In vivo tracking of Campylobacter jejuni by using a novel recombinant expressing green fluorescent protein.
Campylobacter jejuni is a leading cause of food-borne disease in developed countries. The goal of this study was to develop a plasmid-based reporter system with green fluorescent protein (GFP) to facilitate the study of C. jejuni in a variety of niches. C. jejuni transformants harboring the pMEK91 GFP gene (gfp)-containing vector were readily detectable by both fluorescence microscopy and flow cytometry. Given the ease of detecting these organisms, additional experiments were performed in which BALB/c mice were injected intraperitoneally with C. jejuni harboring the gfp-containing vector. Four hours after injection of the mice, flow cytometry analyses determined that C. jejuni synthesizing GFP were predominantly associated with granulocytes. More specifically, the proportion of CD11b(+) Gr-1(+) lavage neutrophils with green fluorescence ranged from 99.7 to 100%, while the proportion of CD11b(+) Gr-1(-) lavage macrophages ranged from 77.0 to 80.0%. In contrast, few CD11b(-) CD45R(+) B lymphocytes from the lavage of the C. jejuni-injected mice were associated with green-fluorescent C. jejuni (proportions ranged from 0.75 to 0.77%). Cell-free C. jejuni was recovered from tissue homogenates after intraperitoneal injection. Macrorestriction profiling with pulsed-field gel electrophoresis identified a genotypic variant of the C. jejuni F38011 wild-type isolate. In vivo this variant displayed a phenotype identical to that of the wild-type isolate. In summary, we demonstrate that C. jejuni associates with marker-defined cellular subsets in vivo with a novel gfp reporter system and that C. jejuni genotypic variants can be isolated from both in vitro and in vivo systems.
School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234, USA.
Philip F
PF
Mixter
John D
JD
Klena
Gary A
GA
Flom
Amy M
AM
Siegesmund
Michael E
ME
Konkel
D000818:Animals; D001483:Base Sequence; D002169:Campylobacter Infections / Q000382:microbiology; D016123:Campylobacter jejuni / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D016521:Electrophoresis, Gel, Pulsed-Field; D005260:Female; D005434:Flow Cytometry; D005517:Foodborne Diseases / Q000382:microbiology; D017930:Genes, Reporter; D014644:Genetic Variation; D005822:Genetic Vectors; D006098:Granulocytes / Q000382:microbiology; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D017737:Macrophages, Peritoneal / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D009504:Neutrophils / Q000382:microbiology; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D014774:Virulence / Q000235:genetics
D004269:DNA, Bacterial; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins