Global Genomic Analysis of AlgU (ςsgr;(E))-Dependent Promoters (Sigmulon) in Pseudomonas aeruginosa and Implications for Inflammatory Processes in Cystic Fibrosis

The conversion of Pseudomonas aeruginosa to the mucoid phenotype coincides with the establishment of chronic respiratory infections in cystic fibrosis (CF). A major pathway of conversion to mucoidy in clinical strains of P. aeruginosa is dependent upon activation of the alternative sigma factor AlgU (P. aeruginosa ςsgr;(E)). Here we initiated studies of AlgU-dependent global expression patterns in P. aeruginosa in order to assess whether additional genes, other than those involved in the production of the mucoid exopolysaccharide alginate, are turned on during conversion to mucoidy. Using genomic information and the consensus AlgU promoter sequence, we identified 35 potential AlgU (ςsgr;(E)) promoter sites on the P. aeruginosa chromosome. Each candidate promoter was individually tested by reverse transcription and mRNA 5′-end mapping using RNA isolated from algU(+) and algU::Tc(r) mutant cells. A total of 10 new AlgU-dependent promoters were identified, and the corresponding mRNA start sites were mapped. Two of the 10 newly identified AlgU promoters were upstream of predicted lipoprotein genes. Since bacterial lipoproteins have been implicated as inducers of inflammatory pathways, we tested whether lipopeptides corresponding to the products of the newly identified AlgU-dependent lipoprotein genes, lptA and lptB, had proinflammatory activity. In human peripheral blood monocyte-derived macrophages the peptides caused production of interleukin-8, a proinflammatory chemokine typically present at excessively high levels in the CF lung. Our studies show how genomic information can be used to uncover on a global scale the genes controlled by a given ςsgr; factor (collectively termed here sigmulon) using conventional molecular tools. In addition, our data suggest the existence of a previously unknown connection between conversion to mucoidy and expression of lipoproteins with potential proinflammatory activity. This link may be of significance for infections and inflammatory processes in CF.

铜绿假单胞菌（Pseudomonas aeruginosa）向黏液型表型的转化，与囊性纤维化（CF）患者慢性呼吸道感染的建立过程同步发生。临床分离的铜绿假单胞菌菌株向黏液型转化的核心途径，依赖于替代σ因子AlgU（铜绿假单胞菌σ^E）的激活。本研究率先针对铜绿假单胞菌中AlgU依赖型全局表达模式展开探究，以明确在向黏液型转化的过程中，除参与黏液型胞外多糖藻酸盐合成的基因外，是否存在其他被激活的基因。本研究借助基因组信息与AlgU启动子保守序列，在铜绿假单胞菌染色体上共鉴定出35个潜在的AlgU（σ^E）启动子位点。我们利用从algU(+)野生型菌株与algU::Tcr四环素抗性突变株中提取的RNA，通过逆转录（reverse transcription）与mRNA 5'端定位实验，对每一个候选启动子分别进行了验证。最终共鉴定出10个全新的AlgU依赖型启动子，并确定了其对应的mRNA转录起始位点。在这10个新鉴定的AlgU依赖型启动子中，有2个位于预测的脂蛋白（lipoprotein）基因的上游调控区域。鉴于细菌脂蛋白已被证实可作为炎症通路的诱导因子，本研究针对新鉴定的AlgU依赖型脂蛋白基因lptA与lptB的编码产物设计脂肽（lipopeptide），检测其是否具备促炎活性（proinflammatory activity）。在人外周血单核细胞来源的巨噬细胞（human peripheral blood monocyte-derived macrophages）中，该脂肽可诱导白细胞介素-8（interleukin-8, IL-8）的产生——IL-8作为一种促炎性趋化因子（chemokine），在CF患者的肺组织中通常呈现异常高表达水平。本研究展示了如何借助常规分子生物学技术，结合基因组信息在全局范围内鉴定某一σ因子所调控的全部基因（本文中将此类基因集合统称为σ因子调控子（sigmulon））。此外，本研究数据表明，铜绿假单胞菌向黏液型的转化与具有潜在促炎活性的脂蛋白表达之间，存在此前未被报道的关联。这一关联或许对CF患者的感染与炎症进程具有重要的研究价值与临床意义。