Robert E Hancock

Professor

Relevant Degree Programs

 

Postdoctoral Fellows

Great Supervisor Week Mentions

Each year graduate students are encouraged to give kudos to their supervisors through social media and our website as part of #GreatSupervisorWeek. Below are students who mentioned this supervisor since the initiative was started in 2017.

 

Bob is a #GreatSupervisor for challenging me to do my best, encouraging me to take advantage of opportunities for collaboration and publication, and setting a high standard for intellectual knowledge and achievement. He is incredibly honest and is always motivating his students to develop as independent researchers. Thanks Bob!

Corrie (2017)

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
A novel peptide-based treatment for bacterial abscess infections (2018)

Methicillin-resistant Staphylococcus aureus (MRSA), a major causative agent of skin infections, is a serious public threat causing a variety of hospital and community-acquired infections. In addition to its high-level mutational resistance, MRSA can form aggregated multicellular communities called biofilms, causing 10- to 1000-fold higher adaptive resistance to conventional antibiotics as compared to free-growing bacteria. Despite biofilm infections representing 65% of all human infections, there is a lack of antibiofilm agents in the drug development pipeline. To combat this issue, I conducted a high-throughput screen on a large peptide library to find peptides with potent anti-MRSA biofilm activity. This led to the identification of a small synthetic cationic peptide composed of D-enantiomeric amino acids, DJK-5, that could inhibit and eradicate pre-formed MRSA biofilms. In a murine cutaneous infection model, DJK-5 drastically reduced the abscess size and dermonecrosis. The peptide also demonstrated broad-spectrum activity in a P. aeruginosa and E. cloacae skin infection murine model. Interestingly, DJK-5 had modest antimicrobial activity and reduced the bacterial loads in abscesses by roughly 10-fold, inferring a mechanism of action distinct from bactericidal activity. Exploring the mechanism of action, it was found that DJK-5 interfered with the production of ppGpp, a conserved stringent response signal employed by bacteria to cope and adapt to environmental stresses. Through transcriptomic analysis, major pathways were shown to be dysregulated by nutritional stress revealing a link between the stringent response, biofilm formation and S. aureus pathogenesis. Importantly, it was demonstrated that the stringent response was critical in mediating lesion formation and that DJK-5 could reduce abscess pathology by interfering with the production of stringent response regulated cutaneous toxins, phenol soluble modulins. DJK-5 revealed a dynamic effect in synergizing with common antibiotics used to treat Gram-positive and Gram-negative infections. These findings provided new insights into the mechanisms governing abscess formation and created a new paradigm for treating multidrug resistant cutaneous abscesses.

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Combating Pseudomonas aeruginosa lung infections using synthetic host defense peptides (2017)

Pseudomonas aeruginosa is a Gram negative bacterium found frequently in the environment. It can infect immunocompromised patients and is a major cause of nosocomial infections. Of particular concern are its roles in lung infections as a causative agent for pneumonia and in respiratory infections in patients with cystic fibrosis and chronic obstructive pulmonary disease. Treatment of P. aeruginosa lung infections is difficult due to its formation of biofilms and the development of multi-drug resistant P. aeruginosa strains. Synthetic derivatives of host defense peptides (HDPs) called innate defense regulators (IDRs) are alternatives to antibiotics that modulate the immune response rather than directly targeting the bacteria, thus limiting the development of antibiotic resistance. IDRs have shown success against many bacteria but had not previously been tested in P. aeruginosa lung infection models. In this work, IDR-1002 reduced the production of inflammatory cytokines by macrophages in response to P. aeruginosa lipopolysaccharide. IDR-1002 also limited the toxicity caused by live P. aeruginosa to macrophages and bronchial epithelial cells. Importantly, IDR-1002 did not show any toxic effects in vitro, unlike the HDP LL-37. In an acute in vivo P. aeruginosa lung infection model, IDR-1002 significantly decreased the bacterial burden as well as the concentrations of MCP-1, KC, and IL-6 in the lungs. In another in vivo P. aeruginosa lung infection model using alginate to mimic a chronic infection, IDR-1002 decreased the infiltration of cells to the infection site and significantly decreased IL-6 levels in the lungs. To improve drug delivery, peptide IDR-1018, which has a strong aggregation propensity, was tested with various formulations, and its combination with a hyperbranched polyglycerol reduced the production of inflammatory cytokines in vitro and trended towards reducing cytokines in vivo in the acute P. aeruginosa lung infection model. Finally, RNA-Seq and downstream bioinformatics were performed on both lung and blood samples from the acute P. aeruginosa lung infection model, providing insights into the impact of P. aeruginosa during infection and the protective mechanisms of IDR-1002 via its anti-inflammatory effects. These data suggest the strong potential of IDR-1002 for the treatment of P. aeruginosa lung infections.

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Linking cellular metabolism and Innate Defence Regulator peptide function (2016)

Appropriate cellular metabolism is essential to immune cells to survival and their ability to mount effective and appropriate responses to the pathogen and host derived insults they encounter. Malnutrition, caused by nutrient deficiency or excess, can result in significant dysregulation of immune cell activity. Immune cells undergo metabolic reprogramming in response to pathogen and host-derived signals found in their environment. These changes modulate the type of response they will mount. Innate defence regulatory (IDR) peptides, synthetic derivatives of host defence peptides, were developed as anti-infectives that modulate host immune system responses however, much of the mechanisms behind their activity are unknown. In this study, IDR-1018 was shown to modulate glycolytic activity in macrophages, which appeared to be important to its immunomodulatory activity. Activation of the ERK signalling pathway, a major regulator of metabolism and inflammatory responses, by IDR-1018 was found to be a possible mechanism by which IDR-1018 induced both glycolysis and chemokine production. Inhibition of glycolysis using 2-deoxy-d-glucose (2DG) suppressed IDR-1018 induced chemokine production. However, 2DG also suppressed IDR-1018 activity through induction of endoplasmic reticulum stress and the unfolded protein response (UPR), specifically the anti-inflammatory PERK arm of the UPR. The anti-endotoxin activity of IDR-1018 was also found to be associated with modulation of glycolysis. IDR-1018 suppressed lipopolysaccharide (LPS)-induced chemokine and cytokine production, possibly through inhibition of LPS-induced glycolysis. Interestingly, dysregulation of both glycolysis and the UPR by 2DG enhanced the anti-endotoxin activity of IDR-1018, suppressing LPS-induced chemokine and cytokine production. Finally, this study identified a potential new activity for IDRs, the modulation of metabolic pathways dysregulated in response to nutrient excess. Specifically, this study showed that IDR-1018 enhanced HDL-mediated cholesterol efflux from macrophages and smooth muscle cells, two important cellular mediators of atherosclerosis. This may have been a result of IDR-1018 interacting with HDL particles found in serum, facilitating their binding to the plasma membrane of cells. The results presented in this study demonstrated that IDR peptides are potent modulators of both immune cell function and cellular metabolism as well as identified a novel mechanism by which IDR peptides exert their immunomodulatory activity.

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A broad-spectrum anti-biofilm peptide that targets a cellular stress response (2014)

Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate increased adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. In this work, I first identified peptide 1037, which inhibited biofilm formation in a broad-spectrum manner and proposed that this activity might be due to the effect of the peptide on biofilm-associated processes. However, these processes are not conserved in bacteria and therefore did not explain the broad-spectrum activity of the peptide. Additional screens identified 1018 as a potent anti-biofilm peptide that prevented biofilm formation and led to the eradication of mature biofilms in both Gram-negative and Gram-positive bacteria. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. To explain the broad-spectrum activity of the peptide, I hypothesized that it acted to inhibit a common stress response, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this notion, increasing (p)ppGpp synthesis led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to reduced biofilm formation. Eliminating (p)ppGpp expression after 2 days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp, and in vitro directly interacted with ppGpp. These results indicate that 1018 targets (p)ppGpp and marks it for degradation, thus providing an explanation for the broad-spectrum activity of the peptide. Further, the peptide was found to be synergistic with different classes of antibiotics to prevent and eradicate bacterial biofilms. Thus the peptide represents a novel strategy to potentiate antibiotic activity against biofilms. Further studies identified even more potent D-enantiomeric anti-biofilm peptides DJK-5 and DJK-6 that also prevented (p)ppGpp accumulation, were highly synergistic with conventional antibiotics and exhibited in vivo activity. Targeting biofilms represents a novel approach against drug-resistant bacterial infections.

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Complex regulations of swarming and surfing motilities in pseudomonas aeruginosa (2013)

To investigate how the complex adaptation process of swarming is regulated, a P. aeruginosa PA14 transposon mutant library was screened for mutants defective in swarming. As a result, 233 mutants exhibiting alterations in swarming phenotypes were identified and 35 of these genes encoded for regulators. Only a few of these regulatory mutants showed significant defects in the production of type IV pili, flagella, or rhamnolipid, each of which is known to be involved in swarming, suggesting that the majority of these regulators control other factors important in swarming. One regulatory mutant with a mutation in the cbrA gene was chosen to be investigated in detail. In addition to swarming motility and carbon source utilization, the sensor kinase CbrA was shown to play regulatory roles in other virulence and virulence-related processes of Pseudomonas, including biofilm formation, cytotoxicity, and antibiotic resistance. Microarray analysis revealed hundreds of dysregulated genes in the cbrA mutant that might contribute to the virulence and virulence-related phenotypes observed in the mutant. Phenotypic and genetic analyses of a cbrB mutant suggested that CbrA modulated swarming, biofilm formation, and cytotoxicity via the CbrB response regulator and that the CrcZ small RNA and the Crc protein are likely downstream of this two-component regulator.Little was known about the mode of motility P. aeruginosa uses to colonize the lungs of patients with cystic fibrosis (CF) since the viscous lung environment in vivo is influenced by mucin in the mucous. To investigate this, the nutritional composition of the CF sputum was mimicked using plates containing synthetic CF medium (SCFM) with mucin. Addition of small amounts (0.05%) of mucin to SCFM-swimming agar led P. aeruginosa to undergo accelerated motility on the surface of the agar. The surface motility colonies in the presence of mucin were circular with a green center surrounded by a thicker white edge. In contrast to swarming, bacterial cells at the edge of the mucin-promoted motility zone appeared piled up and lacked flagella. Using genetic and microscopic methods, it was demonstrated that mucin might be promoting a modified form of swarming or likely a new form of surface motility, termed “surfing”.

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Exploring the development of endotoxin tolerance during sepsis and a possible immunomodulatory therapy (2013)

The immune system responds to bacterial infections by inducing pro-inflammatory mediators, which recruit and activate immune cells to eliminate the invading microbe. However, a systemic and uncontrolled inflammatory reaction may lead to the development of sepsis, which is characterized by organ failure and eventually death. Classical (M1) and alternative (M2) macrophage polarization is known to occur in response to challenges within a microenvironment, like the encounter of a pathogen. Bacterial products like lipopolysaccharide (LPS), can be a potent inducer of inflammation and M1 polarization. LPS can also generate an effect in mononuclear cells known as endotoxin tolerance, defined as the reduced capacity of a cell to respond to LPS activation after an initial exposure to this stimulus. Using systems biology approaches in PBMCs, and macrophages, it was determined here that gene responses during endotoxin tolerance were similar to those found during M2 polarization, including reduced production of proinflammatory mediators, expression of genes involved in phagocytosis, control of oxidative stress, as well as tissue remodelling (Chapter 2). Moreover, an extensive bioinformatic meta-analysis was performed using these findings, characterizing unique LPS and endotoxin tolerance gene signatures. These signatures were compared with transcriptional changes observed in human sepsis cohorts based on our data or from the literature. Very interestingly, it was found that septic patients strongly presented an immunological profile associated with an endotoxin tolerance gene signature, rather than a dominant pro-inflammatory response as commonly believed to occur in early sepsis (Chapter 3). Additionally, a potential immunomodulator for use in infections and sepsis was investigated at the mechanistic level. Here, the effect of synthetic innate defense regulator peptide (IDR1018) on macrophage differentiation was tested. The results obtained suggests that IDR-1018 drives macrophage differentiation towards an intermediate M1-M2 state, enhancing anti-inflammatory functions while maintaining certain pro-inflammatory activities important to the resolution of infection (Chapter 4).In conclusion, the unique endotoxin tolerance gene signature discovered here and found in septic patients, can be used as biomarkers, that allow characterization of the critical immunological status of the septic patient, enabling the application of appropriate immunological therapies that might improve the survival rate during this deadly syndrome.

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Global regulation of the Lon protease of Pseudomonas aeruginosa and its influence on ciprofloxacin resistance and virulence (2012)

This thesis focuses on investigating global regulation, ciprofloxacin resistance and virulence by the ATP-dependent Lon protease of Pseudomonas aeruginosa. Screening a P. aeruginosa PA14 mutant library for non-essential genes involved in altered ciprofloxacin susceptibility identified more than 100 genes (35 involved in intrinsic and 79 in mutational resistance). The identification of known and novel genes involved in resistance through mutant library screening provided new insights into the ciprofloxacin resistome. These mutants provide insights into adaptive resistance mechanisms and the clinical phenomenon of creeping baselines. The ATP-dependent Lon protease, which showed a 4-8 fold increase in ciprofloxacin susceptibility upon mutation, was chosen for more detailed studies. This study showed that Lon protease regulated antibiotic resistance and virulence properties despite the fact that it is not a traditional regulator. The ciprofloxacin susceptible phenotype of a lon mutant could be complemented. Furthermore, the lon mutant was identified to influence cytotoxicity, adhesion, anaerobic growth and metabolism as well as impact on global regulation. Microarray analysis showed that Lon is at the top of a transcriptional hierarchy dysregulating around 200 genes. Proteomic profiling showed that Lon appeared to be involved in cleaving GroEL, Hfq and KatA amongst others. Furthermore, mechanistic studies on the involvement of Lon protease in the SOS response under sub-inhibitory concentrations of ciprofloxacin revealed that SOS response was less induced in the lon mutant which could be explained by the action of Lon on the key SOS regulator RecA. Lon protease influences virulence in vivo as shown in a lettuce leaf model, an amoeba assay as well as a rat model of chronic infection. The alterations in virulence-related processes in vitro in a lon mutant were also paralleled by defective virulence in vivo. Antibiotic resistance and motility phenotypes were also investigated for other proteases and mutations in pfpI, clpP and clpS had distinct, but overlapping phenotypes cf. the lon mutant. Overall, my results suggest that while the Lon protease is not a traditional regulator, it is still involved in a multitude of cellular processes highlighting its importance for the bacterial cell. Thus, it would be a good target for therapy.

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Mechanisms of immune response regulation by innate defense regulator peptides (2012)

The growing threat of antibiotic-resistant bacteria necessitates the development of new anti-infective therapeutics. Innate defense regulator (IDR) peptides are a novel class of immunomodulatory agents shown to combat bacterial pathogens in murine models of infection via the augmentation of host immune functions, including the stimulation of chemokine production and enhancement of leukocyte recruitment, while suppressing bacterial-induced inflammation. Although IDR-peptides present the potential for future broad-range anti-infective agents, our limited understanding of how they modulate host immunity remains an obstacle in their development as clinical therapeutics. I hypothesized that IDR-peptides impact host immunity by modulating the immune responses of monocytes, a cell population necessary for IDR-mediated protection against infection. In this study, IDR-1002 was found to be a multi-faceted regulator of monocyte migration. IDR-1002 induced the production of monocyte-specific chemokines MCP-1 and MCP-3, as well as neutrophil-specific chemokines, IL-8 and GRO-α in human peripheral blood mononuclear cells (PBMCs), correlating with the activation of the mitogen-activated protein kinases (MAPK), p38 and extracellular-regulated kinase (ERK)-1/2, in monocytes. IDR-1002 was also found to enhance human monocyte migration towards chemokines through the enhancement of β1-integrin-mediated adhesion to fibronectin via regulation of the phosphatidylinositol-3-kinase (PI3K)-Akt signalling pathway. In addition, IDR-1002 increased monocyte responsiveness to the chemokines MIP-1α and RANTES via modulation of CCR5 expression. These results demonstrate an overall promotion of monocyte motility by IDR-1002. In contrast to the immune-strengthening effects of IDR-1002, the production of pro-inflammatory cytokines in human PBMCs stimulated with bacterial lipopolysaccharide (LPS) was suppressed by the peptide, and correlated with a suppression of LPS-induced NFκB and p38 MAPK signalling and activation of PI3K-Akt signalling in monocytes. These results demonstrate that IDR-peptides are potent modulators of human monocyte function via their extensive regulation of monocyte signalling networks, potentially accounting for their multifunctional effects on host immunity in murine models of bacterial infection.

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Regulation of the PhoP-PhoQ two-component system in Pseudomonas aeruginosa and its role in virulence (2012)

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause severeinfections in individuals with underlying medical conditions. P. aeruginosa primarily infects atepithelial surfaces where it interacts initially via type IV pili, flagella and LPS. Two componentregulatory systems control many aspects of pseudomonal physiology and mediate adaptation toenvironmental changes including those that occur in the host. This thesis outlines thecontributions of these systems to the cytotoxicity to epithelial cells and sheds light on theregulation mediated by the two-component sensor PhoQ. Systems that contributed to cytotoxicityfell into several themes including motility, cyclic-di-GMP regulation, and carbon and nitrogenutilization. Several genes controlled by PhoQ were shown to be dysregulated during infection oflung epithelial cells, including upregulation of oprH-phoP-phoQ, the lipid A modification genearnB, and downregulation of a lipid A deacylase, pagL. Consistent with this, lipid A from aphoQ mutant grown in varying magnesium concentrations displayed alterations. LPS of thephoQ mutant revealed increased inflammatory properties as demonstrated by increased secretionof the cytokines IL6, TNFalpha , and IL10 from PBMCs. The decrease in cytotoxicity of a phoQmutant correlated with a decrease in secretion of lipases and proteases when co-incubated withcultured epithelial cells. These results suggest that the PhoP-PhoQ system might adapt thebacterium to lung epithelia and that this might contribute to and be exacerbated by the selectivepressure of inhaled polymyxin therapeutics.Unlike most sensor kinases that phosphorylate their cognate response regulators, PhoQ of P.aeruginosa appears to act only as a phosphatase of its cognate regulator PhoP. Here it wasdemonstrated that PhoP was not activated by PhoQ in a luminescence reporter screen. The sensorkinase, RoxS, involved in regulation of the cyanide insensitive oxidase, was revealed as acandidate phosphodonor to PhoP. Since mutation of roxS was able to reduce but not eliminateexpression from the oprH-phoP-phoQ operon, it is conceivable that other sensors contribute toPhoP phosphorylation. It was also demonstrated that PhoP contributed to the known polymyxinresistance of a phoQ mutant but only partially to cytotoxicity. These results emphasize thecomplexity of the PhoP-PhoQ regulatory system.

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The role of defensins and C-X-C chemokines in mammalian innate immunity (2011)

In humans, defensins constitute the largest group of host defence peptides that are evolutionarily conserved components of innate immunity. Defensins share many structural and functional characteristics with C-X-C chemokines, including a C-X-C amino acid motif, net positive charge, disulphide bonding, three-dimensional shape and chemokine activity. Deficiencies in α-defensins and C-X-C chemokines have been correlated with susceptibility to infection and chronic inflammatory diseases. However the genetics and diversity of defensins and mechanisms underlying these disorders were not well understood. This thesis comprises three separate but overlapping approaches to address these issues.The genomic content of murine α-defensins within the reference C57BL/6J strain was characterized. Novel α-defensin (11) and defensin-related cryptdin (3) genes were found, as were gene duplications and differences in genomic content between strains of mice. A next-generation sequencing method was developed for the quantitative analysis of α-defensin and defensin-related cryptdin gene expression. The α-defensin DEFA1 induced interleukin (IL) 8 and IL10 release from human PBMCs. The mechanism(s) of action of defensins, which appears to involve induction of chemokines and anti-inflammatory cytokines, needs further elucidation in vivo. Consequently, novel murine models of inflammation and immunosuppression were developed. The IL8 and Il10 genes were separately cloned, behind an intestine-specific promoter, into eukaryotic expression vectors, which were used to transfect murine embryonic stem cells. Correct targeting was confirmed for both constructs and germline transmission achieved for the IL8 mice. Conditional homozygous mice were generated, which, upon breeding with Cre-expressing mice, will express IL8, a C-X-C chemokine, in an intestinal-specific manner. This will enable analyses of effects of chemokine overexpression on intestinal infection, and on peptide efficacy in the resolution of infection. In other studies to address innate immune mechanisms, the transcriptional profiles of patients susceptible to Salmonella and mycobacterial infections due to immunodeficiencies in IL12- and interferon-γ-mediated immunity were generated. These data indicated that the chemokines CXCL9 and CXCL10 might mediate immunity to Mycobacteria whereas additional defects in TLR4 responses appeared to underlie susceptibility to Salmonella.The data presented here strengthen our understanding of the murine defensin repertoire and provide tools that enable sophisticated systems level studies of in vivo function.

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Activity of and resistance to cationic antimicrobial agents in Pseudomonas aeruginosa (2010)

This thesis investigates mechanisms of adaptive resistance to the cationic antimicrobial agents tobramycin and polymyxin B in the opportunistic pathogen Pseudomonas aeruginosa. Using a comprehensive mutant library of P. aeruginosa to screen for genes capable of affecting tobramycin susceptibility, 135 genes were identified that caused increased resistance when insertionally inactivated. Transcriptional profiling studies demonstrated downregulation of 53 of these genes in response to tobramycin and significant up-regulation of a number of heat shock genes including an alternative lon protease, AsrA. Induced expression of asrA in trans demonstrated its ability to induce heat shock genes in the absence of tobramycin and also provided protection against tobramycin in the first hour after exposure to a lethal dose of 4 μg/ml. Upregulation of the known efflux pump MexXY was observed after prolonged exposure to sub-inhibitory concentrations of tobramycin but induction of this operon was not observed as part of the immediate response to lethal concentrations of tobramycin. When investigating susceptibility testing methods for polymyxins, 24 P. aeruginosa clinical isolates were observed to have a distinct, reproducible phenotype in which skipped wells were observed during microbroth dilution testing for polymyxin B. Possible mechanisms underlying this phenotype were investigated in two of these isolates and one isolate demonstrating a constitutive resistance phenotype. The effects of varying concentrations of polymyxin B on growth, on expression of the resistance genes phoQ, arnB and PA4773 (pmrAB operon), and on outer membrane permeability were assessed. The isolates presenting the skipped well phenotype demonstrated adaptations in growth, gene expression and membrane permeabilization in response to specific concentrations of polymyxin B consistent with the involvement of Lipid A modifications in the adaptive resistance phenotype.The results of this thesis highlight the complexity of the bacterial response to cationic antimicrobial agents, as we have demonstrated that adaptation conferring immediate protection (induction of heat shock) differs from that providing long term protection (induction of efflux and Lipid A modifications). Furthermore, the regulatory systems involved in conferring resistance through Lipid A modifications, PhoPQ and PmrAB, are complex and may vary between strains as they adapt further to the pressures imposed by antimicrobial treatment.

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Master's Student Supervision (2010 - 2018)
Network-based integrative analysis of multi-omic data (2018)

The rise of high-throughput biology has brought an increase in generation of large datasets such as genomics, transcriptomics, proteomics, and metabolomics: “omics” data. While many biological studies now assay multiple omics types to assess biological function, the analysis of these datasets is typically undertaken separately, contrary to our understanding of how biological systems function. While efforts have been undertaken to integrate these data types, intuitive methodologies that take advantage of modern curated biological databases are lacking. Here I present a methodology for network-based integrative analysis of multi-omic data. This method leverages the power of curated interactome databases and biological network analysis to produce multi-omic biological interaction networks for integrative analysis. The integration of metabolomics data with transcriptomics and proteomics data was enabled by identifying metabolite-protein interactions using MetaBridge, a novel tool that I developed, described here. Identification of these metabolite-protein interactions was shown to facilitate the leveraging of powerful curated protein-protein interaction (PPI) databases such as InnateDB to generate metabolome-centric PPI networks. Such PPI networks accurately encapsulate biological function and enable downstream analysis and dimensionality reduction using proven network analysis techniques. These metabolomics-derived PPI networks could then be integrated with proteomics and transcriptomics data to create multi-omic networks, which provided insights into biological function and could be mined for novel biological insights that would not otherwise be captured by any single omics type. I demonstrated two applications of this methodology to multi-omic datasets. First, I showed how separate gene expression and metabolite signatures for predicting sepsis could be integrated to reveal novel targets for study, demonstrating the utility of this method for hypothesis generation. Second, I demonstrated tri-omic integration of metabolomics, proteomics, and transcriptomics data from neonates in the first week of life. This revealed that network-based multi-omic integration provided consensus on commonly dysregulated biological functions and facilitated novel insights into biological changes.

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Peptide 1018 inhibits swarming motility and dysregulates transcriptional regulators of swarming in Pseudomonas aeruginosa (2018)

Pseudomonas aeruginosa is a Gram-negative environmental pathogen responsible for considerable human morbidity and mortality, especially in vulnerable hospital populations and individuals with cystic fibrosis. Much of this impact stems from its enormous capacity to adapt, colonize, and thrive in a broad variety of host and environmental niches. In P. aeruginosa, adaptive behaviours like biofilm formation and swarming motility can confer significant but conditionally reversible multiple antibiotic resistance, and considerably reduce the efficacy of many clinical antibiotics. Swarming motility is a transitory adaptive behaviour that is induced under stringent conditions (e.g., nutrient limitation, medium viscosity) and has been linked to both in vivo virulence and acute infection in P. aeruginosa. A small, synthetic host defense peptide, 1018, with weak bactericidal activity inhibits the adaptive behaviour biofilm formation at low concentrations in a broad spectrum of Gram-negative and Gram-positive pathogenic bacteria. It also shows synergy with a number of conventional antibiotics. This study aimed to investigate the effect of this peptide on swarming motility. Peptide 1018 inhibited swarming motility at low concentrations in P. aeruginosa and disrupted the expression of seventy-four regulatory genes, including ten of the thirty-five genes identified as swarming regulators. Peptide treatment of bacteria also induced a gene expression profile with significant similarity (67.7%) to cells with a stationary, biofilm-like phenotype. A moderate number of P. aeruginosa mutants with single gene interruptions showed weak tolerance to peptide 1018, and the majority of these interrupted genes were linked to adaptation and survival under stringent conditions. The tolerance phenotype associated with two of these genes, rhlB and anr, was confirmed by complementation. Enhancing the bacterial stringent response through induced amino acid starvation appeared to improve the tolerance of P. aeruginosa to peptide 1018 in a swarming environment. Under these conditions, the wild-type strain and the peptide-tolerant mutants showed respective rescued and enhanced swarming motility when treated with peptide 1018. This study thus supports a link between the mechanism of action of peptide 1018 and the stringent response and demonstrates that peptide 1018 inhibits and broadly dysregulates swarming motility, an adaptive behaviour promoting enhanced antibiotic resistance.

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Inverse regulation of biofilm formation and swarming motility in Pseudomonas aeruginosa by the transcriptional regulator GbuR (2015)

Pseudomonas aeruginosa inversely regulates biofilm formation and swarming motility, which provides the advantage of allowing it to adapt to environmental conditions. These two surface-associated behaviors represent distinct infection states, with swarming being associated with an acute lifestyle, and biofilm formation exemplifying a chronic lifestyle. Thus the inverse regulation of biofilm formation and swarming motility has important implications for the mode of infection, which in turn influences the interaction between P. aeruginosa and an affected host. Recent studies have also shown that the inverse regulation of biofilm formation and swarming motility is under the control of a number of regulatory genes. Characterization of these genes will therefore provide insights into this regulatory phenomenon and its effect on virulence in P. aeruginosa. The aim of this study was to investigate the inverse regulation of biofilm formation and swarming motility by the transcriptional regulator GbuR. It was shown that mutation in gbuR resulted in a severe swarming defect, while biofilm formation was enhanced. Transcriptome analysis defined the modest regulon of gbuR, revealing a number of genes potentially involved in inversely regulating biofilm formation and swarming motility.

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Involvement of regulatory non-coding RNA in motility, biofilm formation, and adaptive antibiotic resistance in Pseudomonas aeruginosa (2014)

Small, non-coding RNA (sRNA) transcripts are emerging as a major mechanism for regulating translational expression in bacteria. Since the discovery of 6S RNA acting to regulate translation of RNA polymerases in Escherichia coli, our understanding of sRNA regulation of translation has expanded, and sRNAs are now known to have a broad range of functions in bacteria ranging from metabolic regulation to virulence determination. The Gram-negative bacterium Pseudomonas aeruginosa is commonly found in natural microbiomes, and is also an opportunistic pathogen as it causes disease in immunocompromised individuals. P. aeruginosa displays a high level of resistance to numerous clinically relevant antibiotics, and is capable of developing biofilms on multiple surfaces in hospital environments. P. aeruginosa is also capable of swarming which is a complex motility involving rhamnolipid surface whetting agents, flagella and type IV pili. This work investigated the involvement of 32 sRNA species in adaptive resistance to antibiotics, swarming motility, and biofilm formation in P. aeruginosa. Unique expression profiles under conditions of swarming and biofilm formation for 27 previously uncharacterized sRNAs were found. It was also found that the sRNAs prrF1, prrF2 and phrS are involved in swarming motility and/or biofilm formation. Compared to free-swimming, planktonic growth expression of the prrF gene loci was up-regulated 163- and 13-fold under swarming and biofilm conditions, respectively, and mutants lacking the entire locus demonstrated modest decreases in swarming while prrF1 mutants demonstrated increased biofilm formation. A transposon insertion mutant in phrS in P. aeruginosa PA14 wildtype displayed a deficiency in swarming motility and biofilm formation. phrS was also found to be involved in the development of adaptive resistance to polymyxin B by impacting on the translation of a lipid A modification operon. Together this work demonstrates that sRNA regulation plays a critical role in swarming motility, biofilm formation and the development of adaptive resistance in P. aeruginosa.

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Immunomodulatory effects of synthetic peptide IDR-1018 in human keratinocytes (2013)

The diverse immunomodulatory properties of naturally occurring host defence peptides have gained prominence over the past decade. There is large interest in creating small synthetic peptides with similar or enhanced immunomodulatory activities. The epithelia are vital components of the human innate immune system, offering protection against potential pathogens by acting as a physical barrier and actively participating in the immune response. The aim of this study was to examine the direct effects of synthetic host defence peptides on the immune response in keratinocytes, the primary cell type in skin, in the context of wound healing, and to evaluate their ability to modulate the keratinocyte immune response in the presence of other immune mediators.The synthetic peptides HHC-36 and IDR-1018 were shown to have a positive effect on keratinocyte proliferation and caused a dose-dependent induction of IL-6 and IL-8. Neither peptide was able to influence keratinocyte migration on its own. The immunomodulatory effects of IDR-1018 in human keratinocytes were further investigated by co-stimulating cells with IDR-1018 in the presence of immune mediators or TLR agonists. Co-treatment of keratinocytes with IDR-1018 and either the TLR3 agonist poly(I:C) or IL-1β resulted in a synergistic induction of IL-8. This synergy could be seen transcriptionally 8 hours post-stimulation and was associated with increased levels of phosphorylated CREB. Synergistic IL-8 induction was not observed when IDR-1018 was given with Pam3CSK4, flagellin or GM-CSF. Pre-treatment of keratinocytes with inhibitors of p38 MAPK, NF-κB or Src-family kinases suppressed the IDR-1018-induced synergistic IL-8 production in the presence of poly(I:C) or IL-1β. PKC was shown to play a role in the synergy induced by IDR-1018 and IL-1β, but not poly(I:C). The results of this study offer insights into the immunomodulatory properties needed to effectively enhance the protective abilities of the skin, and highlight the complexity of the mechanism of action of IDR-1018.

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Immunomodulatory and wound-healing effects of the host defence peptide LL-37 and related innate defence regulators (2012)

LL-37, the only known human cathelicidin peptide, possesses a variety of immunomodulatory properties that extend its role in host defence far beyond its original classification as an antimicrobial peptide. Recently, work has been underway to elucidate signalling pathways initiated by LL-37, with the aim of further understanding this peptide’s role in the immune system. The aim of this study was to further uncover the role of transcription factors during the responses of immune cells to LL-37 and related innate defence regulator peptides. Secondary aims were to investigate potential wound-healing properties of these peptides and to compare host defence peptides with chemokines in terms of immunomodulatory function. Here, I demonstrated involvement of AP-1 in LL-37-induced wound healing. I also showed a functional overlap between chemokine CXCL9/MIG and host defence peptide LL-37 and demonstrated similarities between LL-37 and the antibiotic azithromycin.

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