strategy is not well defined. METHOD: This study investigates the use of a histone deacetylase inhibitor, azelaic bishydroxamic acid (ABHA), for its ability to effectively kill EBV-transformed lymphoblastoid cell lines. RESULTS: In vitro treatment of lymphoblastoid cell lines with ABHA showed that they were effectively killed by low doses of the drug (ID50 2-5 microg/ml) within 48 hr. As well as being effective against polyclonal B-cell lines, ABHA was also shown to be toxic to seven of eight clonal Burkitt's lymphoma cell lines, indicating that the drug may also be useful in the treatment of late-occurring clonal PTLD. In addition, ABHA treatment did not induce EBV replication or affect EBV latent gene expression. CONCLUSION: These studies suggest that ABHA effectively kills both polyclonal and clonal B-cell lines and has potential in the treatment of PTLD.

Chromosome sequence maps of the Giardia lamblia assemblage A isolate WB.

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.

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 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.

Characterization of the transcriptional repressor RBP in Epstein-Barr virus-transformed B cells.

RBP, a transcriptional repressor, is intricately involved in Epstein-Barr virus (EBV) transformation of human B cells. The EBV nuclear proteins EBNA-2, -3, -4 and -6 all utilize RBP to regulate the transcription of both cellular and viral genes. This study investigates the isoforms of the RBP protein in Burkitt's lymphoma (BL) cells and in EBV-transformed lymphoblastoid cell lines (LCLs). Two-dimensional gel electrophoresis showed the presence of two different cellular isoforms of RBP; the molecular masses and isoelectric points of these two isoforms corresponded to RBP-Jkappa and RBP-2N. Fractionation studies and green fluorescent protein (GFP)-tagged expression studies demonstrated that both RBP isoforms were located predominantly in the cell nucleus. Interestingly, GFP-tagged RBP-Jkappa showed diffuse, uniform nuclear staining, whereas GFP-tagged RBP-2N showed a discrete nuclear pattern, demonstrating differences between the two isoforms. Within the nuclear fraction of EBV-negative BL cells, RBP existed both in a free form and bound to chromatin, whereas in LCLs the intranuclear RBP was predominantly chromatin-bound. Expression of the EBV latent proteins was found to lead to the sequestering of RBP from the cytoplasm into the cell nucleus and to an increase in the chromatin-bound forms of RBP.

The Epstein-Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein.

The Epstein-Barr virus nuclear antigen (EBNA)-6 protein is essential for Epstein-Barr virus (EBV)-induced immortalization of primary human B-lymphocytes in vitro. In this study, fusion proteins of EBNA-6 with green fluorescent protein (GFP) have been used to characterize its nuclear localization and organization within the nucleus. EBNA-6 associates with nuclear structures and in immunofluorescence demonstrate a punctate staining pattern. Herein, we show that the association of EBNA-6 with these nuclear structures was maintained throughout the cell cycle and with the use of GFP-E6 deletion mutants, that the region amino acids 733-808 of EBNA-6 contains a domain that can influence the association of EBNA-6 with these nuclear structures. Co-immunofluorescence and confocal analyses demonstrated that EBNA-6 and EBNA-3 co-localize in the nucleus of cells. Expression of EBNA-6, but not EBNA-3, caused a redistribution of nuclear survival of motor neurons protein (SMN) to the EBNA-6 containing nuclear structures resulting in co-localization of SMN with EBNA-6.