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.

Data compiled by the Centers for Disease Control demonstrates that African American women, in particular young people between the ages of 14-25, have an increased incidence of infection with the sexually transmitted pathogen Neisseria gonorrhoeae. Estradiol is a key regulatory hormone in female reproductive function. It has been studied extensively in the cardiovascular field, and has been linked to breast and endometrial cancers in women. However, its impact on infectious diseases is largely unknown. Given what is known about the effect of estradiol on immunologic and inflammatory disorders in women, I hypothesize that estradiol alters the infectivity of Neisseria gonorrhoeae in the female reproductive tract by altering the host inflammatory immune response. This may explain a risk factor for increased rates of infection in some populations. I sought to develop a relevant in vitro model. After screening a number of candidate cell lines, I selected the human endometrial adenocarcinoma cell line, Ishikawa. These cells express specific estrogen receptors and respond to exogenous estrogen stimulation. Estrogen treatment of Ishikawa cells did not have an impact on the invasion of N. gonorrhoeae, nor did it impact bacterial growth. However, gonococcal induced chemokine secretion was reduced by estrogen, as measured by interleukin-8 secretion. I conclude that estrogen blunts the inflammatory response to Neisseria gonorrhoeae without altering bacterial infectivity.

Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be treated with readily available antibiotics, but patients in low-resource settings often do not return to the clinic for results. As current NG diagnostic gold standards suffer from slow turnaround time to result, a rapid, sensitive molecular diagnostic would help increase appropriate treatment at the point-of-care. Here, we report on the design and development of a minimally-instrumented paper-fluidic POC diagnostic that incorporates patient swab sample lysis, isothermal nucleic acid capture, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and lateral flow visual detection. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and device clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept sensitivity and specificity study. This proof-of-principle paper-fluidic diagnostic, which can be completed within 80 minutes, selectively amplifies and detects NG in multiplex with NGIC. It has a clinically relevant limit of detection of 1000 NG cells per device. In urethral swab sample trials (N=20), the device approaches current gold standard NG diagnostic capabilities with 90% sensitivity and 100% specificity and vaginal swab sample trials (N=20) surpass the gold standard with 100% sensitivity and 100% specificity.