RNA-Seq Transcriptomic Responses of Full-Thickness Dermal Excision Wounds to Pseudomonas aeruginosa Acute and Biofilm Infection

Abstract

Pseudomonas aeruginosa infections of wounds in clinical settings are major complications whose outcomes are influenced by host responses that are not completely understood. Herein we evaluated transcriptomic changes of wounds as they counter P. aeruginosa infection—first active infection, and then chronic biofilm infection. We used the dermal full-thickness, rabbit ear excisional wound model. We studied the wound response: towards acute infection at 2, 6, and 24 hrs after inoculating 106 bacteria into day-3 wounds; and, towards more chronic biofilm infection of wounds similarly infected for 24 hrs but then treated with topical antibiotic to coerce biofilm growth and evaluated at day 5 and 9 post-infection. The wounds were analyzed for bacterial counts, expression of P. aeruginosa virulence and biofilm-synthesis genes, biofilm morphology, infiltrating immune cells, re-epithelialization, and genome-wide gene expression (RNA-Seq transcriptome). This analysis revealed that 2 hrs after bacterial inoculation into day-3 wounds, the down-regulated genes (infected vs. non-infected) of the wound edge were nearly all non-coding RNAs (ncRNAs), comprised of snoRNA, miRNA, and RNU6 pseudogenes, and their down-regulation preceded a general down-regulation of skin-enriched coding gene expression. As the active infection intensified, ncRNAs remained overrepresented among down-regulated genes; however, at 6 and 24 hrs they changed to a different set, which overlapped between these times, and excluded RNU6 pseudogenes but included snRNA components of the major and minor spliceosomes. Additionally, the raw counts of multiple types of differentially-expressed ncRNAs increased on post-wounding day 3 in control wounds, but infection suppressed this increase. After 5 and 9 days, these ncRNA counts in control wounds decreased, whereas they increased in the infected, healing-impaired wounds. These data suggest a sequential and coordinated change in the levels of transcripts of multiple major classes of ncRNAs in wound cells transitioning from inflammation to the proliferation phase of healing.

Pseudomonas aeruginosa transcriptome adaptations from colonization to biofilm infection of skin wounds

“…1 . Our previous report describes the transcriptomic response of the rabbit ear wound tissue to this infection as well as the P. aeruginosa viable counts and morphology at each post-infection timepoint, including biofilm morphology characterized as aggregates on Days 5 and 9 that was confirmed by gene expression 21 .

Figure 1 Study design .

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“…After residing in the wounds for 24 h, the PAO1 inoculum increased in viable counts by 2 logs 21 . Coincidently, the COGs related to elevated metabolism and cell replication that were down-regulated at 2 h became mostly up-regulated after 24 h in the wound (Fig.…”

“…Here we have analyzed the P. aeruginosa transcriptome after log-phase bacteria were seeded into wounds and first colonized and then developed acute infection and finally biofilm infection. We previously reported the characterization of the wounds and the transcriptomic response of the wound tissue to this infection 21 .…”

Recent advances in understanding Pseudomonas aeruginosa as a pathogen

“…Thanks to the application of omics technologies, we have gained insight into P. aeruginosa ’s genome organisation and diversity 8 , 10 , 11 , 22 , 100 , habitat-specific transcriptome 101 – 105 , proteome 106 – 108 , and metabolome 108 – 112 and the co-evolution of P. aeruginosa with competitors in human habitats such as Staphylococcus aureus 113 – 115 . The function of hundreds of previously uncharacterised “conserved hypotheticals” 11 and the structure of secretory nanomachines have been resolved 37 , 41 , 116 – 121 and knowledge has been gained about the complex regulation of the release of exopolysaccharides, secondary metabolites, and virulence effectors 1 .…”

Repair Process Impairment by Pseudomonas aeruginosa in Epithelial Tissues: Major Features and Potential Therapeutic Avenues

“…Wound repair can also be assessed using more physiological models by performing wounds (of determined diameters), ex vivo , on explanted epithelial tissues, as well as in vivo , on live animals. It has been shown that P. aeruginosa laboratory or clinical strains decrease the repair rate of cutaneous wounds in rabbit, murine or porcine in in vivo models (Zhao et al, 2010; Mendes et al, 2013; Pastar et al, 2013; Brandenburg et al, 2015; Karna et al, 2016; Chaney et al, 2017). Conversely, Gao et al observed that purified P. aeruginosa flagellin significantly accelerated the wound closure of porcine eye wounds (Gao et al, 2010).…”