Cortactin Is Involved in the Entry of Coxiella burnetii into Non-Phagocytic Cells

Abstract

Background 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. Coxiella burnetii , the etiologic agent of Q fever, is internalized by host cells through a molecular mechanism that is poorly understood. Methodology/Principal Finding Here we analyzed the role of different cortactin motifs in the internalization of C. burnetii by non-phagocytic cells. C. burnetii 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 C. burnetii , 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 C. burnetii 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 C. burnetii 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. Conclusions/Significance Our results suggest that the SH3 domain of cortactin is implicated in C. burnetii entry into HeLa cells. Furthermore, cortactin phosphorylation at serine and dephosphorylation at tyrosine favor C. burnetii internalization. We present evidence that ERK and Src kinases play a role early in infection by this pathogen.

Analysis of theCaenorhabditis elegansinnate immune response toCoxiella burnetii

“…37 Previous studies have also shown that MAPKs are utilized by vertebrates to signal the presence of C. burnetii . 61,82–85 Furthermore, MAPKs of Saccharomyces cerevisiae yeast are modulated by C. burnetii ’s Icm/Dot type IVB secretion system effector proteins. 86 …”

Trypanosoma cruziextracellular amastigotes trigger the protein kinase D1-cortactin-actin pathway during cell invasion

“…Although SFK phosphorylation of cortactin is important in host invasion by Shigella spp., Staphylococcus aureus and Neisseria meningitidis (Hoffmann et al, 2001;Bougnères et al, 2004;Agerer et al, 2005), several recent reports have depicted involvement of serine phosphorylation of cortactin in pathogen internalization. ERK phosphorylation and SFK dephosphorylation of cortactin are important in HeLa cell invasion by Coxiella burnetii and for cortactin capacity to bind N-WASP (Rosales et al, 2012). Similarly, Campylobacter jejuni induces and requires cortactin phosphorylation by ERK, but not by SFK, and host cell invasion is inhibited in cells depleted of cortactin and N-WASP (Samuelson and Konkel, 2013).…”

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…RhoA appears particularly important considering the activity of the downstream effectors ROCK, a protein kinase and mDia1, an actin nucleating factor, are required for optimal internalization [53]. Src-family kinases [54] and the F-actin binding protein cortactin [55] also play roles in internalization of Coxiella by p hagocytic and nonphagocytic cells, respectively.…”