Cortactin Is Involved in the Entry of <em>Coxiella burnetii</em> into Non-Phagocytic Cells

<div><h3>Background</h3><p>Cortactin is a key regulator of the actin cytoskeleton and is involved in pathogen-host cell interactions. Numerous pathogens exploit the phagocytic process and actin cytoskeleton to infect host cells. <em>Coxiella burnetii</em>, the etiologic agent of Q fever, is internalized by host cells through a molecular mechanism that is poorly understood.</p> <h3>Methodology/Principal Finding</h3><p>Here we analyzed the role of different cortactin motifs in the internalization of <em>C. burnetii</em> by non-phagocytic cells. <em>C. burnetii</em> internalization into HeLa cells was significantly reduced when the cells expressed GFP-cortactin W525K, which carries a mutation in the SH3 domain that renders the protein unable to bind targets such as N-WASP. However, internalization was unaffected when the cells expressed the W22A mutant, which has a mutation in the N-terminal acidic region that destroys the protein’s ability to bind and activate Arp2/3. We also determined whether the phosphorylation status of cortactin is important for internalization. Expression of GFP-cortactin 3F, which lacks phosphorylatable tyrosines, significantly increased internalization of <em>C. burnetii</em>, while expression of GFP-cortactin 3D, a phosphotyrosine mimic, did not affect it. In contrast, expression of GFP-cortactin 2A, which lacks phosphorylatable serines, inhibited <em>C. burnetii</em> internalization, while expression of GFP-cortactin SD, a phosphoserine mimic, did not affect it. Interestingly, inhibitors of Src kinase and the MEK-ERK kinase pathway blocked internalization. In fact, both kinases reached maximal activity at 15 min of <em>C. burnetii</em> infection, after which activity decreased to basal levels. Despite the decrease in kinase activity, cortactin phosphorylation at Tyr421 reached a peak at 1 h of infection.</p> <h3>Conclusions/Significance</h3><p>Our results suggest that the SH3 domain of cortactin is implicated in <em>C. burnetii</em> entry into HeLa cells. Furthermore, cortactin phosphorylation at serine and dephosphorylation at tyrosine favor <em>C. burnetii</em> internalization. We present evidence that ERK and Src kinases play a role early in infection by this pathogen.</p>