Other possibilities that might involve in flucytosine resistance in C. tropicalis

tropicalis

Although this study has confirmed the expression of FCY2 mRNA and the attribution of this gene to 5FC resistance in C. tropicalis, it could not completely explain the 5FC susceptibility in all derivatives. For example, one of the derivatives of the isolate YM060088 carrying 145 T/T genotype exhibited an intermediate phenotype, and some derivatives were resistant to 5FC without disrupting any protein that was known to involve in 5FC resistance mechanisms. A BLASTP search has indicated the existence of another homolog gene CTRG_00460. The amino acid sequence of this gene shared an identity of 43% with the C. tropicals Fcy2p, deduced from the FCY2^S allele. Therefore, it is possibly that this FCY2-like gene contributed to the 5FC toxicity as well. It was similar to that found in C. lusitaniae and C. albicans,

where two FCY2-like genes were identified so far (Chapeland-Leclerc et al., 2005;

Hope et al., 2004). In C. albicans, there was no evidence that either one or both genes (i.e., FCY21 and/or FCY22) took part in encoding a functional PCP. However, in C.

lusitaniae, the expression of mRNA was detected only in one of the genes, FCY2. The other gene was considered to be a pseudogene (Chapeland-Leclerc et al., 2005).

According to multiple sequence alignment analysis, C. tropicalis Fcy2p shared 65%

amino acid sequence identity with C. lucitaniae Fcy2p and shared 43% identity with C. lucitaniae Fcy21p, suggesting that Fcy2p in C. tropicalis might correspond to the functional PCP. The research also demonstrated disruption of FCY2 gene alone could confer the 5FC resistance in C. lusitaniae (Chapeland-Leclerc et al., 2005). However, another report has implied that Fcy2p is not solely responsible for 5FC uptake in S.

cerevisiae. Four other FCY2 homologues, FCY21, FCY22, TPN1 and FUR4, were also contributed to 5FC toxicity, although in a less effective way (Paluszynski et al., 2006).

All in all, the studies of S. cerevisiae and C. lusitaniae have indicated that there was only one functional PCP in each yeast species, although the existence of more than one FCY2-like pseudogene and could contribute to the 5FC toxicity. Accordingly, it would be necessary to study the expression of the FCY2-like genes in C. tropicalis to confirm that apart from FCY2 gene whether other FCY2-like genes such as its paralogous gene CTRG_00460 may contribute to 5FC toxicity.

4.4 The cause of differential mRNA levels of FCY2 in yeasts carrying a nonsense mutation

The mRNA levels of the FCY2 gene in the G145T single mutation strains and the FCY2^R/FCY2^R homozygous mutants were lower than those in the clinical isolates, homozygous susceptible strains and the T145G single mutation strains. It indicated the involvement of the nonsense mutation in the decrease of the FCY2 mRNA level.

Since the polymorphic nucleotide was located in the open reading frame instead of the promoter region, it is surprising to detect the influence of this SNP on the mRNA level.

In fact, this phenomenon might be associated with a mechanism called nonsense- mediated mRNA decay, which leads to the initiation of mRNA decay (Jacobson and Peltz, 1996). The event that nonsense mutation in a gene reduced the abundance of the mRNA transcribed from that gene happened in both prokaryotes and eukaryotes

(Maquat, 1995; Peltz et al., 1994). The position where nonsense mutation occurred was important for the triggering of the rapid decay of mRNA. Previous researches have shown that nonsense mutation located within the first two-thirds to three- quarters of the coding region would facilitate the decay rate of the encoded transcripts up to 20 folds (Hagan et al., 1995; Yun and Sherman, 1995). In this study, the observed nonsense codon was located at amino acid position 49 in the deduced Fcy2p (509 a.a.), so it was within the range where a nonsense codon may increase the degradation rate of the FCY2 transcript. Supposedly, one of the functions of nonsense- mediated mRNA decay is to minimize the possibility of any harmful products derived from the transcripts with a nonsense mutation. This mRNA surveillance function raised the question why the nonsense mutation maintained in a significant number of the clinical isolates (24.7%), as the cells worked so hard to protect themselves from the unnecessary deleterious transcripts. Previous researches have implied the activation of the salvage pathway, which could cause the accumulation of high concentration of pyrimidine nucleotides in cells, might be toxic for the cells (Blondel et al., 2004; Seron et al., 1999). Therefore, the existence of this nonsense mutation in FCY2 gene, resulting in the inactivation of PCP, could give the mutation cells a selective advantage over other cells that did not possess this mutation.

4.5 Characterization of the loss of heterozygosity events

In this study, while one clinical isolate YM020291 remained heterozygous in all the polymorphic regions, the other isolate YM060800 showed consecutive homozygous regions on the left end side of the chromosome, extending up to 1.56 Mb.

This large region of homozygosity also reported in another C. tropicalis strain MYA-3404, as well as other Candida species, such as C. albicans, L. elongisporus (Butler et al., 2009). Because of the large chromosomal regions devoid of informative SNP markers, the identification of LOH events on the left end side of the chromosome was limited in the derivatives of YM060800. Apart from the absence of the useful SNP markers, sequence quality could also cause a problem in the SNP mapping process. For instance, the existence of mononucleotide repeats typically disturbed the analysis of nucleotides that were after the 3′ end of the repeat, which led to unclear or weak signals. These poor sequence data sometimes resulted in a false discovery of

SNP markers, and therefore produced a misjudgment of the polymorphic state. As a result, the polymorphic states of regions surrounding the LOH boundaries require additional sequencing check, such as using different primers to examine the corresponding regions or choosing adjacent areas to reevaluate the polymorphic states.

The sequencing profile also revealed a region, sometimes followed mononucleotide repeats, exhibited abundant of superimposed peaks which extended for a long distance.

For example, nucleotide sequence downstream of the gene CTRG_02086 in the clinical isolate YM060800 (from position 2208744 to 2208817, designated according to their relative location in the supercontig 2) has shown the extended superimposition pattern, whereas the corresponding region in its derivative YM060800-2 did not exhibit this pattern. The nucleotide sequence was compared between the clinical isolate YM060800 and its derivative YM060800-2. It has indicated that the cause of this sequencing profile was a deletion of adenine at position 2208754 of supercontig 2, and therefore generated a deletion polymorphism. Accordingly, while the polymorphic site at position 2208754 was heterozygous in the parental strain, it was homozygous in its derivative. In this case, it implied a smaller LOH boundary of 512 bp long (Figure 30).

SNP mapping has shown that four strains (YM020291-1, YM020291-2, YM060800-1 and YM060800) exhibited an extended homozygosity to the end of the supercontig 2. Although genome assemblies of C. tropicalis have not been mapped to chromosome yet, the distance from the 5’ end of the supercontig 2 to the telomeric repeat was estimated to be approximately 4,930 bases (Butler et al., 2009), suggesting the end of the supercontig 2 was likely compatible to the end of the chromosome in C.

tropicalis. As a result, the LOH events that encompassed a large distal region (from 0.7 to 2.25 Mb) in four strains might be due to break-induced replication (BIR) or allelic recombination, whereas the small LOH region observed in the strain YM060800-2 might be caused by gene conversion (Cullen et al., 2007). The majority of LOH events reported in this study were attributed to BIR/allelic recombination;

however, the number of strains analyzed here could not draw any conclusion to the frequency of different LOH events regarding to the generation of 5FC resistance to C.

tropicalis in vitro.

Six LOH boundaries were further investigated to identify features that might be involved in recombination. According to Baudat and Nicolas (1997), regions tending to introduce a double strand break (DSB), the initiation event of homologous

recombination, mostly located in intergenic promoter containing intervals (either one promoter/one terminator combination or two divergent promoters) (Baudat and Nicolas, 1997). In this study, two LOH boundaries (YM020291-1 and the left border in YM060800-2) fell in these intervals while three boundaries, covering a larger area, included such regions. However, it was surprising to observe one LOH boundary was falling within a coding region (CTRG_02123). In conclusion, of the six LOH boundaries, five were likely to be located in regions tending to initiate homologous recombination. However, except for the location of the boundaries, no strong evidence supports other features relating to the recombination was associated with the boundaries acquired in this study.

Chapter 5. Future work

5.1 Evaluation of the mechanism of 5FC resistance in C. tropicalis

The mechanisms of 5FC resistance of the clinical isolate YM060607 and the derivatives of seven clinical isolates (YM020438, YM020671, YM060075, YM060097, YM060210, YM060369 and YM060616) require further investigation.

Based on the nucleotide sequence analysis, the 5FC resistant clinical isolate, YM060607, has a heterozygous SNP (C/T) at the position 431 of the FUR1 gene, and a heterozygous SNP (A/G) at the position 775 of the URA3 gene. Both single nucleotide variations (C431T in FUR1 and G775A in URA3) lead to single amino acid changes (T144I in UPRT and A259T in ODCase), which might influence the functions of proteins involved in the 5FC resistance mechanisms. The contribution of these nucleotide changes to the 5FC resistance can be further investigated by constructing the point mutation strains. Genes, other than URA3, encoding proteins that are involved in the de novo pathway may play a role in 5FC resistance. Therefore, these genes can be examined if there are correlations between particular polymorphic nucleotides in the coding region and the resistance to 5FC. Also, alternations on gene expression, which may be due to changes in the 5’ or 3’UTR region, or the interference of regulators of genes associated with 5FC resistance, could result in 5FC resistance. Accordingly, the mRNA level of these genes should be investigated. In addition, as mentioned in the discussion 4.3, the FCY2-like gene, CTRG_00460, might involve in 5FC toxicity and, therefore, the disruption of this gene is likely to influence the 5FC resistance in C. tropicalis. However, the contribution of this homolog gene to the 5FC resistance needs to be examined.

5.2 Examination of the features of the boundaries that might be related to recombination event.

At least three known factors might be associated with recombination and therefore relate to the LOH boundaries (i.e., the GC content, the SNP frequency and the distance to tRNA). Previous research has implied that high GC contents at local

scales might stimulate recombination rate (Marsolier-Kergoat and Yeramian, 2009).

Nevertheless, a gene is often characterized by having a higher GC content relative to the background GC content for the entire genome and the length of a gene is directly proportional to a higher GC content (Pozzoli et al., 2008). Therefore, the GC content of the boundaries with a comparable length scale could compare to either the background GC content or the GC content of the coding regions. Furthermore, the frequency of SNPs also plays a role in the recombination, which is considerable higher in hot spots relative to the genomic average (Brandstrom et al., 2008). SNP frequency of one of the LOH boundaries, located in a coding region, is higher (one SNP per 67 bases) than the average rate of polymorphism in C. tropicalis (one SNP per 576 bases) (Pozzoli et al., 2008); however, whether the SNP frequency around this boundary is actually higher than other regions of supercontig 2 needs to be determined by additional static analyses. Apart from the location of the LOH boundary and the SNP frequency, several researches have proposed the association of tRNA with gross chromosomal rearrangements (Diogo et al., 2009; Dunham et al., 2002; Dunn et al., 2005). Here, two tRNA genes are found near the boundary of YM060800 (the closest distance of which was 3 kb), but their associations with the boundary should be determined by additional static analyses. Last but not the least, the sequence of each boundary can be compared and analyzed to search for hot spots of the recombination.

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