Uropathogenic Escherichia coli invades host cells via an HDAC6-modulated microtubule-dependent pathway - PubMed (original) (raw)
Uropathogenic Escherichia coli invades host cells via an HDAC6-modulated microtubule-dependent pathway
Bijaya K Dhakal et al. J Biol Chem. 2009.
Abstract
Strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili that promote bacterial colonization and invasion of the bladder epithelium. Type 1 pilus-mediated interactions with host receptors, including alpha3beta1 integrin, trigger localized actin rearrangements that lead to internalization of adherent bacteria via a zipper-like mechanism. Here we report that type 1 pilus-mediated bacterial invasion of bladder cells also requires input from host microtubules and histone deacetylase 6 (HDAC6), a cytosolic enzyme that, by deacetylating alpha-tubulin, can alter the stability of microtubules along with the recruitment and directional trafficking of the kinesin-1 motor complex. We found that disruption of microtubules by nocodazole or vinblastine treatment, as well as microtubule stabilization by taxol, inhibited host cell invasion by UPEC, as did silencing of HDAC6 expression or pharmacological inhibition of HDAC6 activity. Invasion did not require two alternate HDAC6 substrates, Hsp90 and cortactin, but was dependent upon the kinesin-1 light chain KLC2 and an upstream activator of HDAC6, aurora A kinase. These results indicate that HDAC6 and microtubules act as vital regulatory elements during the invasion process, possibly via indirect effects on kinesin-1 and associated cargos.
Figures
FIGURE 1.
Microtubule-dependent invasion of bladder cells by UPEC. 5637 bladder cell monolayers were treated with the indicated concentrations of (a) nocodazole, (b) taxol, or (c) vinblastine for 1 h prior to infection with the UPEC isolate UTI89. After a 2-h infection in the continued presence of drugs, intracellular (a–c) and total cell-associated (d) bacterial titers were determined. Data are expressed relative to untreated controls as the means ± S.E. of at least three independent experiments carried out in triplicate. e, confocal micrographs of bladder cells treated for 1 h with the indicated drugs or (f) infected for 30 min with UTI89 prior to staining to visualize microtubules (green), F-actin (red), and UPEC (blue). The boxed area in f was enlarged to show detail. Scale bars:(e) 20 μm and (f) 5 μm.
FIGURE 2.
Rho GEF-H1 activity is not necessary during UPEC entry. a, levels of activated RhoA-GTP, as determined by pull-down assays using the Rho binding domain of Rhotekin, were quantified relative to total RhoA in 5637 bladder cells following 30 min treatments with carrier alone (control), nocodazole (66 μ
m
), taxol (40 μ
m
), or vinblastine (40 μ
m
), or 3 h treatments with 20 μ
m
of either niltubacin or tubacin. b, Western blot showing levels of GEF-H1 in bladder cells 72 h after transfection with either control nonspecific siRNA or GEF-H1-specific siRNA. Blots were also probed using an anti-actin antibody to verify equal protein loading. Quantification of (c) internalized and (d) total cell-associated bacteria following infection of GEF-H1-silenced bladder cells by UTI89, with or without 33 μ
m
nocodazole treatment. Data are expressed relative to control nonspecific siRNA-transfected samples as the means ± S.E. of at least three independent experiments carried out in triplicate.
FIGURE 3.
HDAC6 promotes UPEC entry into host bladder cells. 5637 bladder cells were pretreated for 3 h with (a and b) 300 n
m
TSA, 5 m
m
NA, or 5 m
m
NaB, or with (c and d) the indicated concentrations of tubacin and niltubacin. Cells were then infected with UTI89 for 2 h and (a and_c_) intracellular and (b and d) total cell-associated bacterial titers were determined relative to controls treated with carrier alone. e, Western blot analysis of bladder cells treated with the indicated inhibitors shows levels of both total and acetylated (Ac) α-tubulin. f, levels of HDAC6 in bladder cells 72 h after transfection with either control nonspecific siRNA or HDAC6-specific siRNA, as detected by Western blot. Blots were also probed using an anti-actin antibody to verify equal protein loading. Following a 2 h infection with UTI89, (g) intracellular and (h) total cell-associated bacterial titers recovered from siRNA-transfected cells were determined. Data in the graphs are expressed relative to appropriate controls as the means ± S.E. of at least three independent experiments carried out in triplicate.
FIGURE 4.
Aurora A kinase-dependent invasion of host cells by UPEC. a and b, 5637 bladder cells were pretreated for 3 h with 10 μ
m
of AKI II or carrier alone. Cells were then infected with UTI89 for 2 h in the continued presence of drug or carrier after which (a) intracellular and (b) total cell-associated bacterial titers were determined. Similarly, numbers of (c) intracellular and (d) total host cell-associated bacteria were calculated 2-h post-infection of AurA-silenced and control siRNA-treated bladder cells.Inset in c shows levels of AurA and actin in bladder cells transfected with either AurA-specific or control non-specific siRNA. Data are expressed relative to controls as the means ± S.E. of at least three independent experiments carried out in triplicate.
FIGURE 5.
Differential requirements for HDAC6 by other invasive bacterial pathogens. Bladder epithelial cells were treated with carrier alone (control), 33 μ
m
nocodazole, or 300 n
m
TSA prior to infection with the indicated pathogens. Invasion indices (calculated by dividing the number of gentamicin-protected intracellular bacteria by the total number of cell-associated bacteria) are presented relative to controls as the means ± S.E. of at least three independent experiments carried out in triplicate.
FIGURE 6.
Kinesin-1 promotes host cell invasion by UPEC. Bladder cells were pre-treated with aurintricarboxylic acid (ATA, 50 μ
m
) for 3 h or with sodium orthovanadate (Na3VO4, 100 μ
m
) for 1 h prior to infection with UTI89. After 2 h in the continued presence of drugs (a) intracellular and (b) total cell-associated bacterial titers were calculated and are expressed relative to controls treated with only carrier. Quantification of (c) internalized and (d) total cell-associated bacteria following infection of KLC2-silenced bladder cells by UTI89. e, semi-quantitative RT-PCR showing KLC2 and GAPDH message levels in bladder cells 72 h after transfection with either control nonspecific siRNA or KLC2-specific siRNA. Data in graphs are expressed relative to appropriate controls as the means ± S.E. of at least three independent experiments carried out in triplicate. f, 5637 bladder cells, following transfection with pKLC2_FLAG, were treated with either DMSO alone (D) or 300 n
m
TSA (T) for 3 h prior to lysis. The Western blot shows levels of FLAG-tagged KLC2, α-tubulin, acetylated tubulin, and actin present in total cell lysates (input) or in supernatants (Sup) recovered after polymerizing and spinning out microtubules (pellets). Similar results were obtained using hemagglutinin-tagged KLC2.
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