Baarda Benjamin I, Emerson Sarah, Proteau Philip J, Sikora Aleksandra E
Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, USA.
Department of Statistics, Oregon State University, Corvallis, Oregon, USA.
J Bacteriol. 2017 Aug 8;199(17). doi: 10.1128/JB.00037-17. Print 2017 Sep 1.
The function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such as A proteomics-driven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates-NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344-in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a Δ mutant (Δ) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains.
细胞包膜蛋白的功能和细胞外定位使其成为开发针对细菌性疾病疫苗的有吸引力的候选对象，这些疾病包括具有挑战性的耐药病原体，如淋病奈瑟菌。一种蛋白质组学驱动的反向疫苗学方法已经产生了多种淋病疫苗候选物；然而，其中许多候选物的生物学功能仍有待阐明。在此，利用表型芯片在1056种条件下对6种淋病疫苗候选物——NGO2121、NGO1985、NGO2054、NGO2111、NGO1205和NGO1344——在细胞包膜稳态中的功能进行了探究，并以Δ突变体（Δ）作为外膜完整性受到干扰的参考。开发了在限定液体培养基中进行表型芯片测定的最佳生长条件，这在其他应用中可能有用，包括快速和全面的抗菌药物敏感性评估。我们的研究揭示了91种对其中一个被检测突变体有独特的正向或负向影响的条件。对37种和57种分别常见的有益和有害化合物进行的聚类分析揭示了三个不同的表型组：NGO2121和NGO1985；NGO1344和BamE；以及NGO1205、NGO2111和NGO2054组成的三联体，其中最后一种蛋白质形成了该聚类的一个独立分支。在高度耐抗生素的世界卫生组织X菌株中，这些疫苗候选物的缺失与相似的表型相关。基于它们广泛的敏感性表型，NGO1985和NGO2121似乎是最有前景的疫苗候选物。这项研究确立了表型芯片可以成功应用于像淋病奈瑟菌这样挑剔的细菌生物体的原则。需要创新方法来开发针对常见和被忽视的性传播感染（如淋病）的疫苗。在此，我们在首次针对淋病奈瑟菌的此类研究中利用了表型芯片，以探究蛋白质组衍生的疫苗候选物在细胞包膜稳态中的功能。从这种筛选中获得的信息可以通过深入了解这些蛋白质在膜通透性、完整性和整体生理学中所起的作用，为疫苗候选决策树提供参考。除了揭示临床菌株和实验室菌株之间的表型差异外，优化的筛选方案还可应用于对在不断增加的全基因组序列中发现的淋病奈瑟菌其他假设蛋白质功能的研究。
