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. 2001 Apr;69(4):2520-6.
doi: 10.1128/IAI.69.4.2520-2526.2001.

Activation of protein tyrosine kinases by Coxiella burnetii: role in actin cytoskeleton reorganization and bacterial phagocytosis

S Meconi  1 , C Capo, M Remacle-Bonnet, G Pommier, D Raoult, J L Mege
Affiliations

Activation of protein tyrosine kinases by Coxiella burnetii: role in actin cytoskeleton reorganization and bacterial phagocytosis

S Meconi et al. Infect Immun. 2001 Apr.

Expression of concern in

Abstract

Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) by C. burnetii in THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflects C. burnetii virulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation in C. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced by C. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, prevented C. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulent C. burnetii but had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation by C. burnetii negatively regulates bacterial uptake by interfering with cytoskeleton organization.

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Figures

FIG. 1
FIG. 1
Tyrosine phosphorylations stimulated by C. burnetii. THP-1 cells were incubated with virulent (A) or avirulent C. burnetii (B) at a bacterium-to-cell ratio of 200:1 for different periods at 37°C. Tyrosine phosphorylations were revealed by using antiphosphotyrosine MAb, peroxidase-conjugated secondary Ab, and an enhanced chemiluminescence kit. The molecular masses of phosphoproteins were determined using standards of known molecular mass (right margin, in kilodaltons). Arrowheads at the left indicate positions of phosphorylated proteins. Panels A and B are representative of five distinct experiments. (C) Tyrosine phosphorylation levels of 55-, 66-, and 120-kDa proteins were assessed by densitometric scanning. Results are expressed as relative increase over the unstimulated value and are the mean ± SE of five distinct experiments.
FIG. 2
FIG. 2
Src-related kinases in C. burnetii-stimulated cells. THP-1 cells were stimulated with virulent or avirulent C. burnetii for different periods, homogenized, and incubated with 1 μg of anti-Hck, -Lyn, or -Fgr MAb. Proteins were immunoprecipitated (IP), electrophoresed, and transferred onto nitrocellulose sheets. Immunoblotting was performed with antiphosphotyrosine MAb (PY). Membranes were stripped and reprobed with anti-Hck, -Lyn, and -Fgr MAbs, respectively. Each blot is representative of three distinct experiments.
FIG. 3
FIG. 3
Colocalization of tyrosine phosphoproteins and F-actin. THP-1 cells were stimulated with virulent (A) or avirulent (B) C. burnetii for the times (minutes) indicated. Tyrosine phosphoproteins and F-actin were labeled with antiphosphotyrosine MAb and rhodamine-conjugated F(ab′)2 anti-mouse IgG and with bodipy phallacidin, respectively. Cells were examined with a laser scanning confocal fluorescence microscope, and representative cells are shown. The colocalization of tyrosine phosphoproteins and F-actin appears in yellow.
FIG. 4
FIG. 4
Distribution of PTK activity in cell fractions. THP-1 cells were pretreated with cytochalasin D (1 μg ml−1) or not pretreated and stimulated by virulent (A) or avirulent (B) C. burnetii for 10 min. Cells were then lysed by 1% Triton X-100. Triton-soluble and -insoluble fractions were incubated with poly(Glu, Tyr) and 1 μCi of [γ-32P]ATP. Radioactivity was measured with a scintillation counter; cpm in Triton-soluble fraction before stimulation = 29,500 ± 5,870; cpm in Triton-insoluble fraction before stimulation = 25,660 ± 5,110. Results are expressed as the ratio of counts after stimulation to counts before stimulation and represent the mean ± SE of five distinct experiments. ∗, P < 0.05.
FIG. 5
FIG. 5
Effect of PTK inhibitors on F-actin organization. (A) THP-1 cells were pretreated with 10 μM PTK inhibitors for 30 min before stimulation with virulent C. burnetii for 10 min. F-actin was labeled with bodipy phallacidin, and its reorganization was examined with fluorescence microscopy. a, control cells; b, C. burnetii-stimulated cells; c, cells preincubated with lavendustin A; d, cells preincubated with lavendustin A and stimulated by C. burnetii; e, cells preincubated with PP1; f, cells preincubated with PP1 and stimulated by C. burnetii. (B) Percentage of cells with protrusions rich in F-actin.
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