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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
11

Characterization of the PilS-PilR two component regulatory system of Pseudomonas aeruginosa

Kilmury, Sara LN 11 1900 (has links)
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.
12

Pseudomonas aeruginosa minor pilins regulate virulence via modulation of FimS-AlgR activity

Marko, Victoria January 2017 (has links)
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.
13

The Type IV Pilus Assembly ATPase PilB as a Regulator of Biofilm Formation and an Antivirulence Target

Dye, Keane 02 June 2022 (has links)
Bacterial type IV pili (T4P) are filamentous surface appendages with a variety of functions including motility, surface attachment, and biofilm formation. In many species of bacteria a clear understanding of how the functions of T4P in lifestyle switching are regulated remains to be elucidated. Here, we focus on understanding the regulation of the T4P assembly ATPase PilB. We examined its interactions with the secondary messenger cyclic-di-GMP (cdG). Specifically we investigated how cdG binding regulates PilB functions not only as the assembly ATPase, but also as an EPS signaling molecule in Myxococcus xanthus biofilm regulation. Chapter 2 focuses on the development of a microplate-based biofilm assay for M. xanthus. This new assay allows for the analysis of the M. xanthus submerged biofilms under vegetative conditions in a high throughput format which has been absent in the published literature. M. xanthus biofilm formation tightly correlates with EPS production, suggesting that the assay can be used as a convenient method of examining EPS production. Chapter 3 examines the regulation of M. xanthus PilB (MxPilB) by cdG binding in vivo. We carried out a mutational analysis of the MshEN cdG binding domain in MxPilB. Mutations were created that either diverge with or converge from the MshEN consensus sequence. These two classes of MxPilB variants are expected to either decrease or increase cdG binding affinity, respectively. We examined the motility, EPS production, and piliation phenotypes of these mutants. Our results were consistent with a model where the function of MxPilB is altered in response to cdG binding, and suggesting that PilB responds to different thresholds of cdG concentration. In Chapter 4, we examine the ligand binding to the N-terminal cdG binding domain and C-terminal ATPase domain of Chloracidobacterium thermophilum PilB (CtPilB) in vitro. Our results confirm that these two domains bind to their respective ligands specifically, and demonstrate these domains communicate with each other in response to ligand binding. The results from all of the studies help us to establish a model where cdG binding fine tunes the functions of PilB to regulate the switch of bacteria between the motile and planktonic states. In addition to their roles in motility and biofilm formation, T4P are key virulence factors in many significant human pathogens. Antivirulence chemotherapeutics are considered to be a promising alternative to antibiotics, as they target disease processes rather than bacterial viability. Because PilB is essential for T4P biogenesis, we sought to identify PilB inhibitors for the development of antivirulence therapies. In Chapter 5, we describe the development of the first high throughput screen (HTS), for PilB inhibitors. This assay is uses the reduction of the binding of a fluorescent ATP analog to CtPilB in vitro, leading to the discovery of the plant flavonoid quercetin as a PilB inhibitor. Using M. xanthus as a model a bacterium, quercetin was found to inhibit T4P-dependent motility and T4P assembly in vivo. Builds on this initial success with CtPilB, Chapter 6 describes the development and implementation of a second HTS based on the inhibition of CtPilB as an ATPase. Screening a large chemical library led to the identification of benserazide and levodopa as CtPilB inhibitors. We show that both compounds inhibit T4P assembly in M. xanthus without any detrimental effects on bacterial growth. Furthermore we demonstrate that both levodopa and benserazide inhibit T4P-mediated motility in Acinetobacter nosocomialis, a human pathogen, providing the first evidence that the compounds identified with CtPilB can be effective against a pathogenic bacterium. Both of these studies validate the effectiveness not only of our HTSs, with of CtPilB as a model protein for the identification of PilB inhibitors. / Doctor of Philosophy / Bacteria can be motile or sessile. Motile bacteria can use hair like structures on their surface, called pili, to move in their natural environment, whereas sessile bacteria produce intricate structures attached to solid surfaces known as biofilms. Bacteria are able to switch between being motile and sessile states depending on their environment conditions. However, it isn't clear how this switch is controlled in bacteria that use pili to move. To answer this question, we studied how PilB the protein that assembles pili, might control this switching process. We specifically investigated PilB because it has two known roles. The first is that it can assemble pili, to enable pili-mediated motility. The second is that it can stimulate or promote biofilm formation. This places PilB at the intersection of these two lifestyles, suggesting that this protein may play a key role in deciding whether a bacterium is to be motile or sessile. Another important reason to understand how PilB functions is because pili are used by some antibiotic resistant pathogenic bacteria. Since PilB is essential for the formation of pili, if the actions of PilB could be blocked, bacteria would be unable to make pili. This could stop bacteria from causing disease. By searching for new chemicals which stop PilB from creating pili, we can potentially identify new drugs to treat bacterial infections.
14

Use of an Inducible Promoter to Characterize Type IV Pili Homologues in Clostridium perfringens

Hartman, Andrea H. 18 October 2012 (has links)
Researchers of <i>Clostridium perfringens</i>, a Gram-positive anaerobic pathogen, were lacking a tightlyregulated, inducible promoter system in their genetic toolbox. We constructed a lactose-inducible plasmid-based system utilizing the transcriptional regulator, BgaR. Using the <i>E. coli</i> reporter GusA, we characterized its induction in three different strains of <i>C. perfringens</i>. We then used a newly-developed mutation system to create in-frame deletion mutants in three genes with homology to Type IV pilins, and we used the promoter system described above to complement the mutants. We analyzed each pilin for localization and expression, as well as tested each of the mutants for various phenotypes frequently associated with type IV pili (TFP) and type II secretion systems. PilA2, PilA3, and PilA4 localized to the poles of the cells. PilA2 was expressed in the wildtype when <i>C. perfringens</i> was grown on agar plates, and the PilA3 mutant lacked a von Willebrand factor A domain-containing protein in its secretome. We used our promoter system to express GFP-tagged versions of the TFP ATPase homologues and view them in cells growing on surfaces. We saw that PilB1 and PilB2 co-localized nearly all of the time, while a portion of PilT was independent of the PilB proteins. PilT appeared necessary for the localization of PilB, and it localized independently of TFP proteins in <i>Bacillus subtilis</i>. PilT's typical localization in <i>Bacillus subtilis</i> was disrupted when the GTPase and polymerization activity of cell division protein FtsZ was blocked, suggesting that PilT associates with cell division proteins. / Master of Science
15

Characterization of Type IV Pilus System Genes and Their Regulation in Clostridium perfringens

Murray, Samantha Rose 06 June 2017 (has links)
Clostridium perfringens is a Gram-positive (Gr+) anaerobic pathogen that was found to contain Type IV pilus (T4P) system genes within the genomes of all its sequenced strains. T4P are widely used in Gram-negative organisms for aggregation, biofilm formation, adherence, and DNA uptake. Because few examples of T4P-utilizing Gram-positive bacteria are studied to date, we wanted to characterize the T4P system in this Gr+ bacterium. To understand the regulation of T4P genes and therefore better understand their expression, we employed the highly powerful next-generation sequencing tool RNA-seq in a variety of conditions. RNA-seq uncovered previously unknown regulatory mechanisms surrounding T4P genes as well as provided transcriptional information for most of the genes in the C. perfringens strain 13 genome. We also utilized reporter gene assays to look at post-transcriptional regulation of T4P promoters. The wealth of RNA-seq data acted as a jumping-off point for many smaller projects involving transcriptional regulators that may influence T4P expression. We investigated a novel small RNA in close proximity to the major T4P operon, as well as two little-characterized transcriptional regulators that function in the same conditions as T4P genes. RNA-seq also provided data to develop a method for protein purification from C. perfringens without induction. / Master of Science
16

Type IV Pili-Dependent Secretion of Biofilm Matrix Material Proteins in Clostridium perfringens

Kivimaki, Sarah Elise 21 January 2022 (has links)
Clostridium perfringens is a Gram-positive bacterium that secretes a biofilm matrix material. The goal of these experiments was to identify pilin mutants that are needed for secretion of the biofilm matrix and develop a functional model for a type II secretion system (T2SS) in C. perfringens. Protein tagging, western blot, and slot blot experiments were done to quantify protein secretion. After performing experiments using a CPE0515-FLAG construct, it was concluded from immunoblot densitometry data that, except for the pilA1 deletion mutant, none of the 18 tested pilin mutants had a statistically significant difference from the wild type (WT) with regard to protein secretion. From slot blot densitometry assays, it was concluded that the pilA1 and CPE2280 mutants showed statistically significant lower values than the WT but the pilA2 and CPE1841 mutants had values that were higher than the wild type. Testing the construct containing only CPE0514 and CPE0515-FLAG showed that CPE0516 and CPE0517 are not needed for secretion of the protein CPE0515. HA-tagged CPE0516 qualitative immunoblots showed that, unlike CPE0515, oligomerization of CPE0516 is not occurring, and that this protein likely forms a heat stable dimer. Overall, the data did not allow us to construct a T2SS model, since there were not enough proteins revealed to be involved to create a complete Type II secretion system. / Master of Science / The methods by which C. perfringens can persist and survive in environmental conditions is something that would be useful to learn more about. One of the methods that many bacteria use to survive is by creating a biofilm matrix material, which provides protection for the bacteria from environmental stresses. In this study, the goal was to determine which specific proteins are needed for the secretion of the biofilm matrix material. Using molecular biology techniques, the proteins thought to be involved in biofilm formation quantified. The results showed that while two proteins ultimately appeared to be needed for secretion, there were not enough proteins involved to create a complete model for a functional secretion system in C. perfringens.
17

Development of a novel genetic system for generation of markerless deletions in Clostridium difficile

Theophilou, Elena Stella January 2014 (has links)
C. difficile is an obligate anaerobic, Gram-positive, rodshaped and spore-forming bacterium. It is a well-recognised causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis. C. difficile has emerged as an important nosocomial pathogen in recent years, associated with considerable morbidity, mortality and economic burden. Despite its importance, functional genomic studies have been lagging behind in comparison to other enteric pathogens. This is attributed to the fact that C. difficile is difficult to manipulate genetically and the lack of robust, reproducible mutagenesis systems for many years. The ideal mutation for robust functional genomic studies is a markerless, in-frame deletion of the gene of interest. All systems developed for C. difficile, up to the start of this study, involve insertional inactivation of the gene of interest. This study describes the development of a novel genetic system for C. difficile, to create precise and markerless chromosomal deletions, using the meganuclease ISceI. For validation of the system, the addBA genes in C. difficile were deleted. The AddAB enzyme complex is important in the survival of many bacteria, since it maintains genome integrity, by the repair of double-strand breaks. Deletion of addBA in C. difficile did not significantly affect growth and viability, but the mutant strains were sensitive to DNA damaging agents. In addition, it was shown that C. difficile is capable of initiating the SOS response after DNA damage and that AddAB is not necessary for the induction of this response. The genetic system was further optimised to delete type IV pili (TFP)- associated genes, particularly pilT (CD3505) and pilA (CD3507), to investigate twitching motility. TFP are important in virulence and pathogenesis of many bacteria and twitching motility is often involved. TFP in C. difficile may be expressed in vivo during infection and may be involved in biofilm formation and colonization. To study potential TFP-mediated motility, a non-flagellated C. difficile strain was first constructed by deleting the fliC gene. The pilT gene, predicted to encode a protein involved in TFP retraction, was then deleted in the ΔfliC strain. A ΔpilT strain was also generated. Preliminary experimental work using these strains did not show any evidence for twitching motility and no difference between the ΔpilT strains and the parental strains. Examination of cells from the ΔfliC strain, under various conditions, did not reveal any pili, which indicates that TFP are regulated in C. difficile and that the TFP locus might be repressed at the transcriptional level. Preliminary work to investigate an intergenic region located upstream of the TFP locus in C. difficile, that might be involved in regulation, suggested that transcription is being initiated within a 500 bp region upstream of the CD3513 gene.
18

Identification of new virulence factors in Francisella tularensis

Forslund, Anna-Lena January 2010 (has links)
Francisella tularensis, the causative agent of tularemia, is a highly virulent bacterium with an infection dose of less than ten bacteria. The ability of a pathogen to cause infection relies on different virulence mechanisms, but in Francisella tularensis relatively few virulence factors are known. Two F. tularensis subspecies are virulent in humans; the highly virulent subspecies tularensis, also referred to as type A, and the less virulent subspecies holarctica, also called type B. The aim of this thesis has been to improve the knowledge regarding the ability of Francisella to cause disease, with the emphasis on surface located and membrane associated proteins and structures. In addition I have also investigated how virulence is regulated by studying the role of the small RNA chaperone, Hfq. The genome of Francisella appears to encode few regulatory genes. In my work I found that Hfq has an important role in regulation of virulence associated genes in Francisella. Similar to what has been found in other pathogens, Hfq functions in negative regulation, and this is the first time a negative regulation has been described for genes in the Francisella pathogenicity island. Another protein with a key role in virulence is a homologue to a disulphide oxidoreductase, DsbA, which was identified as an outer membrane lipoprotein in Francisella. A dsbA mutant was found to be severely attenuated for virulence and also induced protection against wild-type infections, thus making it a candidate for exploration as a new live vaccine. Additional genes with homology to known virulence determinants include a type IV pilin system. The pilin homologue, PilA, was identified to be required for full virulence in both type A and type B strains. In addition, genes involved in pili assembly and secretion, pilC and pilQ, were also found to be virulence associated in the type A strain. In summary, dsbA, hfq and type IV pili associated genes were indentified to be virulence determinants in F. tularensis. DsbA is a potential target for drug development and a dsbA mutant a candidate for a new live vaccine strain. Furthermore the identification of Hfq as a novel regulatory factor opens new insights into the virulence regulatory network in Francisella.
19

Characterization of PilP from the Type IV Pilus System of Pseudomonas aeruginosa

Tammam, Stephanie 16 December 2013 (has links)
Pathogenic bacteria employ a number of mechanisms to induce infection and survive in host tissues, including toxin secretion and the formation of protective multicellular structures called biofilms. Type IV Pili (T4P) are highly conserved organelles essential for both the establishment of infection and biofilm maturation. The goal of this research is to gain a molecular level understanding of the function of the highly dynamic T4P of Pseudomonas aeruginosa. The pilMNOPQ operon encodes 5 members of a transmembrane complex that facilitates pilus function. While PilQ is the putative outer membrane secretin through which the pilus exits the cell, the roles of the PilM/N/O/P proteins are less well defined. Using both in vivo and in vitro techniques our characterization of PilP has provided significant insight into organization of the apparatus. PilP is an inner membrane lipoprotein essential for T4P function, but lipidation is dispensable, suggesting that its interactions with other T4P components are sufficient for PilP function. We showed that PilN/O/P form a stable heterotrimer when expressed in E. coli, and we suggest that they form a similar subcomplex in P. aeruginosa. Additionally we were able to show that PilP is also able to interact with a periplasmic fragment of the outer membrane pore protein PilQ. Structural and bioinformatics studies suggest that the organization of PilN/O/P/Q complex is similar to that of the transenvelope complex of another important Gram-negative virulence factor – the Type II Secretion System (T2SS). Our structural and functional characterization of PilP, the PilN/O/P complex and the striking similarities between the T4P and T2S systems, as well as important differences that make each molecular machine unique, will be presented.
20

Characterization of PilP from the Type IV Pilus System of Pseudomonas aeruginosa

Tammam, Stephanie 16 December 2013 (has links)
Pathogenic bacteria employ a number of mechanisms to induce infection and survive in host tissues, including toxin secretion and the formation of protective multicellular structures called biofilms. Type IV Pili (T4P) are highly conserved organelles essential for both the establishment of infection and biofilm maturation. The goal of this research is to gain a molecular level understanding of the function of the highly dynamic T4P of Pseudomonas aeruginosa. The pilMNOPQ operon encodes 5 members of a transmembrane complex that facilitates pilus function. While PilQ is the putative outer membrane secretin through which the pilus exits the cell, the roles of the PilM/N/O/P proteins are less well defined. Using both in vivo and in vitro techniques our characterization of PilP has provided significant insight into organization of the apparatus. PilP is an inner membrane lipoprotein essential for T4P function, but lipidation is dispensable, suggesting that its interactions with other T4P components are sufficient for PilP function. We showed that PilN/O/P form a stable heterotrimer when expressed in E. coli, and we suggest that they form a similar subcomplex in P. aeruginosa. Additionally we were able to show that PilP is also able to interact with a periplasmic fragment of the outer membrane pore protein PilQ. Structural and bioinformatics studies suggest that the organization of PilN/O/P/Q complex is similar to that of the transenvelope complex of another important Gram-negative virulence factor – the Type II Secretion System (T2SS). Our structural and functional characterization of PilP, the PilN/O/P complex and the striking similarities between the T4P and T2S systems, as well as important differences that make each molecular machine unique, will be presented.

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