Purpose

Previously, we showed that tear fluid protects corneal epithelial cells against Pseudomonas aeruginosa without suppressing bacterial viability. Here, we studied how tear fluid affects bacterial gene expression.

Methods

RNA-sequencing was used to study the P. aeruginosa transcriptome after tear fluid exposure (5 h, 37 ^oC). Outcomes were further investigated by biochemical and physiological perturbations to tear fluid and tear-like fluid (TLF) and assessment of bacterial viability following tear/TLF pretreatment and antibiotic exposure.

Results

Tear fluid deregulated ~180 P. aeruginosa genes ≥8 fold versus PBS including downregulating lasI, rhlI, qscR (quorum sensing/virulence), oprH, phoP, phoQ (antimicrobial resistance) and arnBCADTEF (polymyxin B resistance). Upregulated genes included algF (biofilm formation) and hemO (iron acquisition). qPCR confirmed tear down-regulation of oprH, phoP and phoQ. Tear fluid pre-treatment increased P. aeruginosa resistance to meropenem ~5-fold (4 μg/ml), but enhanced polymyxin B susceptibility ~180-fold (1 μg/ml), the latter activity reduced by dilution in PBS. Media containing a subset of tear components (TLF) also sensitized bacteria to polymyxin B, but only ~22.5-fold, correlating with TLF/tear fluid Ca²⁺ and Mg²⁺ concentrations. Accordingly, phoQ mutants were not sensitized by TLF or tear fluid. Superior activity of tear fluid versus TLF against wild-type P. aeruginosa was heat resistant but proteinase K sensitive.

Conclusion

P. aeruginosa responds to human tear fluid by upregulating genes associated with bacterial survival and adaptation. Meanwhile, tear fluid down-regulates multiple virulence-associated genes. Tears also utilize divalent cations and heat resistant/proteinase K sensitive component(s) to enhance P. aeruginosa sensitivity to polymyxin B.

中文翻译：

---

人泪液调节铜绿假单胞菌转录组以改变抗生素敏感性

目的

以前，我们表明泪液可以保护角膜上皮细胞免受铜绿假单胞菌的侵害，而不会抑制细菌的活力。在这里，我们研究了泪液如何影响细菌基因表达。

方法

RNA 测序用于研究泪液暴露后（5 小时，37  ^o C）的铜绿假单胞菌转录组。通过对泪液和泪样液 (TLF) 的生化和生理扰动以及泪液/TLF 预处理和抗生素暴露后细菌活力的评估，进一步研究了结果。

结果

与 PBS 相比，泪液解除了 ~180 个铜绿假单胞菌基因的 8 倍以上，包括下调lasI、rhlI、qscR（群体感应/毒力）、oprH、phoP、phoQ（抗菌素耐药性）和arnBCADTEF（多粘菌素 B 耐药性）。上调基因包括algF（生物膜形成）和hemO（铁获取）。qPCR 证实了oprH、phoP和phoQ的撕裂下调。泪液预处理增加铜绿假单胞菌对美罗培南的耐药性约为 5 倍（4 微克/毫升），但增强多粘菌素 B 的敏感性约为 180 倍（1 微克/毫升），后者的活性因在 PBS 中稀释而降低。含有泪液成分子集 (TLF) 的培养基也使细菌对多粘菌素 B 敏感，但仅为 ~22.5 倍，与 TLF/泪液 Ca ²⁺和 Mg ²⁺浓度相关。因此，phoQ突变体不会被 TLF 或泪液致敏。泪液相对于 TLF 对野生型铜绿假单胞菌的优越活性是耐热的，但对蛋白酶 K 敏感。

结论

铜绿假单胞菌通过上调与细菌生存和适应相关的基因来响应人类泪液。同时，泪液下调多个毒力相关基因。眼泪还利用二价阳离子和耐热/蛋白酶 K 敏感成分来增强铜绿假单胞菌对多粘菌素 B 的敏感性。