Grzegorz Gula

The interaction between Pseudomonas aeruginosa cells and cationic PC:Chol:DOTAP liposomal vesicles versus outer-membrane structure and envelope properties of bacterial cell

The interactions between cationic liposomal formulations (PC:Chol:DOTAP 3:4:3) and 23 Pseudomonas aeruginosa strains were tested. The study was undertaken because different antimicrobial results had been obtained by the authors for Pseudomonas aeruginosa strains and liposomal antibiotics (Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz, W., Kozubek, A., 2006. The comparison of in vitro antimicrobial activity of liposomes containing meropenem and gentamicin. Cell. Mol. Biol. Lett., 11, 360-375; Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz W., Kozubek, A., 2006. In vitro antimicrobial activity of liposomal meropenem against Pseudomonas aeruginosa strains. Int. J. Pharm., 315, 59-66). The experiments evaluate the roles of the bacterial outer-membrane structure, especially outer-membrane proteins and LPS, and envelope properties (hydrophobicity and electrostatic potential) in the interactions/fusion process between cells and lipid vesicles. The interactions were examined by fluorescent microscopy using PE-rhodamine-labelled liposomes. Some of the strains exhibited red-light emission (fusion with vesicles or vesicles surrounding the cell) and some showed negative reaction (no red-light emission). The main aim of the study was to determine what kinds of bacterial structure or envelope properties have a major influence on the fusion process. Negatively charged cells and hydrophobic properties promote interaction with cationic lipid vesicles, but no specific correlation was noted for the tested strains. A similar situation concerned LPS structure, where parent strains and their mutants possessing identical ladder-like band patterns in SDS-PAGE analysis exhibited totally different results with fluorescent microscopy. Outer-membrane protein analysis showed that an 18-kDA protein occurred in the isolates showing fusion with rhodamine-labelled vesicles and, conversely, strains lacking the 18-kDA protein exhibited no positive reaction (red emission). This suggests that even one protein may be responsible for favouring stronger interactions between Pseudomonas aeruginosa cells and cationic liposomal formulations (PC:Chol:DOTAP 3:4:3).

Correction: Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.

Pseudomonas aeruginosa PA5oct jumbo phage reduces planktonic and biofilm population and impacts its host virulence through a pseudolysogeny event

In 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 Statement The 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.

Complex Signaling Networks Controlling Dynamic Molecular Changes in Pseudomonas aeruginosa Biofilm

The environment exerts strong influence on microbes. Adaptation of microbes to changing conditions is a dynamic process regulated by complex networks. Pseudomonas aeruginosa is a life-threating, versatile opportunistic and multi drug resistant pathogen that provides a model to investigate adaptation mechanisms to environmental changes. The ability of P. aeruginosa to form biofilms and to modify virulence in response to environmental changes are coordinated by various mechanisms including two-component systems (TCS), and secondary messengers involved in quorum sensing (QS) and c-di-GMP networks (diguanylate cyclase systems, DGC). In this review, we focus on the role of c-di-GMP during biofilm formation. We describe TCS and QS signal cascades regulated by c-di-GMP in response to changes in the external environment. We present a complex signaling network dynamically changing during the transition of P. aeruginosa from the free-living to sessile mode of growth.