Role of Rac1 in Escherichia coli K1 invasion of human brain microvascular endothelial cells

Abstract

Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) requires the reorganization of host cytoskeleton at the sites of bacterial entry. Both actin and myosin constitute the cytoskeletal architecture. We have previously shown that myosin light chain (MLC) phosphorylation by MLC kinase is regulated during E. coli invasion by an upstream kinase, p21-activated kinase 1 (PAK1), which is an effector protein of Rac and Cdc42 GTPases, but not of RhoA. Here, we report that the binding of only Rac1 to PAK1 decreases in HBMEC upon infection with E. coli K1, which resulted in increased phosphorylation of MLC. Overexpression of a constitutively active (cAc) form of Rac1 in HBMEC blocked the E. coli invasion significantly, whereas overexpression of a dominant negative form had no effect. Increased PAK1 phosphorylation was observed in HBMEC expressing cAc-Rac1 with a concomitant reduction in the phosphorylation of MLC. Immunocytochemistry studies demonstrated that the inhibition of E. coli invasion into cAc-Rac1/HBMEC is due to lack of phospho-MLC recruitment to the sites of E. coli entry. Taken together the data suggest that E. coli modulates the binding of Rac1, but not Cdc42, to PAK1 during the invasion of HBMEC.

Fig. 1

Assessment of Rac1 and Cdc42 GTPase activity in HBMEC infected with E. coli by PBD-GST bead pull-down assay. HBMEC grown in 100 mm dishes were treated with E. coli for varying periods as indicated, the treated cells were washed with ice-cold PBS, and lysed in 500 μl of lysis buffer. Lysates were centrifuged to remove particulates, 750 μg proteins was incubated with PBD-GST beads in the presence of protease inhibitor cocktail for 60 min at 4 °C. The beads were washed three times with ice-cold PBS; the bound proteins were eluted with SDS sample buffer, and separated by SDS polyacrylamide gel electrophoresis. The blot was stained with Ponceau for few minutes, washed and subjected to Western blotting using a polyclonal antibody against Rac1. The same blot was stripped and reprobed with anti-Cdc42 antibody. In separate experiments, total proteins (20 μg) were separated and immunoblotted with anti-β-actin antibody. (B) The densities of the bands were calculated using ImageJ software after scanning the blots, normalized to the density of PBD-GST, and graphed.

Fig. 2. Rac1 binding to PAK1 decreases in HBMEC upon infection with OmpA+ E. coli

A. Total cell lysates (300 μg proteins) of HBMEC, infected with OmpA+ E. coli for different periods of time as indicated, were immunoprecipitated with anti-Rac1 or anti-Cdc42 antibody, the immune complexes were subjected to Western blot analysis with anti-Rac1 antibody or anti-Cdc42 (mouse). Lower portion of the blot was immunoblotted with anti-phospho-PAK1 antibody. B. Total cell lysates of HBMEC infected with E. coli for varying periods of time were immunoprecipitated with anti-PAK1 antibody and the immune complexes were separated on 12% SDS polyacrylamide gel. The proteins were transferred to nitrocellulose and the upper portion of the blot was probed with anti-phospho-PAK1 and the lower half of the same blot with anti-Rac1 antibody. The blots were stripped and reprobed with either anti-PAK1 or anti-Cdc42 antibodies, respectively.

Fig. 3. Inhibition of E. coli K1 invasion in HBMEC transfected with cAc-Rac1

A. Total cell lysates (20 μg) of either cAc-Rac1/HBMEC or DN-Rac1/HBMEC or pcDNA3/HBMEC were separated on SDS polyacrylamide gels and were analyzed by immunoblotting with anti-Rac1 or anti-β-actin antibody. B. HBMEC overexpressing the Rac1 mutants along with pcDNA3 were incubated with OmpA+ E. coli (E44) and the invasion assays were carried out as described in Section 2. OmpA– E. coli (E91) was also used in these assays as a negative control. The invasion of E44 into these transfectants was expressed as a percent invasion of pcDNA3/HBMEC being taken as 100%. These data are from at least three separate experiments performed in triplicate. Error bars indicate the standard deviation from the mean. The inhibition of E. coli invasion into cAc-Rac1/HBMEC is significantly lower when compared to control cells. (P < 0.01 by unpaired, two-tailed t-test). Similarly, the total cell associated bacteria was represented as binding for each transfectant.

Fig. 4. Phosphorylation levels of MLC and PAK1 in HBMEC overexpressing DN-Rac1 or cAc-Rac1

Total cell lysates of pcDNA3/HBMEC (A), DN-Rac1/HBMEC (B) or cAc-Rac1/HBMEC (C) infected with OmpA+ E. coli for varying periods as indicated were analyzed by Western blot analysis using either anti-pMLC or anti-MLC antibodies. In separate experiments, the total cell lysates were immunoprecipitated with anti-phospho-PAK1 antibody and the immune complexes were analyzed by anti-phospho-PAK1 antibody. Arrows indicate either phospho-PAK1 or phospho-MLC bands. (D). The densities of phospho-PAK1 were determined from the scanned blot using ImageJ software, normalized to the densities of IgG bands, and graphed.

Fig. 5. Inhibition of phospho-MLC and actin accumulation at the E. coli entry site in cAc-Rac1/HBMEC

HBMEC transfected with pcDNA3 were transfected with OmpA+ (A–D) or OmpA– E. coli (U–X) and HBMEC transfected with cAc-Rac1 or DN-Rac1 were infected with OmpA+ E. coli for 15 min (I–L) and (Q–T) or left uninfected (E–H) and (M–P), respectively. The cells were washed, fixed and stained with rhodamine phalloidin (B, F, J, N, R and V) and anti-pMLC antibody (C, G, K, O, S and W). The localization of phospho-MLC was achieved by incubating the anti-pMLC antibody followed by Cy2-conjugated secondary antibody and mounted with anti-fade solution containing DAPI. The bacteria were visualized using transmitted light optics equipped with a blue filter (A, E, I, M, Q and U). The cells were also visualized with dual filter mode for both red and green fluorescence (D, H, L, P, T and X). The arrows indicate the localized bacteria, actin or p-MLC in the infected monolayers. The magnification of panels containing bacteria is 60×, whereas other panels are of 40×.

Fig. 6

Activation PKC-α and PI3-K in HBMEC overexpressing cAc-PAK1. A. Total cell lysates (20 μg of proteins) of DN-Rac1/HBMEC and cAc-Rac1/HBMEC infected with OmpA+ E. coli for varying periods of time were analyzed for PKC-α activity by a PepTag non-radioactive assay. B. Total cell lysates of HBMEC either non-transfected, overexpressing DN-Rac1 or cAc-Rac1 infected with OmpA+ E. coli were separated by SDS polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-phospho-Akt antibody to examine the activation of PI3K.