Prasit Palittapongarnpim << Department of Microbiology, Faculty of Science, Mahidol University

Prasit Palittapongarnpim, M.D., Ph.D.
Associate Professor of Microbiology

B.Sc. (Medical Sciences, First Class honor), Mahidol University 1979
B.Sc. (Mathematics), Ramkamhang University 1991
M.D. (First Class honor) Mahidol University 1981
Certificate of Proficiency in Paediatrics, Chiangmai University 1989
Canadian International Development Agency Postdoctoral Scholarship to University of Alberta 1992
Thai Research Fund's Research Scholarship 1994-9
National Science and Development Agency's Research Carrier Development Award 1997-2002

E-mail: prasit@biotec.or.th

[Research interests] | [Collaborations] | [Publications] | [Students]

MICROBIOLOGY AND MOLECULAR BIOLOGY OF MYCOBACTERIA

Our researches include molecular epidemiology of tuberculosis (2,5-8,13,17-18, 22-24, 26-27,30), studies of the physiological consequences of the genetic polymorphism (1,14), discovery of drug targets, screening of natural compounds for antituberculous activity (9-10,12,15-16, 19-21) as well as developing better methods of antituberculous drug screening (11) and testing of drug candidates(3). We also involved in the development of methods for differentiating species of mycobacteria (25,29)

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Molecular epidemiology of tuberculosis in Thailand.

Southern hybridization with IS6110

	The standard methods for strain characterization of M. tuberculosis is Southern hybridization using the insertion sequence IS6110 as probe. Using the method we found that a significant proportion of M. tuberculosis in Thailand belonged to two families of strains, namely the Beijing family and the Nonthaburi family (fingerprints shown in Fig 1)(7,27). The Beijing family, some of which are also known as the W-like strains, has been reported world wide but is notoriously prevalent in China and nearby countries, including Thailand. A significant proportion of Thai isolates contained a single or only few copies of IS6110 (fingerprints shown in Fig 2). In these cases the discriminatory power of the standard method is not adequate. They must be differentiated by other methods such as spoligotyping, the Southern hybridization with probes that are specific to the 36 bp Direct Repeat and the Polymorphic GC-rich Repetitive Sequence (27) or VNTR typing (6). The Southern hybridization method was used to demonstrate transmission among inmates in a few prisons in Thailand (13,18) and to confirm relapses after treatment (8).
Fig 1 The IS6110-hybridization patterns of some M. tuberculosis isolates in Thailand. Lane 1 and 17 are the standard strains MT14323. Lane 2-6 are the members of the Nonthaburi group. Lane 8-12 and 14-16 are the members of the Beijing family.
Fig 2 The IS6110 -hybridization patterns of M. tuberculosis isolates in Thailand with single copy and a few copies of IS6110. They, together, contribute to almost half of the tuberculosis cases in Thailand.
VNTR typing
Three loci of VNTR of M. tuberculosis that contain the direct repetitive sequences have been cloned (31) and sequenced in our laboratory (26). One is within the coding sequence of the alpha-isopropyl malate synthase gene (leuA) (14). Another one was found at about 500 bp upstream from the coding sequence of the cyclopropanated mycolic acid synthase-2 gene (cma2) (26). The amplified products of the regions containing repetitive sequences were found to have different length in various strains of M. tuberculosis. In the case of leuA , the VNTR results in the production of polymorphic LeuA proteins (1,14). Using the sequences of the three VNTR as probes, we are able to identify 45 more putative VNTR loci (22) and confirmed that 39 of them are polymorphic with variable degrees (6), as exemplified in Fig 3. The degrees of polymorphism can be indicated in several ways, such as PIC (polymorphic information content= 1-Σpi2). Combination of the 10 highest polymorphic VNTR loci offered the same level of discriminating power as IS6110-RFLP for strain differentiation (6). The levels of PIC at different VNTR loci between each IS6110-RFLP group may be different. The phylogenetic tree generated from the results of VNTR typing confirmed the lineage distinction of the Beijing family as demonstrated by many other studies.

Fig 3 The amplified products of various strains of M. tuberculosis. The upper fragment is the amplified products of VNTR4155 (mpp8 segment) while the lower is of VNTR0595 (msx4 segment). The lanes on both sides are molecular markers.
Screening of natural compounds for antituberculous activity. Searching for new antituberculous drugs is a long term goal of our laboratory. At present, we involve in screening natural compounds for antituberculous activity. We currently used the Microplate Alamar Blue Assay (MABA) developed by Dr. Scott G. Franzblau, currently in University of Illinois at Chicago. The screening work is done in the National Center for Genetic Engineering and Biotechnology in Thailand Science Park. More than 20,000 samples have been screened with many interesting compounds being identified (15,19,21). The screening test is provided as a service to scientific communities. Our customers included the Gazi University (9-10,12,16,20) and WHO.
Identification of drug targets. Known antituberculous drugs act on a limited number of targets. We also work on identifying potential new drug targets and their validations, by computational methods, by working on isolates with unusual sensitivity to antibiotics and by comparing the expression profiles when exposed and unexposed to new active compounds (4).
Development of antituberculous screening methods. In collaboration with Dr. Scott G. Franzblau, we developed an in vitro assay for antituberculous activity based on the detection of green fluorescence protein (GFP), which is expressed by M. smegmatis acetamidase promoter (11). The promoter and the gfp gene is present in a recombinant plasmid, pFPCA1 and result in a fluorescence level much higher than the widely used pFPV2 (see Fig 4). The values of MIC of a few recombinant M. tuberculosis strains against 18 drugs determined by the GFP microplate assay were comparable to the MABA (11). The potentials of the reporter system to simplify screening tests for antituberculous activity are being explored.

Fig 4 The fluorescence microscopic image of recombinant M. tuberculosis H37Ra containing pFPCA1 (left) and their colonies in 7H11 agar containing kanamycin (middle), compared to the colonies of original H37Ra in 7H11 agar.
Identification of species of mycobacteria. Determining the species of mycobacteria is a laborious and time-consuming task that can be accomplished only in specialized laboratories. We have developed a PCR-based methods for determining the species by amplification and restriction enzyme analysis of the spacer between the 16S rRNA and 23S rRNA genes (25). Probes specific to the spacers of several species of mycobacteria have also been developed (29). The results of the probes specific to M. tuberculosis and M. avium perfectly conformed to results of Accuprobe (25).
Collaborations Collaborations with the National Center for Genetic Engineering and Biotechnology (BIOTEC) Our laboratory is in close collaboration with the Antituberculous Drug Discovery Laboratory with several personnel sharing projects and facility: Dr. Wandee Yindeeyoungyeon (PhD. Microbiology [Molecular Genetics], University of Georgia, Athens, Georgia, USA) e-mail: wandee@biotec.or.th Dr. Saradee Warit (Ph.D., Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), Manchester, United Kingdom) e-mail: saradee@biotec.or.th Dr. Therdsak Prammananan (Dr. rer. biol. hum., Medical School Hannover, Germany) e-mail: therdsak@biotec.or.th Dr. Nat Smittipat (Ph.D. Microbiology, Faculty of Science, Mahidol University) e-mail: nat.smi@biotec.or.th Dr. Kanchana Dokladda e-mail: kanchana.dok@biotec.or.th Dr. Kamolchanok Rukseree (Ph.D. Microbiology, Faculty of Science, Mahidol University) e-mail: kamolchanok@biotec.or.th Ms. Pamaree Billamas (M.Sc. Microbiology, Faculty of Science, Mahidol University) e-mail: pamaree@biotec.or.th Mr. Tada Juthayothin e-mail: tada.jut@biotec.or.th
Other Current Collaborators
Thai Collaborators
Department of Microbiology, Faculty of Medicine Siriraj Hospital , Mahidol University : Dr. Angkana Chaiprasert. (http://www.si.mahidol.ac.th/siweb_2007/department/microbiology/eng/dept_main.asp) Department of Pathology, Faculty of Medicine at Ramathibodi Hospital , Mahidol University : Mrs. Poonpilas Hongma Tuberculosis Division, Department of Disease Control, Ministry of Public Health: Mr. Somsak Rienthong, Mrs Dhanida Rienthong. National Institute of Health , Ministry of Public Health , Thailand : Dr. Benjawan Phetsuksiri. Department of Chemistry, Faculty of Science, Ramkamhang University: Dr. Apichart Suksumran. Department of Biology, Faculty of Science, Ramkamhang University : Dr. Wimol Chanchaem. Department of Microbiology, Faculty of Science, Prince of Songkla University : Dr. Varaporn Vudhakul. Department of Microbiology, Faculty of Science, Thammasat University: Dr. Sumalee Kondo.
International Collaborators
Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, USA : Dr. Scott G. Franzblau (http://www.tuberculosisdrugresearch.org) Stanford Center for Tuberculosis Research, Stanford University , USA : Dr. Peter Small (http://molepi.stanford.edu/tb.www.html ) Research Institute of Tuberculosis , Japan Antituberculosis Association, Japan : Dr. Norio Doi, Dr. Norio Yamada, and others (http://www.jata.or.jp/eindex.htm ) @ > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > @ back to top
Representative publications (1993 - present) Thong-On A,Sumittipat N, Juthayothin T, Yanai H, Yamada N, Yorsangsukkamol J, Chaiprasert A, Rienthong D, Billamas P, Palittapongarnpim P. Variable -number tandem repeats typing of Mycobavterium tuberculosis isolates with low copy numbers of IS6110 in Thailand. Tuberculosis. 2010, vol. 3, pp.9-15. Yorsangsukkamol J, Chaiprasert A, Prammananan T, Palittapongarnpim P, Limsoontarakul S, Prayoonwiwat N. Molecular analysis of Mycobacterium tuberculosis from tuberculous meningitis patients in Thailand. Tuberculosis (Edinb). 2009 Jul;89(4):304-9. Yindeeyoungyeon W, Likitwiwattanawong S, Palittapongarnpim P. Characterization of alpha-isopropylmalate synthases containing different copy numbers of tandem repeats in Mycobacterium tuberculosis. BMC Microbiol. 2009 Jun 9;9(1):122. Rukseree K, Thammarongtham C, Palittapongarnpim P. One-step purification and characterization of a fully active histiding-taggd Class II fructose-1, 6-bisphosphate aldolase from Mycobacterium tuberculosis. G Model. 2008 Rudeeaneksin J,Srisungngam S,Sawanpanyalert P,Sittiwakin T, Likanonsakul S,Pasadorn S, Palittapongarnpim P, Brennan J. P, Phetsuksiri B. Light Cycler TM real-time PCR for rapid detectionand quantitation of Mycobacterium leprae in skin specimens. FEMS Immunol Med Microbiol. (2008) 263–270 Tongsima W, Tongsima S, Palittapongarnpim P. Outlook on Thailand's Genomics and Computational Biology Research and Development. PLoS Comput Biol. 2008 Jul 25;4(7):e1000115. Chanchaem W, Palittapongarnpim P. The significance and effect of tandem repeats within the Mycobacterium tuberculosis leuA gene on alpha-isopropylmalate synthase. FEMS Microbiol Lett. 2008 Sep;286(2):166-70. Warit S, Stateva LI, Palittapongarnpim P. Cloning and heterologous expression of Cryptococcus neoformans CnSRB1 cDNA in Saccharomyces cerevisiae.Southeast Asian J Trop Med Public Health. 2008 May;39(3):484-91. Ruangrit U, Srikummool M, Assawamakin A, Ngamphiw C, Chuechote S, Thaiprasarnsup V, Agavatpanitch G, Pasomsab E, Yenchitsomanus PT, Mahasirimongkol S, Chantratita W, Palittapongarnpim P, Uyyanonvara B, Limwongse C, Tongsima S. Thailand Mutation and Variation Database (ThaiMUT).HUMAN MUTATION Mutation in Brief #1011, 29:E68-E75, 2008 (Online) Viratyosin W, Ingsriswang S,Pacharawongsakda E, Palittapongarnpim P. Genome-wide subcellular localization of putative outer membrane and extracellular proteins in Leptospira interrogans serovar Lai genome using bioinformatics approaches. BMC Genomics. 2008 Apr 21;9:181. Billamas P, Smittipat N, Juthayothin T, Thong-On A, Yamada N, Yanai H, Palittapongarnpim P.Evolution of some variable-number tandem repeat loci among a group of Beijing strains of Mycobacterium tuberculosis.Tuberculosis. 2007 Nov;87(6):498-501. Hongmanee P, Rukseree K, Buabut B, Somsri B, Palittapongarnpim P. In vitro activities of cloxyquin (5-chloroquinolin-8-ol) against Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2007 Mar; 51(3):1105-6. Bhoopong P, Palittapongarnpim P, Pomwised R, Kiatkittipong A, Kamruzzaman M, Nakaguchi Y, Nishibuchi M, Ishibashi M, Vuddhakul V. Variability of properties of Vibrio parahaemolyticus strains isolated from individual patients. J Clin Microbiol. 2007 May;45(5):1544-50. Jeerayut Chaijaruwanich, Jamlong Khamphachua, Sukon Prasitwattanaseree, Saradee Warit, Prasit Palittapongarnpim Application of Factor Analysis on Mycobacterium tuberculosis Transcriptional Responses for Drug Clustering, Drug Target and Pathway Detections. Lecture Notes in Computer Science 2006; 4093: 835-844. Chaiprasert A, Yorsangsukkamol J, Prammananan T, Palittapongarnpim P, Leechawengwong M, Dhiraputra C. Intact pks15/1 in non-W-Beijing Mycobacterium tuberculosis isolates. EID 2006; 112(5): 772-774. Smittipat N, Billamas P, Palittapongarnpim M, Thong-On A, Temu M, Thanakijcharoen P, Karnkawinpong O, Palittapongarnpim P. Study on the polymorphism of multiple loci of variable number of tandem repeats in Mycobacterium tuberculosis. J Clin Microbiol 2005; 43(10):5034-5043. Rienthong D, Ajawatanawong P, Rienthong S, Chaiprasert A, Smithtikarn S, Pasakorn A, Palittapongarnpim P. Restriction fragment length polymorphism study of nation-wide samples of Mycobacterium tuberculosis in Thailand, 1997-1998. Int J Tuberc Lung Dis 2005; 9(5): 576-581. Yoshiyama T, Yanai H, Rhientong D, Palittapongarnpim P, Nampaisan O, Supawitgul S, Uthaivorawit W, Mori T. Development of acquired drug resistance in recurrent tuberculosis patients with various previous treatment outcomes. Int J Tuberc Lung Dis 2004; 8(1):1-8. Tosun F, Aky?z Kızılay ?, Şener B, Vural M, Palittapongarnpim P. Antimycobacterial Activity of Some Turkish Plants Pharmaceutical Biology 2004;42(1):39-43. Tosun F, Aky?z Kızılay ?, Şener B, Vural M, Palittapongarnpim P. Antimycobacterial screening of some Turkish plants J Ethnopharmacology 2004;95(2-3):273-275. Changsen C, Fransblau S, Palittapongarnpim P. Improved green fluorescent protein reporter gene-based microplate screening for anti-tuberculosis compounds by utilizing an acetamidase promoter. Antimicrob Agents Chemother 2003;47(12):3682-3687. Erdemoglu N, Sener B, Palittapongarnpim P. Antimycobacterial Activity of Taxus baccata. Pharmaceutical Biology 2003;41(8):614 – 615. Sretrirutchai S, Silapapojakul K, Palittapongarnpim P, Phongdara A, Vuddhakul V. Tuberculosis in Thai prisons: magnitude, transmission and drug susceptibility. Int J Tuberc Lung Dis 2002 Mar;6(3):208-14 Chanchaem W, Palittapongarnpim P. Variable number of tandem repeats result in polymorphic ?-isopropylmalate synthase in Mycobacterium tuberculosis. Tuberculosis 2002; 82: 1-6. Seephonkai P, Isaka M, Kittakoop P, Palittapongarnpim P, Kamchonvongpaisan S, Jaturapat A, Sangvichien E, Tanticharoen M, Thebtaranonth Y. Evaluation of antimycobacterial and antiplasmodial activities and cytotoxicity of preussomerins isolated from the lichen fungus Microsphaeropsis sp. BCC3050. Planta Medica 2002; 68: 45-48. Orhan I, Sener B, Atici T, Palittapongarnpim P. In vitro Antimycobacterial potential of some fresh-water macroalgae and aqueous plants. Pharmaceutical Biology 2002;40(8): 568–569. Fang Z, Kenna DT, Doig C, Smittipat DN, Palittapongarnpim P, Watt B, Forbes KJ. Molecular evidence for independent occurrence of IS6110 insertions at the same sites of the genome of Mycobacterium tuberculosis in different clinical isolates. J Bacteriol. 2001 Sep;183(18):5279-84. Mongkonjit S, Saringcaringul S, Smittipat N, Thong-On A, Palittapongarnpim P. An evidence for the transmission of tuberculosis in a prison in Nakhon Ratchasima, Thailand. Int J Tuberc Lung Dis 2001;5(6):586-7. Boonphong S, Kittkoop P, Isaka M, Palittapongarnpim P, Jaturapat A, Danwisetkanjana K, Tanticharoen M, Thebtaranonth Y. A new antimycobacterial, 3b-acetoxy-15a,22-dihydroxyhopane from the insect pathogenic fungus Aschersonia tubulata. Planta Med 2001;67:279-281. Deliorman D, Ergun F, Sener B, Palittapongarnpim P. Evaluation of antimycobacterial activity of Viscum album subspecies. Pharmaceutical Biol 2001; 39(5):381-383. Nilanonta C, Isaka M, Kittakoop P., Palittapongarnpim P, Kamchonwongpaisan S, Pittayakhajonwut D, Tanticharoen M, Thebtaranonth Y. Antimycobacterial and Antiplasmodial Cyclodepsipeptides from the Insect Pathogenic Fungus Paecilomyces tenuipes BCC 1614. Planta Medica 2000; 66: 1-2. Smittipat N, Palittapongarnpim P. Identification of variable number of tandem repeats in the Mycobacterium tuberculosis. Tubercle Lung Dis 2000;80(2):69-74. Kremer K, van Soolingen D, Frothingham R, Haas WH, Hermans PWM, Martin C, Palittapongarnpim P, Plikaytis BB, Riley LW, Yakrus MA, Musser JM, van Embden JDA. Comparison of molecular epidemiologic markers for Mycobacterium tuberculosis complex: an interlaboratory study on differentiation and reproducibility. J Clin Microbiol 1999;37(8):2607-2618. Fang Z, Doig C, Kenna DT, Smittipat N, Palittapongarnpim P, Watt B, Forbes KJ. IS6110-Mediated deletions of wild-type chromosomes of Mycobacterium tuberculosis. J Bacteriol 1999 Feb;181(3):1014-20 Sansila A, Hongmanee P, Chuchottaworn C, Rienthong S, Rienthong D, Palittapongarnpim P. Differentiation between Mycobacterium tuberculosis and Mycobacterium avium by amplification of the 16S-23S ribosomal DNA spacer. J Clin Microbiol 1998 Sep;36(9):2399-403. Namwat W, Luangsuk P, Palittapongarnpim P. The genetic diversity of Mycobacterium tuberculosis strains in Thailand studied by amplification of DNA segments containing direct repetitive sequences. Int J Tuberc Lung Dis 1998 Feb;2(2):153-9. Palittapongarnpim P, Luangsook P, Tansuphaswadikul S, Chuchottaworn C, Prachaktam R, Sathapatayavongs B. Restriction fragment length polymorphism study of Mycobacterium tuberculosis in Thailand using IS6110 as probe. Int J Tuberc Lung Dis 1997 Aug;1(4):370-6 Chowdhury, ZU; Palittapongarnpim, P. DNA fingerprinting of Mycobacterium tuberculosis isolates from Bangladesh by polymerase chain reaction. J. Infect. Dis. Antimicrobial agents 1997;14(1):1-3. Lappayawichit P, Rienthong S, Rienthong D, Chuchottaworn C, Chaiprasert A, Panbangred W, Saringcarinkul H, Palittapongarnpim P. Differentiation of Mycobacterium species by restriction enzyme analysis of amplified 16S-23S ribosomal DNA spacer sequences. Tuber Lung Dis 1996 Jun;77(3):257-63 Palittapongarnpim P, Rienthong S, Panbangred W. Comparison of restriction fragment length polymorphism of Mycobacterium tuberculosis isolated from cerebrospinal fluid and sputum: a preliminary report. Tuber Lung Dis 1993 Jun;74(3):204-7 Palittapongarnpim P, Chomyc S, Fanning A, Kunimoto D. DNA fragment length polymorphism analysis of Mycobacterium tuberculosis isolates by arbitrarily primed polymerase chain reaction. J Infect Dis 1993 Apr;167(4):975-8. Palittapongarnpim P, Chomyc S, Fanning A, Kunimoto D. DNA fingerprinting of Mycobacterium tuberculosis isolates by ligation-mediated polymerase chain reaction. Nucleic Acids Res 1993 Feb 11;21(3):761-2. @ > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > @ back to top
Students Current Students Ph.D. students >> Ms. Praveenuch Penpassakarn >> Ms. Pornpen Tantivithayakul, M.D. M.Sc. students >> Mr. Sathish Moosa
Graduates Ph.D. students Dr. Wimol Chanchaem 2000 now in Department of Biology, Faculty of Science, Ramkamhang University Dr. Nat Smittpat 2003 now in National Center for Genetic Engineering and Biotechnology Dr. Chartchai Changsen 2004 now in Department of Microbiology, Faculty of Science, Mahidol University Dr. Kamolchanok Rukseree 2007 now in Institute of Tuberculosis Research, University of Illinois at Chicago, USA
M.Sc. students Mrs. Poonpilas Hongmanee 1995, Department of Pathology, Faculty of Medicine Ramathibodi Hospital , Mahidol University Dr. Wises Namwat 1996, Ph. D. ( Osaka University ) Department of Microbiology, Faculty of Medicine, Khon Kaen University Mr. Praphan Luangsuk 1997, Department of Microbiology, Faculty of Medical Technology, Chiangmai University Mrs. Arunnee Sanga 1997, Department of Microbiology, Faculty of Medical Technology, Khon Kaen University Ms. Hataichanok Saringcaringul , Colorado State University , Fort Collins , Colorado. Ms. Waralee Torsuwan 1999, Department of Biology, faculty of Science, Payap University , Chiangmai. Mr. Mansuet Michael Temu 1999, National Institute for Medical Research , Tanzania. Mrs. Dhanida Rienthong , Tuberculosis Division, Department of Disease Control, Ministry of Public Health. Ms. Pamaree Bilamas 2003, National Center for Genetic Engineering and Biotechnology. Ms. Supaporn Likitwiwatanawong 2003, Institute of Molecular Biology and Genetics, Mahidol University. Ms. Arunee Thong-on 2005, Department of Microbiology, Faculty of Science, Ubonratchathani University Ms. Siribun Panapruksachat 2005, Medical Molecular Biology Unit, Faculty of Medicine Siriraj Hospital, Mahidol University.
International Trainees Md. Zafar Ullah Chowdhury 1996, NIPSOM, Mohakhali , Dhaka , Bangkladesh Md. Akhtarun Nahar 1998, Microbiology Department, Sir Salimullah Medical College , Mitford , Dhaka , Bangkladesh Ms. Steena Tong 1999 Grantham Hospital , Hong Kong , China. @ > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > @ back to top