Human tear fluid modulates the Pseudomonas aeruginosa transcriptome to alter antibiotic susceptibility

Lauren M Tabor; Melinda R Grosser; Matteo M M E Metruccio; Naren G Kumar; Yvonne T Wu; Vincent Nieto; David J Evans; Suzanne M J Fleiszig

doi:10.1016/j.jtos.2021.07.004

Human tear fluid modulates the Pseudomonas aeruginosa transcriptome to alter antibiotic susceptibility

Ocul Surf. 2021 Oct:22:94-102. doi: 10.1016/j.jtos.2021.07.004. Epub 2021 Jul 28.

Authors

Lauren M Tabor¹, Melinda R Grosser¹, Matteo M M E Metruccio¹, Naren G Kumar¹, Yvonne T Wu¹, Vincent Nieto¹, David J Evans², Suzanne M J Fleiszig³

Affiliations

¹ School of Optometry, University of California, Berkeley, CA, USA.
² School of Optometry, University of California, Berkeley, CA, USA; College of Pharmacy, Touro University California, Vallejo, CA, USA.
³ School of Optometry, University of California, Berkeley, CA, USA; Graduate Groups in Vision Science, Microbiology, and Infectious Diseases & Immunity, University of California, Berkeley, CA, USA. Electronic address: fleiszig@berkeley.edu.

Abstract

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.

Keywords: Antimicrobial susceptibility; Polymyxin B; Pseudomonas aeruginosa; RNA-Sequencing; Tear fluid; Tear-like fluid; arn operon; oprH-phoP/phoQ.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Anti-Bacterial Agents / pharmacology
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Gene Expression Regulation, Bacterial
Humans
Pseudomonas aeruginosa* / genetics
Transcriptome*

Substances

Anti-Bacterial Agents
Bacterial Proteins

Grants and funding

R01 EY024060/EY/NEI NIH HHS/United States