Real-time reverse transcription PCR for the quantification of the mntH expression of Salmonella enterica as a function of growth phase and phagosome-like conditions.
This article presents an experimental design for measuring the mRNA expression in Salmonella enterica of the mntH gene in phagosome-mimicking conditions. The expression of mntH was quantified by real-time reverse transcription PCR for different S. enterica strains of porcine origin under different biological growth conditions which mimicked the environment inside the phagosome. The expression of mntH and the different control genes (16S rRNA, rpoD and gmk) varied according to the growth phase. For mntH a maximum in the expression was detected in the early exponential phase. To obtain an accurate quantification and reliable comparison of the mntH expression in different S. enterica strains under various biological conditions, the ratio mntH mRNA level to the normalization factor was determined. The latter is the geometric mean of the RNA level of three housekeeping genes 16S rRNA, rpoD and gmk calculated by the geNorm program. MntH was basally expressed in all tested S. enterica strains and induced by hydrogen peroxide (H(2)O(2)) or ethylenediaminetetraacetic acid (EDTA) in Brain Heart Infusion. Under the nutrient limiting conditions of Sauton medium, the basal mntH expression was higher than in BHI, whereas H(2)O(2) induced the expression 40 times. A similar induction was obtained for Salmonella in porcine peripheral blood monocytes (PBM).
Botteldoorn
Nadine
N
Centre for Agricultural Research-Ghent, Department for Animal Product Quality and Transformation Technology, Brusselsesteenweg 370, 9090 Melle, Belgium. N.Botteldoorn@clo.fgov.be
Van Coillie
Els
E
Grijspeerdt
Koen
K
Werbrouck
Hadewig
H
Haesebrouck
Freddy
F
Donné
Eef
E
D'Haese
Eva
E
Heyndrickx
Marc
M
Pasmans
Frank
F
Herman
Lieve
L
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D027682:Cation Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D004492:Edetic Acid / Q000494:pharmacology; D015870:Gene Expression; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000382:microbiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012481:Salmonella Infections, Animal / Q000097:blood / Q000382:microbiology*; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics; D013552:Swine; D013553:Swine Diseases / Q000097:blood / Q000382:microbiology*
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; C433793:MntH protein, bacteria; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012808:Sigma Factor; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D006162:Guanylate Cyclase