Pathogen-associated gene discovery workflows for novel antivirulence therapeutic development.

Novel therapeutics to manage bacterial infections are urgently needed as the impact and prevalence of antimicrobial resistance (AMR) grows. Antivirulence therapeutics are an alternative approach to antibiotics that aim to attenuate virulence rather than target bacterial essential functions, while minimizing microbiota perturbation and the risk of AMR development. Beyond known virulence factors, pathogen-associated genes (PAGs; genes found only in pathogens to date) may play an important role in virulence or host association. Many identified PAGs encode uncharacterized hypothetical proteins and represent an untapped wealth of novel drug targets. Here, we review current advances in antivirulence drug research and development, including PAG identification, and provide a comprehensive workflow from the discovery of antivirulence drug targets to drug discovery. We highlight the importance of integrating bioinformatic/genomic-based methods for novel virulence factor discovery, coupled with experimental characterization, into existing drug screening platforms to develop novel and effective antivirulence drugs.

Salt and surfactant coated filters with antiviral properties and low pressure drop for prospective SARS-CoV2 applications.

The COVID-19 pandemic motivated research on antiviral filtration used in personal protective equipment and HVAC systems. In this research, three coating compositions of NaCl, Tween 20 surfactant, and NaCl-Tween 20 were examined on polypropylene spun-bond filters. The pressure drop, coverage, and crystal size of the coating methods and compositions were measured. Also, in vitro plaque assays of the Phi6 Bacteriophage on Pseudomonas syringae as a simulation of an enveloped respiratory virus was performed to investigate the antiviral properties of the coating. NaCl and NaCl-Tween 20 increased the pressure drop in the range of 40-50 Pa for a loading of 5 mg/cm2. Tween 20 has shown an impact on the pressure drop as low as 10 Pa and made the filter surface more hydrophilic which kept the virus droplets on the surface. The NaCl-Tween 20 coated samples could inactivate 108 plaque forming units (PFU) of virus in two hours of incubation. Tween 20 coated filters with loading as low as 0.2 mg/cm2 reduced the activity of 108 PFU of virus from 109 to 102 PFU/mL after 2 h of incubation. NaCl-coated samples with a salt loading of 15 mg/cm2 could not have antiviral properties higher than reducing the viral activity from 109 to 105 PFU/mL in 4 h of incubation.

The Small RNAs PA2952.1 and PrrH as Regulators of Virulence, Motility, and Iron Metabolism in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that undergoes swarming motility in response to semisolid conditions with amino acids as a nitrogen source. With a genome encoding hundreds of potential intergenic small RNAs (sRNAs), P. aeruginosa can easily adapt to different conditions and stresses. We previously identified 20 sRNAs that were differentially expressed (DE) under swarming conditions. Here, these sRNAs were overexpressed in strain PAO1 and were subjected to an array of phenotypic screens. Overexpression of the PrrH sRNA resulted in decreased swimming motility, whereas a ΔprrH mutant had decreased cytotoxicity and increased pyoverdine production. Overexpression of the previously uncharacterized PA2952.1 sRNA resulted in decreased swarming and swimming motilities, increased gentamicin and tobramycin resistance under swarming conditions, and increased trimethoprim susceptibility. Transcriptome sequencing (RNA-Seq) and proteomic analysis were performed on the wild type (WT) overexpressing PA2952.1 compared to the empty vector control under swarming conditions, and these revealed the differential expression (absolute fold change [FC] ≥ 1.5) of 784 genes and the differential abundance (absolute FC ≥ 1.25) of 59 proteins. Among these were found 73 transcriptional regulators, two-component systems, and sigma and anti-sigma factors. Downstream effectors included downregulated pilus and flagellar genes, the upregulated efflux pump MexGHI-OpmD, and the upregulated arn operon. Genes involved in iron and zinc uptake were generally upregulated, and certain pyoverdine genes were upregulated. Overall, the sRNAs PA2952.1 and PrrH appeared to be involved in regulating virulence-related programs in P. aeruginosa, including iron acquisition and motility.IMPORTANCE Due to the rising incidence of multidrug-resistant (MDR) strains and the difficulty of eliminating P. aeruginosa infections, it is important to understand the regulatory mechanisms that allow this bacterium to adapt to and thrive under a variety of conditions. Small RNAs (sRNAs) are one regulatory mechanism that allows bacteria to change the amount of protein synthesized. In this study, we overexpressed 20 different sRNAs in order to investigate how this might affect different bacterial behaviors. We found that one of the sRNAs, PrrH, played a role in swimming motility and virulence phenotypes, indicating a potentially important role in clinical infections. Another sRNA, PA2952.1, affected other clinically relevant phenotypes, including motility and antibiotic resistance. RNA-Seq and proteomics of the strain overexpressing PA2952.1 revealed the differential expression of 784 genes and 59 proteins, with a total of 73 regulatory factors. This substantial dysregulation indicates an important role for the sRNA PA2952.1.

Loss of the Two-Component System TctD-TctE in Pseudomonas aeruginosa Affects Biofilm Formation and Aminoglycoside Susceptibility in Response to Citric Acid.

The two-component system TctD-TctE is important for regulating the uptake of tricarboxylic acids in Pseudomonas aeruginosa TctD-TctE accomplishes this through derepression of the gene opdH, which encodes a tricarboxylic acid-specific porin. Previous work from our lab revealed that TctD-TctE in P. aeruginosa also has a role in resistance to aminoglycoside antibiotics. The aim of this study was to further characterize the role of TctD-TctE in P. aeruginosa in the presence of citric acid. Here it was found that deletion of P. aeruginosa PA14 TctD-TctE (ΔtctED) resulted in a 4-fold decrease in the biofilm bactericidal concentrations of the aminoglycosides tobramycin and gentamicin when citric acid was present in nutrient media. Tobramycin accumulation assays demonstrated that deletion of TctD-TctE resulted in an increase in the amount of tobramycin retained in biofilm cells. The PA14 wild type responded to increasing concentrations of citric acid by producing less biofilm. In contrast, the amount of ΔtctED mutant biofilm formation remained constant or enhanced. Furthermore, the ΔtctED strain was incapable of growing on citric acid as a sole carbon source and was highly reduced in its ability to grow in the presence of citric acid even when an additional carbon source was available. Use of phenotypic and genetic microarrays found that this growth deficiency of the ΔtctED mutant is unique to citric acid and that multiple metabolic genes are dysregulated. This work demonstrates that TctD-TctE in P. aeruginosa has a role in biofilm development that is dependent on citric acid and that is separate from the previously characterized involvement in resistance to antibiotics.IMPORTANCE Nutrient availability is an important contributor to the ability of bacteria to establish successful infections in a host. Pseudomonas aeruginosa is an opportunistic pathogen in humans causing infections that are difficult to treat. In part, its success is attributable to a high degree of metabolic versatility. P. aeruginosa is able to sense and respond to varied and limited nutrient stress in the host environment. Two-component systems are important sensors-regulators of cellular responses to environmental stresses, such as those encountered in the host. This work demonstrates that the response by the two-component system TctD-TctE to the presence of citric acid has a role in biofilm formation, aminoglycoside susceptibility, and growth in P. aeruginosa.

High-throughput detection of RNA processing in bacteria.

BACKGROUND: Understanding the RNA processing of an organism's transcriptome is an essential but challenging step in understanding its biology. Here we investigate with unprecedented detail the transcriptome of Pseudomonas aeruginosa PAO1, a medically important and innately multi-drug resistant bacterium. We systematically mapped RNA cleavage and dephosphorylation sites that result in 5'-monophosphate terminated RNA (pRNA) using monophosphate RNA-Seq (pRNA-Seq). Transcriptional start sites (TSS) were also mapped using differential RNA-Seq (dRNA-Seq) and both datasets were compared to conventional RNA-Seq performed in a variety of growth conditions. RESULTS: The pRNA-Seq library revealed known tRNA, rRNA and transfer-messenger RNA (tmRNA) processing sites, together with previously uncharacterized RNA cleavage events that were found disproportionately near the 5' ends of transcripts associated with basic bacterial functions such as oxidative phosphorylation and purine metabolism. The majority (97%) of the processed mRNAs were cleaved at precise codon positions within defined sequence motifs indicative of distinct endonucleolytic activities. The most abundant of these motifs corresponded closely to an E. coli RNase E site previously established in vitro. Using the dRNA-Seq library, we performed an operon analysis and predicted 3159 potential TSS. A correlation analysis uncovered 105 antiparallel pairs of TSS that were separated by 18 bp from each other and were centered on single palindromic TAT(A/T)ATA motifs (likely - 10 promoter elements), suggesting that, consistent with previous in vitro experimentation, these sites can initiate transcription bi-directionally and may thus provide a novel form of transcriptional regulation. TSS and RNA-Seq analysis allowed us to confirm expression of small non-coding RNAs (ncRNAs), many of which are differentially expressed in swarming and biofilm formation conditions. CONCLUSIONS: This study uses pRNA-Seq, a method that provides a genome-wide survey of RNA processing, to study the bacterium Pseudomonas aeruginosa and discover extensive transcript processing not previously appreciated. We have also gained novel insight into RNA maturation and turnover as well as a potential novel form of transcription regulation. NOTE: All sequence data has been submitted to the NCBI sequence read archive. Accession numbers are as follows: [NCBI sequence read archive: SRX156386, SRX157659, SRX157660, SRX157661, SRX157683 and SRX158075]. The sequence data is viewable using Jbrowse on www.pseudomonas.com .

Novel roles for two-component regulatory systems in cytotoxicity and virulence-related properties in Pseudomonas aeruginosa.

The rapid adaptation of the opportunistic bacterial pathogen Pseudomonas aeruginosa to various growth modes and environmental conditions is controlled in part through diverse two-component regulatory systems. Some of these systems are well studied, but the majority are poorly characterized, even though it is likely that several of these systems contribute to virulence. Here, we screened all available strain PA14 mutants in 50 sensor kinases, 50 response regulators and 5 hybrid sensor/regulators, for contributions to cytotoxicity against cultured human bronchial epithelial cells, as assessed by the release of cytosolic lactate dehydrogenase. This enabled the identification of 8 response regulators and 3 sensor kinases that caused substantial decreases in cytotoxicity, and 5 response regulators and 8 sensor kinases that significantly increased cytotoxicity by 15-58% or more. These regulators were additionally involved in motility, adherence, type 3 secretion, production of cytotoxins, and the development of biofilms. Here we investigated in more detail the roles of FleSR, PilSR and WspR. Not all cognate pairs contributed to cytotoxicity (e.g. PhoPQ, PilSR) in the same way and some differences could be detected between the same mutants in PAO1 and PA14 strain backgrounds (e.g. FleSR, PhoPQ). This study highlights the potential importance of these regulators and their downstream targets on pathogenesis and demonstrates that cytotoxicity can be regulated by several systems and that their contributions are partly dependent on strain background.

A novel small RNA is important for biofilm formation and pathogenicity in Pseudomonas aeruginosa.

The regulation of biofilm development requires multiple mechanisms and pathways, but it is not fully understood how these are integrated. Small RNA post-transcriptional regulators are a strong candidate as a regulatory mechanism of biofilm formation. More than 200 small RNAs in the P. aeruginosa genome have been characterized in the literature to date; however, little is known about their biological roles in the cell. Here we describe the identification of the novel regulatory small RNA, SrbA. This locus was up-regulated 45-fold in P. aeruginosa strain PA14 biofilm cultures. Loss of SrbA expression in a deletion strain resulted in a 66% reduction in biofilm mass. Furthermore, the mortality rate over 72 hours in C. elegans infections was reduced to 39% when infected with the srbA deletion strain compared to 78% mortality when infected with the parental wild-type P. aeruginosa strain. There was no significant effect on culture growth or adherence to surfaces with loss of SrbA expression. Also loss of SrbA expression had no effect on antibiotic resistance to ciprofloxacin, gentamicin, and tobramycin. We conclude that SrbA is important for biofilm formation and full pathogenicity of P. aeruginosa.

Interconnection of post-transcriptional regulation: The RNA-binding protein Hfq is a novel target of the Lon protease in Pseudomonas aeruginosa.

Besides being a major opportunistic human pathogen, Pseudomonas aeruginosa can be found in a wide range of environments. This versatility is linked to complex regulation, which is achieved through the action of transcriptional regulators, and post-transcriptional regulation by intracellular proteases including Lon. Indeed, lon mutants in this species show defects in motility, biofilm formation, pathogenicity and fluoroquinolone resistance. Here, the proteomic approach stable isotope labeling by amino acids in cell culture (SILAC) was used to search for novel proteolytic targets. One of the proteins that accumulated in the lon mutant was the RNA-binding protein Hfq. Further experiments demonstrated the ability of Lon to degrade Hfq in vitro. Also, overexpression of the hfq gene in the wild-type strain led to partial inhibition of swarming, swimming and twitching motilities, indicating that Hfq accumulation could contribute to the phenotypes displayed by Lon mutants. Hfq overexpression also led to the upregulation of the small regulatory RNA PhrS. Analysis of the phenotypes of strains lacking or overexpressing this sRNA indicated that the Lon protease might be indirectly regulating the levels and activity of sRNAs via Hfq. Overall, this study revealed new links in the complex regulatory chain that controls multicellular behaviours in P. aeruginosa.

Safety and efficacy of composite collagen-silver nanoparticle hydrogels as tissue engineering scaffolds.

The increasing number of multidrug resistant bacteria has revitalized interest in seeking alternative sources for controlling bacterial infection. Silver nanoparticles (AgNPs), are amongst the most promising candidates due to their wide microbial spectrum of action. In this work, we report on the safety and efficacy of the incorporation of collagen coated AgNPs into collagen hydrogels for tissue engineering. The resulting hybrid materials at [AgNPs] < 0.4 µM retained the mechanical properties and biocompatibility for primary human skin fibroblasts and keratinocytes of collagen hydrogels; they also displayed remarkable anti-infective properties against S. aureus, S. epidermidis, E. coli and P. aeruginosa at considerably lower concentrations than silver nitrate. Further, subcutaneous implants of materials containing 0.2 µM AgNPs in mice showed a reduction in the levels of IL-6 and other inflammation markers (CCL24, sTNFR-2, and TIMP1). Finally, an analysis of silver contents in implanted mice showed that silver accumulation primarily occurred within the tissue surrounding the implant.

Antibiotic resistance in Pseudomonas aeruginosa biofilms: towards the development of novel anti-biofilm therapies.

The growth of bacteria as structured aggregates termed biofilms leads to their protection from harsh environmental conditions such as physical and chemical stresses, shearing forces, and limited nutrient availability. Because of this highly adapted ability to survive adverse environmental conditions, bacterial biofilms are recalcitrant to antibiotic therapies and immune clearance. This is particularly problematic in hospital settings where biofilms are a frequent cause of chronic and device-related infections and constitute a significant burden on the health-care system. The major therapeutic strategy against infections is the use of antibiotics, which, due to adaptive resistance, are often insufficient to clear biofilm infections. Thus, novel biofilm-specific therapies are required. Specific features of biofilm development, such as surface adherence, extracellular matrix formation, quorum sensing, and highly regulated biofilm maturation and dispersal are currently being studied as targets to be exploited in the development of novel biofilm-specific treatments. Using Pseudomonas aeruginosa for illustrative purposes, this review highlights the antibiotic resistance mechanisms of biofilms, and discusses current research into novel biofilm-specific therapies.

The Lon protease is essential for full virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO1 lon mutants are supersusceptible to ciprofloxacin, and exhibit a defect in cell division and in virulence-related properties, such as swarming, twitching and biofilm formation, despite the fact that the Lon protease is not a traditional regulator. Here we set out to investigate the influence of a lon mutation in a series of infection models. It was demonstrated that the lon mutant had a defect in cytotoxicity towards epithelial cells, was less virulent in an amoeba model as well as a mouse acute lung infection model, and impacted on in vivo survival in a rat model of chronic infection. Using qRT-PCR it was demonstrated that the lon mutation led to a down-regulation of Type III secretion genes. The Lon protease also influenced motility and biofilm formation in a mucin-rich environment. Thus alterations in several virulence-related processes in vitro in a lon mutant were reflected by defective virulence in vivo.

Novel genetic determinants of low-level aminoglycoside resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa strains isolated from patients with persistent lung infections and cystic fibrosis have been found to gradually develop aminoglycoside resistance over time. The aim of this study was to identify potential contributors to low-level aminoglycoside resistance, which may cause such graduated increases in resistance. The Harvard P. aeruginosa PA14 nonredundant library, consisting of approximately 5,800 mutants, was screened for twofold or greater increases in tobramycin resistance. Mutants carrying mutations in a total of 135 unique genes were identified and confirmed to have reduced susceptibility to tobramycin. Many of these genes were involved predominantly in energy metabolism; however, most of these mutants did not exhibit growth defects under the conditions tested, although some exhibited the small-colony phenotype and/or defects in growth under anaerobic conditions. Lipopolysaccharide mutants were also identified, and it was found that tobramycin had a reduced ability to permeabilize the outer membranes of these mutants. The results of this study emphasize the complexity of the interactions that tobramycin may have within the bacterial cell and introduce a large number of novel genes which may play a role in tobramycin resistance.