Second Joint Congress of the Federation of Infectious Diseases Societies of Southern African (FIDSSA), held in Stellenbosch, SA from 28 – 31 October 2007. Staff from the Laboratory of Sexually Transmitted Diseases within the Department of Medical Microbiology for identifying LGV C.
INTRODUCTION
A caspase assay along with Transwell ® permeable media was used to investigate whether the cytotoxic effects were apoptotic or not and to determine whether this occurred through cell contact or via a secreted molecule. To investigate the spatial relationship between dying cells and infected cells using the TUNEL assay with counterstaining for C.
LITERATURE REVIEW
Historical aspects and classification of Chlamydia trachomatis
Although the family Chlamydiaceae was previously thought to include a single genus, Chlamydia, which included four species: C. Each biovar is further subdivided into several serovars or serotypes, based on differences in the epitopes carried by the major outer membrane protein (MOMP) ( district 2002).
Clinical manifestations and epidemiology
In South Africa, LGV is treated according to local syndromic treatment guidelines for genital ulcers (Moodley et al 2003). These guidelines recommend the use of erythromycin and benzathine penicillin for the treatment of genital ulcers.
Laboratory diagnosis
Due to the fastidious nature of the organism, proper specimen collection and transport is essential. It is the characteristic morphology of the inclusions with this fluorescent stain that is responsible for the almost 100% specificity.
Biology of the organism
- Elementary Body
- Reticulate body
- Architecture of the cell envelope
During replication, RB is located on the periphery of the inclusion in close association with the inclusion membrane ( Peterson and de la Maza, 1988 ). During this time, the area of the RB in contact with the inclusion membrane exhibits an apparently rigid structure (Peterson and de la Maza, 1988).
Pathogenesis
- Inclusion membrane
- Type III secretion system
- Acquisition of energy and nutrients
- Reorganisation of the host cytoskeleton and organelles
- Chlamydial toxin
These include CopN which is part of the chlamydial type III secretion system (Fields and Hackstadt 2000) and Cap1 (Fling et al. 2001). Host cell lysis causes host cell death while exudation does not (Hybiske and Stephens 2007).
Interaction with the immune system
Th2 cells increase antibody production but do not assist in clearance of the organism (Perry et al. 1997, Hawkins et al. 2002). Like CD4+ T cells, CD8+ T cells can produce INF-γ to mediate intracellular death of the organism.
Cell death
- Initiation of apoptosis
- Execution of apoptosis
- Inhibition of apoptosis
- Induction of cell death
In the cell death literature, necrosis is the term used to describe non-apoptotic or accidental cell death. In the absence of direct activation of the effector caspases, this is sufficient to inhibit apoptosis (Fan et al, 1998, Fischer et al, 2004b).
Keratinocytes
Dumrese et al (2005) have suggested that cell death at the end of the chlamydial cycle occurs through a process they call HaCaT cells are used in studies of cell cycle (Chaturvedi et al, 1999) and carcinogenesis (Boukamp et al. 1994).
Cervical Cells
The ME-180 cell line is a spontaneously immortalized human cervical cell line developed in 1967 by Sykes and co-workers (Sykes et al. 1970). The ME-180 cell line is widely used in carcinogenesis studies and is sensitive to etoposide-induced cytotoxicity (Tanaka et al, 2006).
MATERIALS AND METHODS
Cell Culture
The cells were seeded in an equilibrated 75cm2 tissue culture flask containing cell culture medium supplemented with 10% FBS. The number of viable (colorless) and non-viable (blue) cells was counted in the 5 primary squares: each corner square and the center square. If the cells in each primary square were too numerous to count, the cell suspension was diluted with PBS, and the trypan blue dye exclusion assay was repeated.
Bacterial Culture
- Monolayer preparation
- Infection
- Isolation and storage
EB-SPG suspensions from all vials of the same batch were pooled, mixed, and aliquoted into sterile Eppendorf tubes with a safety seal for storage at −70°C. One hundred microliters of SPG was used for the negative control instead of the 100µl EB-SPG suspension. Since the conversion factors are affected by the surface area of the well and the area of the field of view visualized with each objective, the conversion factors differ and are calculated by dividing the area of the field of view by the area of the well.
Fluorescent Microscopy
After the fixation step, the excess fixative was aspirated and the monolayers were rinsed once with PBS to remove any unattached cell debris. While the monolayer was still moist, 30 µl of balanced staining reagent (Appendix) was applied to the coverslips and incubated for 30 min at 37 °C in a humidified chamber. The monolayer had to be moist so that the staining reagent covered the entire coverslip, and the coverslips were incubated in a humid chamber to prevent drying of the stains on the monolayer, which would result in nonspecific binding.
Collection and propagation of clinical isolates
The material was suspended in 1 ml of chlamydia transport medium and the plastic loop was left in the container. Ulcer samples that were PCR positive for Chlamydia and LGV biovar restriction sample were plated on McCoy cell monolayers in CGM-P. Slides were examined using a fluorescent microscope with a filter system for FITC to confirm the presence of chlamydial inclusions in the samples and the absence of chlamydial inclusions in the negative control.
Lactate Dehydrogenase Detection
A culture medium background control, comprising only cell culture medium without cells, corrects for serum-contributed LDH activity in the cell culture medium as well as the presence of phenol red. Each day after infection, 1 plate from each cell line and both temperatures for the HaCaT cell line was removed from the incubator for use in the LDH assay and chlamydial growth curve study. In the LDH assay, lysis solution was added to correct for volume and control peak LDH release.
Chlamydial Growth Curves
The ethanol was removed and the plates were allowed to dry before being frozen at -70°C until use. Each image was separated into the red, green and blue components and the new image of the green component was selected. The results were displayed in a Microsoft Excel spreadsheet and the inclusion parameters of the chlamydia strains were compared.
Transmission electron microscopy (TEM)
Once confluent cells were trypsinized and 1 x 105 cells were seeded in 24 well plates (Greinier) in which 12mm round Thermanox coverslips (Nunc) were placed in each well. Cells were infected with 100 µl of chlamydial suspension at an MOI of 0.25, which was equivalent to 1 chlamydial EB per 4 cells to ensure the presence of both infected and uninfected exposed cells in each well. Cells were fixed with 2% gluteraldehyde in EMEM for 30 minutes, then washed with EMEM twice for 5 minutes.
MTT Assay
Ten microliters of MTT saline (5 µg/ml in PBS) was added to each well, and the cells were incubated for 2 hours and 30 minutes at the experimental temperature. The culture medium was aspirated and the purple formazan crystals were dissolved in 150 µl DMSO by pipetting up and down 5 times. This solution was transferred to a new 96-well plate and the absorbance data was read at a test wavelength of 570 nm with a reference wavelength of 630 nm.
TUNEL assay
The remaining inoculum was aspirated, replaced with 1 ml of CGM-E, and monolayers were incubated at 37°C or 33°C for 2 or 5 days. Slides were examined using bright-field microscopy to identify cells with fragmented DNA and DAB-positive nuclei; then switched to fluorescence (excitation wavelength 450–490 nm; emission wavelength 520 nm) and the same field of view was examined for the presence and location of C. For each field of view, DAB-positive cells were captured in a bright field photograph, and the same field of view captured under fluorescence.
Caspase detection in uninfected but exposed cells
On the same day, 2 x 104 ME-180 or HaCaT cells were seeded in the upper chambers of Transwell® Permeable Supports. In the late afternoon, transwell ® permeabilized supports were transferred to 24-well plates containing ME-180 or HaCaT cells incubated at 37 or 33°C. Two hundred microliters of staining solution (1µl FITC-VAD-fmk per 300µl CGM-E) was added to the wells of a fresh 24-well plate.
Statistical analyses
The membrane was placed on a glass slide with 2 drops of washing buffer and covered with a glass cover slip. For the positive caspase control, the coverslip was washed twice with 300 µl wash buffer for 5 min each directly in the original well of the 24-well plate. The round glass cover was removed from the well and placed cell side down on a glass slide.
RESULTS
Chlamydial growth curves
There was an overall significant difference between the strains for both area occupied by chlamydia and inclusions per 10 thousand cells (P<0.001 for both). There was an overall significant difference between the strains for both area covered by chlamydia and inclusions per 10 thousand cells (P = 0.001 and P = 0.019 respectively). In the HaCaT cells at 33°C, the area occupied by chlamydia increased from day 1 to day 5 (figure 4E).
Transmission electron microscopy
There were also highly significant differences between strains at each day post-infection (P < 0.001). In monolayers infected with serovar E, myelin numbers indicative of degenerated mitochondria were present at 3 hours post-infection (Figure 13B). In contrast, organelle integrity was maintained in monolayers infected with serovar L2 for a longer period of time—myelin levels were first observed 18 hours post-infection (Figure 14B).
MTT assay
Null hypothesis 7: There is no difference in the level of mitochondrial activity in HaCaT cells at 37°C when infected with different strains of C. Alternative hypothesis 7: There is a difference in the level of mitochondrial activity in HaCaT cells at 37°C when infected with different strains of C .Alternative hypothesis 8: There is a difference in the level of mitochondrial activity in HaCaT cells at 33 °C when infected with different strains of C.
Lactate dehydrogenase detection
Null hypothesis 8: There is no difference in the level of mitochondrial activity in HaCaT cells at 33 °C when infected with different strains of C. In HaCaT cells at 33 and 37 °C, the mean percentage of cytotoxicity did not exceed 2% and 4%, respectively, while when C. In general, there are differences in the percentage of cytotoxicity exerted on HaCaT cells at 37 °C by strains of C.
TUNEL Assay
There were approximately the same number of DAB positive cells in the infected and uninfected monolayers. When the incubation step was extended to 5 days, the inclusions were larger, but there was still no increase in the number of DAB positive cells except for serovar L3 which showed a slight increase in the number of DAB positive cells, while the serovar E OG strain showed a slight decrease. At high magnification (X 1000), small non-nuclear DAB positive particles were observed in ME-180 and HaCaT cells monolayers grown at 37°C, but not in HaCaT cells at 33°C.
Caspase detection
In the HaCaT cells at 33°C, there were on average between 1 and 2 caspase-positive cells per 200x field of view for the negative control and both serovar E and L2. Note the well-defined structure in the region of the RB attached to the inclusion membrane (circled). Note the cytopathic changes in the cytoplasm of cells 1 hour after infection (B) compared to the uninfected cells (A).
DISCUSSION
This again indicates a difference in the behavior of reference strains compared to fresh clinical isolates. A study by Schöier et al. (2001) reported an increase in the number of TUNEL positive uninfected cells in C. But only the reference strain L2 caused a significant increase in the number of inclusions per 10 thousand cells.
CONCLUSIONS
Determination of the physical environment of the Chlamydia trachomatis inclusion using ion-selective ratiometric probes. Interaction between a Trachoma strain of Chlamydia trachomatis and mouse fibroblasts (McCoy cells) in the absence of centrifugation. Sensitivity of immunofluorescence with monoclonal antibodies for detection of Chlamydia trachomatis inclusions in cell culture.
High-resolution mapping of serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis. Characterization of the Chlamydia trachomatis vacuole and its interaction with the host endocytic pathway in HeLa cells.
