Evolution of hypervirulence during bloodstream adaptation of CA-MRSA strain USA300. Evolution of hypervirulence during bloodstream adaptation of CA-MRSA strain USA300

The epidemic community-acquired methicillin-resistant S. aureus (CA-MRSA) clone USA300 has recently become a leading cause of hospital-associated bloodstream infections (BSI). Leveraging this recent introduction into hospitals and the limited genetic variation across the USA300 strains, we combined microbial comparative genomics with phenotypic analyses to discover adaptive mutations. USA300 isolates from BSI were found to have independently evolved single nucleotide variants in the transcriptional regulator sarZ. sarZ inactivation lead to altered expression of virulence factors, resulting in increased lethality in a murine model of BSI. Thus, USA300 strains can optimize their fitness in hospitals through evolution of higher virulence. Overall design: To establish the SarZ regulon in the USA300 background we performed RNA-Seq experiments in sarZ wild-type, mutant and complement strains during logarithmic or stationary growth. The laboratory-constructed strains AH-LAC + vector, AH-LAC sarZ::bursa + vector, and AH-LAC sarZ::bursa + sarZWT were used. In addition we selected two clinical USA300 bloodstream infection (BSI) isolates with a wild-type (ER00573) and mutant sarZ locus (ER00594), and constructed sarZ deletion (sarZ::tet) and complement strains. Each strain was profiled, in triplicate, after culturing for 3h (logarithmic growth) and 5h (stationary phase) in 5mL tryptic soy broth (TSB).流行性社区获得性耐甲氧西林金黄色葡萄球菌（community-acquired methicillin-resistant S. aureus, CA-MRSA）克隆株USA300近期已成为医院相关性血流感染（bloodstream infections, BSI）的主要致病原。依托该菌株新近侵入医院环境以及USA300菌株间有限的遗传变异这一研究基础，我们结合微生物比较基因组学与表型分析手段，以发掘适应性突变。研究发现，从血流感染病例分离得到的USA300菌株，均在转录调控因子sarZ中独立演化出单核苷酸变异（single nucleotide variants）。sarZ基因失活会导致毒力因子的表达水平发生改变，进而在血流感染小鼠模型中提升了菌株的致死性。由此可见，USA300菌株可通过演化出更强的毒力，优化其在医院环境中的生存适配能力。总体实验设计：为明确USA300遗传背景下的SarZ调控子，我们在对数生长期与稳定生长期，对sarZ野生型、突变型及互补型菌株开展了RNA测序（RNA-Seq）实验。本研究使用实验室构建的AH-LAC+空载体、AH-LAC sarZ::bursa+空载体以及AH-LAC sarZ::bursa+sarZWT菌株。此外，我们选取了两株临床来源的USA300血流感染分离株：其中ER00573的sarZ基因座为野生型，ER00594的sarZ基因座携带突变，并构建了sarZ基因缺失株（sarZ::tet）与互补株。所有菌株均在5mL胰蛋白胨大豆肉汤（tryptic soy broth, TSB）中分别培养3小时（对应对数生长期）与5小时（对应稳定期）后，进行三次生物学重复的转录组测序分析。