Measuring Replicative Lifespan in Cryptococcus neoformans.

Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic yeast Cryptococcus neoformans is essential because old yeast cells with age-related phenotypes accumulate during infection and are associated with increased virulence and antifungal tolerance. Microdissection and microfluidic devices are valuable tools for continuously tracking cells at the single-cell level. In this chapter, we describe the features of these two platforms and outline technical limitations and information to study aging mechanisms while assessing the lifespan of yeast cells.

Cryptococcal dissemination to the central nervous system requires the vacuolar calcium transporter Pmc1.

Cryptococcus neoformans is a basidiomycetous yeast and the cause of cryptococcosis in immunocompromised individuals. The most severe form of the disease is meningoencephalitis, which is one of the leading causes of death in HIV/AIDS patients. In order to access the central nervous system, C. neoformans relies on the activity of certain virulence factors such as urease, which allows transmigration through the blood-brain barrier. In this study, we demonstrate that the calcium transporter Pmc1 enables C. neoformans to penetrate the central nervous system, because the pmc1 null mutant failed to infect and to survive within the brain parenchyma in a murine systemic infection model. To investigate potential alterations in transmigration pathways in these mutants, global expression profiling of the pmc1 mutant strain was undertaken, and genes associated with urease, the Ca2+ -calcineurin pathway, and capsule assembly were identified as being differentially expressed. Also, a decrease in urease activity was observed in the calcium transporter null mutants. Finally, we demonstrate that the transcription factor Crz1 regulates urease activity and that the Ca2+ -calcineurin signalling pathway positively controls the transcription of calcium transporter genes and factors related to transmigration.

Low-grade sulfadoxine-pyrimethamine resistance in Plasmodium falciparum parasites from Lubango, Angola.

BACKGROUND: Malaria is a major parasitic disease, affecting millions of people in endemic areas. Plasmodium falciparum parasites are responsible for the most severe cases and its resistance to anti-malarial drugs is notorious. This is a possible obstacle to the effectiveness of intermittent preventive treatment (IPT) based on sulfadoxine-pyrimethamine (SP) cures administrated to pregnant women (IPTp) during their pregnancy. As this intervention is recommended in Angola since 2006, it has assessed, in this country, the molecular profiles in P. falciparum dhfr and dhps, two polymorphic genes associated to pyrimethamine and sulfadoxine resistance, respectively. METHODS: Blood samples from 52 falciparum patients were collected in Lubango, Angola and pfdhfr and pfdhps polymorphisms were analysed using nested-PCR and DNA sequencing. RESULTS: In the pfdhfr gene, the 108N mutation was almost fixed (98 %), followed by 59R (63 %), 51I (46 %), 50R and 164L (2 %, respectively). No 16V/S mutations were found. The most common double mutant genotype was CNRN (59 + 108; 46 %), followed by CICN (51 + 108; 29 %) whereas IRN (51 + 59 + 108; 15 %), CNRNVL (59 + 108 + 164; 2 %) and RICN (50 + 51 + 108; 2 %) triple mutant genotypes were detected. Investigations of the pfdhps gene showed that the 437G mutation was the most prevalent (97 %). Only two and one samples disclosed the 540E (7 %) and the 436A (3 %), respectively. Single mutant SGKAA (437; 86 %) was higher than SGEAA (437 + 540; 7 %) or AGKAA (436 + 437; 3 %) double mutants genotypes. No polymorphism was detected at codons 581G and 613T/S. Combining pfdhfr and pfdhps alleles two triple mutant haplotypes (double mutant in dhfr and single mutant in dhps) were observed: the ACICNVI/SGKAA in 14 (56 %) samples and the ACNRNVI/SGKAA in five (20 %) samples. One quadruple mutant haplotype was detected (ACIRNVI/SGKAA) in six (24 %) P. falciparum samples. No quintuple pfdhfr-pfdhps mutant was noted. CONCLUSION: pfdhfr and pfdhps gene mutations in isolates from Lubango are suggestive of a low-grade SP resistance and IPT for pregnant women and infant based on SP treatment could be effective. Routine molecular studies targeting polymorphism in these two genes need to be routinely conducted at country level.

Molecular markers of antifolate resistance in Plasmodium falciparum isolates from Luanda, Angola.

BACKGROUND: Plasmodium falciparum malaria remains a leading health problem in Africa and its control is seriously challenged by drug resistance. Although resistance to the sulphadoxine-pyrimethamine (SP) is widespread, this combination remains an important component of malaria control programmes as intermittent preventive therapy (IPT) for pregnant women and children. In Angola, resistance patterns have been poorly characterized, and IPT has been employed for pregnant women since 2006. The aim of this study was to assess the prevalence of key antifolate resistance mediating polymorphisms in the pfdhfr and pfdhps genes in P. falciparum samples from Angola. METHODS: Plasmodium falciparum samples collected in Luanda, in 2007, were genotyped by amplification and DNA forward and reverse sequencing of the pfdhfr and pfdhps genes. RESULTS: The most prevalent polymorphisms identified were pfdhfr 108N (100%), 51I (93%), 59R (57%) and pfdhps 437G (93%). Resistance-mediating polymorphisms in pfdhps less commonly observed in West Africa were also identified (540E in 10%, 581G in 7% of samples). CONCLUSION: This study documents an important prevalence of 4 P. falciparum polymorphisms that predicts an antifolate resistance in Luanda. Further, some samples presented additional mutations associated to high-level resistance. These results suggest that the use of SP for IPT may no longer be warranted in Angola.

Brazilian Plasmodium falciparum isolates: investigation of candidate polymorphisms for artemisinin resistance before introduction of artemisinin-based combination therapy.

BACKGROUND: This study was performed to better understand the genetic diversity of known polymorphisms in pfatpase6 and pfmdr1 genes before the introduction of ACT in Brazil, in order to get a genotypic snapshot of Plasmodium falciparum parasites that may be used as baseline reference for future studies. METHODS: Parasites from P. falciparum samples collected in 2002, 2004 and 2006-2007 were genotyped using PCR and DNA sequencing at codons 86, 130, 184, 1034, 1042, 1109 and 1246 for pfmdr1 gene, and 243, 263, 402, 431, 623, 630, 639, 683, 716, 776, 769 and 771 for pfatpase6 gene. RESULTS: A pfmdr1 haplotype NEF/CDVY was found in 97% of the samples. In the case of pfatpase6, four haplotypes, wild-type (37%), 630 S (35%), 402 V (5%) and double-mutant 630 S + 402 V (23%), were detected. CONCLUSION: Although some polymorphism in pfmdr1 and pfatpase6 were verified, no reported haplotypes in both genes that may mediate altered response to ACT was detected before the introduction of this therapy in Brazil. Thus, the haplotypes herein described can be very useful as a baseline reference of P. falciparum populations without ACT drug pressure.

Plasmodium falciparum isolates from Angola show the StctVMNT haplotype in the pfcrt gene.

BACKGROUND: Effective treatment remains a mainstay of malaria control, but it is unfortunately strongly compromised by drug resistance, particularly in Plasmodium falciparum, the most important human malaria parasite. Although P. falciparum chemoresistance is well recognized all over the world, limited data are available on the distribution and prevalence of pfcrt and pfmdr1 haplotypes that mediate resistance to commonly used drugs and that show distinct geographic differences. METHODS: Plasmodium falciparum-infected blood samples collected in 2007 at four municipalities of Luanda, Angola, were genotyped using PCR and direct DNA sequencing. Single nucleotide polymorphisms in the P. falciparum pfcrt and pfmdr1 genes were assessed and haplotype prevalences were determined. RESULTS AND DISCUSSION: The most prevalent pfcrt haplotype was StctVMNT (representing amino acids at codons 72-76). This result was unexpected, since the StctVMNT haplotype has previously been seen mainly in parasites from South America and India. The CVIET, CVMNT and CVINT drug-resistance haplotypes were also found, and one previously undescribed haplotype (CVMDT) was detected. Regarding pfmdr1, the most prevalent haplotype was YEYSNVD (representing amino acids at codons 86, 130, 184, 1034, 1042, 1109 and 1246). Wild haplotypes for pfcrt and pfmdr1 were uncommon; 3% of field isolates harbored wild type pfcrt (CVMNK), whereas 21% had wild type pfmdr1 (NEYSNVD). The observed predominance of the StctVMNT haplotype in Angola could be a result of frequent travel between Brazil and Angola citizens in the context of selective pressure of heavy CQ use. CONCLUSIONS: The high prevalence of the pfcrt SVMNT haplotype and the pfmdr1 86Y mutation confirm high-level chloroquine resistance and might suggest reduced efficacy of amodiaquine in Angola. Further studies must be encouraged to examine the in vitro sensitivity of pfcrt SVMNT parasites to artesunate and amodiaquine for better conclusive data.

Chloroquine and sulphadoxine-pyrimethamine sensitivity of Plasmodium falciparum parasites in a Brazilian endemic area.

BACKGROUND: The goal of the present study was the characterization of Plasmodium falciparum genes associated to malaria drug resistance (pfcrt, pfdhfr and pfdhps), in samples from two Brazilian localities. METHODS: Parasites from 65 P. falciparum samples were genotyped using nested-PCR and direct DNA sequencing. RESULTS: Six resistant sulphadoxine-pyrimethamine (SP) pfdhfr genotypes and one haplotype associated to SP sensitivity were detected. For pfcrt gene, SVMNT chloroquine (CQ)-resistant genotype was detected as well as the CVMNK CQ-sensitive haplotype in the same sample from Paragominas, that showed a SP-sensitive genotype. CONCLUSION: This study is the first to document the sensitivity of P. falciparum parasites to CQ and SP in Brazilian field samples. The importance of these findings is discussed.