Solution 2241 midterm ii f14 v2

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Solution 2241 midterm ii f14 v2

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The chlamydial protease CPAF is expressed during the replicative phase of the chlamydial developmental cycle and is secreted into the lumen of the Chlamydia-containing vacuole called inclusion.

How the secreted protease is activated in the inclusion lumen is currently not fully understood. We have identified human serine peptidase inhibitor PI15 as a potential host factor involved in the regulation of CPAF activation.

Silencing expression as well as over expression of PI15 affected normal development of Chlamydia. Our findings shed light on a new aspect of chlamydial host co-evolution which involves the recruitment of host cell proteins into the inclusion to control the activation of bacterial proteases like CPAF that are important for the normal development of Chlamydia.

Introduction Chlamydia trachomatis is an obligate intracellular pathogen, which causes the eye disease trachoma and different sexually transmitted diseases.

Due to its intracellular life style and the dependency on host metabolites for replication, Chlamydia has evolved numerous unique characteristics that support its own survival without damaging the host cell.

One of the fascinating aspects of its life cycle is the establishment of a membrane-bound vesicle called inclusion inside which it completes its biphasic developmental cycle and at the same time utilizes it as a barrier to protect itself from cell autonomous defense mechanism of the host Fischer and Rudel, ; Fischer et al.

These bacteria use type III secretion system to release bacterial proteins into the inclusion membrane and beyond into the host cell cytosol and organelles Betts-Hampikian and Fields, On the other side, chlamydial proteins secreted via the bacterial type II secretion system including secreted proteases are kept within the inclusion and away from host cell cytoplasm, although release of such factors into the host cell cytosol has been suggested to occur during the late stage of the developmental cycle Chen et al.

One such widely studied chlamydial protease is Chlamydial Protease-Like Activity Factor CPAFwhich has initially been shown to cleave numerous host cell proteins in infected cells Pirbhai et al.

However, most of these CPAF targets turned out to be cleaved during cell lysis and not in intact cells Chen et al.

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Time specific expression and restricted localization of CPAF inside the inclusion lumen during nearly the whole chlamydial life cycle apparently does not allow cleavage of the host cell targets thus avoiding possible premature lysis of the host cell.

CPAF possesses autocatalytic activity and activates itself in a concentration dependent manner in vitro Huang et al. However, initiation of CPAF catalytic activity under very low concentrations as they are found inside the chlamydial inclusion is not completely understood.

Similarly, nothing is known about the molecular mechanism that controls CPAF activity within the inclusion lumen. During a microarray study of host cell transcriptome, we encountered a human peptidase inhibitor called PI15 also called as p25TI that was differentially regulated during chlamydial infection.

PI15 belongs to the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins CAP superfamily and is a weak serine protease inhibitor Koshikawa et al. PI15 is expressed in a variety of human and other animal tissues Yamakawa et al. Here we show that PI15 is recruited into the chlamydial inclusion and plays a crucial role in initial CPAF activation.

These cells were washed with PBS twice before Chlamydia infection. All the cell types were generally infected with C.

Chlamydia infectious progeny was determined by using EB preparations for a second round of infection and subsequent quantification of the secondary progeny by both immunoblotting and immunostaining techniques Prusty et al.

For immunostaining-based progeny calculation, total number of inclusions were calculated within a frame and were divided by total number of cells counted from DAPI staining to get the average inclusion numbers per cell.

The membranes were then probed with respective primary antibodies and subsequently with HRP-conjugated secondary antibodies.

Proteins were detected by enhanced chemiluminescence ECL, Amersham in the linear response range using X-ray films.

Immunofluorescence Microscopy Cells were seeded in well plates on glass coverslips and were infected with Chlamydia trachomatis for desired durations.

After washing, the cells were permeabilized using 0. Draq5 or DAPI was used for staining cell nuclei. For live cell imaging, HeLa cells were transfected with a construct expressing mCherry tagged PI15 or an empty vector.

Afterwards cells were imaged using Leica SP5 microscope. After washing, cells were permeabilized using 0. Cells were then incubated in 1: After washing three times with PBS a post staining fixation process was performed.

4 Chapter Assignments and Tests This document contains only library issues which are actively being considered by the Library Working Group, i. While some issues will eventually be elevated to official Defect Report status, other issues will be disposed of in other ways.
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All samples were imaged on Structured Illumination platform. To prevent spectral cross talk, all SIM data were acquired in alternating excitation mode.Start studying BUSMGT Midterm 1.

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Solution 2241 midterm ii f14 v2

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