Periodontitis, the primary cause of tooth-loss worldwide, is a bacterially induced chronic inflammatory disease of the periodontium. It is associated with systemic conditions such as cardiovascular disease (CVD). However, pathogenic mechanisms of periodontitis-associated bacteria that may contribute to the CVD association are unclear. The aim of this doctoral thesis project was to characterize bacterial mechanisms that can originate from the periodontal pocket and expose the host to multiple effector proteins, thereby potentially contributing to periodontal tissue degradation and systemic stimulation. As our main model, we have used Aggregatibacter actinomycetemcomitans, a Gram-negative species associated with aggressive forms of periodontitis, and with non-oral infections, such as endocarditis. Since Gram-positive species might be more common in periodontitis than previously believed, we have also investigated mechanisms of the multipotent bacterium, Staphylococcus aureus. Using an ex vivo insert model we showed that free-soluble surface material, released during growth by A. actinomycetemcomitans independently of outer membrane vesicles (OMVs), enhanced the expression of several proinflammatory cytokines in human whole blood. A clear LPS-independent effect suggested the involvement of effector proteins in this cytokine stimulation. This was supported by MALDI-TOF-MS and immunoblotting, which confirmed the release of GroEL and peptidoglycan-associated lipoprotein (PAL), in free-soluble form. We next demonstrated that A. actinomycetemcomitans OMVs could deliver multiple proteins including biologically active cytolethal distending toxin (CDT), a major virulence factor, into human gingival fibroblasts and HeLa cells. Using confocal microscopy, the active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. By using a fluorescent probe, B-R18, it was shown that the OMVs fused with lipid rafts in the plasma membrane. These findings suggest that OMVs can deliver biologically active virulence factors such as CDT into susceptible cells of the periodontium. Using A. actinomycetemcomitans vesicles labeled with the lipophilic dye, PKH26, it was shown that the OMVs can be internalized into the perinuclear region of human cells in a cholesterol-dependent manner. Co-localization analysis supported that the internalized OMVs carried A. actinomycetemcomitans antigens. Inhibition assays suggested that although OMV internalization appeared to have a major role in effector protein delivery, additional interactions such as vesicle membrane fusion may also contribute. The OMVs strongly induced activation of the cytosolic pathogen recognition receptors NOD1 and NOD2 in HEK293T-cells, consistent with a role in triggering innate immunity by carrying PAMPs such as peptidoglycan into host cells. Membrane vesicles (MVs) from S. aureus were found to carry biologically active alpha-toxin, a key virulence factor, which was delivered to host cells and required for full cytotoxicity of the vesicles. Confocal microscopy analysis revealed that these MVs, similar to A. actinomycetemcomitans OMVs, interacted with HeLa cells via membrane fusion. Thus, as S. aureus is frequently found in individuals with aggressive periodontitis, MV production could have potential to contribute to the severity of tissue destruction.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22507号 / 農博第2411号 / 新制||農||1078(附属図書館) / 学位論文||R2||N5287(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 植田 充美, 教授 山口 信次郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Untreatable gonorrhea, caused by fully antimicrobial resistant Neisseria gonorrhoeae (GC), is a major global health threat. While a vaccine would greatly help address this crisis, development of a GC vaccine is complicated by the lack of lab models of symptomatic gonorrhea. We hypothesized that overt disease in animal models of gonorrhea is limited by the human-restriction of gonococcal virulence factors, and the impact of the reproductive hormone cycle (estrus and diestrus phases). We tested these hypotheses by examining the host response to infection in transgenic mice expressing targets of bacterial adhesion, human carcinoembryonic antigen-related cell adhesion molecules (hCEACAMs), in uterine versus vaginal infections, and in different phases of the reproductive cycle (estrus and diestrus phases). hCEACAM expression most impacted estrus phase infections, prolonging colonization in vaginal infection and inducing greater inflammation in uterine. Reproductive phase greatly influenced host response to uterine infection as diestrus infection was more inflammatory than estrus. Phase differences in uterine infection were driven by greater activation of a chemokine-centric common anti gonococcal response and unique induction of type 1 interferons in diestrus. These findings suggest that symptomatic uterine and vaginal GC infection can be modeled by transcervically infected wild-type diestrus mice and transgenic, vaginally-infected estrus mice, respectively. A novel approach to GC vaccine development is also needed. Mono-antigenic vaccines have failed to produce immunity suggesting a poly-antigenic antigen, like natural outer membrane vesicles (nOMVs) may be necessary. It has been shown that any GC vaccine must lack the bacterioprotective antigen, reduction modifiable protein (RMP), and no such nOMV has been previously described. Here we report successful isolation of RMP-deficient nOMVs through sequential size and weight restrictive filtration. Vesicle morphology, proteomics, and bioactivity was characterized via various methods. nOMVs were found to be consistent in size, shape and antigenic load. As antigens, nOMVs induced high serum titers and measurable vaginal levels of antigen and GC specific IgG that recognized several nOMV immunogens supporting the vaccine potential of GC nOMVs. These findings lay the groundwork for protective studies of nOMV vaccines in novel models of active gonorrhea moving the field closer to discovering the mechanism of protective anti-gonococcal immunity.

Brucella abortus is a zoonotic agent that primarily infects cattle and causes brucellosis. B. abortus strain RB51 is a live, attenuated vaccine licensed for cattle. However, there is no available vaccine to prevent human brucellosis. Outer membrane vesicles have been tested as potential vaccines to prevent diseases caused by bacterial species. OMV are constantly released from Gram-negative bacteria. They are comprised principally of the outer membrane components and periplasmic proteins from the bacterial cell envelope. The research in this thesis examined the adjuvant property of non-replicative, metabolically active irradiated strain RB51 and the protective ability of OMV derived from strain RB51. Irradiated B. abortus strain RB51 was assessed for its ability to act as an adjuvant to induce protection against malaria. It was found that irradiated B. abortus strain RB51 administered along with fasciclin related adhesive protein (FRAP) to mice induced a protective immune response and a significant decrease in parasitemia after challenge with Plasmodium berghei. Strain RB51 and strain RB51 over-producing Cu/Zn superoxide dismutase (Cu/Zn SOD) were used to produce OMV. Western blotting and SDS-PAGE gel staining confirmed the presence of OMV and the over-production of Cu/Zn SOD. OMV were delivered to mice using an intraperitoneal route and, in some cases, with aluminum hydroxide adjuvant. The immune response was assessed by antibody isotyping with respect to OMV and measuring splenic clearance (i.e. protection) from a B. abortus strain 2308 challenge. The results demonstrate that OMV from B. abortus strain RB51 or strain RB51 over producing Cu/Zn SOD produced a Th1 polarized immune response as measured by specific OMV antibodies and cytokines but no statistically significant protection was observed. / Master of Science

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is primarily associatedwith aggressive forms of periodontal disease. Additionally, it has occasionally been found to causemetastatic infections in non-oral sites. This requires the ability to evade the bactericidal activity ofthe complement system of the humoral immune system. Outer membrane proteins, namely,Omp100 and OmpA have been connected to normal human serum resistance for several bacteriaspecies. The objective of this study was to investigate if serum-resistant ompA mutants can beobtained, and to detect changes in OMP expression. We used A. actinomycetemcomitansserotype a strain D7SS and D7SS ompA knockouts. The strains were incubated in 50 % NHS.This resulted in a substantial decrease of survival among D7SS ompA knockouts. D7SS ompAknockouts were exposed to 50 % NHS once more to confirm stable serum resistance. 13 out of14 tested clones showed growth, indicating that serum resistant ompA mutants could begenerated. SDS-PAGE gel of extracted outer membrane vesicles revealed an additional proteinband of approximately 34 kDa in at least 4 of 5 tested serum resistant ompA mutants. This proteinband has been analyzed in the laboratory, and according to LC-MS/MS it contains an OmpAhomologue, which has been named OmpA2. We conclude that OmpA2 expression might be amajor mechanism for serum survival in A. actinomycetemcomitans serotype a strain D7SS ompAknockouts.

We have developed a novel, sensitive and less time-consuming method to detect activity of the SecA-dependent protein-conducting channels. Nanogram levels of E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. The observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein, proOmpA, or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5’-(β, γ-methylene)-diphosphonate, a non-hydrolyzable ATP analog, both of which are known to inhibit SecA protein activity. Channel activity was also stimulated by oocyte endogenous precursor proteins, which could be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides, but not defective signal peptides, stimulated the ionic currents. We also measured SecA-dependent currents with membranes depleted of SecYEG. Wild-type LamB signal peptides, or precursor proteins stimulated ionic currents following a co-injection of SecYEG¯ membranes with puromycin. Excess exogenous SecA stimulated ionic currents through SecYEG¯ membranes. Similar activities of added SecA were observed with reconstituted membranes depleted of SecYEG. Currents through such SecYEG-depleted membranes were also stimulated by addition of defective LamB signal peptides and unfolded mature PhoA protein. In contrast, currents produced by the membranes containing wild-type SecYEG were not so stimulated, but ionic currents were stimulated through mutant strains, similar to PrlA (SecY) suppressors, e.g. PrlA4, or PrlA665 membranes, suggesting that the proofreading function of SecY was bypassed in these membranes. We have observed that azide can inhibit ionic currents when E. coli wild-type MC4100 membranes were injected with proOmpA or LamB signal peptides into Xenopus oocytes. However, such inhibition was lost when observed with oocyte-endogenous signal peptides in the absence of bacterial signal peptides. Moreover, azide did not show complete inhibition upon using SecYEG¯ membranes or SecYEG¯ reconstituted membranes plus excess SecA in the presence or absence of LamB signal peptides. Such conformational alterations reflect different sensitivity in response to azide during the opening of protein-conducting channels.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22495号 / 農博第2399号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5275(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 木岡 紀幸 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

The bacterium Streptococcus pneumoniae (pneumococcus), is considered to be a leading cause of morbidity and mortality globally, particularly in infants and the elderly. It is one of the most frequent causes of respiratory tract infections, which sporadically have the potential to develop into serious invasive symptoms including sepsis and meningitis. The development of effective vaccination against this pathogen is essential for reducing the morbidity and mortality it causes since the currently available vaccines can protect against only a limited number of the 100 pneumococci serotypes which target the polysaccharidic capsule of the bacterium. The potential use of conserved protein antigens could provide a defense to a wider range of serotypes and clonal types. The immunogenic properties of the proteins MalX and PrsA as well as their role in vital biological functions of S. pneumoniae have made them stand out as potential targets. MalX is a crucial membrane protein involved in the metabolism of maltose, whereas PrsA is a chaperone-like protein that is connected to the cell envelope. To understand these proteins' potential as vaccine candidates, it is essential to understand their immunogenic characteristics and physiological roles. In this project, we tried to characterize the two antigens to determine the functional significance of different regions and domains in antigen recognition and their expression dynamics in bacterial host. A better understanding of the antigenic properties of the PrsA and MalX proteins will drive the construction of improved versions of antigens for vaccine prototypes. Some approaches were used to clarify the structural characteristics and antigenic determinants associated with these proteins including, protein expression, purification, and structural characterization. Additionally, their expression in E. coli was examined using immunological assays including ELISA and Western blot. The identification of antigenic regions of these proteins also provides insight into how to develop epitope-based vaccinations that specifically target S. pneumoniae. This project discusses the possibility of using membrane vesicles (MVs) as a platform for vaccination. Membrane vesicles made from bacterial cells have innate immunogenic qualities that expose the immune system to a wide variety of antigens. Incorporating MalX and PrsA into such vesicles can improve the vaccine candidate's overall immunogenicity and effectiveness and trigger a stronger immune response against S. pneumoniae.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23952号 / 農博第2501号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5387(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM