Molecular characterisation of 12 Chlamydophila felis polymorphic membrane protein genes.
A group of genes thought to encode members of the unique chlamydial polymorphic membrane protein (pmp) family were recently described in the Chlamydophila felis genome. This study aimed to commence characterisation of a subset of 12 of these putative pmp genes by developing and using gene-specific real-time (Q)PCR assays to confirm their presence in a wide range of C. felis field isolates and laboratory strains, and to look for pmp mRNA expression during in vitro infection. Sequencing of 525-698 base pair regions of pmp genes 7, 9-11, 13-20 for two laboratory strains of C. felis and alignment with the published Fe/C-56 sequence found only a single nucleotide polymorphism present in pmp9. Following the development of gene-specific (Q)PCR assays, analysis of genomic DNA extracted from 40 C. felis field isolates and 4 laboratory strains found that all 12 pmp genes were represented in all cases. Reverse transcription (RT)-QPCR analysis of RNA extracted from cell cultures at 24 and 48 h post inoculation with 1 of 5 different strains of C. felis detected transcripts for all 12 pmp genes at both time points. Analysis of the relative levels of pmp gene transcription suggested that down-regulation of the expression of multiple C. felis pmp genes occurs between 24 and 48 h post inoculation. This study provides the first evidence that 12 of the putative pmp C. felis genes are transcribed during in vitro infection, and shows that these genes are present in a large range of C. felis field isolates and multiple passage laboratory-grown strains.
Harley
Ross
R
School of Clinical Veterinary Science, University of Bristol, Langford House, Langford, Bristol BS40 5DU, United Kingdom. ross.harley@bristol.ac.uk
Herring
Alan
A
Egan
Kathy
K
Howard
Pam
P
Gruffydd-Jones
Tim
T
Azuma
Yoshinao
Y
Shirai
Mutsumori
M
Helps
Chris
C
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002371:Cat Diseases / Q000382:microbiology*; D002415:Cats; D002460:Cell Line; D023501:Chlamydophila / Q000235:genetics*; D023521:Chlamydophila Infections / Q000382:microbiology / Q000662:veterinary*; D004269:DNA, Bacterial / Q000737:chemistry; D015536:Down-Regulation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D020641:Polymorphism, Single Nucleotide; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D016415:Sequence Alignment; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases