Silva Inês N, Ramires Marcelo J, Azevedo Lisa A, Guerreiro Ana R, Tavares Andreia C, Becker Jörg D, Moreira Leonilde M
IBB, Institute for Bioengineering and Biosciences, Instituto Superior Técnico (IST), Lisbon, Portugal.
Instituto Gulbenkian de Ciência, Oeiras, Portugal.
Appl Environ Microbiol. 2017 Sep 15;83(19). doi: 10.1128/AEM.01343-17. Print 2017 Oct 1.
LysR-type transcriptional regulators (LTTRs) are the most commonly found regulators in complex, comprising opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. Despite LTTRs being global regulators of pathogenicity in several types of bacteria, few have been characterized in Here, we show that gene of encoding an LTTR is cotranscribed with encoding a d-lactate dehydrogenase and evaluate their implication in virulence traits such as exopolysaccharide (EPS) synthesis and biofilm formation. A comparison of the wild type (WT) and its isogenic Δ mutant grown in medium with 2% d-glucose revealed a negative impact on EPS biosynthesis and on cell viability in the presence of LdhR. The loss of viability in WT cells was caused by intracellular acidification as a consequence of the cumulative secretion of organic acids, including d-lactate, which was absent from the Δ mutant supernatant. Furthermore, LdhR is implicated in the formation of planktonic cellular aggregates. WT cell aggregates reached 1,000 μm in size after 24 h in liquid cultures, in contrast to Δ mutant aggregates that never grew more than 60 μm. The overexpression of d-lactate dehydrogenase LdhA in the Δ mutant partially restored the formed aggregate size, suggesting a role for fermentation inside aggregates. Similar results were obtained for surface-attached biofilms, with WT cells producing more biofilm. A systematic evaluation of planktonic aggregates in CF clinical isolates showed aggregates in 40 of 74. As CF patients' lung environments are microaerophilic and bacteria are found as free aggregates/biofilms, LdhR and LdhA might have central roles in adapting to this environment. Cystic fibrosis patients often suffer from chronic respiratory infections caused by several types of microorganisms. Among them are the complex bacteria, which cause progressive deterioration of lung function that, in some patients, might develop into fatal necrotizing pneumoniae with bacteremia, known as "cepacia syndrome." pathogenesis is multifactorial as they express several virulence factors, form biofilms, and are highly resistant to antimicrobial compounds, making their eradication from the CF patients' airways very difficult. As is commonly found in CF lungs in the form of cell aggregates and biofilms, the need to investigate the mechanisms of cellular aggregation is obvious. In this study, we demonstrate the importance of a d-lactate dehydrogenase and a regulator in regulating carbon overflow, cellular aggregates, and surface-attached biofilm formation. This not only enhances our understanding of pathogenesis but can also lead to the development of drugs against these proteins to circumvent biofilm formation.
赖斯氏菌属转录调节因子（LTTRs）是复杂细菌中最常见的调节因子，其中包括导致囊性纤维化（CF）患者慢性呼吸道感染的机会致病菌。尽管LTTRs是几种细菌致病性的全局调节因子，但在[具体细菌名称]中对其进行表征的却很少。在此，我们表明编码LTTR的[细菌名称]基因与编码d - 乳酸脱氢酶的基因共转录，并评估它们在诸如胞外多糖（EPS）合成和生物膜形成等毒力特性中的作用。对在含2% d - 葡萄糖的培养基中生长的野生型（WT）及其同基因Δ突变体进行比较，结果显示在存在LdhR的情况下，对EPS生物合成和细胞活力有负面影响。WT细胞活力的丧失是由于包括d - 乳酸在内的有机酸累积分泌导致的细胞内酸化所致，而Δ突变体上清液中不存在d - 乳酸。此外，LdhR与浮游细胞聚集体的形成有关。在液体培养24小时后，WT细胞聚集体大小达到1000μm，相比之下，Δ突变体聚集体大小从未超过60μm。在Δ突变体中过表达d - 乳酸脱氢酶LdhA部分恢复了形成的聚集体大小，表明聚集体内部发酵起到了作用。对于表面附着的生物膜也获得了类似结果，WT细胞产生的生物膜更多。对[具体细菌名称]CF临床分离株中的浮游聚集体进行系统评估显示，74株中有40株存在聚集体。由于CF患者的肺部环境是微需氧的，并且细菌以游离聚集体/生物膜的形式存在，LdhR和LdhA可能在适应这种环境中起核心作用。囊性纤维化患者经常遭受由几种微生物引起的慢性呼吸道感染。其中包括[具体细菌名称]复合菌，它们会导致肺功能逐渐恶化，在一些患者中可能发展为伴有菌血症的致命坏死性肺炎，即“洋葱伯克霍尔德菌综合征”。[具体细菌名称]的发病机制是多因素的，因为它们表达多种毒力因子、形成生物膜并且对抗菌化合物具有高度抗性，这使得从CF患者气道中根除它们非常困难。由于[具体细菌名称]在CF肺部中通常以细胞聚集体和生物膜的形式存在，因此显然需要研究细胞聚集的机制。在本研究中，我们证明了d - 乳酸脱氢酶和一种调节因子在调节碳溢流、细胞聚集体和表面附着生物膜形成中的重要性。这不仅增强了我们对[具体细菌名称]发病机制的理解，还可能导致开发针对这些蛋白质的药物以规避生物膜形成。
