Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, United States of America.
Department of Microbiology and Immunology, F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
PLoS Pathog. 2019 Mar 7;15(3):e1007385. doi: 10.1371/journal.ppat.1007385. eCollection 2019 Mar.
The six-component maintenance of lipid asymmetry (Mla) system is responsible for retrograde transport of phospholipids, ensuring the barrier function of the Gram-negative cell envelope. Located within the outer membrane, MlaA (VacJ) acts as a channel to shuttle phospholipids from the outer leaflet. We identified Neisseria gonorrhoeae MlaA (ngo2121) during high-throughput proteomic mining for potential therapeutic targets against this medically important human pathogen. Our follow-up phenotypic microarrays revealed that lack of MlaA results in a complex sensitivity phenome. Herein we focused on MlaA function in cell envelope biogenesis and pathogenesis. We demonstrate the existence of two MlaA classes among 21 bacterial species, characterized by the presence or lack of a lipoprotein signal peptide. Purified truncated N. gonorrhoeae MlaA elicited antibodies that cross-reacted with a panel of different Neisseria. Little is known about MlaA expression; we provide the first evidence that MlaA levels increase in stationary phase and under anaerobiosis but decrease during iron starvation. Lack of MlaA resulted in higher cell counts during conditions mimicking different host niches; however, it also significantly decreased colony size. Antimicrobial peptides such as polymyxin B exacerbated the size difference while human defensin was detrimental to mutant viability. Consistent with the proposed role of MlaA in vesicle biogenesis, the ΔmlaA mutant released 1.7-fold more membrane vesicles. Comparative proteomics of cell envelopes and native membrane vesicles derived from ΔmlaA and wild type bacteria revealed enrichment of TadA-which recodes proteins through mRNA editing-as well as increased levels of adhesins and virulence factors. MlaA-deficient gonococci significantly outcompeted (up to 16-fold) wild-type bacteria in the murine lower genital tract, suggesting the growth advantage or increased expression of virulence factors afforded by inactivation of mlaA is advantageous in vivo. Based on these results, we propose N. gonorrhoeae restricts MlaA levels to modulate cell envelope homeostasis and fine-tune virulence.
脂质不对称性（Mla）系统的六组分维持负责磷脂的逆行运输，确保革兰氏阴性细胞包膜的屏障功能。MlaA（VacJ）位于外膜内，作为将磷脂从外叶层穿梭的通道。我们在高通量蛋白质组学挖掘针对这种医学上重要的人类病原体的潜在治疗靶标时，发现了淋病奈瑟菌 MlaA（ngo2121）。我们随后的表型微阵列揭示，缺乏 MlaA 会导致复杂的敏感性表型。在此，我们专注于 MlaA 在细胞包膜生物发生和发病机制中的功能。我们证明在 21 种细菌物种中存在两种 MlaA 类，其特征是存在或缺乏脂蛋白信号肽。纯化的截短淋病奈瑟菌 MlaA 引发的抗体与不同奈瑟菌的抗体发生交叉反应。关于 MlaA 的表达知之甚少；我们提供了第一个证据，即 MlaA 水平在静止期和厌氧条件下增加，但在缺铁时减少。在模拟不同宿主小生境的条件下，缺乏 MlaA 会导致细胞计数增加；然而，它也显著降低了菌落大小。抗菌肽如多粘菌素 B 加剧了大小差异，而人防御素对突变体的生存力有害。与 MlaA 在囊泡生物发生中的作用一致，ΔmlaA 突变体释放的膜泡增加了 1.7 倍。来自ΔmlaA 和野生型细菌的细胞包膜和天然膜泡的比较蛋白质组学显示， TadA 的丰度增加 - 通过 mRNA 编辑重新编码蛋白质-以及粘附素和毒力因子的水平增加。MlaA 缺陷淋病奈瑟菌在小鼠下生殖道中的竞争能力比野生型细菌高（高达 16 倍），这表明 mlaA 失活赋予的生长优势或增加的毒力因子表达在体内是有利的。基于这些结果，我们提出淋病奈瑟菌限制 MlaA 水平以调节细胞包膜内稳态并微调毒力。
