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Type IV pili (T4P) are long adhesive surface filaments produced by bacteria and are a key virulence factor for many pathogens. T4P are produced by a dynamic intracellular nanomachine that facilitates the assembly (extension) and disassembly (retraction) of pili. Pilus dynamics are enabled by the motor subcomplex of the nanomachine, where cytoplasmic ATPases power pilus assembly (PilB) and disassembly (PilT and PilU). In many, but not all, T4P expressing bacteria – including our model organism Pseudomonas aeruginosa – two retraction ATPases are required for functional retraction, which can be assessed by measuring twitching motility. Deletion of pilT results in loss of twitching and phage susceptibility (another hallmark of pilus function) while deletion of pilU results in loss of twitching but retention of phage susceptibility, indicating pili can still be retracted. We hypothesized that PilU adds to the force of pilus retraction, facilitating disassembly when the fiber is under tension. We mutated ΔpilU and pilU::Tn5 strains with ethyl methanesulfonate and screened for gain-of-twitching mutants. Whole genome sequencing revealed multiple point mutations in the major pilin protein PilA or the pilus adhesin, PilY1. These point mutations were recapitulated in a ΔpilU strain and restored twitching to varying degrees. Complementation of pilA point mutants with pilU in trans influenced the twitching zone of only one mutant, and in trans expression of wild-type pilA resulted in a significant reduction in twitching in most. The contribution of PilU to the force of pilus retraction was further investigated by a polyacrylamide micropillar assay, where no pulling events could be detected for either ΔpilT or ΔpilU mutants. Exopolysaccharide production, a proxy for surface sensing, was uncoupled from twitching motility in the pilA point mutants. These results are a significant step forward to understanding what PilU does and, provides insight to the dynamics of the pilus fiber. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a bacterium that causes serious infections. P. aeruginosa uses adhesive, “grappling hook” filaments called Type IV pili (T4P) to stick to its hosts. T4P can be repeatedly extended and retracted, allowing the bacteria to crawl on surfaces (twitching) but making them susceptible to bacteriophages, viruses that attach to pili then kill the bacterial cells. The motor proteins PilT and PilU are required for twitching, but only PilT is essential for phage killing, implying that pili are retracted even when PilU is missing. Here we hypothesized that PilU is important for twitching because it helps generate force for retraction when pili are under tension. We isolated multiple mutations in pilus components that restored twitching in the absence of PilU, and propose that these mutations allow for easier retraction of pili. This information helps us understand how T4P help the bacteria to spread during infection.

Two-component regulatory systems are an important means for most prokaryotes to adapt quickly to changes in their environment. Canonical systems are composed of a sensor kinase, which detects signals that trigger autophosphorylation, and a response regulator, which imparts changes within the cell, usually through transcriptional regulation. The opportunistic pathogen, Pseudomonas aeruginosa, expresses a plethora of two-component systems including the PilS-PilR sensor-regulator pair, which directs transcription of the major component of the type IV pilus (T4P) system, pilA, in response to an unknown signal. T4P are surface appendages that are required for full virulence, as they perform several important functions including twitching motility, cell surface attachment, surface sensing, and biofilm formation. While loss of pili is known to decrease virulence, the effect of surplus surface pili on pathogenicity was unknown. In other T4P-expressing bacteria, PilR regulates the expression of non-T4P related genes, but its regulon in P. aeruginosa was undefined. Here, we identify PilA as an intramembrane signal for PilS, regulating its own expression. When PilS-PilR function is altered through the use of activating point mutations, which induce hyperpiliation, pathogenicity in C. elegans was significantly impaired compared to both wild type and non-piliated strains of P. aeruginosa. This phenotype could be recapitulated using other hyperpiliation-inducing mutations, providing evidence that over production of surface pili likely prevents productive engagement of contact-dependent virulence factors. Last, transcriptomic analyses revealed that expression of over 50 genes – including several involved in flagellar biosynthesis and function – is modulated by PilSR, suggesting coordinate regulation of motility in P. aeruginosa. Together, this work provides new information on the control of pilA transcription and suggests novel roles for surface pili and the PilSR two component system in virulence and swimming motility, respectively. The knowledge gained from this work could be applied to the development of a PilS or PilR based anti-virulence therapeutic. / Thesis / Doctor of Philosophy (PhD) / Pseudomonas aeruginosa is a Gram negative bacterium and a common cause of hospital acquired infections. The World Health Organization recently ranked P. aeruginosa as one of the top “priority pathogens” for which new treatments are desperately needed, in part due to its intrinsic resistance to many antibiotics. Among the key features that contribute to the infectivity of P. aeruginosa are its Type IV pili (T4P), which are flexible, retractile surface appendages involved in cell surface attachment, movement across solid surfaces and other important functions. Production of the major pilin protein, PilA, which forms most of the pilus, is tightly controlled by the two-component regulatory system, PilS-PilR, where PilS is a sensor and PilR is a regulator that directly controls pilin expression. The aim of this work was to identify the signal(s) detected by the sensor, as well as additional genes or systems under PilSR control. We showed that the pilin protein interacts directly with the sensor to control its own expression, and that dysregulation of the PilS-PilR two-component system impairs both pathogenicity and other forms of motility. Together, the data presented here provide insight into how PilS-PilR control expression of systems required for virulence of P. aeruginosa and highlight the potential of these proteins as possible therapeutic targets.

The type IV pilus is a motility organelle found in a range of bacteria, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The principle structural protein of the pilus is the major pilin, PilA, while a set of low abundance “minor pilins” are proposed to constitute the pilus tip. The minor pilins, FimU and PilVWXE, along with the non-pilin protein PilY1, prime assembly of surface-exposed pili. The fimU-pilVWXY1E operon is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 is an adhesin and mechanosensor that, along with PilW and PilX, triggers virulence upon surface attachment. Here, we aimed to uncover the mechanism for PilWXY1-mediated virulence. We hypothesized that loss of PilWXY1 would relieve feedback inhibition on FimS-AlgR, resulting in increased transcription of the minor pilin operon and dysregulation of virulence factors in the AlgR regulon. Caenorhabditis elegans slow killing assays revealed that pilW, pilX, and pilY1 mutants had reduced virulence relative to a pilA mutant, implying a role in virulence independent of pilus assembly. FimS-AlgR were required for the increased promoter activity of the minor pilin operon upon loss of pilV, pilW, pilX, or pilY1. Overexpression or hyperactivation of AlgR by point mutation led to reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants were dependent on FimS-AlgR expression. We propose that PilWXY1 inhibit their own expression at the level of FimS-AlgR, such that loss of pilW, pilX, or pilY1 leads to FimS-mediated activation of AlgR, and reduced expression of acute-phase virulence factors. Accumulation of mutations in the minor pilin operon may represent an evolutionary strategy for P. aeruginosa populations in chronic lung infections, as loss of PilWXY1 would upregulate the expression of AlgR-dependent virulence factors – such as alginate – characteristic of such infections. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a bacterium that causes dangerous infections, including lung infections in cystic fibrosis patients. The bacteria use many strategies to infect their hosts, one of which involves a grappling hook-like fibre called the type IV pilus. There are many components involved in assembly and function of the pilus, including five proteins called “minor pilins” and a larger protein called PilY1 that may help the pilus detect surface attachment. We used a roundworm infection model to show that loss of PilY1 and specific minor pilins leads to delayed killing, while loss of other pilus proteins has no effect on worm survival. This effect was due to increased activation of a regulatory system called FimS-AlgR that inhibits expression of other factors used by this bacterium to infect its hosts. By studying how P. aeruginosa causes infection, we can design better strategies to disarm it and reduce the severity of infections.

Oxidative stress-inducing chemical contamination in the environment is a significant concern for public health. The depletion of antioxidants by these chemicals results in oxidative stress which may cause detrimental effects in many cell types. For example, multiple stress responses may be activated in bacteria and several disorders including neurodegenerative disorders may occur in mammalian organisms. Oxidative chemicals also have negative effects on engineered water systems as an oxidative stress response in bacteria has been implicated to cause process failure in wastewater treatment facilities. Therefore, it is essential to monitor oxidative chemical contamination in water environments to provide early warning of potential negative effects. Whole-cell biosensors that indicate bacterial stress responses to oxidative toxic agents can be powerful tools in environmental monitoring. An oxidative stress response found in many Gram-negative heterotrophic bacteria called the glutathione-gated potassium efflux (GGKE) mechanism is a good biological indicator to be used in a biosensor designed to detect the presence of oxidative chemicals in water. The authors of this study propose the development of a GGKE biosensor using an environmental strain of Pseudomonas aeruginosa. The abundance of the global antioxidant glutathione, the gating compound in GGKE, in various cell types suggests that there may be connections between the responses of the different cell types to oxidative stress. In this study, specific oxidative stress responses in two distantly related cell types were studied: the GGKE mechanism in Gram-negative heterotrophic bacteria, and mitochondrial dysfunction in rat brain cells. Furthermore, the use of an octanol-based emulsification method for the immobilization of P. aeruginosa in calcium alginate microbeads was evaluated for long-term mechanical stability, viability, and GGKE response of the immobilized cells. The immobilization of cells is an important factor in the design of a whole-cell biosensor, and must yield viable and active cells over time. This study showed that the dose-dependent responses of GGKE in Pseudomonas aeruginosa cells and of mitochondrial dysfunction in a mixed culture of rat brain cells to a model oxidative electrophilic chemical, N-ethylmaleimide, correspond well to each other. We also showed that both responses are accompanied by the depletion of intracellular glutathione, which precedes the GGKE response in P. aeruginosa as well as mitochondrial damage in rat brain cells. Thus, this study suggests that bacterial responses to oxidative stress involving glutathione, such as GGKE, could potentially be used as an early warning to predict the presence of bioavailable oxidative chemicals that can induce oxidative stress in eukaryotic systems. Although further research is needed, this suggests that bacterial stress response biosensors may be used to predict oxidative stress responses in mammalian brain cells. The octanol-based emulsification method produced P. aeruginosa encapsulated alginate microbeads with an average diameter of 200 μm. The microbeads were mechanically stable in solutions containing up to 20 mg/L K+ for 15 days. LIVE/DEAD® and specific oxygen uptake rate (SOUR) analyses showed that the microbead-immobilized cells recovered their membrane integrity within 5 days but not their net respiration potential. The microbead immobilized cells had no net GGKE potential in response to 50 mg/L N-ethylmaleimide after 14 days whereas water-based alginate bead (2mm) immobilized cells did, albeit at a reduced level to planktonic cells. Confirmation experiments revealed that octanol impeded cellular activities of the immobilized cells. Overall, this study showed that the octanol-based emulsification method is not suitable for the immobilization of P. aeruginosa for use in the GGKE biosensor and other microscale immobilization methods should be evaluated. / Master of Science

Microbes in nature live collaboratively in adherent communities, known as biofilms. Biofilms can be contextually beneficial or detrimental. In medical implants, biofilms cause infections leading to additional healthcare costs of billions of dollars. Studies have found that micro/nanoscale surface topography can significantly alter (i.e., promote or hinder) the process of biofilm formation. The formation of biofilm starts with planktonic microbes attach to the surface. To further understand the biophysical underpinning of this process, the effect of aligned nanoscale surface structures on microbial adhesion was studied. To this end, aligned nanofiber coating with controlled fiber diameter and edge-to-edge spacing were manufactured using the Spinneret-based Tunable Engineered Parameters (STEP) techniques. The effect of surface topography on bacterial near-surface motility was studied. The experimental results showed that the bacterial attachment and near-surface motion can be greatly impacted by surface topography. Furthermore, the finding was applied to ureteral stents. The results showed that the aligned nanofiber can significantly reduce the biofilm formation process on ureteral stents. / Master of Science / Many microbes in nature live in adherent communities called biofilm. Biofilms contain individual microbes inside polymeric matrix which protect them from environmental stressors such as antibiotics. Biofilms are a significant contributor to the infection of implantable medical devices, which leads to additional healthcare costs of billions of dollars annually in the U.S. alone. Studies have found that sub-micron scale surface topography can significantly promote or hinder biofilm formation; however, the exact mechanism remains poorly understood. To further understand this process, the effect of aligned nanoscale surface structures on microbial adhesion was studied. The formation of microbial biofilm starts with swimming bacteria sensing the liquid-solid interface and attaching to the surface. Microbes are more likely to settle on a surface if a surface is favorable to attach. However, the decision-making process has not been fully understood. Our experimental results showed that the bacterial attachment and near-surface motion can be greatly influenced by surface topography. Furthermore, the finding was applied to ureteral stents, which is a type of medical implants used to maintain the flow of urine in the urinary tract. Ureteral stents serve great for medical purposes, but as foreign bodies, they also lead to urinary tract infection. The results showed that some types of aligned fiber coating increased microbial attachment density, while other types of aligned fiber coating reduced the bacterial surface coverage by up to 80%, which provides directions for future studies.

The opportunistic pathogen Pseudomonas aeruginosa ranks among the leading causes of nosocomial infections.  The type III secretion system (T3SS) aids acute P. aeruginosa infections by injecting potent cytotoxins (effectors) into host cells to suppress the host's innate immune response.  Expression of all T3SS-related genes is strictly dependent upon the transcription factor ExsA.  Consequently, ExsA and the biological processes that regulate ExsA function are of great biomedical interest.  The ExsA-ExsC-ExsD-ExsE signaling cascade ties host cell contact to the up-regulation of T3SS gene expression.  Prior to T3SS induction, the antiactivator protein ExsD binds to ExsA and blocks ExsA-dependent transcription by interfering with ExsA dimerization and promoter interactions.  Upon host cell contact, ExsD is sequestered by the T3SS chaperone ExsC, resulting in the release of ExsA and an up-regulation of the T3SS. ExsA is an AraC/XylS-type transcriptional regulator and belongs to a subfamily of activators that regulate the T3SS in a variety of Gram-negative pathogens.  These regulators are characteristically difficult to purify due to the low solubility of their C-terminal DNA binding domains.  A new method for purifying ExsA was developed and produced ExsA with improved solubility.  The interaction of ExsA and its PexsD promoter was examined using fluorescence anisotropy.  An in vitro transcription assay was developed and it was determined that ExsA is sufficient to activate T3SS transcription. Next, the ExsD--ExsA inhibitory mechanism was examined.  It was demonstrated for the first time that ExsD alone is sufficient to inhibit ExsA-dependent transcription in  vitro without the aid of any other cellular factors.  More significantly and contrary to previously published results, it was discovered that independently folded ExsD and ExsA are capable of interacting, but only at 37 degrees C and not at 30 degrees C.  Guided by the crystal structure of ExsD, a monomeric variant of the protein was designed to demonstrate that ExsD trimerization prevents ExsD from inhibiting ExsA-dependent transcription at 30 degrees C. To further elucidate the ExsD-ExsA inhibitory mechanism, the ExsD-ExsA interface was examined.  ExsD variants were generated and used to determine which region of ExsD interacts with ExsA.  Interestingly, ExsD was also found to bind DNA, although it is unclear whether or not this plays a role in ExsA inhibition.  Fully understanding the mechanism by which ExsD inhibits ExsA may enable the development of drugs that target ExsA in order to shut down the T3SS, thereby eliminating P. aeruginosa infection. / Ph. D.

Bacterial biofilms are communities of micro-organisms encased a self-produced extracellular matrix. While they form readily in a nature, biofilm formation in man-made systems have economic and health consequences. Prior research demonstrated that topographical features comprised of uniform, micro-meter sized particles hindered the biofilm formation of Pseudomonas aeruginosa (P. aeruginosa), an opportunistic human pathogen. The goal of the present work is to 1) further develop a potential anti-biofilm coating by improving its robustness and 2) study the mechanism(s) by which surface topography hinders biofilm formation. The robustness of a topographical coating comprised of an array of silica particles is improved by the introduction of silica bridges through a sol-gel reaction. To study the mechanism(s), specifically, we hypothesized that the motion, or surface motility, of P. aeruginosa is hindered by the presence of micro-meter scale obstacles via physical obstruction. To test this, we analyzed the behavior of single P. aeruginosa cells at micron-scale spatial resolutions using time-lapse fluorescence microscopy, image analysis, and particle tracking techniques. We fabricated various types of micron-scale topography with curvature (particle arrays) and recti-linear features (vertical steps) and varied the critical dimension within the range of 0.5 – 10 µm which spans the dimensions of a typical P. aeruginosa cell. We found that there was a threshold feature size of 1-2 µm at which bacterial surface motility is drastically impacted. On positively curved topography (particle arrays), we found that the frequent obstacles reduced the average speed of a bacterium from 6.2  0.3 µm per 5 min on a flat surface to 2.1  0.3 µm per 5 min on an array of 2 µm particles. Furthermore, we observed that bacteria often move in-between particles, suggesting that bacteria have difficulty climbing over tall obstacles. To further investigate P. aeruginosa's ability to cope with topography, we examined the effect of recti-linear features (vertical steps) on surface motility. We found that step heights > 0.9 µm drastically reduced the probability of crossing and that the average speed when approaching the step is reduced by a factor of 2. Interestingly, we find that bacteria have a slight preference to traverse down which is against the direction of gravity in our system. In summary, these results offer insights into how a surface motile bacterium copes with a topographical surface. Our data indicate that the topography of a surface can impede the surface motility of bacterium and thus, may be an important mechanism by which topography prevents biofilm formation. / Doctor of Philosophy / Bacteria and other micro-organisms can grow on surfaces such as medical devices and cause infections. Other examples of where bacteria can grow are on drains and pipes causing clogging, and on the hulls of ships, thus increasing drag. The goal of the current work is to investigate material coatings that resist the attachment and growth of bacteria on surfaces. We demonstrate that changing the roughness of the surface can reduce the number of bacteria found on the surface. More specifically, we have made surfaces covered with spheres that are approximately the same size as a bacterium, about 1 micrometer (10x smaller than the diameter of hair). We find that the spheres act as physical obstacles that block bacteria from moving on a surface. These results suggest that changing the micro-scale geometry of a surface may reduce the rate of infections on medical devices or hinder the growth of bacteria in other systems