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Novel genetic mutations in pyrimidine salvage pathway enzymes from Cryptococcus neoformans contribute to reduced susceptibility against 5-fluorocytosine

ABSTRACT: The most common cause of cryptococcal meningitis is Cryptococcus neoformans. The addition of 5-fluorocytosine to the treatment of patient with this potentially fatal infection significantly improves disease outcome. However, developing resistance to this drug is a serious problem in patient management. In this study, we investigated the molecular mechanisms causing 5-FC resistance in three clinical isolates of C. neoformans using genomic and proteomic approaches. Our findings show that genetic alterations in genes involved in the pyrimidine salvage pathway caused 5-FC resistance in these isolates. Specifically, one isolate had a missense mutation (E64G) in the uracil phosphoribosyl transferase gene (FUR1), another isolate had a large deletion spanning the cytosine deaminase gene (FCY1), and the third isolate had a point mutation in the same gene leading to a truncated protein at amino acid 164 (STOPW164*). Proteomic data confirmed that the genetic changes led to the absence or decreased the amount of the enzymes in the 5-FC resistant isolates. Transformation experiments confirmed that the genetic changes ultimately affected 5-FC susceptibility, providing further evidence of the effect of the genetic changes on 5-FC susceptibility.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Cryptococcus Neoformans Var. Neoformans Cryptococcus Neoformans Var. Grubii

SUBMITTER: Alfredo Cabrera-Orefice

LAB HEAD: Paul Verweij

PROVIDER: PXD044249 | Pride | 2024-07-29

REPOSITORIES: Pride

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			Action	DRS
	20210417_Cneof_Fatima_1C_01.raw	Raw
	20210417_Cneof_Fatima_1C_02.raw	Raw
	20210417_Cneof_Fatima_1C_03.raw	Raw
	20210417_Cneof_Fatima_1C_04.raw	Raw
	20210417_Cneof_Fatima_1C_05.raw	Raw

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Project description:Among the most central questions in Leishmania research is why some species remain in the skin dermis at the site of infection by the sand fly vector whereas other species migrate to visceral organs where they cause fatal visceral leishmaniasis. Although L. donovani is the species typically responsible for visceral leishmaniasis, an atypical L. donovani strain is the etiologic agent for cutaneous leishmaniasis in Sri Lanka. To identify molecular determinants for visceral disease, we have analysed the phenotype and genotype of two L. donovani clinical isolates from Sri Lanka where one isolate was derived from a cutaneous leishmaniasis patient (CL) and the other from a visceral leishmaniasis patient (VL). These isolates cause dramatically different pathology when introduced into mice; notably the CL isolate has lost the ability to survive in visceral organs while the VL isolate was highly virulent in visceral organs of BALB/c mice. Whole genome sequencing of the CL and VL isolates revealed that these genomes were very similar as there were no gene deletions and few individual gene amplifications. Indels resulting in frame shifts and loss/gain of stop codons resulted in 13 distinct pseudogenes present in each of the CL and VL isolates. There were 154 non-synonymous SNPs specific to the CL isolate and 193 non-synonymous SNPs specific to the VL isolate. Genome wide gene expression analysis revealed several transcript level differences, including the A2 virulence gene resulting in higher expression of A2 proteins in the VL isolate than in the CL isolate. Genotypic variations relevant to pathology and tropism in Leishmania can be interrogated by reverse genetics. Experimentally increasing A2 expression in the CL isolate through gene transfer significantly increased it’s ability to survive in the spleen of BALB/c mice and conversely, down-regulating A2 expression in the VL isolate abrogated attenuated its survival in BALB/c mice. These observations reveal that there are relatively few genetic differences between the CL and VL isolates apart from the A2 genes, but collectively these have profound effects on human disease and experimentally infected mice.

Project description:Among the most central questions in Leishmania research is why some species remain in the skin dermis at the site of infection by the sand fly vector whereas other species migrate to visceral organs where they cause fatal visceral leishmaniasis. Although L. donovani is the species typically responsible for visceral leishmaniasis, an atypical L. donovani strain is the etiologic agent for cutaneous leishmaniasis in Sri Lanka. To identify molecular determinants for visceral disease, we have analysed the phenotype and genotype of two L. donovani clinical isolates from Sri Lanka where one isolate was derived from a cutaneous leishmaniasis patient (CL) and the other from a visceral leishmaniasis patient (VL). These isolates cause dramatically different pathology when introduced into mice; notably the CL isolate has lost the ability to survive in visceral organs while the VL isolate was highly virulent in visceral organs of BALB/c mice. Whole genome sequencing of the CL and VL isolates revealed that these genomes were very similar as there were no gene deletions and few individual gene amplifications. Indels resulting in frame shifts and loss/gain of stop codons resulted in 13 distinct pseudogenes present in each of the CL and VL isolates. There were 154 non-synonymous SNPs specific to the CL isolate and 193 non-synonymous SNPs specific to the VL isolate. Genome wide gene expression analysis revealed several transcript level differences, including the A2 virulence gene resulting in higher expression of A2 proteins in the VL isolate than in the CL isolate. Genotypic variations relevant to pathology and tropism in Leishmania can be interrogated by reverse genetics. Experimentally increasing A2 expression in the CL isolate through gene transfer significantly increased itM-bM-^@M-^Ys ability to survive in the spleen of BALB/c mice and conversely, down-regulating A2 expression in the VL isolate abrogated attenuated its survival in BALB/c mice. These observations reveal that there are relatively few genetic differences between the CL and VL isolates apart from the A2 genes, but collectively these have profound effects on human disease and experimentally infected mice. 6 Samples in total, 3 each from VL and CL causing isolates were analyzed by Splice Leader RNASeq. These three samples from each of the isolates were grown to form one of the following three lifestages, Promastigotes, Macrophage derived Amastigotes, Axenic Amastigotes.

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Novel genetic mutations in pyrimidine salvage pathway enzymes from Cryptococcus neoformans contribute to reduced susceptibility against 5-fluorocytosine

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