Montemari Anna Lisa, Marzano Valeria, Essa Nour, Levi Mortera Stefano, Rossitto Martina, Gardini Simone, Selan Laura, Vrenna Gianluca, Onetti Muda Andrea, Putignani Lorenza, Fiscarelli Ersilia Vita
Department of Diagnostics and Laboratory Medicine, Unit of Microbiology and Diagnostic Immunology, Unit of Cystic Fibrosis Diagnostics, Bambino Gesù Children's Hospital IRCCS, Rome, Italy.
Multimodal Laboratory Medicine Research Area, Unit of Human Microbiome, Bambino Gesù Children's Hospital IRCCS, Rome, Italy.
Front Med (Lausanne). 2022 Mar 9;9:818669. doi: 10.3389/fmed.2022.818669. eCollection 2022.
Cystic fibrosis (CF) is the most common rare disease caused by a mutation of the gene encoding a channel protein of the apical membrane of epithelial cells leading to alteration of Na and K transport, hence inducing accumulation of dense and sticky mucus and promoting recurrent airway infections. The most detected bacterium in CF patients is (PA) which causes chronic colonization, requiring stringent antibiotic therapies that, in turn induces multi-drug resistance. Despite eradication attempts at the first infection, the bacterium is able to utilize several adaptation mechanisms to survive in hostile environments such as the CF lung. Its adaptive machinery includes modulation of surface molecules such as efflux pumps, flagellum, pili and other virulence factors. In the present study we compared surface protein expression of PA multi- and pan-drug resistant strains to wild-type antibiotic-sensitive strains, isolated from the airways of CF patients with chronic colonization and recent infection, respectively. After shaving with trypsin, microbial peptides were analyzed by tandem-mass spectrometry on a high-resolution platform that allowed the identification of 174 differentially modulated proteins localized in the region from extracellular space to cytoplasmic membrane. Biofilm assay was performed to characterize all 26 PA strains in term of biofilm production. Among the differentially expressed proteins, 17 were associated to the virulome (e.g., Tse2, Tse5, Tsi1, PilF, FliY, B-type flagellin, FliM, PyoS5), six to the resistome (e.g., OprJ, LptD) and five to the biofilm reservoir (e.g., AlgF, PlsD). The biofilm assay characterized chronic antibiotic-resistant isolates as weaker biofilm producers than wild-type strains. Our results suggest the loss of PA early virulence factors (e.g., pili and flagella) and later expression of virulence traits (e.g., secretion systems proteins) as an indicator of PA adaptation and persistence in the CF lung environment. To our knowledge, this is the first study that, applying a shaving proteomic approach, describes adaptation processes of a large collection of PA clinical strains isolated from CF patients in early and chronic infection phases.
囊性纤维化（CF）是最常见的罕见病，由上皮细胞顶端膜通道蛋白编码基因突变引起，导致钠和钾转运改变，进而引起浓稠且黏性的黏液积聚，并促使反复发生气道感染。在CF患者中检测到的最常见细菌是铜绿假单胞菌（PA），它会导致慢性定植，需要严格的抗生素治疗，而这又会诱导多重耐药性。尽管在首次感染时尝试根除该细菌，但它能够利用多种适应机制在如CF肺部这样的恶劣环境中存活。其适应机制包括调节表面分子，如外排泵、鞭毛、菌毛和其他毒力因子。在本研究中，我们比较了分别从患有慢性定植和近期感染的CF患者气道中分离出的PA多重耐药和泛耐药菌株与野生型抗生素敏感菌株的表面蛋白表达。用胰蛋白酶处理后，在高分辨率平台上通过串联质谱分析微生物肽，从而鉴定出174种差异调节蛋白，这些蛋白位于从细胞外空间到细胞质膜的区域。进行生物膜测定以表征所有26株PA菌株的生物膜产生情况。在差异表达的蛋白质中，17种与病毒组相关（例如Tse2、Tse5、Tsi1、PilF、FliY、B型鞭毛蛋白、FliM、PyoS5），6种与耐药组相关（例如OprJ、LptD），5种与生物膜库相关（例如AlgF、PlsD）。生物膜测定表明，慢性抗生素耐药分离株作为生物膜产生者比野生型菌株弱。我们的结果表明，PA早期毒力因子（例如菌毛和鞭毛）的丧失以及后期毒力特征（例如分泌系统蛋白）的表达是PA在CF肺部环境中适应和持续存在的指标。据我们所知，这是第一项应用刮削蛋白质组学方法描述从CF患者早期和慢性感染阶段分离出的大量PA临床菌株适应过程的研究。
