Pseudomonas aeruginosa toxin ExoS: analysis of interaction with 14-3-3 and the intracellular
signal trandsduction pathways involve in ExoS mediated cell death.
Pseudomonas aeruginosa is a gram-negative opportunistic pathogen, affecting patients with Cystic Fibrosis, AIDS, severe burns, neutropenia and ocular infections. Using type III secretion system four toxins from P. aeruginosa named ExoT, ExoU, ExoY and ExoS are translocated into the eukaryotic cell cytosol. ExoS is a bifunctional toxin which possesses N-terminal domain Rho GTPase Activity protein (GAP) activity, which disrupt actin microfilament structure in vitro and the C-terminal domain has been shown to contain an ADP-ribosylating activity, which is lethal to eukaryotic cell by blocking receptor-stimulated Ras activation in vivo. Previously in our lab following works has been done;
· ExoS modifies and thus blocks activation of Ras, a small GTP binding protein, in vivo.
· Further, we have identified 14-3-3, a co-factor, as necessary for the ADP-ribosylation activity and also has shown that the binding site for 14-3-3 is situated in the C-terminal part of ExoS.
· During analysis of the related protein ExoT it has been observed that ExoT induce disruption of actin microfilaments in the cell similar to that of an ADP-ribosylation defective ExoS. ExoT exhibits GAP activity on RhoA in vitro, a logical explanation for the observed disruption of actin cytoskeleton in vivo.
I am interested to find more about the interaction between 14-3-3 and ExoS and the intracellular signal transduction pathways involve in ExoS mediated cell death.
The overall importance of this analysis is to increase our understanding of P. aeruginosa infection at a molecular level which may establish the basis of therapeutic strategies and treatment of P. aeruginosa infections, which may improve the quality of life for those with immuno-compromised defense mechanisms, such as patient with cystic fibrosis and leukemia.
Specific goals:
A. During bacterial infection by Pseudomonas aeruginosa, our observations has been shown that the normal proto-oncogene Ras activity is
disturbed. How this disturbance affects intracellular signal transduction pathway?
This part of our research has importance in developing medical treatments for immunocomprimised hosts, such as Aids and cystic fibrosis
patients.
B. To pin point the interaction between ExoS and 14-3-3 down to the last amino acid at C terminal end.
Previous publication on this project:
1. A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo.Eur J Biochem. 2002 Oct;269(20):4921-9. Henriksson ML, Francis
MS, Peden A, Aili M, Stefansson K, Palmer R, Aitken A, Hallberg B.
2. Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo.Biochem J. 2002
Nov 1;367(Pt 3):617-28. Henriksson ML, Sundin C, Jansson AL, Forsberg A, Palmer RH, Hallberg B.
3. Exoenzyme T of Pseudomonas aeruginosa elicits cytotoxicity without interfering with Ras signal transduction.Cell Microbiol. 2001 Apr;3(4):237-46.
Sundin C, Henriksson ML, Hallberg B, Forsberg A, Frithz-Lindsten E.
4. 14-3-3 proteins are required for the inhibition of Ras by exoenzyme S.
Biochem J. 2000 Aug 1;349 Pt 3:697-701. Henriksson ML, Troller U, Hallberg B.
5. Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras. Biochem J. 2000 Apr 1;347 Pt 1:217-22. Henriksson ML, Rosqvist R, Telepnev M, Wolf-Watz H, Hallberg B.
Another previous Research:
Mitochondrial ribosomes in a trypanosome
Trypanosomatid protozoan parasites of the order Kinetoplastida, such as Trypanosoma and Leishmania, are of considerable medical and veterinary importance. The related trypanosome, Crithidia fasciculata, which is my research target, is a well-studied laboratory model contributing much to understanding trypanosome biology.
The nature, and even the existence, of trypanosome mitochondrial ribosomes (mito-ribosomes) has been the subject of some debate for several reasons. For example, the mito-ribosomal RNAs (mito-rRNAs) in trypanosomes are among the smallest rRNAs known and mito-ribosomes have proved elusive to isolate or define. Further, some key early reports have disagreed on the response of whole trypanosomes and mitochondrial fractions to cycloheximide and chloramphenicol, drugs traditionally used to differentiate mitochondrial and cytosolic translation. All this has served to raise doubts as to the role, and even the presence, of mito-ribosomes in these parasites.
We investigated this further in the insect trypanosome, Crithidia fasciculata. Analysis of sucrose gradients using parasite lysates, mitochondrial ribosomal RNA co-sediments at approximately 35S with nascent peptides synthesized in the presence of the cytosolic translational inhibitor, cycloheximide. Co-sedimenting peptides in this peak are much reduced when the parasites are treated with the bacterial translational inhibitor, chloramphenicol. In CsCl gradients this peak resolves at a buoyant density of 1.42 g/cm(3), a value typical for mito-ribosomes. Electron microscopy of peak material shows particles smaller than cytosolic ribosomes, but with characteristic ribosomal shapes. We propose that these particles represent the parasite's mitochondrial ribosomes.
Molecular typing of Nesseria gonorrhoeae isolated from commercial sex workers (CSWs) in Dhaka City
Sexually transmitted infections (STIs), including gonorrhoea, represent a major public health problem in the developing countries. In Bangladesh, the prevalence of gonoccoccal infections among the general as well as high risk population is not well documented. Neisseria gonorrhoeae is the aetiological agent f gonnorrhoea, a sexually transmitted disease of urogenital tract. The most commonly affected organs are uterus, cervix, rectum, and pharynx. The consequences of gonoccoccal infections are urethritis, proctitis, epididymitis, salpingitis, oophoritis, vulvovaginitis, pelvic inflammatory disease and infection of rectum. Epidemiological studies of gonnorrhoea have been enhanced by gonococcal typing system, which permits the tracing of individual gonococcal phenotypes in population. Here we have done molecular typing of bacteria using PCR-RFLF and PFGE analysis. Some 40 N. gonorrhoeae isolates collected from commercial sex workers (CSWs) were genotyped by PCR-RFLP of por gene using MspAII and PFGE analysis of whole genome using XbaI. Combination of both typing methods provided greater discrimination among these isolates. However the clonal relationship was also studied. Using PCR-RFLP analysis of por gene, 28 PIB strains exhibited 3 polymorphic forms and 12 PIA strains exhibited 2 polymorphic forms. In PFGE analysis, 26 isoates belongs to 7 distinct type and 14 isolates with distinct patterns were obtained. Out of the 40 isolates, 18 isolates are found to derive from common ancestral origin. The remaining 22 strains were heterogeneous.
Current Research:
Ovarian Cancer: Aim at Cell Signalling Pathways
Ovarian cancer is the most frequent cause of gynaecologic cancer-related mortality in women. The overall 5-year survival of ovarian carcinoma is less than 30%, creating a pressing need to understand its biology and develop effective and less toxic therapies for ovarian cancer. Ovarian cancer is associated with a poor prognosis largely due to the advanced stage of disease at the time of diagnosis. These advanced stage tumors typically have peritoneal metastases along with ascites fluid accumulation rendering them principally inoperable and unresponsive to current treatment options. Ovarian carcinomas metastasize by attaching to and invading through the mesothelial lining of the peritoneal cavity. A practical and effective screening program for early detection of ovarian cancer would therefore be a helpful mean. The techniques currently available-transvaginal ultrasound and CA125 (a biomarker of ovarian cancer) serum assays-are problematic for a number of reasons. For example, CA125 is not consistently elevated in serum from patients with early-stage disease and may be elevated in patients with benign gynecologic diseases.
Research has shown that many signal transduction pathways in mammalian cells, which process chemical signals from the cellular environment through the membrane, contain many components that, when altered in quantity or structure, can lead to cancer growth. For example, signal transduction pathway associated with a wide range of malignant behaviours, such as proliferation, cell adhesion, and oncogenic transformation. Such as one of the important pathway is the phosphatidylinositol 3-kinase (PI3-kinase) pathway. The activated PI3-kinase pathway can allow cells to avoid apoptosis (programmed cell death) which is required for healthy growth and development of organisms. A subform of apoptosis known as anoikis, occurs when a cell is dissociated from the matrix as they do in metastasis. Cells have to bypass the process of anoikis to survive. We are in process on investigating cell signal transduction pathways that involve in the development of ovarian cancers by using various drugs and toxin treatment in ovarian cancer cell lines.
The more details about our research will be appearing soon.