Technion - Israel Institute of Technology, Elyachar Central Library

Transcription

1

2 ' ' Index "y Abstract 1 Abbreviations and units 2 1. Scientific background Vibrio cholerae Chironomidae Arthropods as vector of human disease Research objectives 21.? 3. Materials and Methods List of reagents and their suppliers Buffers and culture media 23 " 3.3. Solutions for working with DNA Solutions for working with proteins 26 Methods.."29 f 3.5. Chironomids Bacteria, bacterial identification, and DNA related procedures Biological assays EMDF identification related procedures Characteristicsof the egg mass and its degradation products Transport experiments set up Results 38 y 4.1. Egg mass degradation assay Egg mass degradation factor is secreted by stationary phase cultures 40 t 4.3. Purification and identificationof the EMDF Confirmation of identification 46 * 4.5. Amino acid sequence comparison of HAP 48

3 Index Continued 4.6. Initial characterization of chironomid egg mass composition 51 v 4.7. Cholera toxin effect on hatching from the egg mass Collection of adult chironomids and their egg masses Isolation procedures of V. cholerae from chironomids Assessment of adult chironomid percentage harboring V. cholerae Aerial transfer demonstration of V. c/70/9/ze in the field 60 <f Chironomid adults as transporters of V. cholerae 65 ז A laboratory simulation: adults as carriers of \/. cholerae Field study in notrhern India Discussion Development of the egg mass biodegradation assay Egg mass degradation correlation to stationary phase culture Expression, purification, and identificationof the EMDF Confirmationof the identification of EMDF as HAP Conservation of HAP in aquatic bacteria based on sequence similarity and actual purification The chironomid egg mass Cholera toxin and egg masses Possible role of EMDF in V. <?/?0/e/ze survival in the environment Potential involvement of the chironomid egg masses in disease caused by V. cholerae Presence of V. c/70/erae on adult flying chironomids 80 י Isolation of V. cholerae from adult flying chironomids and its identification 82 t Adult chironomids as transpotrers of environmental V. cholerae strains The potential of chironomids as transpotrers of pathogenic V. cholerae Mechanism of V.c/70/e/3e transpotr by chironomids 85 י

4 Index Continued Magnitude of the aerial transport of V. cholerae Additional observations on the capacity of adult chironomids to carry and transfer V. cholerae 87 ר Adult chironomids as transporters of water associated pathogens Field study in northern India Suggestions for further research 90 y Concluding remarks 90 A / 6. References 92 Appendix 98 Article 1 98 Article II 103

5 Listof Figures Figure 1: Cholera worldwide * Figure 2: Subtyping of V. choleraeq\ 7 Figure 3: Putative conserved domains in the hap gene of V. cholerae 12 Figure 4: Organisms suspected of harboring V. cholerae 15 י. Figure 5: Evolutionary treeof the Culicimorpha 16 Figure 6: Life cycle of chironomids 17 Figure 7: Swarming of non biting midges 17 Figure 8: Collectionof egg masses 30 Figure 9: Conceptual drawingof the field transport experiment 37 Figure 10: Sizeof the nets used in comparison to adult chironomids 37 "' Figure 11: Egg mass degradation under the microscope 39 ר'* Figure 12: Egg mass degradation bioassay in 96 well microplate 40 Figure 13: Growth curve of V. choelrae 09 and EMDF activity of the culture supernatant 41 Figure 14: Gel filtration chromatogramof the soluble proteins secreted from V. cholerae 43 Figure 15: SDS PAGE stained with CBB of fraction containing EMDF activity 44 Figure 16: Identificationof the EMDF protein as HAP from V. cholerae 45 * Figure 17: Hemagglutination and proteolytic activities of fractions obtained from gel exclusion chromatography 46 Figure 18: Analysis of ha/p null mutant and its parental strain supernatant effect on chironomid egg masses 48 Figure 19: Partial amino acid alignment of proteolytic enzymes with high similarity to HAP 50 Figure 20: Selective stainingof egg masses 52 * Figure 21 : Chironomid egg mass stained by acid Schiff reagent 53 Figure 22: Selective staining of central fibers 53 Figure 23: Thin layer chromatographyof the egg mass degradation products 54 י\ Figure 24: Swarm of chironomids over WSP in northern Israel 56 Figure 25: V. cholerae streaked on a TCBS plate 57 Figure 26: V. cholerae identification by PCR 58 Figure 27: Map of the area surrounding the WSP 61 * Figure 28: Field transport experiment set up 61

6 Listof Figures conn. Figure 29: Chironomid adults visit and lay egg mass on the perimeter of the * experimental pools 62 Figure 30: V. cholerae CFU with time, experimental pools 63 Figure 31: Tubes of chironomid larvae found in the experimental pools 64 י, Figure 32: Other fresh water insects found in the experimental pools 90 days or more from set up 64 Figure 33: Egg masses laid by adult chironomids in a sterile vessel 66 Figure 34: GFP tagged V. cholerae 09 on an adult'chironomid 68 Figure 35: GFP tagged V. cho\eraeo\ on adult chironomids 69 Figure 36: Map of northern India 71 "' Figure 37: Female, adult chironomids attached to egg masses 88 r?.a <+ s r

7 Listof Tables Table 1 : Comparison of adult chironomids capture techniques applied in this study 29 v Table 2: Typical PCR reaction mix for the identification of V. cholerae 33 Table 3: Purificationof the EMDF produced by V.cholerae Table 4: Masses of peptides obtained from trypsin digestionof the purified EMDF by י, f,'f * 0 MALDI TOF 42 Table 5: Summary of hemagglutination and proteolytic activity of the purified EMDF produced by V. cholerae 09 47

8 n Abstract The bacterium Vibrio cholerae is the causative agentof the diarrheal disease cholera. Cholera is known not to persist in a chronic state in humans or animals. To date, over two hundred different serogroups are known,of which only two are pandemic causing : serogroups 01 and In contrast to many other human pathogens, the exact natural reservoirof these bacteria is not well characterized and is referred to as the "aquatic environment", Not least puzzling is the manner by which these pathogenic bacteria are transferred between separate bodies of water. The finding that points to the chironomids (Diptera: Chironomidae) as a possible natural reservoir of these bacteria in the aquatic environment is the basis of the work described herein. The egg masses of the non biting midge Chironomus sp. (Diptera) were shown to harbor and serve as a nutritive source for.v cholerae, thereby providing a natural reservoir for the organism. Here it is shown that.v cholerae 09 or 01 or 0139 supernatants lyses the gelatinous matrixof the chironomid egg mass, and inhibits eggs from hatching. The extra cellular factor responsible for the degradation of chironomid egg masses was puirfied from.v cholerae 09 (environmental), as well as from pathogenic strains of.v cholerae, and identifiedas the major secreted hemagglutinin / protease (HAP) of V. cholerae. The substrate of this enzyme, in the egg mass, was partially characterized. The substrate, in addition to its protein qualities, has unique glycan properties and as such is a proteoglycan. These findings show that HAP plays an important role in the interaction of.v cholerae and chironomid egg masses. In addition, this study contributes to the understanding of.v cholerae dissemination. The theoretical modes of dissemination of the bacteria include direct contact of human sewage with drinking water, seaborne currents, and mairne transportation. Herein, evidence is presented that flying, non biting midges (Diptera: Chironomidae), collected in the air, carry viable bacteria belonging to the non 01 non 0139 serogroups of.v cholerae. In simulated field experiments, the transfer of environmental V. cholerae by adult midges from the aquatic environment into water pools that did not contain the bacteira previously, was recorded. In 1 laboratory experiments, flying adult midges that emerged from.v cholerae (01 or 09 or * O139) inoculated water transferred the pathogenic bacteira from one laboratory flask to another. These findings show that aerial transfer by flying chironomids plays a role in the dissemination of.v cholerae in nature. 1