School of Optometry, University of California, Berkeley, CA, USA.
School of Optometry, University of California, Berkeley, CA, USA; College of Pharmacy, Touro University California, Vallejo, CA, USA.
Ocul Surf. 2021 Oct;22:94-102. doi: 10.1016/j.jtos.2021.07.004. Epub 2021 Jul 28.
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.
RNA-sequencing was used to study the P. aeruginosa transcriptome after tear fluid exposure (5 h, 37 C). 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.
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 Ca and Mg 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.
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.
之前，我们表明泪液在不抑制细菌活力的情况下保护角膜上皮细胞免受铜绿假单胞菌的侵害。在这里，我们研究了泪液如何影响细菌的基因表达。
使用 RNA 测序研究了铜绿假单胞菌在泪液暴露（5 小时，37°C）后的转录组。通过对泪液和类似泪液（TLF）的生化和生理干扰以及在泪液/TLF 预处理和抗生素暴露后评估细菌活力，进一步研究了结果。
与 PBS 相比，泪液使约 180 个铜绿假单胞菌基因的表达下调了≥8 倍，包括下调了 lasI、rhlI、qscR（群体感应/毒力）、oprH、phoP、phoQ（抗微生物）和 arnBCADTEF（多粘菌素 B 耐药性）。上调的基因包括 algF（生物膜形成）和 hemO（铁摄取）。qPCR 证实了 oprH、phoP 和 phoQ 的泪液下调。泪液预处理使铜绿假单胞菌对美罗培南的耐药性增加了5 倍（4μg/ml），但使多粘菌素 B 的敏感性增加了180 倍（1μg/ml），后者的活性通过在 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~22.5 倍，与 TLF/泪液中的 Ca 和 Mg 浓度相关。因此，TLF 或泪液不能使 phoQ 突变体敏感。与 TLF 相比，泪液对野生型铜绿假单胞菌的活性具有耐热性但对蛋白酶 K 敏感。
铜绿假单胞菌对人泪液的反应是上调与细菌存活和适应相关的基因。同时，泪液下调了多个与毒力相关的基因。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。
