Escherichia coli K1 RS218 interacts with human brain microvascular endothelial cells via type 1 fimbria bacteria in the fimbriated state

Abstract

Escherichia coli K1 is a major gram-negative organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the central nervous system in vivo. In the present study, we showed using DNA microarray analysis that E. coli K1 associated with HBMEC expressed significantly higher levels of the fim genes compared to nonassociated bacteria. We also showed that E. coli K1 binding to and invasion of HBMEC were significantly decreased with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1 strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e., fimbriated) bacteria. Taken together, we showed for the first time that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC and that type 1 fimbria phase-on E. coli is the major population interacting with HBMEC.

FIG. 1.

The expression levels of the fim genes in HBMEC-bound bacteria compared to those in free bacteria after 90 min incubation of E. coli K1 strain RS218 with HBMEC. The expression levels in the HBMEC-bound bacteria are presented as the percentage in comparison to those in the free bacteria (which was set at 100% arbitrarily). Data shown are the means ± standard deviations derived from three independent experiments. *, P < 0.01 compared to free bacteria.

FIG. 2.

The RS218-ΔfimH isogenic mutant showed decreased association and invasion rates in comparison to the wild-type RS218 (A). The defects of the mutant were restored by complementation with the fimH gene (B). pCHT5, which was derived from the vector plasmid pACYC184, harbors fimH from E. coli K1 RS218. Hatched and filled bars indicate relative association and invasion rates, respectively, compared with the wild-type strain RS218, which was set at 100%. The invasion frequencies of the wild-type RS218 (A) and the pACYC184-harboring wild-type RS218 (B) were 0.81% ± 0.11% and 0.57% ± 0.09% of the original inocula, respectively, while their association frequencies were 18% ± 4% (A) and 15% ± 3% (B) of the original inocula. Data shown are representative of three independent experiments done in triplicate. Results are shown as means ± standard deviations.

FIG. 3.

Characterization of the locked-on and locked-off mutants compared to the wild-type RS218 and RS218-ΔfimH isogenic mutant. The genotypes of the mutants were confirmed with the invertible element orientation assay. Lane a, the locked-on mutant; lane b, the locked-off mutant; lane c, the wild-type RS218 (A). After overnight cultivation in BHI, no FimH was expressed in the locked-off and the RS218-ΔfimH isogenic mutants, while the locked-on mutant and the wild-type strain showed similar levels of FimH expression (B). OmpA expression was similar among the mutants and the wild-type RS218 (C). Lane 1, the wild-type RS218; lane 2, the locked-on mutant; lane 3, the locked-off mutant; lane 4, the RS218-ΔfimH isogenic mutant.

FIG. 4.

The locked-on mutant exhibited significantly higher association and invasion rates than the wild-type RS218, while the locked-off mutant showed significantly lower invasion and association rates than the wild-type RS218. Hatched and filled bars indicate relative association and invasion rates, respectively, compared with the wild-type strain, which was set at 100%. The invasion and association frequencies of the wild-type RS218 were 0.7% ± 0.09% and 19% ± 3% of the original inocula, respectively. Data shown are representative of three independent experiments done in triplicate. Results are shown as means ± standard deviations of the triplicate. WT, wild type.

FIG. 5.

Mannose (methyl α-d-mannopyranoside) dose dependently decreased association with HBMEC of the locked-on mutant (A) but did not affect the association of the locked-off mutant (B). The association rates are presented as percentages of the rates obtained with 0.1 mM of methyl α-d-mannopyranoside, which was set at 100% arbitrarily. The association frequencies of the locked-on and locked-off mutants in 0.1 mM of methyl α-d-mannopyranoside were 28% ± 2% and 8% ± 1% of their original inocula, respectively. Results shown are representative of three independent experiments done in triplicate. Results are shown as means ± standard deviations.

FIG. 6.

Type 1 fimbria piliation of E. coli K1 RS218 interacting with HBMEC. (A) To assess type 1 fimbria piliation of unbound bacteria, the unbound bacteria were detected with light microscopy (left panel) and their type 1 fimbria piliation was detected with immunofluorescence microscopy with a rabbit antiserum directed against type 1 fimbria and fluorescein isothiocyanate-conjugated anti-rabbit IgG antibodies (right panel). (B) To asses type 1 fimbria piliation of HBMEC-bound bacteria, double immunofluorescence microscopy was performed. The HBMEC-bound bacteria were detected with anti-O18 antibody and rhodamine-conjugated anti-mouse IgG antibodies (left panel), and their type 1 fimbria piliation was detected as described in panel A (right panel). (C) The percentages of type 1 fimbria piliated bacteria in the unbound and HBMEC-bound bacteria population were determined. Data shown are the means ± standard deviations of three independent determinations from different fields of the microscope. One hundred bacteria were examined for each determination.