Battisti James M, Sappington Kate N, Smitherman Laura S, Parrow Nermi L, Minnick Michael F
Division of Biological Sciences, The University of Montana, Missoula, MT 59812, USA.
Infect Immun. 2006 Jun;74(6):3251-61. doi: 10.1128/IAI.00245-06.
Of all bacteria, Bartonella quintana has the highest reported in vitro hemin requirement, yet an explanation for this remains elusive. To produce diseases such as trench fever, endocarditis, and bacillary angiomatosis, B. quintana must survive and replicate in the disparate environments of the Pediculus humanus corporis (body louse) gut and the human vasculature. We previously identified a five-member family of hemin binding proteins (Hbps) synthesized by B. quintana that bind hemin on the outer surface but share no similarity to known bacterial heme receptors. In the present study, we examine the transcription, regulation, and synthesis of this virulence factor family by cultivation of the bacterium in environments that simulate natural heme, oxygen, and temperature conditions encountered in the host and insect vector. First, quantitative real-time PCR data show that hbpC expression is regulated by temperature, where a >100-fold increase in transcript quantity was seen at 30 degrees C relative to 37 degrees C, suggesting that HbpC synthesis would be greatest in the cooler temperature of the louse. Second, cultivation at human bloodstream oxygen concentration (5% relative to 21% atmospheric) significantly decreases the transcript quantity of all hbp genes, indicating that expression is influenced by O2 and/or reactive oxygen species. Third, a differential expression pattern within the hbp family is revealed when B. quintana is grown in a range of hemin concentrations: subgroup I (hbpC and hbpB) predominates in a simulated louse environment (high heme), and subgroup II (hbpA, hbpD, and hbpE) is preferentially expressed in a simulated human background (low heme). By using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry fingerprinting, we demonstrate that synthesis of HbpA correlates with hbpA transcript increases observed at low hemin concentrations. Finally, an hbpA promoter-lacZ reporter construct in B. quintana demonstrates that a transcriptional regulator(s) is controlling the expression of hbpA through a cis-acting regulatory element located in the hbpA promoter region.
在所有细菌中，五日热巴尔通体在体外对血红素的需求量据报道是最高的，但对此的解释仍然难以捉摸。为了引发诸如战壕热、心内膜炎和杆菌性血管瘤病等疾病，五日热巴尔通体必须在人体虱肠道和人体脉管系统等不同环境中存活并繁殖。我们之前鉴定出了一个由五日热巴尔通体合成的包含五个成员的血红素结合蛋白（Hbps）家族，这些蛋白在外表面结合血红素，但与已知的细菌血红素受体没有相似性。在本研究中，我们通过在模拟宿主和昆虫载体中自然血红素、氧气和温度条件的环境中培养该细菌，来研究这个毒力因子家族的转录、调控和合成。首先，定量实时PCR数据表明hbpC的表达受温度调节，在30℃时转录本数量相对于37℃增加了100倍以上，这表明在虱子较凉爽的温度下HbpC的合成量最大。其次，在人体血液氧气浓度（相对于大气中的21%为5%）下培养会显著降低所有hbp基因的转录本数量，表明表达受氧气和/或活性氧物种的影响。第三，当五日热巴尔通体在一系列血红素浓度下生长时，hbp家族内呈现出差异表达模式：I亚组（hbpC和hbpB）在模拟虱子环境（高血红素）中占主导，II亚组（hbpA、hbpD和hbpE）在模拟人体背景（低血红素）中优先表达。通过使用二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、免疫印迹和基质辅助激光解吸电离飞行时间质谱指纹图谱，我们证明HbpA的合成与在低血红素浓度下观察到的hbpA转录本增加相关。最后，五日热巴尔通体中的一个hbpA启动子-lacZ报告构建体表明，一个转录调节因子通过位于hbpA启动子区域的顺式作用调节元件控制hbpA的表达。
