Evaluation of the model malaria elimination strategy in Mandla district along with its neighbouring districts: a time series analysis from 2008 to 2020.

BACKGROUND: Compared to 2017, India achieved a significant reduction in malaria cases in 2020. Madhya Pradesh (MP) is a tribal dominated state of India with history of high malaria burden in some districts. District Mandla of MP state showed a considerable decline in malaria cases between 2000 and 2013, except in 2007. Subsequently, a resurgence of malaria cases was observed during 2014 and 2015. The Malaria Elimination Demonstration Project (MEDP) was launched in 2017 in Mandla with the goal to achieve zero indigenous malaria cases. This project used: (1) active surveillance and case management using T4 (Track fever, Test fever, Treat patient, and Track patient); (2) vector control using indoor residual sprays and long-lasting insecticidal nets; (3) information education communication and behaviour change communication; and (4) regular monitoring and evaluation with an emphasis on operational and management accountability. This study has investigated malaria prevalence trends from 2008 to 2020, and has predicted trends for the next 5 years for Mandla and its bordering districts. METHODS: The malaria prevalence data of the district Mandla for the period of January 2008 to August 2017 was obtained from District Malaria Office (DMO) Mandla and data for the period of September 2017 to December 2020 was taken from MEDP data repository. Further, the malaria prevalence data for the period of January 2008 to December 2020 was collected from DMOs of the neighbouring districts of Mandla. A univariate time series and forecast analysis was performed using seasonal autoregressive integrated moving average model. FINDINGS: Malaria prevalence in Mandla showed a sharp decline [- 87% (95% CI - 90%, - 84%)] from 2017 to 2020. The malaria forecast for Mandla predicts zero cases in the next 5 years (2021-2025), provided current interventions are sustained. By contrast, the model has forecasted a risk of resurgence of malaria in other districts in MP (Balaghat, Dindori, Jabalpur, Seoni, and Kawardha) that were not the part of MEDP. CONCLUSION: The interventions deployed as part of MEDP have resulted in a sustainable zero indigenous malaria cases in Mandla. Use of similar strategies in neighbouring and other malaria-endemic districts in India could achieve similar results. However, without adding extra cost to the existing intervention, sincere efforts are needed to sustain these interventions and their impact using accountability framework, data transparency, and programme ownership from state to district level.

A comparative assessment of the community frontline health workers for their knowledge and practices of malaria diagnosis and treatment in three contiguous districts Mandla, Balaghat, and Dindori of Madhya Pradesh, India.

BACKGROUND: Global malaria cases rose by 14 million, and deaths by 69,000, in 2020. In India, a 46% decline has been reported between 2020 and 2019. In 2017, the Malaria Elimination Demonstration Project conducted a needs-assessment of the Accredited Social Health Activists (ASHAs) of Mandla district. This survey revealed the inadequate level of knowledge in malaria diagnosis and treatment. Subsequently, a training programme was launched for enhancing malaria-related knowledge of ASHAs. The present study was conducted in 2021 to evaluate the impact of training on malaria-related knowledge and practices of ASHAs in Mandla. This assessment was also done in two adjoining districts: Balaghat and Dindori. METHODS: A cross-sectional survey using a structured questionnaire was administered to ASHAs to measure their knowledge and practices related to malaria etiology, prevention, diagnosis, and treatment. A comparison of information collected from these three districts was performed using simple descriptive statistics, comparison of means and multivariate logistic regression analysis. RESULTS: Significant improvement was noted amongst ASHAs of district Mandla between 2017 (baseline) and 2021 (endline) in knowledge related to malaria transmission, preventive measures, adherence to the national drug policy, diagnosis using rapid diagnostic tests, and identification of age group-specific, colour-coded artemisinin combination therapy blister packs (p < 0.05). The multivariate logistic regression analysis revealed that odds of Mandla baseline was 0.39, 0.48, 0.34, and 0.07 times lower for malaria-related knowledge on disease etiology, prevention, diagnosis, and treatment, respectively (p < 0.001). Further, participants in districts Balaghat and Dindori showed significantly lower odds for knowledge (p < 0.001) and treatment practices (p < 0.01) compared to Mandla endline. Education, attended training, having a malaria learner's guide, and minimum 10 years' work experience were potential predictors for good treatment practices. CONCLUSION: The findings of the study unequivocally establishes significant improvement in overall malaria-related knowledge and practices of ASHAs in Mandla as a result of periodic training and capacity building efforts. The study suggests that learnings from Mandla district could be helpful in improving level of knowledge and practices among frontline health workers.

Study of relapse and failure cases of CAT I retreated with CAT II under RNTCP--an eleven year follow up.

OBJECTIVE: To analyse the treatment outcome of Cat I smear positive relapse and failure cases and their fate when treated with Cat II regimen under RNTCP. METHODS: All Cat I smear positive relapse and failure TB patients treated with Category II regimen from 1994 to 2005 in a chest clinic of Delhi were analysed in this retrospective study. The re-treatment outcome data for relapse and failure cases of Cat I when treated with Cat II regimen was reviewed. RESULTS: The study population included 5576 registered as Cat I sputum positive cases in Gulabi Bagh chest clinic from 1994 to 2005. A total of 190 (3.4%) failed on Cat I regimen. Further out of 4905 (87.9%) successfully treated Cat I patients, 442 (9%) presented as relapses. The treatment success rate for relapse and failure cases of Cat I when subsequently treated with Cat II regimen were 76.4% and 48.8% respectively, with a significantly higher failure rate (27.6%) among Cat I failures subsequently treated with Cat II regimen. CONCLUSION: The failure cases of Cat I subsequently treated with Cat II were observed to have a significantly lower success rates (p < 0.05) as compared to relapse cases. The need for reappraisal of Cat II re-treatment regimen for failure cases among Cat I is suggested.

Genetic engineering of Escherichia coli for enhanced uptake and bioaccumulation of mercury.

Synthetic phytochelatins (ECs) are a new class of metal-binding peptides with a repetitive metal-binding motif, (Glu-Cys)(n)Gly, which were shown to bind heavy metals more effectively than metallothioneins. However, the limited uptake across the cell membrane is often the rate-limiting factor for the intracellular bioaccumulation of heavy metals by genetically engineered organisms expressing these metal-binding peptides. In this paper, two potential solutions were investigated to overcome this uptake limitation either by coexpressing an Hg(2+) transport system with (Glu-Cys)(20)Gly (EC20) or by directly expressing EC20 on the cell surface. Both approaches were equally effective in increasing the bioaccumulation of Hg(2+). Since the available transport systems are presently limited to only a few heavy metals, our results suggest that bioaccumulation by bacterial sorbents with surface-expressed metal-binding peptides may be useful as a universal strategy for the cleanup of heavy metal contamination.

Malaria during pregnancy and infancy, in an area of intense malaria transmission in central India.

The clinico-epidemiological pattern of malarial infection in a cohort of pregnant women and infants was analysed during a malaria epidemic (1997-1998). The subjects were all members of tribal communities in an isolated and almost inaccessible area of central India. Overall, 151 (55%) of the 274 pregnant women investigated were found to have malarial infections at some time during the study, with Plasmodium falciparum predominating (88% of infections). All of the women investigated, whether primigravidae (42% found infected), secundigravidae (68%) or multigravidae (54%), were at great risk of developing severe malaria. When trimesters were compared, the highest prevalence of P. falciparum infection was recorded in the second (59% infected), irrespective of parity. Of the women found infected with P. falciparum, 3% had abortions, 4% stillbirths and 2% had babies who died while neonates. The small number of P. vivax infections observed prevented similar analyses for this species of parasite. Malarial infection was also seen in 218 (41%) of the 535 infants investigated. The values of age-specific prevalences revealed that > 30% of the infants examined at 2 months of age were then found to have P. vivax and/or P. falciparum parasitaemias. At 1 year of age, overall malaria prevalence was 50%, with P. vivax representing 25% of the infections and P. falciparum the rest. Subsequent follow-up revealed that three of the infants investigated, each of whom had had P. falciparum infections previously, died before their first birthdays. Re-infections (or treatment failures) were found to be common, both in the infants and the pregnant women. Pregnant women and infants from the study area clearly require systematic intervention to reduce their malaria-attributable morbidity.

Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins.

A novel strategy using synthetic phytochelatins is described for the purpose of developing microbial agents for enhanced bioaccumulation of toxic metals. Synthetic genes encoding for several metal-chelating phytochelatin analogs (Glu-Cys)(n)Gly (EC8 (n = 8), EC11 (n = 11), and EC20 (n = 20)) were synthesized, linked to a lpp-ompA fusion gene, and displayed on the surface of E. coli. For comparison, EC20 was also expressed periplasmically as a fusion with the maltose-binding protein (MBP-EC20). Purified MBP-EC20 was shown to accumulate more Cd(2+) per peptide than typical mammalian metallothioneins with a stoichiometry of 10 Cd(2+)/peptide. Cells displaying synthetic phytochelatins exhibited chain-length dependent increase in metal accumulation. For example, 18 nmoles of Cd(2+)/mg dry cells were accumulated by cells displaying EC8, whereas cells exhibiting EC20 accumulated a maximum of 60 nmoles of Cd(2+)/mg dry cells. Moreover, cells with surface-expressed EC20 accumulated twice the amount of Cd(2+) as cells expressing EC20 periplasmically. The ability to genetically engineer ECs with precisely defined chain length could provide an attractive strategy for developing high-affinity bioadsorbents suitable for heavy metal removal.

Zinc-histidine as nucleation centers for growth of ZnS nanocrystals.

Histidine is a chelator of zinc, most notably in zinc-finger proteins (zinc coordinated by cysteine and histidine) and in hyperaccumulator plants. Sulfide incorporation into molecules containing metal-cysteinyl complexes has been shown to occur in vivo in certain yeasts, leading to enhanced metal tolerance. Demonstrated here for the first time is incorporation of sulfide into zinc-histidine, resulting in histidine-ZnS nanocrystals (NCs) having unique optical properties. Sulfide complexation occurred optimally at alkaline pH into zinc-(histidine)2 species, and UV/Vis absorption maxima were red-shifted as increasing sulfide addition occurred. Intermediate sulfide concentrations led to multiple, thermodynamically preferred NC species within a sample. Fluorescence of histidine-ZnS NCs was greater than ZnS prepared previously with cysteinyl peptides. Transmission electron microscopy and selected-area electron diffraction indicated hexagonal ZnS crystals having an average size of 4.2 nm. A photocatalytic application of histidine-ZnS NCs was shown by efficient degradation of p-nitrophenol and paraquat in the presence of UV irradiation.

Effects of stocking density and concentrate supplementation of grazing dairy cows on milk production, composition and processing characteristics.

The effects on milk composition and processing characteristics of varying grass supply by changing stocking density and of offering a concentrate supplement were investigated. The experiment was conducted over 28 weeks of the lactation (April-October) using 48 spring-calved Friesian-Holstein cows. Three herds each of 16 cows were offered a restricted grass supply, a standard grass supply and a standard grass supply with a supplement of 3 kg concentrate/d. Treatment groups were grazed separately with a residence time of 3 d/paddock. Milk production, composition and processing characteristics such as renneting properties, ethanol stability and plasmin activity were measured weekly. Increasing stocking density above the standard system resulted in significant reductions in milk fat and protein yields, the concentrations of total protein, casein and whey proteins, and a deterioration in most processing characteristics. Imposing concentrate supplementation on the standard system increased total protein, casein and whey protein concentrations but generally did not improve processing characteristics except for ethanol stability. These results suggest that the standard grass supply in a rotational grazing paddock system can support efficient production of quality milk, and concentrate supplementation will not improve processing characteristics when an adequate supply of good quality herbage is available.

Malaria and the Narmada-river development in India: a case study of the Bargi dam.

The largest river-valley development to be proposed in India is that in the Narmada valley. The building of the Bargi dam, a multi-purpose irrigation and hydro-electric project, in Jabalpur, in central India, formed part of the first phase of the development of this valley (1974-1988). Many villages and several hectares of land in three districts were submerged as the waters rose behind the dam, the worst affected area being the catchment area of the primary health centre (PHC) at Narayanganj, in Mandla district. Until recently, cases of malaria were relatively rare in Narayanganj. However, an epidemic of malaria in late 1996 claimed hundreds of lives in the area and the outbreak spread, during 1997, to new villages in the region. A review of the records collected by the National Malaria Eradication Programme (NMEP) not only indicated that the slide positivity rate (SPR) for Narayanganj increased > 7.45-fold between 1979 and 1997 but also that the slide falciparum rate (SFR) increased > 32-fold over the same period. The NMEP data available for Mandla district as a whole indicated a doubling in mean SPR and SFR between 1979 and 1997. There is no evidence that a new species of vector has established since 1979. In fact, indoor-resting densities of anophelines and of the most established vector, Anopheles culicifacies, have fallen since the dam was built, but densities of another vector, An. fluviatilis, have increased.

A role for HEM2 in cadmium tolerance.

A Candida glabrata cadmium-sensitive mutant partially defective in glutathione production and exhibiting a complete absence of phytochelatins was used to clone a gene required for Cd tolerance. Transformation of the Cd-sensitive mutant with a genomic library from the wild-type C. glabrata led to the cloning of a gene that restored Cd tolerance and formation of Cd-glutathione and Cd-phytochelatin complexes. The cloned gene showed high levels of nucleic acid and protein sequence homology to the HEM2 genes, encoding porphobilinogen synthases, from several sources. It was shown that the C, glabrata Cd-sensitive mutant indeed exhibited a significant reduction in porphobilinogen synthase levels. The cloned C. glabrata gene complemented a hem2 mutant of Saccharomyces cerevisiae and restored porphobilinogen synthase activity in the mutant. The Cd sensitive mutant predictably showed decreased levels of sulfite reductase that requires siroheme, a metabolite produced in the heme biosynthetic pathway. The addition of cysteine, but not methionine, increased glutathione levels and Cd tolerance of both the wild-type and the mutant strain. However, addition of hemin chloride and methionine together restored Cd tolerance indicating that heme was required for transsulfuration of homocysteine to cysteine.

Characteristics of glutathione-capped ZnS nanocrystallites.

The titration of increasing equivalents of inorganic sulfide into preformed Zn-glutathione led to the appearance of UV/VIS spectral features attributable to ZnS nanocrystallites. Glutathione-ZnS complexes upon irradiation caused reduction of methylviologen confirming their semiconductor properties. Size-fractionation of glutathione-ZnS samples on a gel filtration column showed the formation of a range of complexes whose spectral properties were correlated with the sulfide content. The stoichiometry of Zn:glutathione increased from 1:2 to a maximum of about 7:1 as the sulfide/Zn ratios increase from 0 to approximately 1.0 in Zn-glutathione complex indicating up to 14-fold increase in the Zn-binding capacity of glutathione upon sulfide incorporation. Spectral characteristics of GSH-capped ZnS nanocrystallites were significantly influenced by pH and by the stoichiometry of Zn, sulfide and glutathione in the complex. Samples containing least glutathione and highest sulfide showed maximal luminescence at pH 6, whereas those with higher glutathione and lower sulfide content showed maximal luminescence at pH 11.

Ag(I)-binding to phytochelatins.

Phytochelatins (PCs) are glutathione-derived peptides with the general structure (gamma-Glu-Cys)nGly, where n varies from 2 to 11. A variety of metal ions such as Cu(II), Cd(II), Pb(II), Zn(II), and Ag(I) induce PC synthesis in plants and some yeasts. It has generally been assumed that the inducer metals also bind PCs. However, very little information is available on the binding of metals other than Cu(I) and Cd(II) to PCs. In this paper, we describe the Ag(I)-binding characteristics of PCs with the structure (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly, and (gamma-Glu-Cys)4Gly. The Ag(I)-binding stoichiometries of these three peptides were determined by (i) UV/VIS spectrophotometry, (ii) luminescence spectroscopy at 77 K, and (iii) reverse-phase HPLC. The three techniques yielded similar results. ApoPCs exhibit featureless absorption in the 220-340 nm range. The binding of Ag(I) to PCs induced the appearance of specific absorption shoulders. The titration end point was indicated by the flattening of the characteristic absorption shoulders. Similarly, luminescence at 77 K due to Ag(I)-thiolate clusters increased with the addition of graded Ag(I) equivalents. The luminescence declined when Ag(I) equivalents in excess of the saturating amounts were added to the peptides. At neutral pH, (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly, and (gamma-Glu-Cys)4Gly bind 1.0, 1.5, and 4.0 equivalents of Ag(I), respectively. The Ag(I)-binding capacity of (gamma-Glu-Cys)2Gly and (gamma-Glu-Cys)3Gly was increased at pH 5.0 and below so that Ag(I)/-SH ratio approached 1.0. A similar pH-dependent binding of Ag(I) to glutathione was also observed. The increased Ag(I)-binding to PCs at lower pH is of physiological significance as these peptides accumulate in acidic vacuoles. We also report lifetime data on Ag(I)-PCs. The relatively long decay-times (approximately 0.1-0.3 msec) accompanied with a large Stokes shift in the emission band are indicative of spin-forbidden phosphorescence.

Optical spectroscopic and reverse-phase HPLC analyses of Hg(II) binding to phytochelatins.

Optical spectroscopy and reverse-phase HPLC were used to investigate the binding of Hg(II) to plant metal-binding peptides (phytochelatins) with the structure (gammaGlu-Cys)2Gly, (gammaGlu-Cys)3Gly and (gammaGlu-Cys)4Gly. Glutathione-mediated transfer of Hg(II) into phytochelatins and the transfer of the metal ion from one phytochelatin to another was also studied using reverse-phase HPLC. The saturation of Hg(II)-induced bands in the UV/visible and CD spectra of (gammaGlu-Cys)2Gly suggested the formation of a single Hg(II)-binding species of this peptide with a stoichiometry of one metal ion per peptide molecule. The separation of apo-(gammaGlu-Cys)2Gly from its Hg(II) derivative on a C18 reverse-phase column also indicated the same metal-binding stoichiometry. The UV/visible spectra of both (gammaGlu-Cys)3Gly and (gammaGlu-Cys)4Gly at pH 7.4 showed distinct shoulders in the ligand-to-metal charge-transfer region at 280-290 mm. Two distinct Hg(II)-binding species, occurring at metal-binding stoichiometries of around 1.25 and 2.0 Hg(II) ions per peptide molecule, were observed for (gammaGlu-Cys)3Gly. These species exhibited specific spectral features in the charge-transfer region and were separable by HPLC. Similarly, two main Hg(II)-binding species of (gammaGlu-Cys)4Gly were observed by UV/visible and CD spectroscopy at metal-binding stoichiometries of around 1.25 and 2.5 respectively. Only a single peak of Hg(II)-(gammaGlu-Cys)4Gly complexes was resolved under the conditions used for HPLC. The overall Hg(II)-binding stoichiometries of phytochelatins were similar at pH 2.0 and at pH 7.4, indicating that pH did not influence the final Hg(II)-binding capacity of these peptides. The reverse-phase HPLC assays indicated a rapid transfer of Hg(II) from glutathione to phytochelatins. These assays also demonstrated a facile transfer of the metal ion from shorter- to longer-chain phytochelatins. The strength of Hg(II) binding to glutathione and phytochelatins followed the order: gammaGlu-Cys-Gly<(gammaGlu-Cys)2Gly<(gammaGlu-Cy s)3Gly<(gamma Glu-Cys)4Gly.

Analysis of copper-induced metallothionein expression using autonomously replicating plasmids in Candida glabrata.

Candida glabrata strains and a stable plasmid were developed that were suitable for analysis of copper-inducible expression from promoters of the three metallothionein (MT) genes. The two homologous MTII genes, MTIIa and MTIIb, encode the same polypeptide but are differentially induced by copper salts. MTIIb is more highly inducible than MTIIa and cells harboring a single MTIIb exhibit a greater resistance to copper salts compared to cells harboring a single MTIIa. The differential copper inducibility was mapped to sequences between -03 and -292 upstream of the MT coding sequences. Expression of MTI is highly Cu-regulated, but this MT gene confers much less resistance than MTII genes.

Chain length-dependent Pb(II)-coordination in phytochelatins.

UV/visible and circular dichroism (CD) spectroscopy have been used to study the binding of Pb(II) to plant metal-sequestering peptides, phytochelatins (PCs), with the structure (gamma Glu-Cys)2Gly, (gamma Glu-Cys)3Gly and (gamma Glu-Cys)4Gly. Saturation of the Pb(II)-induced charge-transfer bands indicated that both (gamma Glu-Cys)2Gly and (gamma Glu-Cys)3Gly bound one metal ion per peptide molecule. However, (gamma Glu-Cys)4Gly formed two distinct species with stoichiometries of one and two Pb(II) ions per peptide molecule, respectively. The optical spectra of Pb(II)1-(gamma Glu-Cys)4Gly were similar to those of Pb(II)1-(gamma Glu-Cys)3Gly, whereas the spectra of Pb(II)2-(gamma Glu-Cys)4Gly were similar to those of Pb(II)1-(gamma Glu-Cys)2Gly. Since cysteinyl thiolates are the likely ligands for Pb(II) in PCs, Pb(II) appears to form two-, three- and four-coordinate complexes with PCs depending on their chain length. Furthermore, Pb(II) may exhibit multiple coordination in longer chain PCs as indicated by the formation of two Pb(II)-binding species of (gamma Glu-Cys)4Gly. The transfer of Pb(II) from glutathione to PCs and from shorter chain to longer chain PCs is also demonstrated.

Glutathione-mediated transfer of Cu(I) into phytochelatins.

Room temperature luminescence attributable to Cu(I)-thiolate clusters has been used to probe the transfer of Cu(I) from Cu(I)-glutathione complex to rabbit liver thionein-II and plant metal-binding peptides phytochelatins (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly and (gamma-Glu-Cys)4Gly. Reconstitutions were also performed using CuC1. The Cu(I)-binding stoichiometry of metallothionein or phytochelatins was generally independent of the Cu(I) donor. However, the luminescence of the reconstituted metallothionein or phytochelatins was higher when Cu(I)-GSH was the donor. This higher luminescence is presumably due to the stabilizing effect of GSH on Cu(I)-thiolate clusters. As expected, 12 Cu(I) ions were bound per molecule of metallothionein. The Cu(I) binding to phytochelatins depended on their chain length; the binding stoichiometries being 1.25, 2.0 and 2.5 for (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly and (gamma-Glu-Cys)4Gly respectively at neutral pH. A reduced stoichiometry for the longer phytochelatins was observed at alkaline pH. No GSH was found to associate with phytochelatins by a gel-filtration assay. The Cu(I) binding to (gamma-Glu-Cys)2Gly and (gamma-Glu-Cys)3Gly occurred in a biphasic manner in the sense that the relative luminescence increased approximately linearly with the amount of Cu(I) up to a certain molar ratio whereafter luminescence increased dramatically upon the binding of additional Cu(I). The luminescence intensity declined once the metal-binding sites were saturated. In analogy with the studies on metallothioneins, biphasic luminescence suggests the formation of two types of Cu(I) clusters in phytochelatins.

Heterologous gene expression and protein secretion from Candida glabrata.

We have examined heterologous protein secretion from Candida glabrata with the aid of a stable C. glabrata vector and a secretion reporter cassette comprising the Saccharomyces cerevisiae PGK-gene promoter and a Kluveromyces Iactis secretion signal to drive secretion of Escherichia coli beta-lactamase. Abundant secretion of beta-lactamase from C. glabrata indicates that the S. cerevisiae PGK promoter functions in C. glabrata. Furthermore, we show that C. glabrata processes the secreted beta-lactamase in a manner similar to, but not identical with, S. cerevisiae and K. lactis. C. glabrata may be a suitable new host for the expression of foreign genes.

Role of CdS quantum crystallites in cadmium resistance in Candida glabrata.

Glutathione-related peptides (gamma-Glu-Cys)nGly, trivially known as phytochelatins (PCs), sequester Cd(II) and other heavy metals in all plants and some yeasts. However, the metal resistance levels may depend on factors such as the PC concentrations, their chain length and ability to incorporate labile sulfide. We show here that a highly Cd(II)-resistant mutant of yeast Candida glabrata exhibited Cd(II)-dependent formation of extremely high levels of PC-coated CdS quantum crystallites. The CdS crystallites were formed in the cytosol but finally accumulated in the vacuoles. Cd(II)-stimulated sulfide production required sulfate and was inhibited by both cysteine and methionine. GSH synthesis inhibition sensitized the resistant strain to Cd(II) indicating that GSH still provided primary defense against the metal ion.

Disruption analysis of metallothionein-encoding genes in Candida glabrata.

Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT-IIa, an amplified locus, and MT-IIb, a single-copy gene, to determine the roles of various MT genes in CuSO4 resistance in C. glabrata. The concentration of CuSO4 required to inhibit the growth by 50% (IC50) of a C. glabrata strain harboring an amplified MT-IIa locus and a single-copy MT-IIb and MT-I genes was 7 mM in a synthetic complete medium. The IC50 decreased to approx. 1 mM when the amplified MT-IIa locus was deleted. The disruption of the MT-IIb gene decreased the IC50 further to 0.1 mM. The CuSO4 resistance in a strain lacking both of the MT-II genes was attributable to MT-I; no evidence was found for the production of (gamma EC)nG isopeptides. The comparison of the nucleotide sequence of MT-IIb to that of MT-IIa revealed the same coding sequence with differences in the 5' region. However, substantial differences were found in the 3' region. MT-IIb was expressed since we were able to purify the protein from the strain that had an intact MT-IIb gene, but a deleted MT-IIa gene. In addition, CuSO4 resistance was provided by MT-IIb. Northern analysis of the total RNA from varied C. glabrata strains indicated no significant changes in the expression of MT-I in the presence or absence of the MT-II genes.

Cloning system for Candida glabrata using elements from the metallothionein-IIa-encoding gene that confer autonomous replication.

The yeast Candida glabrata harbors two distinct gene families that encode metallothioneins (MTs). One of these loci, the MT-IIa locus, exhibits selective and tandem amplification in many wild type strains of C. glabrata. The present paper demonstrates that the amplified MT-IIa gene contains autonomously replicating sequences (ARS). These ARS elements have been used to construct vectors capable of replicating in C. glabrata. The ARS element(s) in the MT-IIa gene were localized to a 457-bp segment downstream from the MT-IIa coding sequence. Although plasmids containing this fragment transform C. glabrata with high frequency, the stability of the transformants and the copy number of the plasmid improve when the entire 1.25-kb MT-IIa gene is used. Transformation of C. glabrata with plasmids carrying the 2 microns circle ARS of Saccharomyces cerevisiae led to the formation of micro-colonies, indicating that the ARS elements of 2 microns plasmids replicate only to a limited extent in C. glabrata. Conversely, a C. glabrata plasmid carrying three copies of the MT-IIa gene was able to transform S. cerevisiae.