Robert E Hancock

Antibiotic therapy frequently fails when treating biofilm-associated infections, which are aggregated bacterial communities embedded in a protective, extracellular matrix. Bacterial biofilms cause 65% of bacterial infections in humans and are inherently resistant to antimicrobials. Despite their clinical and economical significance, there are no clinically approved drugs selectively targeting biofilms. To understand the biofilm regulatory gene network, I set out to identify genes phenotypically affecting biofilm growth in the global priority pathogen Pseudomonas aeruginosa that causes multiple different biofilm infections in humans. A transposon insertion sequencing (TnSeq) approach comparing biofilm and planktonic growth of P. aeruginosa PA14 was employed to identify genes in different models from in vitro hydroxyapatite (HA) and in vivo-like human skin organoids to in vivo murine abscesses. Biofilm genes detected in the HA model indicated the requirement of several one-component transcriptional regulators (OCRs), which are the most abundant and versatile group of bacterial regulators but a poorly studied one in the context of biofilms. This led to the identification of six regulators required for biofilm growth without affecting motility phenotypes, namely yeaG, bosR, arsR, merD, PA14_36180 and PA14_56430. The putative oxidative stress regulator bosR was required for biofilm growth in another divergent phylogenetic P. aeruginosa lineage and further confirmed by deletion and complementation. Biofilm growth on in vivo-like human skin organoids involved several known biofilm regulators of well-established biofilm pathways, such as the Gac-Rsm and the alginate regulatory pathway, that showed discrepancies in mutant phenotypes compared with in vitro HA biofilms, suggesting environment-dependent remodeling of the biofilm gene network. In the murine abscess model, TnSeq was employed to support the presence of P. aeruginosa biofilms. Accordingly, many genes previously implicated in biofilm growth were shown to affect growth in murine abscesses in a similar manner, including two dozen characterized biofilm genes. Furthermore, gene cbfA was identified as a novel biofilm gene required in all three biofilm models used in this study. These findings provided new insights into the mechanisms governing biofilm growth in P. aeruginosa and a broad range of genes representing novel environment-specific targets for the design of antibiofilm therapies.

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Sepsis is a common and very heterogenous syndrome defined as the life-threatening organdysfunction caused by an aberrant host response to infection. In the earliest stages, sepsisdiagnoses are often missed due to non-specific symptomatology resulting in a rapid progression tosevere sepsis. Gene expression signatures that measure host immune responses have been shownto provide more sensitive prognostic tools than existing clinical criteria, permitting early predictionof high-risk patients. We recruited, from six global cohorts, 266 suspected sepsis patients in theemergency room and 82 suspected pulmonary sepsis patients in the intensive care unit with varyingdisease severity, and 44 healthy controls. Most recently, I analyzed 135 patients with Covid-19disease that showed immune responses overlapping with sepsis. From this, I identified candidategene expression signatures reflecting endotypes and severity markers, using the transcriptomicsmethod, RNA-Seq, and statistical and computational methods.I determined that early sepsis patients could be stratified into five endotypes defined by distinctpathobiological mechanisms, including unique gene expression differences and accurate,predictive gene expression pairs. Two of the five endotypes were associated with a higher tendencytowards severe sepsis and mortality, two demonstrated much lower severity, and one was relativelybenign. Diverse molecular responses were also observed independently of endotypes; thus,concomitant cross-cutting severity signatures that directly predicted sepsis-induced organdysfunction and mortality were identified, in addition to dysregulated and co-expressed modulegenes. The endotype signatures were often consistent with cellular shifts in neutrophil numbersand function, whereas dysregulated molecular responses like cellular reprogramming and hyperinflammation reflected prognoses. These signatures were assessed in other conditions (e.g.,pancreatitis, appendicitis, myocardial infarction), which indicated the signatures capturedmechanisms specific to early sepsis/sepsis. A compendium of dysregulated genes and signaturesin sepsis was curated from the literature, confirming that these signatures involved wellcharacterized genes.This study demonstrated that signatures relevant to the development of life-threatening sepsis canbe observed as early as first entry into the ER. These signatures will enable the development ofdiagnostics and targeted therapeutics, and importantly, when used early in the sepsis diseasecourse, could prevent rapid patient deterioration, mortality, and poor long-term outcomes.

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Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial and chronic infections contributing to morbidity and mortality in skin wound and cystic fibrosis patients, respectively. One of the reasons for its success as a pathogen is its ability to adapt to a broad range of circumstances. Here, the regulatory two-component system NtrBC is shown to be involved in adaptive and pathogenic states of P. aeruginosa. Characterization of adaptive lifestyles in vitro confirmed that the double ΔntrBC mutant demonstrated a nearly complete inhibition of swarming motility, a modest decrease and alteration of surfing motility and >40% reduction in biofilm formation; except for swarming, single mutants generally had more subtle or no changes. The P. aeruginosa ΔntrBC mutant also had a major increase (~10-fold) in susceptibility to ciprofloxacin. Transcriptional profiles of deletion mutants were defined using RNA-Seq and unveiled dysregulated expression of hundreds of genes implicated in P. aeruginosa virulence during chronic lung infections, as well as carbon and nitrogen metabolism. The role of NtrBC in host-pathogen and interspecies interactions was also examined. P. aeruginosa mutants exhibited distinct host interactions, including modestly increased cytotoxicity toward human bronchial epithelial cells, reduced virulence factor production and 10-fold increased uptake by macrophages in vitro. In a high-density skin infection model, mutants were reduced in their ability to invade or cause damage to tissue and were more susceptible to ciprofloxacin in vivo. To compare the infectivity of strains across tissues, and pre-clinically screen antimicrobial or immunomodulatory therapies for the treatment of sinusitis, a murine model of upper respiratory tract infection was developed. In contrast to the wild-type levels of colonization observed in the abscess model, P. aeruginosa mutants colonized the respiratory tract less well than wild-type. In contrast to wild-type, ΔntrC and ΔntrBC mutants outcompeted Staphylococcus aureus, a commonly co-isolated species in skin wounds and cystic fibrosis patients, during planktonic and biofilm growth. These results indicate that NtrBC is a global regulatory system involved in both adaptive and pathological processes relevant to the success of P. aeruginosa in infection.

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Novel treatments for inflammatory skin disorders are of great demand as sterile inflammatory skin diseases such as psoriasis and atopic dermatitis are common, long-lasting, and detrimental to people’s quality of life, yet have no cure. Skin biofilm infections caused by Staphylococcus aureus and Pseudomonas aeruginosa are intrinsically and adaptively resistant to antimicrobial agents but lack specific treatments. Natural and synthetic host defense peptides are known to exhibit beneficial biological functions including direct antimicrobial, antibiofilm, immunomodulatory and anti-inflammatory properties. Therefore, I proposed that anti-inflammatory peptide IDR-1002 and antibiofilm peptide DJK-5 could tackle skin inflammation by different underlying mechanisms. IDR-1002 was shown to have promising in vitro and in vivo anti-inflammatory effects. In an animal model, it dampened PMA-induced ear edema, proinflammatory cytokine and reactive oxygen and nitrogen species release and neutrophil recruitment by downregulating G-protein coupled receptors that recognize proinflammatory mediators. IDR-1002 also suppressed the IFN-γ pathway and an interferon regulatory factor-8-regulated network in PMA-induced inflammation. Similarly, lipidated peptidomimetics Pam-(Lys-βNspe)6-NH2 and Lau-(Lys-βNspe)6-NH2 were potent suppressors of PMA-induced sterile skin inflammation comparable to the non-steroidal anti-inflammatory drug indomethacin. To study biofilm skin infection, I established an air-liquid interface epidermal model and showed that DJK-5 significantly reduced 1-day and 3-day Methicillin-resistant S. aureus (MRSA) and P. aeruginosa biofilms. Using this in vivo-like humanized system as a screening platform allowed the identification of novel peptides D-3006 and D-3007 with superior antibiofilm activity and immunomodulatory potential. Skin with thermal wounds had increased susceptibility to MRSA biofilm infection, and DJK-5 treatment significantly reduced bacterial load, cytotoxicity, and pro-inflammatory cytokines. Combination treatment of DJK-5 with IDR-1002 further reduced cytotoxicity and skin inflammation. Transcriptomic analysis revealed that DJK-5 treatment restored skin barrier function, suppressed MRSA intracellular invasion, and dampened TNF-α signalling and transcription factors AP-1, c/EBPB and CREB, leading to reduced production of proinflammatory mediators such as cytokines, prostaglandins, and matrix metalloproteinases. Both IDR-1002 and DJK-5 returned skin to homeostasis by downregulating TNF-α and NF-κB signalling and their negative regulators, and upregulating TSC22D3, an important mediator of glucocorticoid anti-inflammatory effects. These data reveal the intrinsic promise of synthetic peptides in treating inflammation and biofilm infections.

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This research assessed the ability and mechanism of azithromycin (AZM) alone, and in synergy, to treat Pseudomonas aeruginosa grown in physiologically relevant media and in vivo when compared to standard, nutrient rich conditions. When compared to treatment in Mueller Hinton broth (MHB), AZM has a substantially lower minimal inhibitory concentration (MIC) in RPMI ± human serum (RPMI/serum) and demonstrates increased synergy in combination with synthetic host defence peptides. Global transcriptional analysis revealed that genes mediating lipopolysaccharides (LPS) modification were downregulated in host-like media compared to MHB. Inactivation of these genes led to increased susceptibility to AZM and synergy between AZM and other antimicrobial agents. Thus indicating that dysregulation of LPS modification might result in increased AZM uptake in the host environment. Using Tn-Seq, mutants with severe growth defects under physiologically relevant conditions were determined. Genes involved in membrane integrity, iron acquisition, and nucleotide and cobalamin biosynthesis were implicated as required for survival under physiologically relevant conditions. The factors influencing growth of P. aeruginosa in physiologically relevant media were also essential in vivo, and may be used to explore alternative treatments that might take advantage or target these systems in the clinic. Finally, unique sets of genes important for susceptibility of P. aeruginosa to AZM under physiologically relevant media conditions and in vivo were identified using Tn-Seq in medium treated with AZM. Increased AZM susceptibility in murine abscess and human skin organoid models was observed for mutations in integral cell envelope genes, stress signaling and biofilm formation genes, type III secretion system genes, and transcriptional regulators. Most of the AZM susceptibility mutants had predicted interactions with iron acquisition components and increased susceptibility to AZM was induced as a function of iron limitation. Evidently, multiple factors in host-like environments are responsible for the observed changes in susceptibility of P. aeruginosa, including altered membrane permeability, altered iron availability, and stress responses. Understanding the mechanisms that underly bacterial susceptibility to antimicrobials in varying conditions could help change standard approaches to drug testing and lead to more complete and functional assays enabling more predictive drug discovery routes.

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Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that possesses intrinsic antibiotic resistance. Highly adaptable, P. aeruginosa is capable of different forms of motility, including swarming, swimming, twitching and surfing. Swarming motility is a multicellular movement of cells across semisolid surfaces that is associated with complex adaptations including adaptive antibiotic resistance. Here a disc diffusion assay showed that swarming bacteria were resistant to multiple antibiotics, including aminoglycosides, β-lactams, chloramphenicol, ciprofloxacin, macrolides, tetracycline, and trimethoprim. RNA-Seq of swarming cells showed the dysregulation of 1,581 genes, including 104 regulatory factors, upregulated virulence and iron acquisition factors, and downregulated ribosomal genes. Forty-one mutants resistant to tobramycin under swarming conditions were found, including prtN, a regulator of pyocin, and wbpW, involved in LPS biosynthesis. RNA-Seq of swarming cells treated with tobramycin revealed the upregulation of the multidrug efflux pump mexXY. To investigate the role of swarming in vivo, a screen for swarming-specific mutants was performed, revealing ptsP, a regulator of carbon and nitrogen metabolism. The ∆ptsP mutant was deficient specifically in swarming but not swimming or twitching motility. Interestingly, ∆ptsP also had greatly reduced organ invasion in a mouse infection model, suggesting a likely role for swarming in vivo. Besides ptsP, small RNAs also regulated swarming motility, typically via post-transcriptional means. A screen of sRNA overexpressing strains revealed an sRNA, PA0805.1 that influenced diverse bacterial behaviours including swarming, swimming, twitching, cytotoxicity, adherence and tobramycin resistance. RNA-Seq and proteomics uncovered a broad regulatory profile with 1,121 differentially expressed genes and 925 proteins, including 118 regulatory factors, downregulated pilus genes, upregulated adherence and virulence factors, and upregulated multidrug efflux systems including mexXY and mexGHI-opmD. Another sRNA, PA2952.1, when overexpressed influenced swarming, swimming, and tobramycin, gentamicin and trimethoprim resistance. Transcriptomics and proteomics showed differential abundance of 784 genes and 445 proteins, encompassing 82 regulatory factors, downregulated pili, dysregulated flagellar genes, upregulated mexGHI-opmD and the upregulated arn operon involved in LPS modification. Overall this thesis has shown that swarming motility is a complex adaptation conferring multiple antibiotic resistance, that is regulated by sRNAs and coupled to virulence adaptations in vivo.

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Different forms of macrophage activation or polarization are relevant in the pathogenesis of a variety of diseases from inflammatory conditions to infections. It has been previously established that classically activated or M1 macrophages such as those produced by IFNγ stimulation are non-permissive for intracellular Salmonella infection, while alternatively activated or M2 macrophages such as those produced by IL-4 stimulation are permissive for Salmonella growth. It is not known whether endotoxin tolerant macrophages (primed with endotoxin stimulation), such as those observed in sepsis, are permissive for Salmonella growth. A gentamicin protection assay was performed for these three types of differently polarized human monocyte-derived macrophages (MDM) in vitro, and bacterial load measured through colony counts and microscopy. Endotoxin primed MDM (MEP) had a similar bacterial load to M1 macrophages at the initial and 2-hour time-points, but became more susceptible to Salmonella by the 4- and 24-hour time-points. Transcriptomic comparisons using RNA-Seq were performed to generate hypotheses regarding mechanisms for the differences observed between these polarization types, based on differential gene expression. Key immune pathways including JAK-STAT were enriched in uninfected M1 and MEP compared to uninfected M2 macrophages, suggesting a priming effect on these pathways due to polarization. Meanwhile, Salmonella-infected M1 showed increased expression of key inflammasome genes and Salmonella resistance genes compared to M2 and MEP macrophages. These effects were also observed in similarly treated human induced-pluripotent stem cell derived macrophages (iPSDM), further validating the usefulness of iPSDM as a macrophage model in polarization and infection experiments. In order to investigate the mechanistic relevance of these observations, Ruxolitinib was applied to inhibit JAK1-2 during the polarization phase of the experiment. This increased Salmonella permissiveness at the 4-hour time point in resistant M1 macrophages, but not in M2 or MEP macrophages, which are susceptible at this time point. This is consistent with an important role for JAK-STAT priming and resistance to Salmonella infection. These observations provide insights into the effects of polarization on Salmonella resistance in macrophages, and the suitability of iPSDM for macrophage study.

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Pseudomonas aeruginosa is an opportunistic pathogen associated with a high incidence of infections in hospitalized and cystic fibrosis (CF) patients. P. aeruginosa is highly adaptable and exhibits diverse lifestyle adaptations depending on its surrounding environment. Here I studied a complex motility lifestyle termed surfing that occurs in the presence of mucin, a glycoprotein that is found in large abundance in the CF lung, and showed that surfing was associated with broad-spectrum antibiotic resistance, conserved in several bacterial species, and regulated by a complex networks of regulators. RNA-Seq revealed ~1,024 genes dysregulated in P. aeruginosa under surfing conditions, while a screen of the PA14 transposon mutant library revealed 192 mutants that exhibited surfing deficiency, 40 of which were regulatory genes, including the putative chemotaxis regulator, PA1463, and two-component regulator, pfeR. Both PA1463 and pfeR were found to be master regulators of P. aeruginosa surfing and mutants in these genes demonstrated dysregulation of the majority of other regulators influencing surfing. Using disk diffusion assays, I investigated the adaptive antibiotic resistance associated with surfing motility. P. aeruginosa surfing cells were significantly more resistant to several antibiotics including all tested aminoglycosides, carbapenems, polymyxins, fluoroquinolones, and trimethoprim, tetracycline, and chloramphenicol. To identify the genes mediating surfing-dependent antibiotic resistance, transposon mutants in antibiotic susceptibility genes that were dysregulated under surfing conditions were screened for altered susceptibility under surfing conditions. This revealed 65 mutants, including mutants in armR, recG, atpB, clpS, nuoB, that exhibited changes in susceptibility to one or more antibiotics, consistent with a contribution to the observed adaptive resistance. It was further demonstrated that other motile bacterial species, including Escherichia coli, Salmonella enterica, Vibrio harveyi, Enterobacter cloacae, Proteus mirabilis, and Bacillus subtilis, exhibited similar characteristics of surfing as observed for P. aeruginosa in the presence of mucin, including rapid surface growth, dependence on flagella, and broad- spectrum adaptive resistance. Therefore, surfing is a conserved motile lifestyle regulated by complex networks of regulators and leads to broad spectrum adaptive antibiotic resistance.

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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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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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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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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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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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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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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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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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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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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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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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