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.
Mixter
Philip F
PF
School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234, USA.
Klena
John D
JD
Flom
Gary A
GA
Siegesmund
Amy M
AM
Konkel
Michael E
ME
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