Plasmodium vivax serine repeat antigen (SERA) multigene family exhibits similar expression patterns in independent infections
Plasmodium vivax serine repeat antigen (SERA) multigene family exhibits similar expression patterns in independent infections
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DOI：
10.1016/j.molbiopara.2006.07.006
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发表时间：
2006-12-01
影响因子：
1.5
通讯作者：
Horii, Toshihiro
中科院分区：
文献类型：
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作者：
Palacpac, Nirianne Marie Q.;Leung, Betty W. Y.;Horii, Toshihiro
采集P.间日疟原虫感染的无并发症成人（≥16岁）患者，咨询泰国西北部的梅索疟疾诊所。提取寄生虫总RNA，合成第一链互补DNA，进行实时聚合酶链反应（PCR），提取基因组DNA。用于基因分型的引物扩增500-800 bp的短C-末端片段。将扩增产物克隆到pCR-Blunt II-TOPO中并测序。每个基因座使用至少两个独立的扩增。当发现单突变时，从原始模板重复双向测序以进行验证。进行核苷酸和推导的氨基酸序列比对。所有患者样本的感染红细胞的实时PCR分析显示几乎相似的转录谱，尽管转录水平在一定程度上取决于单个分离株，如观察到的PfSERA家族成员。PvSERA 4总是比其他SERA基因更强地转录; PvSERA 3和-7是转录最少的成员。还注意到PvSERA 2、-5、-10和-11的相对高的转录。PvSERA 4在所有菌株中的最高转录水平与P.恶性疟原虫。在评估SERA多样性时，使用PvSERA 4、-5和-8的C末端序列进行分析，并以PvMSP 1作为对照。PvSERA C-末端区域的异质性与PfSERA 5 C-末端的完整序列保守性形成鲜明对比。PvSERA基因的每个位点的可变区可分为2-3个主要等位基因形式，每个等位基因又表现出由点突变或缺失引起的多态性。PvSERA 4等位基因组与缺失和插入以及不同数量的QG（P/S）（P/S）分散重复序列显示出41-67%的成对同一性。PvSERA 5等位基因在以甘氨酸不完全重复（等位基因5A）或GVGVA（P/T）重复（等位基因5 B）为特征的基本序列类型之间具有50-80%的成对序列同一性。PvSERA 8等位基因的同源性为63-72%，其中一组（等位基因8A）为多态性，另一组（等位基因8B）相对于Sal-1型有多处替换; PvMSP 1在测序区域内也基本上为多态性。大多数寄生虫是Sal-1序列类型。PvSERA 4、-5和-8的单倍型多样性相当高，范围为0.78至0.94。无论基因家族成员是否高表达，这3个位点的单倍型多样性均无显著差异，且其多样性水平与PvMSP 1相当。所有的感染都是复杂的，根据PvSERA和PvMSP 1，每个感染平均有2-3种基因型。不同PvSERA等位基因的流行和基因型感染的多样性很可能是PvSERA 4和-5在不同分离株和不同采样时间观察到的重组类型的来源。因此，在aP中存在高水平的PvSERA等位基因杂合性。来自泰国的vivax人口，那里疟疾传播率低。
Blood samples were collected fromP. vivax-infected uncomplicated adult (≥16 years old) patients consulting the Mae Sot Malaria Clinic, Northwestern Thailand. Parasite total RNA was isolated, first-strand complementary DNA was synthesized, real-time polymerase chain reaction (PCR) was performed and genomic DNA was isolated. Primers for genotyping amplified short C-terminal fragments of 500-800 bp. Amplified products were cloned into pCR-Blunt II-TOPO and sequenced. At least two independent amplifications for each gene locus were used. When singleton mutation was found, bi-directional sequencing was repeated from the original template for verification. Nucleotide and deduced amino acid sequence alignments were performed. Real-time PCR analysis of infected erythrocytes from all patient samples show almost similar transcription profiles, though transcription levels varied to some extent depending on the individual isolates as observed forPfSERAfamily members.PvSERA4was always more strongly transcribed than otherSERAgenes;PvSERA3and -7, the least transcribed members. Relatively high transcription was also noted forPvSERA2, -5, -10, and -11. The highest transcription ofPvSERA4in all isolates parallels that ofPfSERA5, the predominantly expressed gene member inP. falciparum. In assessingSERAdiversity, C-terminal sequences ofPvSERA4, -5, and -8, withPvMSP1as control, were used for analyses. The heterogeneity in the C-termini region ofPvSERAwas in sharp contrast to the complete sequence conservation in the C-termini ofPfSERA5. At each locus ofPvSERAs, variable regions can be grouped into 2-3 major allelic forms and each allele in turn shows polymorphism resulting from point mutations or deletions.PvSERA4allele groups showed 41-67% pairwise identity with deletions and insertions as well as differing numbers of QG(P/S)(P/S) dispersed repeats. One recombinant type (Allele 4R) was identified.PvSERA5alleles had 50-80% pairwise sequence identity between basic sequence types characterized by having either glycine imperfect repeats (Alleles 5A) or GVGVA(P/T) repeats (Alleles 5B). One recombinant type (5R) was also identified.PvSERA8alleles had 63-72% pairwise identity, with dimorphic SNPs in one group (Alleles 8A) and another group having a number of substitutions relative to Sal-1 type (Alleles 8B).PvMSP1polymorphism was also essentially dimorphic in the region sequenced. Majority of the parasites are of the Sal-1 sequence type. Haplotype diversity ofPvSERA4, -5, and -8was substantially high, ranging from 0.78 to 0.94. The haplotype diversity in this three loci did not differ significantly from each other whether the gene family member was highly expressed or not; and their diversity levels were comparable toPvMSP1. All infections were complex, having a mean of 2-3 genotypes per infection based onPvSERAandPvMSP1, respectively. The prevalence of diversePvSERAalleles and multiplicity of genotype infections may well be the source of the recombinant types observed inPvSERA4and -5from different isolates and sampling periods. Thus, there is a high level of heterozygosity ofPvSERAalleles in aP. vivaxpopulation from Thailand where malaria transmission is low.