by Paula A. Livingston. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web. / Two protein tyrosine phosphatases, dual specificity phosphatase PVP and low molecular weight phosphatase WZB were purified and characterized. PVP was expressed as inclusion bodies and a suitable purification and refolding method was devised. Enzyme kinetics revealed that p-nitrophenylphosphate and (Sb(B-naphthyl phosphate were substrates with KM of 4.0mM and 8.1mM respectively. PVP showed no reactivity towards phosphoserine. Kinetic characterization of WZB showed that only pnitrophenylphosphate was a substrate with no affinity for Ç-naphthyl phosphate and phosphoserine. Optimal conditions for activity with PNPP were found at a pH of 5 with a KM of 1.1mM, kcat of 35.4s-1 and kcat/KM of 32.2s-1mM-1. Inhibition studies showed that phosphate, fluoride, and molybdate were competitive inhibitors with Ki of 3.2mM, 71.7mM, and 50.4(So(BM respectively and hydrogen peroxide abolished activity. Active site mutants of WZB Cys9Ser and Asp115Asn showed no activity.

In summary, the present findings have demonstrated the important roles of CFTR in prostatitis and cancer development, which may provide new insight into the understanding of the prostate in health and disease. The present findings may also have potential application in diagnosis and prognosis of cancer. / In the first part of the study, the possible role and a bacterial killing mechanism involving CFTR-mediated bicarbonate secretion in prostatitis were investigated in a rat prostate model. CFTR was found to be expressed in the epithelium of rat ventral prostate. Experiments using cultured rat primary prostate epithelial cells demonstrated that CFTR was involved in mediating bicarbonate extrusion across the prostate epithelium. The expression of CFTR and carbonic anhydrase II (CAII), a key enzyme involved in cellular HCO 3- production, along with several pro-inflammatory cytokines including IL-6, IL-1beta, TNF-alpha, was significantly up-regulated in the primary culture of rat prostate epithelial cells upon E.coli-LPS challenge. Inhibition of CFTR function in vitro or in vivo resulted in reduced bacterial killing by prostate epithelial cells or the prostate. High HCO3- content (>50mM), rather than alkaline pH, was found to be responsible for bacterial killing. The direct action of HCO 3- on bacterial killing was confirmed by its ability to suppress bacterial initiation factors in E coli. The relevance of the CFTR-mediated HCO3- secretion in human was demonstrated by the upregulated expression of CFTR and CAII in human prostatitis tissues. The present results have demonstrated that CFTR plays a previously undefined role in prostatitis and could be up-regulated during the inflammation in prostate as a host defense mechanism to increase bicarbonate secretion for bacterial killing. / In the second part of the study, the possible role of CFTR in prostate cancer development and the underlying mechanisms were investigated. Our results showed that the expression of CFTR and CAII in prostate was remarkably decreased in aged rat prostate. We observed that testosterone could up-regulate the expression of CFTR and CAII in vitro and in vivo , indicating that the declined male hormones during aging may be responsible for the observed age-dependent expression of CFTR. In the present study, we found that inhibition of CFTR enhanced cell proliferation/anti-apoptosis in the prostate primary epithelial cells. CFTR was detected in all examined prostate cell lines, but with relatively higher expression levels in immortalized cell lines (PZ-HPV-7, PNT1A, PNT2C2) than in cancer cell lines (PC-3, DU-145, LNCaP). Immunohistological studies showed that the expression of CFTR was dramatically reduced in prostate cancer specimens as compared to that in normal prostate tissues. Furthermore, our gain and loss of function studies showed that knockdown of CFTR profoundly enhanced cell proliferation, cell adhesion, invasion and migration, while inhibited apoptosis in prostate cancer cell lines, overexpression of CFTR dramatically suppressed tumorigenic phenotype of cancer cells. Soft agar anchorage-independent growth assay showed that knockdown of CFTR in prostate cancer cells increased the number of colonies formed in soft agar. More importantly, we demonstrated that CFTR knockdown promoted the tumor growth in vivo and forced overexpression of CFTR in prostate cancer cells and ultrasound-mediated gene transfer of CFTR inhibited xenograft tumor growth in vivo. Mechanistically, multiple mechanisms were identified to contribute to the CFTR- mediated tumor suppressive effects. Firstly, CFTR chloride channel function was implicated in the regulation of apoptosis in prostate cancer cells. Secondly, CFTR up-regulated the transcription level of miR-34a and miR-193b, both of which have been indicated as tumor suppressors in multiple cancers. Thirdly, 11 cancer-related genes were found to be up- or down-regulated by CFTR using PCR-array. These data demonstrated that CFTR may play an important role in prostate cancer development by acting as a tumor suppressor. / The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel conducting both Cl- and HCO3 -. It is expressed in epithelial cells of a wide variety of tissues. CFTR is also known to be expressed in human prostate; however, the physiological role of CFTR in the prostate and related diseases remains largely unknown. This thesis explored the biological roles of CFTR in prostatitis and cancer development. / Xie, Chen. / Adviser: Chan LiShaw Chang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 175-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Type-A Î3-amino-butyric acid receptors (GABA_ARs) are pentameric ligand-gated ion channels (pLGICs), which mediate the majority of fast inhibitory neurotransmission in the animal central nervous system. Their dysfunction is related to numerous conditions including epilepsy, insomnia, anxiety, panic disorders, depression and schizophrenia. GABA_ARs are therefore major targets of clinically important drugs, including benzodiazepines and the intravenous general anaesthetics etomidate and propofol, as well as endogenous modulators, for example neurosteroids. Despite recent progress in structural biology of pLGICs, GABA_AR structures remain notoriously elusive. Structural information available at the beginning of this project was limited to the benzamidine-bound homopentameric GABA_AR-Î23, in a desensitised conformation. A large number of fundamental questions, including the molecular architecture of physiological, heteromeric GABA_ARs, their signalling mechanisms, the binding and action modes of their numerous ligands, remained to be answered. During this DPhil project, I employed structural biology techniques (X-ray crystallography and single particle cryo-electron microscopy) to further the molecular understanding of human GABAARs. I used subunit-specific llama nanobodies to aid crystallization of homomeric GABA_A-β3 receptors, which led to a 3.16 Å structure in complex with the general anaesthetic etomidate. This structure elucidates the binding mode of the etomidate, the basis for its subunit selectivity and illustrates conformational changes it triggers. I then used cryo-electron microscopy to determine the first structure of a heteromeric GABA_AR, the human α1b3g2, bound to an activating llama nanobody at a medium (5.2 Å) resolution. The numerous other insights obtained range from unambiguously establishing the subunit arrangement and stoichiometry, to proposing a mechanism for receptor assembly and discovering an unexpected role played by N-linked glycans in this process. The work described here opens multiple avenues for future research. Immediate opportunities include high resolution structural characterization of heteromeric GABA_ARs, via cryo-electron microscopy, further development of nanobodies as novel, high affinity and subunit specific tools to modulate GABA-ergic signalling, and structural characterization of numerous small-molecule modulators, of clinical and physiological relevance, bound to human GABA_ARs.

Ion channels are membrane proteins that facilitate electrical signaling in important physiological processes, such as the rhythmic contraction of the heart. KCNQ1 is the pore-forming subunit of a voltage-gated potassium channel that assembles with the β-subunit KCNE1 in the heart to generate the IKs current, which is critical to cardiac action potential repolarization and electrical conduction in the heart. Mutations in IKs subunits can cause potentially lethal arrhythmia, including long QT syndrome, short QT syndrome, and atrial fibrillation. Each channel consists of four voltage-sensing domains and a central pore through which ions permeate. Voltage-dependent gating occurs when movement of voltage sensors cause pore opening/closing through coupling mechanisms. Although KCNQ1 by itself is able to form a voltage-dependent potassium channel, its assembly with KCNE1 is essential to generating the physiologically critical cardiac IKs current, characterized by a delay in the onset of activation, an increase in current amplitude, and a depolarizing shift in the current-voltage relationship. KCNE1 is thought to have multiple points of contact with KCNQ1 that reside within both the voltage-sensing domain and the pore domain, allowing for extensive modulation of channel function. Atrial fibrillation is the most common cardiac arrhythmia and affects more than 3 million adults in the United States. Much rarer, genetic forms of atrial fibrillation have been associated with gain-of-function mutations in KCNQ1, such as two adjacent mutations, S140G and V141M. Both mutations drastically slow channel deactivation, which underlies their pathophysiology. Deactivation slowing causes accumulation of open channels in the context of repeated stimulation, which abnormally increases the repolarizing K+ current, excessively shortens the action potential duration, and predisposes to re-entry arrhythmia such as atrial fibrillation. Although both mutations are located in the voltage-sensing domain, their mechanisms of action remain unknown. Understanding the gating mechanisms underlying deactivation slowing may provide key insights for the development of mechanism-based pharmacologic therapies for arrhythmias associated with KCNQ1 mutations. In addition to gain-of-function mutations, molecular activators of KCNQ1 can slow deactivation and increase channel activity. An existing problem in the pharmacologic treatment of arrhythmia is that many antiarrhythmic drugs do not have specific targets and cause undesired side effects such as additional arrhythmia. Thus, developing mechanism-based therapies may optimize clinical treatment for patients with specific forms of channel dysfunction. Two KCNQ1 activators, ML277 and R-L3, have been previously shown to slow current deactivation, but the underlying gating mechanisms remain known. Although these modulators are unlikely to serve directly as antiarrhythmic therapy, investigating their mechanisms will likely provide fundamental insights on channel modulation and guide future efforts to develop personalized therapies for arrhythmia, such as congenital long QT syndrome. Given the central importance of deactivation slowing in both pathophysiology and pharmacology, we focused on investigating gating mechanisms that underlie deactivation slowing. To this end, we utilized voltage clamp fluorometry, a technique that simultaneously assays for voltage sensor movement and ionic current through the channel pore. In Chapter 1, we begin our study by examining the gating mechanisms of KCNQ1 atrial fibrillation mutations in the absence of KCNE1. We show that S140G slows voltage sensor deactivation, which indirectly slows current deactivation. On the other hand, V141M neither slows voltage sensor nor current deactivation. This is followed by Chapter 2, where we examine the gating mechanisms underlying deactivation slowing by atrial fibrillation mutations in the presence of KCNE1. We show that both S140G and V141M slow IKs deactivation by slowing pore closing and altering voltage sensor-pore coupling. Based on these findings, we proposed a molecular mechanism in which both mutations disrupt the orientation of KCNE1 relative to KCNQ1 and thus impede pore closing, implying that future efforts to modulate KCNQ1 function can benefit from targeting the β-subunit. Finally, in Chapter 3, we explore the gating mechanisms underlying deactivation slowing for two small-molecule activators of KCNQ1. We show that ML277 predominantly slows pore transitions, whereas R-L3 slows voltage sensor deactivation, which indirectly slows current deactivation. Taken together, these studies guide future efforts to develop mechanism-based therapies for arrhythmia.

Single-particle cryogenic electron microscopy (cryo-EM) has become a powerful mainstay tool in high resolution structural biology thanks to advances in hardware, software and sample preparation technology. In my thesis, I utilized this technique to unravel the function of various challenging biological macromolecules. My first focus was bacterial ribosomal biogenesis: understanding how bacteria assemble their ribosomes. Ribosomes are the factories of the cell, responsible for manufacturing all proteins. Ribosomes themselves are huge, with the bacterial version made of 52 proteins and 4566 RNA nucleotides. How these components assemble has long been a mystery. Early groundbreaking work sketched out a biogenesis pathway using purified components in vitro – but under non-physiological conditions. We sought to understand how the bacterial ribosome – specifically the large subunit 50S – is built inside the cell. To achieve this, we engineered a conditional knock-out bacterial strain that lacked one specific ribosomal protein (L17). This caused the cells to accumulate incomplete intermediates along the 50S biogenesis pathway. These intermediates were purified and examined with mass spectrometry and single-particle cryo-EM. Two major hurdles arose in this project: firstly, the biogenesis intermediates exhibited a preferred orientation when vitrified for cryo-EM analysis. This means that instead of showing many different views required for reconstruction of the 3D structure, the intermediates only adopted one view on the cryo-EM grid. To overcome this problem, we engineered a method to induce additional views on the microscope by tilting the stage. Using another test protein that also exhibited preferred orientation (hemagglutinin), we optimized and characterized this new tilt methodology and showed it was generally applicable to overcoming preferred orientation, regardless of type of specimen. We also created a software tool, called 3DFSC (3dfsc.salk.edu), for other microscopists to calculate the degree of directional anisotropy in their structures due to preferred orientation. Using this tilt strategy finally enabled the structural elucidation of our 50S intermediates. The second challenge in the project was the large amount of heterogeneity present in the sample. Through hierarchical 3D classification schemes using the latest software tools, we obtained 14 different 50S intermediate structures, all from imaging a single cryo-EM grid. By analyzing the missing components of each intermediate, and corroborating these observations with mass spectrometry data, we outlined the first in vivo 50S assembly pathway, and showed that ribosome assembly occurs step-wise and in parallel pathways. My second focus was on pushing the resolution limits of single-particle cryo-EM using adeno-associated virus (AAV) serotype 2 homogeneous virus-like particles (VLPs) that lack DNA. Exploiting several technical advances to improve resolution, including use of gold grids, per-particle CTF refinement, and correction for Ewald sphere curvature, we managed to obtain a 1.86 Å resolution reconstruction of the AAV2L336C variant VLP, the highest resolution icosahedral virus reconstruction solved by single-particle cryo-EM to date. Using our structure, we were able to show improvements using Ewald sphere curvature correction and shed light on the mechanistic basis as to why the L336C mutation resulted in defects in genome packaging and infectivity compared to the WT viral particles. My third focus was the understanding of small membrane proteins involved in infectious diseases. Membrane proteins are a challenge to work with due to the need for them to be extracted from the lipid bilayer for studies as compared to soluble proteins. Infectious diseases have a huge burden on society, with the top three infectious agents accounting for 2.7 million deaths in 2016. The third most deadly infectious disease is malaria, a mosquito-borne parasite which kills 450,000 people annually. One drug used early on for treating malaria was chloroquine but its usefulness waned due to development of resistance. Chloroquine resistance is mediated by the chloroquine resistance transporter (PfCRT). Although small (49 kDa) for single-particle cryo-EM, we solved its structure by using fragment antibody technology to add mass and help with image alignment and 3D reconstruction. The 3.2 Å structure resembles other drug metabolite transporters, and the chloroquine resistance mutations map to a ring around the central cavity, suggesting this central pore as the drug binding site. Tuberculosis (TB) is the top killer, above malaria and HIV/AIDS, being responsible for 1.3 million deaths. In TB, a common antibiotic target is the bacterium’s cell wall synthesis machinery. One family of such enzymes is the arabinosyltransferases, which synthesize the critical arabinose sugars. Using single-particle cryo-EM, we solved two high resolution structures of one such essential enzyme, AftD. Due to the low yield of the protein, a picoliter automated sample dispensing robot was crucial to allow for initial cryo-EM analysis. We then performed mutagenesis studies in M. smegmatis, a TB model organism, which uncovered the critical amino acid residues in the active site and determined that a bound acyl-carrier-protein was likely involved in allosteric inhibition of AftD’s active site. Another member of the family, EmbB, is the target of a widely used frontline TB drug called ethambutol. We have solved the high resolution structures of the apo and putative drug-bound states of EmbB, allowing us to map out, for the first time, both the active site and drug-resistance mutations of this crucial enzyme. The atomic structures of the functional pockets of Mycobacterial AftD and malarial PfCRT will hopefully enable structure-based drug design to improve existing drugs or potentially even develop new treatments against these infectious maladies. In conclusion, the continual and breathtaking improvements in single-particle cryo-EM methodology has been instrumental in allowing the elucidation of the aforementioned biological macromolecules from ribosome biogenesis intermediates, to AAV2 vehicle, Plasmodium drug resistance transporter to mycobacterial glycosyltransferases – structures of which help explain biological function.

Uroplakin 1B (UPKIB) is an integral structural protein interacting with uroplakins 1A, 2 and 3 to form hexameric plaques along the bladder lumen in the asymmetric unit membrane of urothelial umbrella cells in humans and other mammals. UPKIB mRNA expression is deregulated in transitional cell carcinomas (TCCs), however the mechanisms of regulation of UPKIB have not been established. Using genome databases, a Xenopus UPKIB homologue was identified. Maximum Parsimony and BAMBE (Bayesian Analysis in Molecular Biology and Evolution) data support a close evolutionary relationship between mammalian and amphibian UPKIB mRNA. Using Unigene, UPKIB human expressed sequence tags were identified in tissues including brain, skeletal muscle and liver, suggesting the relatively widespread distribution of this membrane protein. The UPKIB genomic structure was also deduced using genome databases. Contig AC083800, identified in a high throughput genomic sequence database, spanned UPKIB and 9 exons and 8 introns were defined. A 67bp 5' untranslated region was identified using 5' rapid amplification of cDNA ends. This product was sequenced and a putative UPKIB promoter and transcription start site was deduced. Contig AC083800 spanned the transcription start site and putative promoter. Transcription factor binding motif prediction programs detected no TATA box, but did predict a CCAAT box and several binding motifs including 4 Sp-1 sites and a NFKB site. A weak CpG island was identified within a 0.5kb region including the putative promoter, exon 1 and intron 1, which was 54% GC rich with CpG:GpC ratio of 0.46, containing 15 CpG dinucleotides. Seven TCC cell lines and five peripheral blood lymphocyte samples were analysed for UPKIB expression using RT-PCR and two cell lines expressed UPKIB transcripts. Eleven CpG sites in the putative promoter were investigated for methylation using bisulfite modification analysis in normal PBL, TCC cell lines and patient TCC samples. An inverse correlation was established in TCC cell lines between UPKIB mRNA expression and degree of methylation. 5-Aza-2'deoxycytidine induced UPKIB mRNA expression in T24 cells, previously observed not to express UPKIB. Sequence analysis of patient samples revealed more complex CpG methylation patterns, reflecting tumour heterogeneity. In summary, the uroplakin 1B gene has been characterised and one mechanism of regulation of gene expression involves methylation. / Thesis (Ph.D.)--Dept of Surgery, 2003.

Genomic analysis is a new approach for the characterization and investigation of novel genes, gene clusters, the function of uncharacterized proteins, and genetic diversity in microorganisms. These approaches are important for the study of chlamydiae, a system in which several genomes have been sequenced but in which techniques for genetic manipulation are not available. The objective of this thesis is to combine computer-based analysis of chiamydial inclusion membrane proteins (Incs) with cellular and molecular biological analysis of the bacteria. Three different experimental lines of investigation were examined, focusing on Incs of C. trachomatis and C. pneumoniae. Chlamydiae are obligate intracellular bacteria that develop within a nonacidified membrane bound vacuole termed an inclusion. Putative Inc proteins of C. trachomatis and C. pneumoniae were identified from genomic analysis and a unique structural motif. Selected putative Inc proteins are shown to localize to the inclusion membrane. Chiamydia trachomatis variants with unusual multiple-lobed, nonfusogenic, inclusion were identified from a large scale serotyping study. Fluorescence microscopy showed that IncA, a chiamydial protein localized to the inclusion membrane, was undetectable on non-fusogenic inclusions of these variants. Sequence analysis of incA from non-fusogenic variant isolates revealed a defective incA in most of the variants. Some variants lack not only IncA on the inclusion membrane but also CT223p, an additional Inc protein. However, no correlation between the absence of CT223p and distinctive inclusion phenotype was identified. Nucleotide sequence analysis revealed sequence variations of C. trachomatis incA and CT223 in some variant and wild type isolates. Comparative analyses of the three recently published C. pneumoniae genomes have led to the identification of a novel gene cluster named the CPn1O54 gene family. Each member of this family encodes a polypeptide with a hydrophobic domain characteristic of proteins localized to the inclusion membrane. These studies provided evidence that gene variation might occur within this single collection of paralogous genes. Collectively, the variability within this gene family may modulate either phase or antigenic variation, and subsequent physiologic diversity, within a C. pneumoniae population. These studies demonstrate the genetic diversity of Inc proteins and candidate Inc proteins, within and among the different chiamydial species. This work sets the stage for further investigations of the structure and function of this set of proteins that are likely critical to chlamydial intracellular growth. / Graduation date: 2003

Membrane proteins are vital components in the cell and crucial for the proliferation of all living organisms. Unfortunately our collective knowledge of structures of membrane proteins is very limited, as compared to the information available on soluble proteins. This is to a large extent due to the outstanding challenge of working with membrane proteins and the relatively high cost associated with determining a membrane protein structure.  Therefore, the establishment of efficient methods and means for the production and crystallization of membrane proteins is urgently needed. The two methods explored in this thesis  are aimed to achieve rapid optimization of expression and purification conditions of membrane proteins, thereby allowing for the rapid production of more suitable samples for crystallization trials. Despite the challenges in membrane protein structure determination two structures are presented in the thesis: The first structure determined is of the CorA magnesium transporter from Thermotoga maritima will be the focus of this thesis. The CorA revealed a pentameric protein in a closed state. The presence of two regulatory metal binding sites is suggested, as well as a putative magnesium ion bound in the ion conductive pathway. The second structure is of the human enzyme LTC4-synthase, which catalyzes the pivotal step in eicosanoid synthesis by the conjugation of glutathione to LTA4, a reactive epoxide-containing derivative from arachidonic acid. The products of this step, the so-called cysteinyl leukotrienes are potent inflammatory mediators making this enzyme a potential drug target. The structure reveals a charged binding pocket for a horseshoe-shaped glutathione, and a hydrophobic binding pocket for a lipophilic LTA4 molecule. Based on the structure a key residue for catalysis has been identified, Arg 104, which is proposed to play a critical role in activating the thiol group of glutathione for the nucleophilic attack on LTA4.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a Gram-negative coccobacillus of the Pasteurellaceae family. It is implicated in periodontitis, a common low-grade bacterial infection, but it can also cause non-oral infections. The main aim of this project was to identify and characterize in A. actinomycetemcomitans novel cell surface components bearing virulence potential that could contribute to systemic immunoinflammatory burden. We first established and evaluated a method for preparing homogeneous cell suspensions of autoaggregating clinical isolates of A. actinomycetemcomitans. The chosen method is based on a gradual dispersion of bacterial colonies into solution, which generated homogeneous suspensions without losing cell viability or fimbriation. When sera from two patients with A. actinomycetemcomitans-associated infections were used to probe A. actinomycetemcomitans outer membrane protein (OMP) preparations in western blot, strong reactions were found at 17 kDa. Interestingly, antiserum against CsgA, a major subunit of Eschirichia coli curli, also reacted with A. actinomycetemcomitans OMP preparations at 17 kDa size, that is the size of E. coli CsgA, suggesting antigenic crossreactivity. The 17 kDa A. actinomycetemcomitans OMP was subsequently identified as peptidoglycan-associated lipoprotein (PAL; AaPAL) by using immunoproteomics methods. Studies on the pal gene and its gene product showed that they were conserved among the clinical A. actinomycetemcomitans isolates representing all currently known serotypes. AaPAL expression was shown under different nutritional and atmospheric conditions that resembled those in periodontal pockets. PAL deficiency in turn led to pleiotropic effects on the phenotype of A. actinomycetemcomitans, such as cell elongation and decreased growth rate. To purify AaPAL we employed affinity chromatography using anti-AaPAL peptide antibodies. The extensive characterization of the purified AaPAL by SDS-PAGE gel staining and mass spectrometry demonstrated that the final purification product did not contain other bacterial proteins than AaPAL. The protein had not lost its antigenicity during purification, since it was recognized by sera from patients with A. actinomycetemcomitans-associated oral and nonoral infections. AaPAL also appeared to be a strongly immunoreactive antigen in patients with periodontitis whose serum IgG antibodies recognized in western blot a 17 kDa OMP in the parental strain but not in the pal-deficient mutant. In addition to its immunogenicity, AaPAL also induced proinflammatory cytokine and chemokine response from human whole blood as determined by a cytokine antibody array. A cell culture insert model was designed to study how bacterial components could be introduced to the host in infections. The experiments demonstrated that live bacteria released extracellularly free-soluble AaPAL, but also other components, via an unknown outer membrane vesicle-independent mechanism. The immunogenicity and proinflammatory potential of the previously uncharacterized outer membrane lipoprotein of A. actinomycetemcomitans, AaPAL, suggests that it contributes to the pathogenicity of this bacterium. That live A. actinomycetemcomitans cells released free-soluble cell components may represent a new pathogenic mechanism.

A vast number of studies have been completed on the virulence determinants of Yersinia spp.; however, the focus of many of these studies has been on the virulence plasmid and the plasmid-encoded Type three secretion system. Nevertheless, many chromosomal genes whose products are directly involved in virulence have also been identified. Some of these critical virulence determinants are outer membrane proteins. Outer membrane proteins of Gram-negative bacteria often have important physiological roles; however, some have also been found to be important for pathogenesis. In this thesis, we investigated two Yersinia. pestis outer membrane proteins, Ail and OmpA, and their roles in virulence. We provide evidence that Y. pestis Ail is a highly expressed outer membrane protein that is absolutely essential for Y. pestis to resist the killing action of the complement system present in human blood and tissues, as well as the blood and tissues of other mammalian hosts. Furthermore, Ail was important for virulence in a Y. pestis-Canorhabditis elegans model of infection.The work in this thesis also provided the first evidence that another surface-exposed outer membrane protein, termed OmpA, is required for both Yersinia pseudotuberculosis and Y. pestis to survive and proliferate intracellularly in macrophages. Finally, we provide evidence that Y. pestis has a functional small RNA MicA that controls the expression of OmpA. This is the first demonstration of sRNA-mediated regulation of a Yersinia virulence factor. This work has paved the way for future studies on the role of outer membrane proteins in virulence, particularly the role of Ail and OmpA.