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Pseudomonas aeruginosa, like other pathogens, adapts to the limiting nutritional environment of the host by altering patterns of gene expression and utilizing alternative pathways required for survival. Understanding the genes essential for survival in the host gives insight into pathways that this organism requires during infection and has the potential to identify better ways to treat infections. Here, we used a saturated transposon insertion mutant pool of P. aeruginosa strain PAO1 and transposon insertion sequencing (Tn-Seq), to identify genes conditionally important for survival under conditions mimicking the environment of a nosocomial infection. Conditions tested included tissue culture medium with and without human serum, a murine abscess model, and a human skin organoid model. Genes known to be upregulated during infections, as well as those involved in nucleotide metabolism, and cobalamin (vitamin B 12 ) biosynthesis, etc., were required for survival in vivo - and in host mimicking conditions, but not in nutrient rich lab medium, Mueller Hinton broth (MHB). Correspondingly, mutants in genes encoding proteins of nucleotide and cobalamin metabolism pathways were shown to have growth defects under physiologically-relevant media conditions, in vivo, and in vivo- like models, and were downregulated in expression under these conditions, when compared to MHB. This study provides evidence for the relevance of studying P. aeruginosa fitness in physiologically-relevant host mimicking conditions and identified metabolic pathways that represent potential novel targets for alternative therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Belanger, Dostert, Blimkie, Lee, Dhillon, Wu, Akhoundsadegh, Rahanjam, Castillo-Arnemann, Falsafi, Pletzer, Haney and Hancock.)

Biofilms are the most common cause of bacterial infections in humans and notoriously hard to treat due to their ability to withstand antibiotics and host immune defenses. To overcome the current lack of effective antibiofilm therapies and guide future design, the identification of novel biofilm-specific gene targets is crucial. In this regard, transcriptional regulators have been proposed as promising targets for antimicrobial drug design. Therefore, a Transposon insertion sequencing approach was employed to systematically identify regulators phenotypically affecting biofilm growth in Pseudomonas aeruginosa PA14 using the TnSeq analysis tools Bio-TraDIS and TRANSIT. A screen of a pool of 300,000 transposon insertion mutants identified 349 genes involved in biofilm growth on hydroxyapatite, including 47 regulators. Detection of 19 regulatory genes participating in well-established biofilm pathways validated the results. An additional 28 novel prospective biofilm regulators suggested the requirement for multiple one-component transcriptional regulators. Biofilm-defective phenotypes were confirmed for five one-component transcriptional regulators and a protein kinase, which did not affect motility phenotypes. The one-component transcriptional regulator bosR displayed a conserved role in P. aeruginosa biofilm growth since its ortholog in P. aeruginosa strain PAO1 was also required for biofilm growth. Microscopic analysis of a chromosomal deletion mutant of bosR confirmed the role of this regulator in biofilm growth. Overall, our results highlighted that the gene network driving biofilm growth is complex and involves regulators beyond the primarily studied groups of two-component systems and cyclic diguanylate signaling proteins. Furthermore, biofilm-specific regulators, such as bosR , might constitute prospective new drug targets to overcome biofilm infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dostert, Belanger, Pedraz, Alford, Blimkie, Falsafi, Bains, Dhillon, Haney, Lee and Hancock.)

Engineered liposomes composed of the naturally occurring lipids sphingomyelin (Sm) and cholesterol (Ch) have been demonstrated to efficiently neutralize toxins secreted by Gram-positive bacteria such as Streptococcus pneumoniae and Staphylococcus aureus . Here, we hypothesized that liposomes are capable of neutralizing cytolytic virulence factors secreted by the Gram-negative pathogen Pseudomonas aeruginosa . We used the highly virulent cystic fibrosis P. aeruginosa Liverpool Epidemic Strain LESB58 and showed that sphingomyelin (Sm) and a combination of sphingomyelin with cholesterol (Ch:Sm; 66 mol/% Ch and 34 mol/% Sm) liposomes reduced lysis of human bronchial and red blood cells upon challenge with the Pseudomonas secretome. Mass spectrometry of liposome-sequestered Pseudomonas proteins identified the virulence-promoting hemolytic phospholipase C (PlcH) as having been neutralized. Pseudomonas aeruginosa supernatants incubated with liposomes demonstrated reduced PlcH activity as assessed by the p -nitrophenylphosphorylcholine (NPPC) assay. Testing the in vivo efficacy of the liposomes in a murine cutaneous abscess model revealed that Sm and Ch:Sm, as single dose treatments, attenuated abscesses by >30%, demonstrating a similar effect to that of a mutant lacking plcH in this infection model. Thus, sphingomyelin-containing liposome therapy offers an interesting approach to treat and reduce virulence of complex infections caused by P. aeruginosa and potentially other Gram-negative pathogens expressing PlcH ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wolfmeier, Wardell, Liu, Falsafi, Draeger, Babiychuk, Pletzer and Hancock.)

Bacterial biofilms are complex and highly antibiotic-resistant aggregates of microbes that form on surfaces in the environment and body including medical devices. They are key contributors to the growing antibiotic resistance crisis and account for two-thirds of all infections. Thus, there is a critical need to develop anti-biofilm specific therapeutics. Here we discuss mechanisms of biofilm formation, current anti-biofilm agents, and strategies for developing, discovering, and testing new anti-biofilm agents. Biofilm formation involves many factors and is broadly regulated by the stringent response, quorum sensing, and c-di-GMP signaling, processes that have been targeted by anti-biofilm agents. Developing new anti-biofilm agents requires a comprehensive systems-level understanding of these mechanisms, as well as the discovery of new mechanisms. This can be accomplished through omics approaches such as transcriptomics, metabolomics, and proteomics, which can also be integrated to better understand biofilm biology. Guided by mechanistic understanding, in silico techniques such as virtual screening and machine learning can discover small molecules that can inhibit key biofilm regulators. To increase the likelihood that these candidate agents selected from in silico approaches are efficacious in humans, they must be tested in biologically relevant biofilm models. We discuss the benefits and drawbacks of in vitro and in vivo biofilm models and highlight organoids as a new biofilm model. This review offers a comprehensive guide of current and future biological and computational approaches of anti-biofilm therapeutic discovery for investigators to utilize to combat the antibiotic resistance crisis., Competing Interests: RH has invented anti-biofilm peptides, related to those discussed here, and assigned these to his employer the University of British Columbia who filed for patent protection and licensed them to the ABT Innovations Inc., a company owned in part by RH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 An, Choi, Baghela and Hancock.)

Host-defense peptides (HDPs) are vital components of innate immunity in all vertebrates. While their antibacterial activity toward bacterial cells was the original focus for research, their ability to modulate immune and inflammatory processes has emerged as one of their major functions in the host and as a promising approach from which to develop novel therapeutics targeting inflammation and innate immunity. In this review, with particular emphasis on the cathelicidin family of peptides, the roles of natural HDPs are examined in managing immune activation, cellular recruitment, cytokine responses, and inflammation in response to infection, as well as their contribution(s) to various inflammatory disorders and autoimmune diseases. Furthermore, we discuss current efforts to develop synthetic HDPs as therapeutics aimed at restoring balance to immune responses that are dysregulated and contribute to disease pathologies., (Copyright © 2020 Alford, Baquir, Santana, Haney and Hancock.)

Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of nosocomial and chronic infections contributing to morbidity and mortality in cystic fibrosis patients. One of the reasons for its success as a pathogen is its ability to adapt to a broad range of circumstances. Here, we show the involvement of the general nitrogen regulator NtrBC, which is structurally conserved but functionally diverse across species, in pathogenic and adaptive states of P. aeruginosa . The role of NtrB and NtrC was examined in progressive or chronic infections, which revealed that mutants (Δ ntrB , Δ ntrC , and Δ ntrBC ) were reduced in their ability to invade and cause damage in a high-density abscess model in vivo. Progressive infections were established with mutants in the highly virulent PA14 genetic background, whereas chronic infections were established with mutants in the less virulent clinical isolate LESB58 genetic background. Characterization of adaptive lifestyles in vitro confirmed that the double Δ ntrBC mutant demonstrated >40% inhibition of biofilm formation, a nearly complete inhibition of swarming motility, and a modest decrease and altered surfing motility colony appearance; with the exception of swarming, single mutants generally had more subtle or no changes. Transcriptional profiles of deletion mutants under swarming conditions were defined using RNA-Seq and unveiled dysregulated expression of hundreds of genes implicated in virulence in PA14 and LESB58 chronic lung infections, as well as carbon and nitrogen metabolism. Thus, transcriptional profiles were validated by testing responsiveness of mutants to several key intermediates of central metabolic pathways. These results indicate that NtrBC is a global regulatory system involved in both pathological and physiological processes relevant to the success of Pseudomonas in high-density infection., (Copyright © 2020 Alford, Baghela, Yeung, Pletzer and Hancock.)

Prosthesis and joint infections are an important threat in public health, especially due to the development of bacterial biofilms and their high resistance to antimicrobials. Biofilm-associated infections increase mortality and morbidity rates as well as hospitalization costs. Prevention is the best strategy for this serious issue, so there is an urgent need to understand the signals that could induce irreversible bacterial adhesion on a prosthesis. In this context, we investigated the influence of the bone environment on surface adhesion by a methicillin-susceptible Staphylococcus aureus strain. Using static and dynamic biofilm models, we tested various bone environment factors and showed that the presence of Mg 2+ , lack of oxygen, and starvation each increased bacterial adhesion. It was observed that human osteoblast-like cell culture supernatants, which contain secreted components that would be found in the bone environment, increased bacterial adhesion capacity by 2-fold ( p = 0.015) compared to the medium control. Moreover, supernatants from osteoblast-like cells stimulated with TNF-α to mimic inflammatory conditions increased bacterial adhesion by almost 5-fold ( p = 0.003) without impacting on the overall biomass. Interestingly, the effect of osteoblast-like cell supernatants on bacterial adhesion could be counteracted by the activity of synthetic antibiofilm peptides. Overall, the results of this study demonstrate that factors within the bone environment and products of osteoblast-like cells directly influence S. aureus adhesion and could contribute to biofilm initiation on bone and/or prosthetics implants.

Microorganisms continuously monitor their surroundings and adaptively respond to environmental cues. One way to cope with various stress-related situations is through the activation of the stringent stress response pathway. In Pseudomonas aeruginosa this pathway is controlled and coordinated by the activity of the RelA and SpoT enzymes that metabolize the small nucleotide secondary messenger molecule (p)ppGpp. Intracellular ppGpp concentrations are crucial in mediating adaptive responses and virulence. Targeting this cellular stress response has recently been the focus of an alternative approach to fight antibiotic resistant bacteria. Here, we examined the role of the stringent response in the virulence of P. aeruginosa PAO1 and the Liverpool epidemic strain LESB58. A Δ relA /Δ spoT double mutant showed decreased cytotoxicity toward human epithelial cells, exhibited reduced hemolytic activity, and caused down-regulation of the expression of the alkaline protease aprA gene in stringent response mutants grown on blood agar plates. Promoter fusions of relA or spoT to a bioluminescence reporter gene revealed that both genes were expressed during the formation of cutaneous abscesses in mice. Intriguingly, virulence was attenuated in vivo by the Δ relA /Δ spoT double mutant, but not the relA mutant nor the Δ relA /Δ spoT complemented with either gene. Treatment of a cutaneous P. aeruginosa PAO1 infection with anti-biofilm peptides increased animal welfare, decreased dermonecrotic lesion sizes, and reduced bacterial numbers recovered from abscesses, resembling the phenotype of the Δ relA /Δ spoT infection. It was previously demonstrated by our lab that ppGpp could be targeted by synthetic peptides; here we demonstrated that spoT promoter activity was suppressed during cutaneous abscess formation by treatment with peptides DJK-5 and 1018, and that a peptide-treated relA complemented stringent response double mutant strain exhibited reduced peptide susceptibility. Overall these data strongly indicated that synthetic peptides target the P. aeruginosa stringent response in vivo and thus offer a promising novel therapeutic approach.