Snake Venom Research
Snake venom poisonings account for over 125000 deaths worldwide, mostly in developing countries. The most effective treatment is the administration of potent antivenoms (AVs) which are specific for the venoms in each locality. Surprisingly, effective AVs for many parts of Africa and Asia have not been prepared. We have worked out a highly effective yet simple immunization protocol which is now widely used and is recommended by WHO. Currently, we are working on the production of a ‘universal’ antivenom against various neurotoxic snake venoms. A simple, efficient and economical fractionation scheme for large scale fractionation of equine plasma has also been studied.
Snake venoms are rich sources of biologically active compounds, the study of which can lead to the elucidation/characterization of natural pathways/components. These molecules may also be useful as lead compounds in drug development. For example, venom proteins with RGD (Arg-gly-asp) sequence of disintegrins have been used in the design of molecules with antiplatelet activity. It is therefore likely that proteins with important and novel biological activities can be found in the diverse population of venomous snakes in Thailand, and this is one of the areas we are working on.
The Laboratory of Immunology has been involved in developing simple immunodiagnostic testsbased on immunochromatography (IC) for infectious agents that affect Thais and other people of this region. The followings are examples.
1) An IC test for the diagnosis of human Pythiosis, an emerging infectious disease caused by Pythium insidiosum. Due to lack of early diagnosis, the infection causes tissue necrosis of the limbs, often requiring amputation and sometimes resulting in death. The test kit enables early diagnosis and has saved lives and limbs of many patients.
2) Development of an IC test for Fasciola gigantica, a liver parasite in ruminants. This disease causes economic loss of about half a billion Baht annually. The simple IC test of blood specimens, in place of fecal egg count, leads to treatment with anthelmintics which results in higher product value and reduces the spread of the disease.
3) We have also studied the use of biotinylated liposome entrapped with chemiluminescent compounds as a generic signal generator to enhance the detection of specific interactions of antigen-antibody and of nucleotide hybridization. We are also developing tests based on fluorescence polarization immunoassay (FPIA) for the detection of drugs and food-borne toxins.
Microbial Degradation of Pollutants
Actinobacteria are a group of gram-positive, GC-rich bacteria which are well-known producers of biologically active compounds. Many current anticancer and antibiotic drugs are made from actinobacteria, many of them originally identified in tropical soils. We are exploring bioactive products isolated from Actinobacteria which have a wide spectrum of biological activities and numerous therapeutic applications.
Interest in the microbial biodegradation of pollutants has intensified in recent years as mankind strives to find sustainable ways to clean-up contaminated environments. Our newly isolated microorganisms was found to utilize Phthalate esters as carbon source. Insights into molecular biology of key biodegradative pathways to efficiently degrade these toxic compounds by genetically engineered microorganisms is being studied.
Lipases have evolved to be efficient catalysts for lipolytic reactions involving the hydrolysis of ester linkages of mono, di and triglycerides. There are a number of industrial applications of lipases. Lipases can also be used to conduct transesterification reactions producing useful biodiesel from vegetable oils. The conditions that lipases are exposed to in these industrial applications are far from their natural environment. Protein engineering is a useful and necessary tool for improving the lipolytic performance of the lipases i.e. thermo-stability and surface expression of lipase.
Antibiotic resistance in opportunistic pathogenic bacteria
The group of opportunistic pathogens including Stenotrophomonas maltophilia and Pseudomonas aeruginosa are of interested due to it is considered as increasingly nosocomial pathogen and its intrinsic antimicrobial resistance to a variety of antibiotics. These bacteria are reported to be associated with infections of immunocompromised individuals, particularly for patients who have been admitted in hospitals for long period. It causes meningitis, endocarditis, septicemia, infections of respiratory tract, urinary tract, and gastrointestinal tract. Our research is focused on identification of novel genes that required for acquired multidrug resistance as well as resistance to disinfectants in these bacteria.
Oxidative stress response and pathogenicity of plant pathogenic bacteria
The research has been working in Xanthomonas campestris pv. campestris, a causative agent of black rot on cruciferous plants, one of the most serious diseases worldwide. Oxidative stress has been recognized as an initial component of plant defense system to combat pathogens during host-microbe interaction both compatible and incompatible ones. The genes that involved in oxidative stress response and protection in this bacterium will be isolated and functionally characterized to identify genes essential for bacterial pathogenicity. The knowledge would be applied to efficiently control this plant pathogen without the use of toxic chemicals.
Response of human pathogens to oxidative stress and drug resistance mechanism
Pseudomonas aeruginosa is one of the most important opportunistic bacterial pathogens that cause a variety of diseases, often serious, in patients with impaired immune response or in a critical condition. The incidence of P. aeruginosa nosocomial infection has increased worldwide. The ability of a pathogen to successfully invade the host is often linked to its capacity to overcome host defense including the oxidative burst during the host-pathogen interactions. We are interesting in the study of the defense machinery of P. aeruginosa against oxidative stress and how the expression of the defense systems are regulated. Example of the defense systems are OhrR-ohr and OspR-gpx.
Recently, we have shown in Acinetobacter spp, another human pathogen, the link between exposure to a biocide and an increase in both the resistance to the biocide and oxidative stress resistance by the exposed bacteria. The detailed molecular mechanism of how the cross-protection occurred is under investigation.
Knowledge of how these microorganisms sense and overcome these stresses will help unravel the complexities of the infection and disease processes in humans.
Iron homeostatsis in pathogenic bacteria
We are interested in the regulation of iron homeostasis and oxidative stress response in Agrobacterium tumefaciens, a plant pathogen that causes crown gall tumor disease and belongs to the alpha-proteobacteria. Iron is an essential nutrient for nearly all forms of life but excessive amounts of iron can damage cells through the production of highly deleterious hydroxyl radicals via the Fenton reaction. Therefore, intracellular iron levels are tightly regulated and mostly occur at the transcription level. Iron regulation in alpha-proteobacteria very differs from most other bacteria. The transcriptional regulators RirA (rhizobial iron regulator) and Irr (iron response regulator) evolved to function in the regulation of iron-responsive genes involved in iron uptake, storage and utilization for maintaining iron homeostasis.
Iron deprivation and oxidative bursts are environmental stresses that bacteria inevitably encountered during interaction with their host. Bacterial pathogens have to overcome these stressful conditions in order to survive and successfully cause diseases. A. tumefaciens can serve as a model to study the roles RirA and Irr during host-pathogen interactions in members of alpha-proteobacteria including related animal and human pathogens. Understanding the important roles of RirA and Irr on the virulence of bacteria, in controlling iron homeostasis and stress response, and their mechanisms of gene regulation may provide a potential target for controlling diseases.
The research particularly focuses on the following questions: 1) How are genes regulated at the molecular level by RirA and Irr in response to metals? 2) Structure-function relationship in RirA and Irr proteins. 3) How do cells sense and respond to peroxide stress and nitric oxide stress via RirA and Irr? 4) How are RirA, Irr and other transcriptional regulators interacted to mediate the regulation of gene expression in response to diverse stressful conditions?