Pseudomonas aeruginosa PA5oct jumbo phage impacts planktonic and biofilm population and reduces its host virulence

Abstract

SummaryIn this work we analyzed the impact of jumbo phage PA5oct on the planktonic, cell line adhered, and biofilm population of P. aeruginosa. PA5oct has a broad host-range, able to infect up to 40% of our clinical P. aeruginosa Cystic Fibrosis (CF) collection. In the airway surface liquid (ASL) model, the infection of PA5oct effectively reduced the bacterial population both adhered to epithelial cells, mucus entrapped, and dispersed. The explanation for its infectivity can also be linked to the sensitization of infected bacteria to the innate immune mechanisms and pro-inflammatory effect. Interferometry of a 72-hour old biofilm highlighted the contribution of PA5oct in biofilm matrix degradation. Interestingly, two virion-associated proteins, gp162 and gp205, have been found as putative enzymes that can degrade matrix exopolysaccharides. Two third of biofilm clones developed PA5oct phage-resistance and the cross-resistance to both LPS- and pili-dependent phages. Simultaneously, all clones resistant to phage PA5oct maintain the phage DNA within the population, strongly reducing bacterial virulence in vivo. These properties can be considered as key parameters for the application of this bacterial virus in phage therapy settings.Originality-Significance StatementThe emergence of phage-resistant mutants is a key aspect of lytic phages-bacteria interaction and the main driver for the co-evolution between both organisms. However, this fundamental property also has implications for bacterial eradication in phage therapy settings. Here, we analyze the impact of PA5oct jumbo phage treatment of planktonic/cell line associated and sessile P. aeruginosa population in a preclinical evaluation of this phage for therapeutic applications. Besides its broad-spectrum activity and efficient bacteria reduction in both airway surface liquid (ASL) model, and biofilm matrix degradation, PA5oct appears to persist in most of phage-resistant clones. Indeed, a high percentage of resistance (20/30 clones) to PA5oct is accompanied by the presence of phage DNA within bacterial culture. Moreover, the maintenance of this phage in the bacterial population is correlated to reduced P. aeruginosa virulence, coupled with a sensitization to innate immune mechanisms, and a significantly reduced growth rate. We observed rather unusual consequences of PA5oct infection causing an increased inflammatory response of monocytes to P. aeruginosa. This, phenomenon combined with the loss or modification of the phage receptor makes most of the phage-resistant clones significantly less pathogenic in in vivo model. During phage therapy treatment, phage-resistance is considered as an adverse effect, but our results indicate that it leads to diminished bacterial virulence and increased clearance of the infected host. These findings provide new insights into the general knowledge of giant phages biology and the impact of their application in phage therapy.

Integrative omics analysis of Pseudomonas aeruginosa virus PA5oct highlights the molecular complexity of jumbo phages

“…Specifically, we see a PA5oct‐specific upregulation of the pslE‐J part of the pslA‐L exopolysaccharide operon, the napABCDE operon which encodes the periplasmic nitrate reductase, parts of a Pf1 prophage and the operon PA4918‐4920 involved in NAD biosynthesis. We also observe a downregulation of Type IV pili genes which have been shown to be a necessary receptor to initiate PA5oct infection (Olszak et al ., ). The exhaustive list of differentially expressed genes can be found in the Supporting Information Table S7.…”

“…Since PA5oct downregulates biosynthesis of Type IV pili and indirectly modifies bacterial LPS, the signalling networks might also influence the overproduction of nitrate reductase. As it was proven in our related manuscript, Type IV pili and LPS structures serve as phage PA5oct receptors (Olszak et al ., ). Receptor downregulation possibly functions as a tactic to reduce losses of clonal phages through secondary adsorption to already phage‐infected bacteria (Abedon, ).…”

Chitosan-based matrix as a carrier for bacteriophages

“…Assuming that the antibacterial activity of prepared dressing could be related to the physicochemical properties of phages themselves, we selected viruses differing in taxonomy classification, capsid size, and virion morphology, as well as in recognized receptors (saccharide versus proteinous) to evaluate viral particles’ release profile and the potential interference with chitosan matrix. All selected phages were stable in a wide pH range and tolerant to the environment of the chitosan matrix exhibiting a slightly alkaline pH (Olszak et al 2018 ).…”

The impact of agarose immobilization on the activity of lytic Pseudomonas aeruginosa phages combined with chemicals

“…Physical laser interferometry was used in parallel. The interferometry system consisted of a two-beam Mach–Zehnder interferometer with a He–Ne laser type HN 40P (Zeiss, Oberkochen, Germany), a cuvette (internal dimensions, 70 mm high, 10 mm wide; optical path length, 7 mm) made with optical glass of high uniformity, a TV-CCD camera, and a computer with software for the acquisition and processing of interference images (interferograms) (Arabski et al 2007 ; Olszak et al 2018 ; Rewak‐Soroczynska et al 2021 ). The agarose-immobilized phages were placed at the bottom of the cuvette which was then filled with water (initial substance concentration C 0 = 0).…”