fliP influences Citrobacter koseri macrophage uptake, cytokine expression and brain abscess formation in the neonatal rat

Department of Pathology, Childrens Hospital Los Angeles 1 and University of Southern California Keck School of Medicine, Los Angeles 2 , CA 90027, USA Correspondence Stacy M. Townsend stacy.townsend{at}ntu.ac.uk Received 27 February 2006 Accepted 8 August 2006 Citrobacter koseri causes neonatal meningitis frequently complicated with multiple brain abscesses. During C. koseri central nervous system infection in the neonatal rat model, previous studies have documented many bacteria-filled macrophages within the neonatal rat brain and abscesses. Previous studies have also shown that C. koseri is taken up by, survives phagolysosomal fusion and replicates in macrophages in vitro and in vivo . In this study, in order to elucidate genetic and cellular factors contributing to C. koseri persistence, a combinatory technique of differential fluorescence induction and transposon mutagenesis was employed to isolate C. koseri genes induced while inside macrophages. Several banks of mutants were subjected to a series of enrichments to select for gfp : : transposon fusion into genes that are turned off in vitro but expressed when intracellular within macrophages. Further screening identified several mutants attenuated in their recovery from macrophages compared with the wild-type. A mutation within an Escherichia coli fliP homologue caused significant attenuation in uptake and hypervirulence in vivo , resulting in death within 24 h. Furthermore, analysis of the immunoregulatory interleukin (IL)-10/IL-12 cytokine response during infection suggested that C. koseri fliP expression may alter this response. A better understanding of the bacteria–macrophage interaction at the molecular level and its contribution to brain abscess formation will assist in developing preventative and therapeutic strategies. Abbreviations: CNS, central nervous system; CSF, cerebrospinal fluid; FACS, fluorescence activated cell sort; i.c., intracranial; IL, interleukin; i.p., intraperitoneal; PMA, phorbol 12-myristate 13-acetate. The GenBank/EMBL/DDBJ accession number for the fliP sequence of Citrobacter koseri mutant SMT350 determined in this study is DQ233671. Present address: School of Biomedical and Natural Sciences, The Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK.