The Pseudomonas aeruginosa type III secretion system (T3SS) needle comprised of multiple PscF subunits is essential for the translocation of effector toxins into human cells, facilitating the establishment and dissemination of infection. Mutations in the pscF gene provide resistance to the phenoxyacetamide (PhA) series of T3SS inhibitory chemical probes. To better understand PscF functions and interactions with PhA, alleles of pscF with 71 single mutations altering 49 of the 85 residues of the encoded protein were evaluated for their effects on T3SS phenotypes. Of these, 37% eliminated and 63% maintained secretion, with representatives of both evenly distributed across the entire protein. Mutations in 14 codons conferred a degree of PhA resistance without eliminating secretion, and all but one were in the alpha-helical C-terminal 25% of PscF. PhA-resistant mutants exhibited no cross-resistance to two T3SS inhibitors with different chemical scaffolds. Two mutations caused constitutive T3SS secretion. The pscF allele at its native locus, whether wild type (WT), constitutive, or PhA resistant, was dominant over other pscF alleles expressed from nonnative loci and promoters, but mixed phenotypes were observed in chromosomal ΔpscF strains with both WT and mutant alleles at nonnative loci. Some PhA-resistant mutants exhibited reduced translocation efficiency that was improved in a PhA dose-dependent manner, suggesting that PhA can bind to those resistant needles. In summary, these results are consistent with a direct interaction between PhA inhibitors and the T3SS needle, suggest a mechanism of blocking conformational changes, and demonstrate that PscF affects T3SS regulation, as well as carrying out secretion and translocation.

中文翻译：

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铜绿假单胞菌III型分泌针蛋白PscF的结构功能抑制分析。

在铜绿假单胞菌由多个PSCF亚基的III型分泌系统（T3SS）针是用于执行毒素进入人类细胞易位必不可少的，促进感染的建立和传播。pscF基因中的突变为T3SS抑制性化学探针的苯氧乙酰胺（PhA）系列提供抗性。为了更好地了解PscF的功能以及与PhA的相互作用，pscF的等位基因用71个单突变改变了编码蛋白的85个残基中的49个残基对T3SS表型的影响。在这些蛋白中，有37％消除了分泌，63％保持了分泌，两者的代表均匀分布在整个蛋白质上。14个密码子的突变在不消除分泌的情况下赋予了一定程度的PhA抗性，除一个密码子外，其他所有密码子都位于PscF的25％的α螺旋C末端。抗PhA的突变体对具有不同化学支架的两种T3SS抑制剂无交叉抗性。两个突变导致组成型T3SS分泌。无论是野生型（WT），组成型还是PhA抗性，其天然位点的pscF等位基因均优于其他pscF等位基因由非天然位点和启动子表达，但是在具有野生型和突变等位基因的染色体ΔpscF菌株中观察到混合表型。一些抗PhA的突变体表现出降低的易位效率，并以PhA剂量依赖性的方式得到改善，这表明PhA可以与那些抗性针结合。总之，这些结果与PhA抑制剂与T3SS针头之间的直接相互作用相一致，表明了阻断构象变化的机制，并证明PscF影响T3SS的调控以及进行分泌和转运。