Neisseria gonorrhoeae is a strict human pathogen and the causative agent of the sexually transmitted disease, gonorrhea. Gonococcal (GC) diseases remain one of the most reported sexually transmitted infections (STI) worldwide, representing a significant threat to reproductive health and burden on global health systems, accounting for 541,987 disability adjusted-life years in the year 2011. Infection by N. gonorrhoeae also increases the likelihood of patient acquisition and transmission of human immunodeficiency virus (HIV). Unfortunately, antibiotic treatment to gonococcal diseases is being threatened by the rapid spread of resistance to third-generation cephalosporins, the remaining treatment option used in clinics. The urgency of the situation is compounded by the relative lack of immunological protection conferred by previous infection by the bacterium. In response to the emergence of multidrug resistance, renewed energies are being directed towards the development of an effective, broadly protective vaccine. Difficulties in vaccine development arise from a lack of known correlates of protective immunity; there is no known broadly cross-protective immunity to GC and a truly reflective animal model has not been available. Nonetheless, previous studies have indicated that porins, neisserial major outer membrane proteins, are promising vaccine candidates. PorB makes up over 60% of the bacterium's outer membrane content and is involved in solute and ion exchange, invasion of target host cells, and evasion of host immunity. Porins from both the gonococcus and the meningococcus have been shown to have immunostimulatory activity, boosting B and dendritic (DC) cell proliferation and maturation in the absence of an exogenous adjuvant as mediated by TLR2 and MyD88. Importantly, as a potential vaccine candidate, PorB has relatively low antigenic variability, and can induce bactericidal antibodies. Gonococcal PorB was purified from a genetically modified strain, MS11delP3, which lacks another outer membrane protein, RMP, which is known to induce bactericidal blocking antibodies. PorB was formed into pure protein micelles, termed proteosomes, to protect the integrity of the native trimeric structure. Our study demonstrated that gonococcal PorB is able to stimulate both human embryonic kidney (HEK) cell line that overexpresses TLR2 and mouse primary macrophages (similar to the meningococcal PorB). To test PorB's immunogenicity, mice were immunized three times at two week intervals with PorB and porin specific IgG levels were measured. Unfortunately, PorB elicited lower levels of porin specific IgG than what was expected, which may be due to technical issues. We are currently investigating various possibilities. In addition, further immunization studies shall be carried out to better contextualize these results.

The obligate human pathogen Neisseria gonorrhoeae is responsible for the widespread sexually transmitted disease gonorrhoea, which in rare cases also leads to the development of disseminated gonococcal infection (DGI). DGI is mediated by PorBIA-expressing bacteria that invade host cells under low phosphate condition by interaction with the scavenger receptor-1 (SREC-I) expressed on the surface of endothelial cells. The interaction of PorBIA and SREC-I was analysed using different in vitro approaches, including surface plasmon resonance experiments that revealed a direct phosphate-independent high affinity interaction of SREC-I to PorBIA. However, the same binding affinity was also found for the other allele PorBIB, which indicates unspecific binding and suggests that the applied methods were unsuitable for this interaction analysis. Since N. gonorrhoeae was recently classified as a “super-bug” due to a rising number of antibiotic-resistant strains, this study aimed to discover inhibitors against the PorBIA-mediated invasion of N. gonorrhoeae. Additionally, inhibitors were searched against the human pathogen Chlamydia trachomatis, which causes sexually transmitted infections as well as infections of the upper inner eyelid. 68 compounds, including plant-derived small molecules, extracts or pure compounds of marine sponges or sponge-associated bacteria and pipecolic acid derivatives, were screened using an automated microscopy based approach. No active substances against N. gonorrhoeae could be identified, while seven highly antichlamydial compounds were detected. The pipecolic acid derivatives were synthesized as potential inhibitors of the virulence-associated “macrophage infectivity potentiator” (MIP), which exhibits a peptidyl prolyl cis-trans isomerase (PPIase) enzyme activity. This study investigated the role of C. trachomatis and N. gonorrhoeae MIP during infection. The two inhibitors PipN3 and PipN4 decreased the PPIase activity of recombinant chlamydial and neisserial MIP in a dose-dependent manner. Both compounds affected the chlamydial growth and development in epithelial cells. Furthermore, this work demonstrated the contribution of MIP to a prolonged survival of N. gonorrhoeae in the presence of neutrophils, which was significantly reduced in the presence of PipN3 and PipN4. SF2446A2 was one of the compounds that had a severe effect on the growth and development of C. trachomatis. The analysis of the mode of action of SF2446A2 revealed an inhibitory effect of the compound on the mitochondrial respiration and mitochondrial ATP production of the host cell. However, the chlamydial development was independent of proper functional mitochondria, which excluded the connection of the antichlamydial properties of SF2446A2 with its inhibition of the respiratory chain. Only the depletion of cellular ATP by blocking glycolysis and mitochondrial respiratory chain inhibited the chlamydial growth. A direct effect of SF2446A2 on C. trachomatis was assumed, since the growth of the bacteria N. gonorrhoeae and Staphylococcus aureus was also affected by the compound. In summary, this study identified the severe antichlamydial activity of plant-derived naphthoquinones and the compounds derived from marine sponges or sponge-associated bacteria SF2446A2, ageloline A and gelliusterol E. Furthermore, the work points out the importance of the MIP proteins during infection and presents pipecolic acid derivatives as novel antimicrobials against N. gonorrhoeae and C. trachomatis. / Neisseria gonorrhoeae ist ein obligat humanpathogenes Bakterium, das für die weltweit verbreitete sexuell übertragbare Krankheit Gonorrhoe verantwortlich ist. In seltenen Fällen kann es auch zur Ausbildung der Disseminierten Gonokokken-Infektion (DGI) kommen, die mit der Expression des Gonokokken Oberflächenproteins PorBIA assoziiert ist. PorBIA-exprimierende Bakterien invadieren in die Wirtszelle unter phosphatfreien Bedingungen, was durch eine Interaktion mit dem zellulären Oberflächenrezeptor scavenger receptor-1 (SREC-I) vermittelt wird. Die direkte Interaktion zwischen PorBIA und SREC-I wurde mittels verschiedenster Methoden analysiert, einschließlich einer Oberflächenplasmonresonanz-analyse, die eine direkte Bindung von PorBIA zu SREC-I in einem phosphatunabhängigen Schritt aufzeigte. Allerdings wurde dieselbe Affinität auch zu PorBIB gefunden, was auf eine unspezifische Bindung hindeutet und dafür spricht, dass die verwendeten Methoden für diese Interaktionsanalyse ungeeignet sind. N. gonorrheae wurde vor kurzem wegen der stetig steigenden Anzahl antibiotikaresistenter Stämme als „Superkeim“ bezeichnet. Aufgrund dessen wurden Inhibitoren gegen die PorBIA-vermittelte Invasion von N. gonorrhoeae, aber auch gegen Chlamydia trachomatis, den humanpathogenen Erreger von sexuell übertragbaren Infektionen und chronisch-follikulärer Bindehautentzündung, gesucht. 68 niedermolekulare Substanzen wurden mittels eines automatisierten Fluoreszenzmikroskopieverfahrens auf ihre inhibitorische Wirkung hin analysiert. Zu den getesteten Substanzen zählten pflanzenabstammende Stoffe, Isolate aus marinen Schwämmen oder Schwamm-assoziierten Bakterien, sowie Pipecolinsäure-Derivate. Gegen N. gonorrheae konnten keine Substanzen identifiziert werden, während sieben antichlamydiale Inhibitoren detektiert wurden. Pipecolinsäurederivate wurden synthetisiert als potentielle Inhibitoren des virulenz-assoziierten Proteins “macrophage infectivity potentiator” (MIP), das eine Peptidyl-Prolyl-cis-trans-Isomerase Aktivität besitzt. Diese Arbeit untersuchte die Rolle des MIP Proteins von N. gonorrhoeae und C. trachomatis während einer Infektion. Die zwei Inhibitoren PipN3 und PipN4 senkten die PPIase Aktivität des rekombinanten Chlamydien und Neisserien MIPs. Beide Substanzen beeinträchtigten das chlamydiale Wachstum und die Entwicklung in Epithelzellen. Ebenso konnte eine tragende Rolle des N. gonorrhoeae MIPs für das Überleben der Bakterien in Gegenwart von Neutrophilen aufgezeigt werden, das durch PipN3 und PipN4 inhibiert wurde. SF2446A2 war einer der Inhibitoren, der einen erheblichen Effekt auf das Wachstum und die Entwicklung von C. trachomatis aufgewiesen hat. Während der Analyse des Wirkmechanismus von SF2446A2 konnte eine Hemmung der mitochondrialen Atmungskette und eine Abnahme der mitochondrialen ATP Produktion in der Wirtszelle festgestellt werden. Allerdings war die Entwicklung von C. trachomatis unabhängig von der Funktionsfähigkeit der Mitochondrien. Eine Verbindung zwischen der antichlamydialen Wirkung von SF2446A2 und der Inhibierung der Mitochondrienatmungskette konnte damit ausgeschlossen werden. Nur das Reduzieren von zellulärem ATP durch Blockieren der Glykolyse und mitochondrialen Atmungskette verursachte eine Beeinträchtigung des Chlamydienwachstums. Eine direkte Auswirkung von SF2446A2 auf C. trachomatis wurde angenommen, da die Substanz auch das Wachstum von anderen Bakterien wie N. gonorrhoeae und Staphylococcus aureus inhibierte. Zusammengefasst identifizierte diese Studie die antichlamydiale Aktivität pflanzenabstammender Naphthochinone und der Isolate aus marinen Schwämmen oder Schwamm-assoziierten Bakterien SF2446A2, ageloline A und gelliusterol E. Ebenso verdeutlicht die Arbeit die Bedeutung der MIP Proteine während der Infektion und legt Pipecolinsäurederivate als mögliche neue Antibiotika gegen N. gonorrhoeae und C. trachomatis nahe.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 78-98).

Clinical and epidemiological studies have provided a large body of evidence supporting a link between HIV and other sexually transmitted co-infections. Co-infections have been associated with promoting HIV transmission and acquisition. One of the closest studied interactions is the co-infection with N. gonorrhoeae, the etiological agent of gonorrhea, yet a clear understanding of this relationship is still elusive. Studies aimed at deciphering how N. gonorrhoeae mediates these effects have provided mixed results with some suggesting co-infection promotes HIV replication, and others suggesting the opposite. The aim of this thesis is to uncover molecular mechanisms that explain these results through in vitro co-infection studies using a combination of mixed peripheral mononuclear blood cells (PBMCs) and isolated human cell types. The results presented here demonstrate that gonococcal co-infection profoundly inhibits HIV replication in co-infected PBMCs. This inhibition is due to both the release of anti-HIV IFN via TLR9-mediated activation of plasmacytoid dendritic cells (pDCs), and the activation of  T cells. In addition, I show that responses between CD4+ T cell lines, such as the Jurkat cell line, and primary CD4+ T cells can differ, which may explain some of the contrasting results seen in published literature. The results in this thesis have implications for understanding the relationship between gonococci and HIV, providing new insight into molecular and immunological interactions that influence viral transmission, and reveal new opportunities to combat HIV.

Highly infectious pathogenic strains of bacteria are becoming increasingly resistant to the current clinical antibiotics which have created a dire need for the development of novel antibiotics. O-Acetylpeptidoglycan esterase 1 (Ape1) is a periplasmic esterase present in several peptidoglycan (PG) O-acetylating pathogenic species of Gram-positive and all Gram-negative bacteria that perform this modification to this essential cell wall polymer. Inhibition of this growth-limiting enzyme may prove the principle that Ape1 has the potential to be the target for the development of a novel class of antibiotics. Ape1 plays a crucial role in bacterial growth by regulating PG turnover through catalytic removal of the C-6 acetyl group from O-acetylPG. This activity is required for the continued metabolism of PG because the major autolytic enzymes involved, the lytic transglycosylases, require a free C-6 hydroxyl group to produce their reaction product, 1,6-anhydromuramic acid. Several of the compounds that have been identified to effectively inhibit Ape1, were re-evaluated by determining their kinetic parameters. Work presented in this thesis explored the inhibitory potential of these compounds, belonging to the anthraquinone (alizarin, quinizarin, quinalizarin, emodin, sennoside A) or tannin (ellagic acid) families of compounds, both in vitro and in vivo, among species of bacteria that are known to O-acetylate their PG. Of the inhibitory compounds tested, ellagic acid was found to be most effective in vitro, with an IC50 value of 0.91 µM ± 0.06, Ki 1.18 ± 0.04 and in vivo it was shown to reduce bacterial growth.

Clinical and epidemiological studies have provided a large body of evidence supporting a link between HIV and other sexually transmitted co-infections. Co-infections have been associated with promoting HIV transmission and acquisition. One of the closest studied interactions is the co-infection with N. gonorrhoeae, the etiological agent of gonorrhea, yet a clear understanding of this relationship is still elusive. Studies aimed at deciphering how N. gonorrhoeae mediates these effects have provided mixed results with some suggesting co-infection promotes HIV replication, and others suggesting the opposite. The aim of this thesis is to uncover molecular mechanisms that explain these results through in vitro co-infection studies using a combination of mixed peripheral mononuclear blood cells (PBMCs) and isolated human cell types. The results presented here demonstrate that gonococcal co-infection profoundly inhibits HIV replication in co-infected PBMCs. This inhibition is due to both the release of anti-HIV IFN via TLR9-mediated activation of plasmacytoid dendritic cells (pDCs), and the activation of  T cells. In addition, I show that responses between CD4+ T cell lines, such as the Jurkat cell line, and primary CD4+ T cells can differ, which may explain some of the contrasting results seen in published literature. The results in this thesis have implications for understanding the relationship between gonococci and HIV, providing new insight into molecular and immunological interactions that influence viral transmission, and reveal new opportunities to combat HIV.

Neisseria meningitidis is the causative agent of potentially life-threatening meningitis and septicaemia. According to W.H.O., meningococcal disease causes at least 500,000 cases and results in 50,000 deaths worldwide each year (W.H.O., 2008). Neisseria gonorrhoeae is causing the second most common sexually transmitted bacterial infection, with a global incidence of 62 million cases per year. Previous studies have shown surface expressed proteins like pilin, the subunit protein that forms pili (Type IV Fimbriae), in N. meningitidis and N. gonorrhoeae are post-translationally modified by O-glycosylation. This modification has been proposed to be of importance in the pathogenesis of these species. Although the exact function of these post-translational modifications are not fully understood, it is suggested that these modification have a role for immune evasion in the host. In this thesis, an additional outer membrane glycoprotein was identified in pathogenic Neisseria, the nitrite reductase AniA. Mass spectrometry analysis showed that AniA is glycosylated in its C-terminal imperfect (AASAP) repeat region by the pilin glycosylation pathway. This is the first report of a general O-glycosylation pathway in a prokaryote. It was shown AniA is surface exposed. To investigate whether AniA is subject to immune selection, a large collection of N. meningitidis and N. meningitidis clinical isolates were sequence analysed and evaluated. Analysis of published AniA 3D structure revealed that AniA displayed polymorphisms in residues that map to the surface of the protein. This suggests that AniA is under immune selection, and that glycosylation may facilitate immune evasion. Sequencing analyses revealed a frame shift mutation that abolished AniA expression in 34% of N. meningitidis strains surveyed. However, all N. gonorrhoeae strains examined are predicted to express AniA, implying a crucial role for AniA in gonococcal biology. In summary, the data presented here suggested that the protein may be under immune selective pressure. The addition of a phase variable glycan to this surface protein may serve as an additional immune evasion strategy. Immune selection on surface proteins in these host-adapted pathogens may have been the driving force for the evolution of this general O-glycosylation pathway. Therefore, the discovery that AniA is a glycoprotein has given insights into the pathogenesis and the host-pathogen interactions of these organisms.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (M.Sc. (Microbiology))--University of Medical Sciences, 1967.

Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: leaves 134-165.