BACKGROUND Plasmodium falciparum cysteine proteases (falcipains) play indispensable roles in parasite infection and development, especially in the process of host erythrocyte rupture/invasion and hemoglobin degradation. No detailed molecular analysis of transcriptional regulation of parasite proteases especially cysteine proteases has yet been reported. In this study, using a combination of transient transfection assays and electrophoretic mobility shift assays (EMSA), we demonstrate the presence of stage specific nuclear factors that bind to unique sequence elements in the 5'upstream regions of the falcipains and probably modulate the expression of cysteine proteases. RESULTS Falcipains differ in their timing of expression and exhibit ability to compensate each other's functions at asexual blood stages of the parasite. Present study was undertaken to study the transcriptional regulation of falcipains. Transient transfection assay employing firefly luciferase as a reporter revealed that a ~1 kb sequence upstream of translational start site is sufficient for the functional transcriptional activity of falcipain-1 gene, while falcipain-2, -2' and -3 genes that exist within 12 kb stretch on chromosome 11 require ~2 kb upstream sequences for the expression of reporter luciferase activity. EMSA analysis elucidated binding of distinct nuclear factors to specific sequences within the 5'upstream regions of falcipain genes. Analysis of falcipains' 5'upstream regulatory regions did not reveal the presence of sequences known to bind general eukaryotic factors. However, we did find parasite specific sequence elements such as poly(dA) poly(dT) tracts, CCAAT boxes and a single 7 bp-G rich sequence, (A/G)NGGGG(C/A) in the 5' upstream regulatory regions of these genes, thereby suggesting the role(s) of Plasmodium specific transcriptional factors in the regulation of falcipain genes. CONCLUSION Taken together, these results suggest that expression of Plasmodium cysteine proteases is regulated at the transcriptional level and parasite specific factors regulate the expression of falcipain genes. These findings open new venues for further studies in identification of parasite specific transcription factors.

中文翻译：

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不同的和阶段特异性的核因子调节恶性疟原虫、恶性疟原虫半胱氨酸蛋白酶的表达。

背景技术恶性疟原虫半胱氨酸蛋白酶(falcipains)在寄生虫感染和发育，特别是在宿主红细胞破裂/侵袭和血红蛋白降解过程中起着不可或缺的作用。尚未报道对寄生虫蛋白酶特别是半胱氨酸蛋白酶的转录调控的详细分子分析。在这项研究中，使用瞬时转染分析和电泳迁移率变化分析 (EMSA) 的组合，我们证明了阶段特异性核因子的存在，这些因子与 falcipains 5' 上游区域的独特序列元素结合，并可能调节半胱氨酸蛋白酶。结果 恶性疟原​​虫的表达时间不同，并且在寄生虫的无性血液阶段表现出补偿彼此功能的能力。本研究旨在研究恶性疟原虫的转录调控。使用萤火虫荧光素酶作为报告基因的瞬时转染分析表明，翻译起始位点上游的 ~1 kb 序列足以满足 falcipain-1 基因的功能性转录活性，而 falcipain-2、-2' 和 -3 基因存在于 12 11 号染色体上的 kb 延伸需要~2 kb 的上游序列来表达报告荧光素酶活性。EMSA 分析阐明了不同核因子与 falcipain 基因 5' 上游区域内特定序列的结合。对恶性疟原虫 5' 上游调控区的分析并未揭示已知结合一般真核生物因子的序列的存在。然而，我们确实发现了寄生虫特定的序列元件，例如 poly(dA) poly(dT) 束，CCAAT 框和单个 7 bp-G 丰富的序列，(A/G)NGGGG(C/A) 在这些基因的 5' 上游调控区，从而表明疟原虫特异性转录因子在调控恶性疟基因。结论 综上所述，这些结果表明疟原虫半胱氨酸蛋白酶的表达在转录水平受到调节，而寄生虫特异性因子调节恶性蛋白酶基因的表达。这些发现为进一步研究鉴定寄生虫特异性转录因子开辟了新的途径。这些结果表明，疟原虫半胱氨酸蛋白酶的表达在转录水平上受到调控，而寄生虫特异性因子调控着恶性蛋白酶基因的表达。这些发现为进一步研究鉴定寄生虫特异性转录因子开辟了新的途径。这些结果表明，疟原虫半胱氨酸蛋白酶的表达在转录水平上受到调控，而寄生虫特异性因子调控着恶性蛋白酶基因的表达。这些发现为进一步研究鉴定寄生虫特异性转录因子开辟了新的途径。