High Mobility Group Nucleosomal Binding Domain 2 Protein Protects Bladder Epithelial Cells from Klebsiella pneumoniae Invasion

Abstract

Due to the predominance of multiple-antibiotic-resistant Klebsiella pneumoniae strains, the search for new approaches for the prevention of K. pneumoniae infections is now under intensive investigation. The objective of the present study was to investigate the effects of high mobility group nucleosomal binding domain 2 (HMGN2) protein, which acts on the bladder epithelial cells T 24, on the invasion of K. pneumoniae 03183 and explore its possible mechanisms. Pretreatment with HMGN2 significantly reduced K. pneumoniae 03183 uptake by T 24 cells. In T 24 cells, there were no detectable cytotoxic effects of HMGN2 at any concentration between 32 and 256 m mg/ml after 2 h incubation. HMGN2 exhibited no appreciable antibacterial activity against K. pneumoniae 03183. Fluorescence microscopy and flow cytometry analysis revealed that HMGN2 blocked K. pneumoniae 03183-induced actin polymerization. K. pneumoniae 03183-induced phosphorylation of extracellular signal-regulated kinase (ERK) and cofilin were prevented by pretreatment with HMGN2. These results indicated that pretreatment with HMGN2 inhibited cofilin phosphorylation and then induced actin disruption which may block ERK phosphorylation. These changes led to inhibition of K. pneumoniae 03183 invasion of T 24 bladder epithelial cells.Key words high mobility group nucleosomal binding domain 2; actin; Klebsiella pneumoniae; extracellular signal-regulated kinase; cofilin Biol. Pharm. Bull. 34(7) 1065-1071 (2011) © 2011 Pharmaceutical Society of Japan * To whom correspondence should be addressed. e-mail: huangpanxiaoxiao@sina.com tein Assay Kit (Pierce, U.S.A.) and the purity was confirmed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with a primary antibody mouse anti-His monoclonal antibody (Roche, U.K.).Invasion Assay For the invasion assay, T 24 cells were seeded at 5ϫ10 4 cells per well in 24-well plates. Cells were preincubated with substances (rHMGN2 or cytochalasin B or latrunculin B or Y27632 or PD98059) for 2 h prior to bacterial infection in order to study the cell structures and processes involved in internalization. The monolayers were washed twice with PBS to remove any substances prior to infection with K. pneumoniae 03183. In each well of a 24-well cell culture cluster, about 5ϫ10 6 mid-log-phase K. pneumoniae 03183 (OD 600 ϭ0.4-0.6) were added to epithelial cells per well. Invasion was allowed to occur for 2 h at 37°C in 5% CO 2 . Before a second 2 h incubation (kill) period under the same conditions but with fresh medium containing 100 mg of gentamicin per ml, monolayers were washed once with PBS (pH 7.4). During the kill period, all extracellular K. pneumoniae 03183 were killed but the viability of internalized bacteria was not affected. Finally, monolayers were washed twice with PBS and lysed with 0.1% Triton X-100 and the released intracellular bacteria were enumerated by plate count. The number of intracellular K. pneumoniae 03183 after invasion in the presence of an inhibitor was divided b...

High-mobility group nucleosome-binding domain 2 protein inhibits the invasion of Klebsiella pneumoniae into mouse lungs in vivo

“…In the K. pneumoniae and BSA groups, F-actin expression and stress fibers were increased and the average fluorescence intensity of F-actin was significantly increased as compared with that in the PBS group, which indicated that following infection, polymerized F-actin was enhanced. Of note, in the HMGN2 group, F-actin expression and average fluorescence intensity of F-actin were decreased, which indicated that HMGN2 inhibited K. pneumoniae 03183 invasion of lung cells directly through reducing the polymerization of F-actin, which is consistent with previous studies by our group (24,25).…”

“…indicated that it inhibited the invasion of K. pneumoniae 03183 neither by inhibiting the growth of bacteria nor by inhibiting the physical progress of pre-treatment with protein (24,25).…”

“…Is was able to inhibit the gene expression and replication of hepatic B virus in vitro (22) and also exhibited anti-cancer potency in HeLa cells (23). Further studies by our group demonstrated that pre-treatment of K. pneumoniae 03183 with 128 µg/ml HMGN2 significantly inhibited the internalization of K. pneumoniae 03183 into human bladder carcinoma T24 cells by decreasing K. pneumoniae-induced actin polymerization (24,25).…”

Antibacterial mechanism of high-mobility group nucleosomal-binding domain 2 on the Gram-negative bacteria Escherichia coli

“…And we also showed that HMGN2 is a novel antimicrobial molecule, having a potent antimicrobial activity against Escherichia coli ML-35p, hepatitis B virus in vitro (Feng et al, 2005;. The antimicrobial effects of HMGN2 on Klebsiella pneumoniae internalization and invasion in bladder epithelial cells Cao et al, 2011), and in E. coli and Pseudomonas aeruginosa have been reported (Feng et al, 2005). However, the antibacterial mechanism of HMGN2 has not been fully elucidated.…”

HMGN2 regulates non‐tuberculous mycobacteria survival via modulation of M1 macrophage polarization

“…31 Otherwise, we found HMGN2 participated in MAPK signalling through activating ERK1/2 and P38, and regulating autophagy by AMPK pathway to reduce Uropathogenic Escherichia coli internalization of bladder epithelial cells. 32 However, the role of HMGN2 for regulating the anti-non-tuberculous mycobacteria innate immunity of macrophage is still largely unknown.…”