Carbon and energy metabolism in Chlamydia trachomatis

by Iliffe-Lee, Emma

Introduction C hlarnydiae Intracelluar Parasitism Biochemistry: Energy and Carbon Metabolism in Chiarnydiae Materiab and Methods Material s Chlamydia trachomatis strains and propagation Ce11 lines and culture conditions Preparation of RB extracts for enzyme assays E. coli strallis used for molecular cloning Construction of degenerate oligonucleotide primers Molecular cloning of C. trachomalis gap, pgk, pyk and zwf a) Construction of probes b) Colony blot hybridization DNA sequencing RT-PCR E. coli strains used for complementation and enzyme studies E. coli culture media Constructionof expression vectors Preparation of competent fi. coli for electroporation Complementationstudies for CTGAPDH, CTPGK, CTPK and CTZWF Preparation of bacterial ce11 extracts for enzyme assays Crude GAPDH? PGK, PK and ZWF enzyme assays Molecuiar clonhg sequencing, and expressionof C. trachomatispfpA mdpfpB Crude ATP-PFK and PPi-PFK enzyme assays Expression and purification of C. trachomatis PK Kinetic analysis of C. frachomatis PK Quantification of glycogen and glucose Incorporation of radiolabeled glucose or glutamate into glycogen of uninfected and infectedoHeLa cells Nucleotide pool rneasurements Infectivity titration assay Results A. Energy Metabolism in C.trachomaris 1. Identification, cloning and charac terization of energy-producing genes in C. trachomatis a) Enzyme assays with crude RB extracts b) Cloning of C. trachomatisgap, pgk and pyk genes C) Characterization of C. trachomatis gap, pgk, pyk and zwf ORFs Southem hybridization Stage-specific expression of C. trachomatis gap, pgk, pyk and mf genes using RT-PCR Complementation studies In vitro enzyme analysis of C. îruchornatis GADPH, PGK, PK and ZWF recombinant enzymes Cloning and characterization of C. trachomatis p/A andpfpB a) Cloning and sequence analysis of C. trachomatispfpA and pfpB genes b) In vitro enzyme anaiysis of C. trachomatis PFPA and PFPB recombinant enzymes RT-PCR analysis of C. trachomatis metabolic genes Enzyme Studies of C. trachowmtis Pyruvate Kinase Expression and purification of CTPK Kinetic analysis a) Activity as a fiuiction of enzyme concentration b) pH optima c) Cofactor requirements d) Nucleotide specificity e) PEP kinetics in the absence and presence of activators f) ADP kinetics in the absence and presence of F26BP g) F26BP kinetics h) Inhibitors i) Effect of F26BP and various inhibitors on CTPK activity Carbon Metaboüsm ia C. Iracliromutis Metabolic pathways in C. trachomatis as inferred tiom the genome sequence Effect of culture conditions containkg various carbon substrates and concentrations on the NTP pool size Effect of various culture conditions on the yield of infectious chlamydia1 EBs Effect of various carbon conditions on glycogen stores of HeLa and C. trachomatis-infected HeLa cells Incorporation of D-W-"C] glucose or L-I[I-'~c] glutamite into glycogen in uninfected and C. trachomatis-infected HeLa cells Evaluation of the expression of C. trachomatis L2 genes involved in carbon metabolism using RT-PCR Discussion 1. Energy and Glucose Metabolism in C. trachomatis 2. Kinetics of C. trachomatis Pyruvate Kinase 3. Glycogen and Carbon Metabolism 4. Summary References Appendix 1. List ofabbreviations