Use of CFSE staining of borreliae in studies on the interaction between borreliae and human neutrophils.
BACKGROUND
Species of the tick-transmitted spirochete group Borrelia burgdorferi sensu lato (B. burgdorferi) cause Lyme borreliosis. Acute borrelial infection of the skin has unusual characteristics with only a mild local inflammatory response suggesting that the interaction between borreliae and the cells of the first-line defence might differ from that of other bacteria. It has been reported that human neutrophils phagocytose motile borreliae through an unconventional mechanism (tube phagocytosis) which is not observed with non-motile borreliae. Therefore, it would be of great interest to visualise the bacteria by a method not affecting motility and viability of borreliae to be able to study their interaction with the cells of the innate immunity. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labelling has been previously used for studying the adhesion of labelled bacteria to host cells and the uptake of labelled substrates by various cells using flow cytometry.


RESULTS
In this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility. Use of CFSE staining allowed subsequent quantification of borreliae associated with human neutrophils with flow cytometry and confocal microscopy. As a result, no difference in association between different borrelial genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii), or between borreliae and the pyogenic bacterium Streptococcus pyogenes, with neutrophils could be detected. Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain.


CONCLUSIONS
These results suggest that the flow cytometric assay using CFSE labelled borreliae is a valuable tool in the analysis of the interaction between borreliae and human neutrophils. The results also indicate a clear difference in the association with neutrophils between virulent and non-virulent borrelial strains.
Tuominen-Gustafsson
Helena
H
Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, FI-20520 Turku, Finland. hetugu@utu.fi
Penttinen
Markus
M
Hytönen
Jukka
J
Viljanen
Matti K
MK
D001898:Borrelia / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D005452:Fluoresceins / Q000032:analysis*; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D009504:Neutrophils / Q000382:microbiology*; D013194:Staining and Labeling / Q000379:methods*; D013297:Streptococcus pyogenes / Q000302:isolation & purification; D013388:Succinimides / Q000032:analysis*; D014774:Virulence
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides