Hcp family proteins secreted via the type VI secretion system coordinately regulate Escherichia coli K1 interaction with human brain microvascular endothelial cells

Abstract

Type VI secretion systems (T6SSs) are involved in the pathogenicity of several gram-negative bacteria. Based on sequence analysis, we found that a cluster of Escherichia coli virulence factors (EVF) encoding a putative T6SS exists in the genome of the meningitis-causing E. coli K1 strain RS218. The T6SS-associated deletion mutants exhibited significant defects in binding to and invasion of human brain microvascular endothelial cells (HBMEC) compared with the parent strain. Hcp family proteins (the hallmark of T6SS), including Hcp1 and Hcp2, were localized in the bacterial outer membrane, but the involvements of Hcp1 and Hcp2 have been shown to differ in E. coli-HBMEC interaction. The deletion mutant of hcp2 showed defects in the bacterial binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SS-dependent manner and induced actin cytoskeleton rearrangement, apoptosis, and the release of interleukin-6 (IL-6) and IL-8 in HBMEC. These findings demonstrate that the T6SS is functional in E. coli K1, and two Hcp family proteins participate in different steps of E. coli interaction with HBMEC in a coordinate manner, e.g., binding to and invasion of HBMEC, the cytokine and chemokine release followed by cytoskeleton rearrangement, and apoptosis in HBMEC. This is the first demonstration of the role of T6SS in meningitis-causing E. coli K1, and T6SS-associated Hcp family proteins are likely to contribute to the pathogenesis of E. coli meningitis.

Fig 1

Schematic diagram of the genetic organization of RS218 T6SS gene clusters. The T6SS whole-gene cluster of ORFs with different colors and different direction arrows is shown in the diagram. RS218 evfC, evfH, evfK, hcp1, hcp2, and evfW are the homologs of known vasK, clpV, dotU, hcp1, hcp2, and vgrG genes in V. cholerae, respectively. The database of Clusters of Orthologous Groups of proteins (COGs) was obtained from the National Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/COG/new/).

Fig 2

T6SS and hcp family genes of E. coli strain RS218 are involved in the bacterial binding to and invasion of HBMEC. (A) T6SS of E. coli strain RS218 is involved in the bacterial binding to and invasion of HBMEC. Both ΔRDI-1 and ΔT6SS mutants of E. coli strain RS218 exhibited significantly decreased association with and invasion of HBMEC. The ΔRDI-1 and ΔT6SS strains exhibited 68.1% ± 5.6% and 36.9% ± 6.2% (means ± standard deviations [SD]), respectively, of the association frequency of the wild-type strain, which was arbitrarily set at 100%, while their relative invasion frequencies were 46.4% ± 5.0% and 39.8% ± 6.2% (means ± SD), respectively. The error bars indicate the SD representing the means from three independent experiments. **, P < 0.01. (B) Hcp2 of E. coli strain RS218 was involved in the bacterial binding to and invasion of HBMEC. The Δhcp2 strain had relative association and invasion rates decreased to 36.2% ± 3.4% and 28.4% ± 2.0% (means ± SD) of those of the wild-type strain, respectively. The error bars indicate the SD representing the means from three independent experiments. *, P < 0.05; **, P < 0.01. (C) The complemented hcp2 strain restored the bacterial binding to and invasion of HBMEC. The hcp2 complementation strain pAC-hcp2/Δhcp2 exhibited 108.8% ± 22.1% and 129.7% ± 22.5% in HBMEC association and invasion (means ± SD) rates, respectively, compared to that of the parental strain with the vector control. The error bars indicate the SD representing the means from three independent experiments. *, P < 0.05.

Fig 3

Hcp1 secretion rather than Hcp2 secretion is dependent on the T6SS in E. coli RS218. (A) Hcp2 is undetected in supernatant from strain RS218. The Hcp2 secretion pathway was demonstrated by Western blotting with Hcp2 and Crp antibodies using the supernatants of E. coli strains RS218 and ΔT6SS. Crp is a cytoplasmic protein marker. (B) Hcp1 is secreted by the T6SS of RS218. Hcp1 secretion was demonstrated by Western blotting with Hcp1 and Crp antibodies using the supernatants from E. coli strains RS218, ΔT6SS, Δhcps, ΔevfC, and pAC-evfC/ΔevfC. Crp is a cytoplasmic protein marker. (C) β-Lactamase activity detection in the supernatants of various strains. pCX-hcp1/ΔT6SS, pCX-hcp2/RS218, and pCX-hcp2/ΔT6SS showed negligible β-lactamase activity, indicating no secretion, while pCX-hcp1/RS218 exhibited considerable β-lactamase activity, supporting Hcp1 secretion. The represented Bla activity data (means ± SD) represent the results from duplicate samples of two independent experiments. ***, P < 0.001. OD 486, optical density at 486 nm.

Fig 4

Effect of Hcp1 on F-actin distribution in HBMEC. The cells showed a time- and dose-dependent increase in stress fiber formation and cytoskeleton rearrangement in response to Hcp1 treatment compared to that of the 1× PBS (pH 7.4) negative control (Mock). Magnification, ×1,024.

Fig 5

Apoptosis and cytokine release induced by Hcp1 in HBMEC. (A) The percentages of the apoptosis of cells exposed to Hcp1 and Hcp2 were determined by flow-cytometric analysis. After 24 h of incubation, the early apoptosis rate was 17% in HBMEC with Hcp1 (100 μg/ml), which was significantly higher than rates of 6% for Hcp2 (100 μg/ml), 4% for PBS, and 4% for BSA and medium negative controls (Mock). The error bars indicate the SD from three different experiments. ***, P < 0.001. (B) Colorimetric caspase 8 detection in total lysates of HBMEC. At 24 and 36 h, caspase 8 activity treated with Hcp1 (100 μg/ml) was increased compared to that of the medium treatment control (Mock). The color change was measured with a microplate reader at 405 nm. The results are represented with error bars from three experiments in triplicate. **, P < 0.01. (C) Western blot analysis revealed cleaved caspase 8 with full-length caspase 8 antibody. Comparing the bands in the negative control (Mock) and BSA control, the HBMEC treated with Hcp1 showed decreased full-length caspase 8 after 24- and 36-h treatments. (D) Cytokine release in HBMEC incubated with Hcp family proteins. Approximately 1.3 ng/ml IL-6 and 3.8 ng/ml IL-8 were released in response to Hcp1 treatment within 6 h. The results are presented as the means ± SD from duplicate samples from two individual studies. ***, P < 0.001.

Fig 6

Model for the role of T6SS in E. coli K1 interaction with HBMEC. We propose a potential model to illustrate the role of T6SS in E. coli K1 interaction with HBMEC. We propose a one-step channel through the periplasm which is mainly composed of Hcp1, Hcp2, EvfC, and other proteins. As one of the T6SS component proteins, Hcp2 is responsible for transporting cytotoxic effectors, such as the Hcp1 protein, through the channel. Once Hcp1 was secreted outside, it was recognized by the specific receptor of HBMEC and led to a series of effects, including cytoskeleton rearrangement, apoptosis, caspase 8 activation, and cytokine release, which can initiate inflammation, including the migration of leukocytes across HBMEC. CP, bacterial cytoplasm; IM, bacterial inner membrane; PP, bacterial periplasm; OM, bacterial outer membrane; ECM, extracellular milieu; and PM, host cell plasma membrane.