|Grant Support||Research Pictures||TEM Core Facility|
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inclusions (green/yellow) in infected genital epithelial cells
microscopy (TEM) photograph
of chlamydial inclusions in
Estrogen Modulates Chlamydial Attachment and Host Cell Physiology
For the most common isolates, C. trachomatis serovars D/E, little is known about the adhesin/receptor complexes mediating entry into genital epithelial cells. The major outer membrane protein (MOMP) is believed to initiate attachment of the negatively charged infectious elementary body (EB) to the negatively charged host cell surface, after which either a high affinity 2° adhesion/receptor interaction occurs or multiple low-affinity events. Using a strategy of stripping off EB attached to biotinylated, polarized apical endometrial epithelial membranes and subjecting this immuno-precipitated EB with their associated labeled receptors to 2D PAGE, a component of the estrogen hormone receptor complex was identified by MALDI-MS sequence analysis. Surface protein disulfide isomerase (PDI) participates in disulfide exchange reactions, which result in a conformational rearrangement to activate the estrogen receptor (but not the progesterone receptor) for higher affinity estrogen binding and in stabilizing the complex for subsequent estrogen-response element-DNA binding. Using selected, characterized hormone-responsive cell lines (HIC, Ishikawa, MCF-7, ICC-1806, HeLa, etc.) and a variety of different techniques, the roles of estrogen receptor (ER) alpha and ER beta in chlamydial entry are being examined. Further, blocking ER function with inhibitors (anti-ER ABYs and tamoxifen) after chlamydial entry alters chlamydial development, indicating an additional important role of estrogen on host cell physiology. These studies are underway by Director of Research, Maria Schell; Research Technician Cheryl Moore; and Post-doctoral Fellow, Dr. Jenny Vanover Hall.
Director of Research
Dr. Jenny Vanover Hall
A Pig Model Confirms Hormone Modulation of Chlamydial Attachment/Entry and Infection
Previous studies in our laboratory clearly
demonstrated significantly enhanced attachment of EB to
estrogen-dominant primary human endometrial epithelial cells and
markedly reduced attachment to progesterone-dominant epithelial
cells. These data correlate with epidemiological observations of
hormone modulation of chlamydial infection in women and with
histological aspects in C. muridarum – infected animal
models in vivo.
To confirm these data,
Postdoctoral Fellow Natalia Guseva, D.V.M./Ph.D. Microbiology,
established a swine-
(S45) ex vivo model. Discarded genital tracts from mature
female pigs, eliminated from breeding programs, were obtained
from a nearby abbatoir and brought to the laboratory for
separation and culture of the luminal and glandular epithelial
cells of the uterine horns. Macroscopic assessment of the ovaries
for hormone cycle staging was confirmed by microscopic
histological analysis so that exogenously supplied hormones
maintained the cultured epithelia in their appropriate
physiological state. Experimental results to date with swine
chlamydiae-infected cells confirmed and enhanced previous
findings with human chlamydiae-infected primary cells, i.e. that
estrogen-dominant swine epithelial cells are more susceptible to
chlamydial infection than are progesterone-dominant epithelia.
Most importantly, chlamydial infection was acute in mature
luminal epithelia where as persistent forms of chlamydiae
predominated in the less-differentiated glandular epithelial
cells, confirming Dr. Shirley Richmond’s original clever
hypothesis that chlamydiae exist in vivo in a persistent form in
these deep, undifferentiated glandular cells.
|Natalia Guseva, D.V.M./Ph.D. Microbiology, Postdoctoral Fellow||Estrogen dominant polarized pig epithelial cultured in vitro|
Early Inflammatory Responses Differ between Non-Invasive Serovar E – and Invasive Serovar LGV-Infected HeLa Cells
Using cDNA microarray technology, Research Assistant Professor Sophie Dessus-Babus discovered that C. trachomatis infection of HeLa cells triggers up-regulation of host cell mRNA for the anti-inflammatory cytokine IL-11 and that the amounts of IL-11 differ in HeLa cells infected with the invasive LGV serovar versus the non-invasive serovar E. Using a chronological 3-stage co-culture system consisting of chlamydiae-infected HeLa cells, chemotactic PMNs and, lastly, monocyte-derived macrophages, the resulting modulation of TNFalpha production was dramatic, being significantly depressed in the presence of the ‘non-invasive’ serovar E and significantly increased (>500 – 1000-fold) in the presence of invasive serovar LGV-2.
|Dr. Sophie Dessus-Babus||
Expert Electron Microscopist
Chlamydial Antigens Escape from the Developing Chlamydial Inclusion via Everted Inclusion Membrane Vesicles, Which Can Fuse with the Endoplasmic Reticulum!
Isolated peripheral blood PMNs, pre-loaded with the antibiotic azithromycin, chemotax through the extracellular matrix and intercellular spaces of polarized monolayers of genital epithelial cells to deliver azithromycin selectively to chlamydiae-infected cells. The azithromycin is transported via the basal membrane and into the membrane-bound chlamydial inclusion, where it eventually kills metabolically-active chlamydiae. Excessive chlamydial envelope blebbing occurs in the antibiotic-induced killing process. The chlamydial blebs, containing MOMP and LPS, appear by transmission electron microscopy to escape from the inclusion via inclusion membrane everted vesicles. Some of the bleb-containing vesicles are trafficked to the infected cell surface where they are likely phagocytosed and presented by PMNs to MØ for processing. Graduate student Davy Giles also demonstrated that the bleb-containing vesicles take alternate trafficking pathways and fuse with intracellular vesicles of the endoplasmic reticulum for epithelial basal membrane MHC Class I complex presentation. This pathway for epithelial antigen presentation might offer a different/complementary route to the presumed Type III secretion-to-cytoplasm-to-proteosome-to-ER “accepted dogma” pathway.
|Davey Giles, Graduate student||
Everted Chlamydial Inclusion Membrane
Vesicles Containing Envelope Blebs
|Postdoctoral Fellow - Melanie Sal|
Research Description - Chlamydial Projections
In the 1970's, Dr. Akira Matsumoto first discovered and described curious surface structures on C. psittaci EB, which he termed "projections". They have since been shown by transmission electron microscopy to be present on all species of Chlamydia in vitro and have been seen on chlamydiae in vivo. There are approximately 16-18 electron photomicrographs of the RB projections penetrating the inclusion membrane and extending beyond the inclusion into the infected host cell cytoplasm; he proposed chlamydiae used these structures to take in nutrients and energy from the host cell. More recently, the projections have been postulated to be the chlamydial Type III Secretion injectisome - equivalent, which could function both during attachment and entry as well as from "inside-to-outside" the inclusion. Genome sequence data indicate that chlamydiae have both TTSS homologues as well as some flagella homologues, which increases their intrigue. However, the actual identity and composition of the projections is currently unknown. Jane Raulston actually devised a protocol for isolation of the projections and Postdoctoral Fellow Melanie Sal, Ph.D. is isolating the C. trachomatis serovar E projections in hopefully sufficient quantity for their identification by mass spectral analysis. Our hypothesis is that the projections are a combination structure of T35 and flagella.