Cell Biology & Molecular Genetics
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Item Roles of Female Sex Hormones in Regulating Neisseria gonorrhoeae Colonization of the Human Cervix(2024) Di Benigno, Sofia; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) is a human-exclusive pathogen that infects the genital tract. Gonococcal infection may present with or without symptoms and can lead to a variety of serious sequelae if left untreated, especially in female patients. Despite this, there are few models that can effectively mimic GC infection in the female reproductive tract (FRT); of these, even fewer consider the impact of the menstrual cycle, an important feature of the FRT, on GC infection. I used the human cervical tissue explant model previously developed in our lab, which can recapitulate GC infection in vivo. Tissue explants were treated with the sex hormones estradiol and progesterone to mimic various stages of the menstrual cycle and examine its impact on GC infectivity. Estradiol was used to mimic the late proliferative phase, and a combination of estradiol and progesterone was used to mimic the middle of the secretory phase. The effects of hormones on GC infectivity were examined after 72 total hours of hormone treatment and 24 hours of inoculation with GC of strain MS11. My results show that treatment with estradiol and with a combination of estradiol and progesterone both increase the level of GC colonization on the endocervix, but not on the ectocervix, compared to controls that were not treated with hormones. However, the hormone treatment did not affect GC penetration of the cervical epithelium. Both hormone treatments increased the number of GC colonies on the endocervical epithelium, and a combination of estradiol and progesterone produced an additional population of large GC colonies, leading to an increase in the average colony size. These increases in colony number and size were not associated with an increase in the expression of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which are the host receptors for GC Opa proteins. In contrast, treatment with estradiol induced a redistribution of CEACAMs from the luminal surface to the inside of epithelial cells. Additionally, estradiol altered the morphology of endocervical epithelial cells from columnar to cuboidal, but the integrity of cell-cell junctions was unchanged. The increase in colonization under high estradiol conditions was correlated with a decrease in levels of certain pro-inflammatory cytokines and chemokines, but this decrease was not sufficient to fully explain the increase in colonization. Next, I investigated the impact of cervical mucus on GC infectivity and interactions, as gel-forming mucin MUC5B but not MUC5AC increases with estradiol at the proliferation phase. Under both hormone treatment conditions, GC were able to establish close interaction with the luminal surface of the endocervical epithelial cells, displacing membrane-spanning mucin MUC1 in the membrane. Furthermore, GC were able to diffuse through an artificial mucin hydrogel and diffused more efficiently through a MUC5AC-dominant than a MUC5B-dominant hydrogel. Gel-forming mucins collected from cervical tissue explants enhanced GC aggregation in vitro, even at very low concentrations. However, mucins collected from estradiol-treated tissues showed less impact on GC aggregation than those collected from untreated tissues or tissues treated with both estradiol and progesterone. MUC5B and MUC5AC purified from cows and pigs also increase GC aggregation in vitro with GC aggregating more in a MUC5AC- than a MUC5B-dominant mucin mixture. Taken together, my research reveals for the first time that female sex hormones regulate GC colonization at the human cervix by changing the composition of the cervical mucus, providing a mechanism of hormonal regulation underlying the varying susceptibility of female patients to mucosal GC.Item Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules(MDPI, 2020-08-20) Stein, Daniel C.; Stocker, Lenea H.; Powell, Abigail E.; Kebede, Salsawi; Watts, David; Williams, Emma; Soto, Nicholas; Dhabaria, Avantika; Fenselau, Catherine; Ganapati, Shweta; DeShong, PhilipIdentification of antigens is important for vaccine production. We tested extraction protocols using cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS) to formulate surfactant vesicles (SVs) containing components from Neisseria gonorrhoeae. Carbohydrate and protein assays demonstrated that protein and carbohydrates were incorporated into the vesicle leaflet. Depending on the extraction protocol utilized, 100–400 µg of protein/mL of SVs solution was obtained. Gel electrophoresis followed by silver staining demonstrated that SV extracts contained lipooligosaccharide and a subset of bacterial proteins and lipoproteins. Western blotting and mass spectral analysis indicated that the majority of the proteins were derived from the outer membrane. Mass spectrometric and bioinformatics analysis of SVs identified 29 membrane proteins, including porin and opacity-associated protein. Proteins embedded in the SVs leaflet could be degraded by the addition of trypsin or proteinase K. Our data showed that the incorporation of CTAT and SDBS into vesicles eliminated their toxicity as measured by a THP-1 killing assay. Incorporation of gonococcal cell surface components into SVs reduced toxicity as compared to the whole cell extracts, as measured by cytokine induction, while retaining the immunogenicity. This process constitutes a general method for extracting bacterial surface components and identification of antigens that might be included in vaccines.Item NEISSERIA GONORRHOEAE MODULATES INFECTIVITY BASED ON PROPERTIES OF HUMAN CERVICAL EPITHELIA AND PHASE VARIABLE BACTERIAL SURFACE STRUCTURES(2019) Yu, Qian; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) infection in the human female reproductive tract causes various clinical outcomes, from no symptom to severe complications. The major barrier to a better understanding of GC infection in women is the lack of experimental system closely mimicking in vivo infection. Here, I developed a human cervical tissue explant model, which maintains the heterogeneity of the cervical epithelium. Using this model, my thesis research examined the impact of the heterogeneity of the cervical epithelium and the phase variation of GC surface structures on GC infectivity. My research revealed that GC preferentially colonize the ectocervix and the transformation zone (TZ), but exclusively penetrate into the subepithelial tissues of the TZ and endocervix. Pili are essential for GC colonization in all regions of the cervix. Expression of Opa isoforms that bind to the host receptors CEACAM (OpaCEA) enhances GC colonization in the ecto/endocervix but inhibits GC penetration into the endocervix. However, GC infectivity in the TZ does not respond to Opa phase variation, due to the low expression level and intracellular location of CEACAMs in the TZ epithelial cells. OpaCEA enhances GC colonization in the ecto/endocervix by inhibiting epithelial exfoliation and suppresses GC penetration into the endocervical subepithelium by inhibiting GC-induced disassembly of the apical junction. Opa-mediated modulation of GC infectivity depends on the immune receptor tyrosine-based inhibitory motif (ITIM) of CEACAM1 and its downstream phosphatase SHP. The effect of epithelial cell polarity on GC invasion was studied using a cell line model. My results show that GC invade more efficiently into non-polarized than polarized epithelial cells without changing the adhesion efficiency. Opa (phase variable) expression enhances both adhesion and invasion in both non-polarized and polarized cells. In non-polarized cells, Opa expression induces F-actin accumulation and microvilli elongation underneath GC microcolonies, suggesting an actin-mediated uptake of GC. In contrast, GC expressing no Opa reduce F-actin and demolish microvilli underneath microcolonies in both polarized epithelial cell line and endocervical epithelial cells potentially by increasing calcium flux, NMII activation and the redistribution of actin nucleation factor Arp2/3 from the apical surface. Taking together, my research demonstrates that both the heterogeneity of the cervical epithelium and the phase variation of bacterial surface structures regulate GC infectivity in the human cervix, either dominated by colonization or penetration, consequently influencing the clinical outcomes of the infection.Item EFFECT OF OPA EXPRESSION ON TRANSMIGRATION OF NEISSERIA GONORRHOEAE ACROSS POLARIZED EPITHELIAL CELLS(2013) Le Van, Luz Angela Adriana; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) is a solely human pathogen that causes gonorrhea. This study examines how Opas, which are surface factors expressed by GC that undergo antigenic and phase variation can affect transmigration across epithelium. Opas are encoded by 11 different genes. A gonococcal variant that lacked all opa genes was constructed to help elucidate the role of Opa in pathogenesis. This variant retained most physical characteristics of the parent strain including growth rate and LOS profile but proved to produce a different interaction with other GC by being unable to bind LOS of adjacent GC and form microcolonies. Lack of Opa expression increased the ability of GC to transmigrate across polarized colonic epithelial cells T84. When the opa deletion variant was not expressing pili, bacteria were observed entering and crossing the polarized epithelia as early as four hours after infection. GC were observed at the bottom of the polarized epithelial monolayer demonstrated by confocal microscopy. While GC transmigrate across the monolayer, they do not appear to disrupt the integrity of tight junction proteins. Transepithelial resistance did not show a significant change and there was no leakage of FITC or HRP. Inhibitors of acid sphingomyelin and F-actin did not cause a redistribution of ZO-1 and did not increase the transmigration of GC. Only in the presence of EGTA, a calcium chelator, were Opa-expressing GC observed crossing the monolayer through visible disruption of the tight junctions. Induction of TNF-α by T84 cells was increase when infected with GC but not the production of IL-8. This study indicates that the lack of expression of Opa and pili leads to an increase in invasion into subepithelial tissues.Item Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae(2009-11-23) Stein, Daniel C; Carrizosa, Esteban; Dunham, StephenBackground: Organisms that are sensitive to nitrofurantoin express a nitroreductase. Since bacterial resistance to this compound results primarily from mutations in the gene encoding nitroreductase, the resulting loss of function of nitroreductase results in a selectable phenotype; resistance to nitrofurantoin. We exploited this direct selection for mutation to study the frequency at which spontaneous mutations arise (transitions and transversions, insertions and deletions). Results: A nitroreductase- encoding gene was identified in the N. gonorrhoeae FA1090 genome by using a bioinformatic search with the deduced amino acid sequence derived from the Escherichia coli nitroreductase gene, nfsB. Cell extracts from N. gonorrhoeae were shown to possess nitroreductase activity, and activity was shown to be the result of NfsB. Spontaneous nitrofurantoin-resistant mutants arose at a frequency of ~3 × 10-6 - 8 × 10-8 among the various strains tested. The nfsB sequence was amplified from various nitrofurantoin-resistant mutants, and the nature of the mutations determined. Transition, transversion, insertion and deletion mutations were all readily detectable with this reporter gene. Conclusion: We found that nfsB is a useful reporter gene for measuring spontaneous mutation frequencies. Furthermore, we found that mutations were more likely to arise in homopolymeric runs rather than as base substitutions.Item Estrogen and Progesterone enhance Neisseria gonorrhoeae Transmigration across a Polarized Monolayer via a Mechanism that Hijacks EGFR(2012) Edwards, Vonetta Lisa; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Gonorrhea, a common sexually transmitted infection, is caused by the gram-negative bacterium Neisseria gonorrhoeae. In the female reproductive tract, gonococci (GC) initiate infection at the apical surface of columnar endocervical epithelial cells. These cells provide a physical barrier against mucosal pathogens by forming continuous apical junctional complexes between neighboring cells. This study examines the interaction of GC with polarized epithelial cells. We show that viable, but not gentamicin killed, GC preferentially localize at the apical side of the cell-cell junction in polarized endometrial and colonic epithelial cells, HEC-1-B and T84, respectively. In GC infected epithelial cells, continuous apical junctional complexes are disrupted, and the junction-associated protein β-catenin is redistributed from the apical junction to the cytoplasm and to GC adherent sites. However, GC inoculation does not change the overall cellular level of junctional proteins. This redistribution of junctional proteins is associated with a decrease in the apical junction's barrier function against the lateral movement between the apical and basolateral membranes, but not against the permeability through the paracellular space. Disruption of the apical junction by removing calcium increases GC transmigration across the epithelial monolayer. GC inoculation induces the phosphorylation of both epidermal growth factor receptor (EGFR) and β-catenin, while inhibition of EGFR kinase significantly reduces both GC-induced β-catenin redistribution and GC transmigration. These results suggest a relationship between junction protein redistribution from the plasma membrane with the resultant weakening of the junctional complex, and an increase in the ability of GC to transmigrate. The presence of the female sex hormones estrogen and progesterone, lead to an increased degree of disruption of the junctional complex and enhance GC transmigration across the monolayer. Therefore, GC are capable of weakening the apical junction and the polarity of epithelial cells via activating EGFR, which facilitates GC transmigration across the epithelium.Item Mechanisms Utilized by Neisseria gonorrhoeae to Persist in the Human Cervical Epithelium: Signaling, Invasion, Intracellular Survival, and Escape(2007-11-07) Bish, Samuel Earl; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The obligate human pathogen Neisseria gonorrhoeae (gonococci) causes the sexually transmitted disease gonorrhea. Interactions between gonococci and human cervical epithelial cells that promote bacterial invasion and post-invasion survival were examined. The data showed that gonococci activated MAPK signaling in cervical epithelial cells prior to invasion. Gonococci triggered prolonged ERK phosphorylation (possibly through EGFR signaling), which could facilitate gonococcal invasion. Furthermore, gonococci expressing lacto-N-neotetraose LOS decreased JNK activation and elicited lower IL-8 production from host cells compared to variants expressing lactosyl LOS. I propose that gonococci can manipulate MAPK signaling to facilitate invasion into cervical epithelial cells and to evade the innate immune response. To investigate gonococcal invasion, I expressed β-lactamase on the outer membrane of gonococcal strain FA1090 to generate the reporter strain FA1090 psi(bla-iga'). Utilizing a β-lactamase reporter assay, I demonstrated that FA1090 psi(bla-iga') only invaded a subpopulation of cervical epithelial cells. Nonviable FA1090 psi(bla-iga') did not invade host cells and failed to recruit F-actin to sites of adherence. Viable gonococci required pili expression but not Opa expression to invade susceptible host cells. These data suggest that viable gonococci elicit invasion-promoting interactions with host cells not triggered by nonviable gonococci. Finally, the data demonstrated that gonococci realize different intracellular fates after entry into cervical epithelial cells. I developed a tannic acid survival assay to show that intracellular gonococci survived within host cells. Intracellular survival did not require catalase or the NfsB nitrogen reductase. A subpopulation of gonococci could escape from cells back into the extracellular milieu. Gonococci utilized an exocytosis pathway to escape after invasion that was blocked by treating cells with tannic acid, but not cytoskeletal inhibitors. I propose a working scenario whereby gonococci undergo cycles of invasion into and escape from a susceptible subpopulation of cervical epithelial cells. Gonococci that survive within host cells may represent bacteria that progress into deeper host tissues to disseminate to secondary sites. Escaping gonococci could invade into other host cells and are well-positioned for transmission to another host. Gonococci interact with the cervical epithelium through invasion, intracellular survival, and escape to establish bacterial persistence in the female host.