Archaeal protein trafficking is a poorly understood process that is only beginning to unfold. Integral to this process are the various signal peptidases. Two types of archaeal signal peptidases are identified thus far: signal peptidase I (SPI) and the preflagellin peptidase. SPI is responsible for processing the majority of secreted proteins. Sequence analysis of archaeal SPI enzymes indicates a Sec-11 type enzyme with two conserved Aspartic acid and a Histidine in place of the conserved Lysine in bacteria. Site directed mutagenesis and in vitro assays identified three conserved residues (Ser52, His122 and Asp148) critical for M. voltae SPI activity, distinguishing the archaeal enzyme from its bacterial and eukaryal counterparts. The archaeal preflagellin peptidase is a type IV prepilin peptidase-like enzyme, initially characterized for its essential role in preflagellin processing prior to flagellar filament assembly. Unusual substrates have been proposed for this enzyme, including preflagellins of peculiar signal peptide lengths in certain archaeal species, as well as sugar binding proteins with extremely short signal peptides in S. solfataricus. In this thesis, in vitro comparisons of the signal peptide length requirements for the two different preflagellin peptidases, FlaK in M. voltae and PibD in S. solfataricus, are presented. While a signal peptide length cut-off of 5 amino acids was found for FlaK below which preflagellins remained unprocessed, substrates with shorter (4 and 3 aa) signal peptides were recognized and properly cleaved by PibD, suggesting a diversification of the preflagellin peptidases among the archaeal species. The ability of FlaK and PibD to complement FlaK activity in an M. maripaludis flaK mutant was evaluated. M. maripaludis flaK is a markerless, stable mutant displaying pili as the sole cell surface appendage, providing the unique opportunity to closely study this structure without the interference of flagella. Here, purification and characterization of the archaeal pili is described for the first time in any archaeon. A putative pilus gene cluster with characteristics of type IV pilus genes was identified in M. maripaludis. In-frame deletion of the putative major pilin gene, MMP0237, resulted in a nonpiliated phenotype that could be restored by complementation, providing a direct link of this gene to piliation. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2007-09-28 12:59:16.801

Thesis (Ph.D)--University of Adelaide, Dept. of Molecular Sciences, 2001. / Corrigenda attached after the bibliography. Bibliography: leaves 166-206.

Bacteria have strain-dependent host attachment mechanisms. Bacteria with pili, also called fimbriae, attach to the host epithelial cells allowing the bacterium time to adhere, form biofilms, and infect the body without being flushed out by the host’s cleansing mechanisms. Haemophilus influenzae type B (Hib) are bacteria that colonize the human nasopharynx by attaching via adhesion pili to the microvilli found in the respiratory tract. To gain insight into the attachment mechanism of Hib bacteria, pilus expression and purification were optimized. To determine the structure of Hib pili, cryogenic electron microscopy (cryo-EM) images were collected of the isolated and purified filaments. Reconstructions were computed and the secondary structure of the Hib subunit (HifA) was predicted and fitted into the reconstruction. The structure of the Hib filament showed the pili to have one-start helical symmetry, with a zigzag shape. The predicted structure of HifA had two regions of interaction with the adjacent subunit, including an N-terminal extension (NTE) and a loop, both extending from the immunoglobulin (Ig)-like domain. These extensive contacts resulted in a stronger interaction between the subunits compared to other types of adhesion pili. Toward the goal of inhibiting pili adherence to the host cells, the effects of salivary peptides were tested. Peptide binding assays were performed and analyzed to determine changes in structure or macromolecular assembly of Hib pili. Our discovery of the presence and absence of pilus three-dimensional bundling and two-dimensional networks can aid in the future prevention of bacterial colonization and infection within the human body thereby preventing Hib related diseases.

Mycobacterium tuberculosis is responsible for nearly three million human deaths every year. Understanding the mechanisms and bacterial factors responsible for M. tuberculosis' ability to cause disease in humans is critical for the development of improved treatment strategies. Using negative staining and transmission electron microscopy it was discovered that mycobacteria, including the human pathogen M. tuberculosis, produce fine surface structures known as pili. Mass spectroscopy analysis demonstrated that purified pili from M. tuberculosis are comprised of protein subunits encoded by the predicted M. tuberculosis H37Rv ORF designated Rv3312A. These pili termed M. tuberculosis pili, Mtp, are highly aggregative 2-5 nm diameter fibers and are recognized by IgG antibodies contained within TB patient sera. These results indicate that Mtp are produced during human infection. Mtp bind to the extracellualr matrix protein laminin in vitro suggesting that Mtp are a newly identified adherence factor for M. tuberculosis.A second pili morphotype that appeared as rope-like bundles were observed for M. tuberculosis and it was found that the M. tuberculosis chromosome contains a type IVB pili gene cluster. The M. tuberculosis type IV pili belong to the Flp sub-family of type IVB pili. RT-PCR analysis reveals that flp is expressed by M. tuberculosis and IF microscopy with Flp-specific antibodies shows the Flp protein is secreted from the bacteria. Evidence presented herein also demonstrates that an Flp-derived peptide is capable of polymerizing into pili-like fibers in vitro over a pH range of 4.5-7.5. Further studies show that the M. tuberculosis type IV pili are encoded by a novel 5-kb genomic island that contains the flp prepilin and putative biogenesis genes. The flp genomic island is characterized by an increased G+C content of 70% (the mean G+C content of the M. tuberculosis chromosome is 65%) and is flanked by multiple direct repeats. The identification of type IV pili in M. tuberculosis is the first report of any classical virulence factor for the bacillus and the genetic characteristics of the locus strongly suggest this chromosomal region was horizontally acquired.

Corrigenda attached after the bibliography. Bibliography: leaves 166-206.

Corrigenda attached after the bibliography. / Bibliography: leaves 166-206. / 206 leaves [6] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Sciences, 2001

Zugl.: Berlin, Humboldt-Univ., Diss., 2009

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 122-143).

Thesis (Ph.D.)--Duquesne University, 2006. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p.157-190) and index.

Type IV pili (T4P) are hair-like adhesins involved in many processes, including surface attachment, twitching, DNA uptake, electron transfer, and pathogenesis. These flexible filaments are expressed in various pathogens, including the opportunistic pathogen Pseudomonas aeruginosa. The pilus fibre is primarily composed of the major pilin structural subunit, PilA, which is rapidly polymerized or depolymerized during pilus extension or retraction, respectively. The transcription of pilA is tightly controlled by the PilS-PilR two-component system, which responds to fluctuating levels of PilA in the inner membrane. In addition to pilA, the response regulator, PilR, also regulates a subset of other non-T4P related genes. Here, we used hyperactivating point mutants in the PilS-PilR two-component system, which induce hyperpiliation without loss of pilus function, to assess the effects of increased surface pili expression on virulence against Caenorhabditis elegans, and to identify additional non-T4P genes regulated by the PilS-PilR two-component system. We hypothesized that dysregulation of the PilS-PilR two-component system impacts the expression of pilA and other genes, which impacts both surface piliation and T4P dynamics, resulting in altered P. aeruginosa virulence. C. elegans slow killing assays revealed that hyperpiliation, independently of T4P function, reduces virulence of model P. aeruginosa strains PAK and PA14. We propose a model whereby a surfeit of pili reduces virulence, potentially through impeding effective engagement of contact-dependent antagonism systems, such as the type III secretion system. Transcriptomic analysis of the hyperactive PilR point mutant also identified a subset of 26 genes, including those related to phenazine biosynthesis, quorum sensing, and ethanol oxidation, regulated by the PilS-PilR two-component system. Last, a T4P cysteine-labelling system was implemented for P. aeruginosa, allowing for the visualization of real-time pilus dynamics. Together, this work provides new insights into the consequences of hyperpiliation and the scope of the PilS-PilR signalling network, as well as novel tools for investigating P. aeruginosa T4P dynamics in vivo. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a major contributor to hospital-acquired infections and is of particular concern due to its intrinsic resistance to many frontline antibiotics. To aid in infection, Pseudomonas encodes an arsenal of virulence factors, including type IV pili (T4P), hair-like adhesins involved in many processes, such as twitching motility and surface attachment. T4P are primarily composed of the major pilin, PilA, whose expression is tightly regulated by the PilS-PilR two-component system. The sensor kinase, PilS, monitors the inner membrane PilA inventory and modifies activity of the response regulator, PilR, to regulate pilA transcription. Here, we demonstrate that P. aeruginosa virulence in a roundworm infection model is reduced when the amount of T4P expressed at the cell surface increases, regardless of the ability of the bacteria to twitch. We propose that inappropriate increases in surface T4P expression may impair pathogenicity-associated systems which require intimate host-cell contact. New genes in the regulon of the PilS-PilR two-component system were also identified. A tool to fluorescently label and image T4P in real-time using microscopy was established in the lab. This work highlights the consequences of increased surface T4P expression, providing potential new targets for antipseudomonal therapeutics which act on components involved in T4P expression and function.