June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
RNA-seq of P. aeruginosa PA14 treated with lacritin bactericidal peptide 'N-104' reveals an underlying disruption of iron and spermidine availability together with other mechanisms
Author Affiliations & Notes
  • Fatemeh Norouzi
    Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia, United States
  • Mohammad Sharifian Gh.
    Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia, United States
  • Gordon W Laurie
    Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia, United States
    Ophthalmology, University of Virginia School of Medicine, Charlottesville, Virginia, United States
  • Footnotes
    Commercial Relationships   Fatemeh Norouzi None; Mohammad Sharifian Gh. None; Gordon Laurie None
  • Footnotes
    Support  EY026171 and EY032956 to GWL, and an unrestricted gift to the Department of Cell Biology from TearSolutions, Inc.
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 4374. doi:
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      Fatemeh Norouzi, Mohammad Sharifian Gh., Gordon W Laurie; RNA-seq of P. aeruginosa PA14 treated with lacritin bactericidal peptide 'N-104' reveals an underlying disruption of iron and spermidine availability together with other mechanisms. Invest. Ophthalmol. Vis. Sci. 2023;64(8):4374.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Non-lytic lacritin bactericidal peptide 'N-104' interrupts molecular transport by FeoB (ferrous iron) and PotH (spermidine) in the pathogenic P. aeruginosa strain PA14 as revealed by unbiased resistance screening followed by several validation studies. Iron and spermidine are necessary for viability yet there exist multiple iron uptake mechanisms. Here, we sought a wider understanding of iron uptake and other processes affected by N-104 as may be inferred by unbiased RNA-sequencing (RNA-seq).

Methods : 2.5x108 PA14 log cells resuspended in 0.1xPBS were incubated without or with 20 μM N-104 or negative control N-80/C-25 for 45 min at 35°C (3 replicates). Cells were resuspended in TE buffer with RNA-protect Bacteria Reagent, lysed in lysozyme, proteinase K and SDS for RNA isolation. Library construction/sequencing were performed by UVA's GATC involving enrichment via the NEBNext® rRNA Depletion Kit (Bacteria) and transcriptome sequencing. Data were analyzed by UVA's Bioinformatics Core.

Results : Heat maps of (i) N-104 treated vs. N-80/C-25 treated samples or (ii) N-104 treated vs. untreated were plotted. Although 28 down- and 36 up-regulated genes were shared between (i) and (ii), particular attention was paid to (i) including decreased expression of: PA14_37490 (putative TonB-dependent receptor (iron uptake)); ssuF (sulfur metabolism on which iron regulation of ilvA1 and ilvD are dependent); speC (catalyzes the generation of the putrescine, a precursor of spermidine); and lcaD (adhesion). In (i), we observed increased expression of tatC (involved in iron uptake) and ccmF (involved in the formation of [Fe-S] clusters). Expression of numerous other genes was affected. Those with decreased expression were involved in biofilm formation (PA14_42090), metabolism, virulence, stress response and resistance (psrA, PA14_40380, 26140, 47520, 27530), and transport of small molecules and peptides (PA14_37460, 37470). Those up-regulated were involved in two-component sensors (dctB, PA14_22960 and 46980), cell morphology and lysis (pbpA), radical activation (PA14_51680) and secretion (xcpX).

Conclusions : RNA-seq suggests that N-104 may affect iron and polyamine metabolism more widely beyond FeoB and PotH, and that numerous other N-104 dependent mechanisms remain to be explored.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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