Transcriptional Profile of Haemophilus influenzae: Effects of Iron and Heme. Haemophilus influenzae Rd KW20

Haemophilus influenzae frequently causes human disease, and humans are it’s sole niche. This bacterium has an absolute requirement for both a porphyrin and an iron source for aerobic growth, and exogenous heme can satisfy both requirements. Heme and iron can be acquired by H. influenzae from free or human protein-bound sources. The ability to selectively regulate the acquisition of heme and iron from physiological sources is a major virulence determinant for this microorganism. We utilized whole genome arrays to identify the full set of H. influenzae Rd KW20 iron and heme regulated genes. Condition specific RNA was derived from cells starved for both heme and iron and cells from the same culture 20 mins after the addition of exogenous iron and heme. The results identified 162 genes with a change in transcription = 1.5 fold. Eighty genes in 42 operons were preferentially expressed under iron/ heme starvation; 82 genes in 50 operons were preferentially expressed under iron/heme replete conditions. In each case, all genes contained within the operon were co-regulated. The former group included genes encoding proteins known to have a role in iron and heme uptake as well as several hypothetical ORFs. Enzymes involved in the gluconeogenesis pathway and glycogen biosynthesis were also upregulated. The genes showing increased transcription immediately after the addition of iron and heme primarily encode proteins involved with aerobic respiration and protein biosynthesis, consistent with a relaxation of starvation. Genomic transcriptional profiling provides a more complete understanding of the effects of iron and heme availability. Keywords: Transcription analyses Overall design: Three replicate cultures were grown in iron and heme depleted media for 90 minutes and samples taken for expression analyses. Fe and heme were supplemented to the remaining cultures and the cultures allowed to incubate an additional 20 minutes. Microarray analyses were used to determine changes in gene expression resulting from transfer from FeHm-deplete to FeHm-replete conditions.

流感嗜血杆菌（Haemophilus influenzae）常引发人类疾病，且人类是其唯一的生存生态位。该细菌在有氧生长过程中，绝对依赖卟啉与铁源，而外源性血红素可同时满足这两种需求。流感嗜血杆菌可从游离状态或人类蛋白质结合状态的底物中获取血红素与铁。从生理底物中选择性调控血红素与铁摄取的能力，是该微生物的主要毒力决定因子。
本研究利用全基因组阵列（whole genome arrays），鉴定了流感嗜血杆菌Rd KW20株中铁与血红素调控基因的完整集合。条件特异性核糖核酸（Ribonucleic Acid, RNA）分别提取自血红素与铁饥饿状态的菌体，以及在添加外源性铁和血红素20分钟后、来自同一培养物的菌体。
实验结果共鉴定出162个转录水平变化达1.5倍的基因。其中，42个操纵子（operon）内的80个基因在铁/血红素饥饿条件下优先表达；50个操纵子内的82个基因在铁/血红素充足条件下优先表达。两种情况下，操纵子内的所有基因均存在共调控现象。前一组基因包括已知参与铁与血红素摄取的蛋白编码基因，以及多个假设型开放阅读框（Open Reading Frame, ORFs）；糖异生途径（gluconeogenesis pathway）与糖原生物合成（glycogen biosynthesis）相关的酶也呈现上调表达。在添加铁与血红素后即刻出现转录上调的基因，主要编码参与有氧呼吸与蛋白质生物合成的蛋白，这与饥饿胁迫的解除相一致。基因组转录谱分析可更全面地解析铁与血红素可获得性对菌体的影响。
关键词：转录分析（transcription analyses）
整体实验设计：将三组重复培养物在铁与血红素耗尽的培养基中培养90分钟后，取样进行表达分析。向剩余培养物中添加铁与血红素，继续孵育20分钟。利用微阵列分析（microarray analyses），确定菌体从铁/血红素耗尽条件转移至铁/血红素充足条件后，基因表达发生的变化。