SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Entry of Streptococcus pyogenes or group A streptococcus (GAS) into host cells is mediated by fibronectin bound to surface proteins, M1 or PrtF1, forming a bridge to α5β1 integrins. This interaction leads to cytoskeletal rearrangement and uptake of streptococci. We postulated that integrin-linked kinase (ILK), which directly associates with integrins, is the universal link between integrins and several bacterial pathogens. We showed that inhibition of ILK expression by siRNA silencing, or ILK kinase activity by chemical inhibitors or expression of a dominant negative form of ILK reduced M1-mediated invasion of epithelial cells up to 80%. To evaluate the ILK requirement for PrtF1-mediated GAS invasion, a M1PrtF1+ recombinant strain within the M1 background was constructed. Inhibition of ILK kinase activity also significantly reduced invasion of epithelial cells by this recombinant and wild-type strain JRS4 that expresses PrtF1. In addition, impaired ILK kinase activity results in significant reduction of integrin-dependent invasion mediated by invasins of two other important pathogens, Staphylococcus aureus and Yersinia spp. This study suggests that bacterial pathogens evolved different molecules and strategies to exploit the host integrin signalling pathway for their survival.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Microbial pathogens have evolved a variety of strategies to invade host cells, either to evade bacteriocins, to avoid innate defences or to circumvent immunity. Efficient entry into host cells requires direct or indirect interactions between the bacteria and eukaryotic cellular surface receptors. Bacterial fibronectin-binding proteins (FnBP), produced by many microbial pathogens (Joh et al., 1999) are able to bind integrins of host cells through a fibronectin (Fn) bridge and hijack cellular machinery essential for their successful survival in the host. Although components of focal adhesion complexes induced by interactions of several bacterial pathogens with epithelial cells have been defined, the intermediate links between integrin receptors engaged by these pathogens and those components are unknown.

Group A streptococcus (GAS) is among the most ubiquitous and versatile of human bacterial pathogens known. They can cause uncomplicated infections, such as pharyngitis or life-threatening illnesses, such as toxic shock and necrotizing fasciitis. This species expresses surface FnBPs, which have been shown to mediate intracellular invasion of epithelial cells (Joh et al., 1999). Our laboratory has extensively studied the M1 protein, which promotes streptococcus survival in human blood (Lancefield, 1969), and binds Fn to mediate invasion of epithelial cells (Cue et al., 1998). Among more than 120 M protein genotypes, a subclone of serotype M1 strain has been frequently associated with severe invasive GAS disease with increased mortality and global dissemination (Cleary et al., 1992). This serotype has predominated epidemiologically for more than 20 years (Bisno et al., 2003). Previous studies in our laboratory demonstrated that high-frequency internalization of epithelial cells by the serotype M1 90-226 strain is dependent upon M1 protein and Fn, and is abrogated by antibodies to α5β1 integrins (Cue et al., 1998; Dombek et al., 1999). Moreover, M1 protein-mediated GAS entry into epithelial cells requires activation of the phosphoinositide 3-kinase (PI3K) pathway (Purushothaman et al., 2003). These and other studies suggest that M1 protein is an important invasin and may indirectly enlarge the human reservoir, which accounts in part for global dissemination of this strain (Cleary et al., 1992).

Fibronectin contributes in part to the insoluble extracellular matrix (ECM) of tissue and is also an abundant soluble component of plasma and other body fluids. It is a ligand for a dozen members of the integrin receptor family, including the classic receptor α5β1 (Pankov and Yamada, 2002). ECM binding causes integrin clustering, leading to signalling cascades that mediate a wide variety of cellular activities, including adhesion, migration, growth and differentiation (Pankov and Yamada, 2002). Increased understanding of integrin signalling achieved in the last few years revealed that the integrin-linked kinase (ILK) is a crucial multifunctional protein in integrin-dependent cellular activities. This ubiquitously expressed serine-threonine protein kinase is able to interact with cytoplasmic domains of β integrins, coupling them to the actin cytoskeleton (Hannigan et al., 1996). It binds to focal adhesion proteins and acts as both a catalytic and structural component for actin cytoskeleton assembly in a PI3K-dependent manner (Persad and Dedhar, 2003).

Ingestion of other bacterial pathogens, such as Staphylococcus aureus (Fowler et al., 2000), Neisseria gonorrhoeae (Dehio et al., 1998), Shigella flexneri (Watarai et al., 1996), Yersinia enterocolitica, Y. pseudotuberculosis (Isberg et al., 2000) and Mycobacterium leprae (Byrd et al., 1993) by epithelial cells is also known to be initiated by either direct or indirect engagement of integrin receptors. As ILK couples integrins and cytoskeletal proteins, we postulated that ILK is a component of the cellular machinery required for bacterial invasion. In this study we focused primarily on GAS, but also evaluated ILK requirement for other integrin-dependent bacterial invasion. Inhibition of ILK activity significantly prevented GAS invasion mediated by either M1 or PrtF1 proteins. Experiments showed that integrin-dependent ingestion of other bacteria such as S. aureus and Yersinia spp. by epithelial cells also requires ILK activity.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Genetic knock-down expression of ILK reduced GAS invasion

M1 protein of GAS is an FnBP and can mediate efficient invasion of epithelial cells by serving as an anchor for a Fn bridge to integrins (Cue et al., 2001). However, events downstream of integrin engagement remain largely undefined. We postulated that reduction of ILK protein expression would likewise reduce ingestion of streptococci. To limit ILK protein expression, siRNA interference assays were performed. Epithelial cells were transfected with a previously described siRNA (ILK-H) that corresponds to the human ILK gene and specifically targets the ILK pleckstrin homology (PH)-like domain (Troussard et al., 2003) and an inverted sequence of ILK-H siRNA (control-HC), used as a control. Transfected cells were infected with 90-226, a serotype M1+ F1 strain, and internalized bacteria were measured by a standard invasion assay (Cue and Cleary, 1997). As shown in Fig. 1, siRNA ILK-H-transfected cells were significantly less able to ingest GAS than cells transfected with control-HC siRNA. To monitor inhibition of ILK expression by ILK siRNA, extracts from transfected cells were examined by Western blot. When equal amounts of total cell proteins were examined (as shown by actin, middle row, lower panel of Fig. 1), ILK protein was significantly less in cells transfected with ILK-H siRNA compared with those transfected with control siRNA (top row, lower panel of Fig. 1). Inhibition of kinase activity was corroborated by ILK kinase assays using a downstream target of ILK, the Akt-GST fusion protein as substrate (Persad et al., 2000). As expected ILK kinase activity had decreased in ILK-H-transfected cells (bottom row of lower panel in Fig. 1). However, as ILK also acts as a structural connection between integrins and focal adhesion proteins, these experiments do not prove that kinase activity per se is required.

image

Figure 1. Genetic knock-down expression of ILK reduced GAS invasion. HEp-2 cells were transfected with equal amounts (80 nM) of ILK-H and control-HC siRNA. Invasion assays were performed 72 h post transfection. Uninfected cells with the same treatment were analysed for efficiency of ILK silencing by Western blot and ILK kinase activity using the Akt-GST fusion protein as substrate. Asterisk (*) indicates significant differences between treatments (P ≤ 0.05). Data are presented as means ± standard deviations (n = 3) for a representative from two separate experiments. The Western blot panel compares ILK protein from control siRNA-transfected cells to that from siRNA ILK-H-transfected cells. The Kinase assay panel compares the amount of P-Akt-GST formed in reactions containing immunoprecipitated ILK from control and siRNA ILK-H transfected cells.

Download figure to PowerPoint

Chemical and genetic inhibition of ILK kinase activity reduced GAS invasion

To assess a requirement for ILK kinase activity for invasion, HEp-2 cells were infected with strain 90-226 after pretreatment with KP-392, a chemical inhibitor of ILK kinase activity with high selectivity (D’Amico et al., 2000). Ingestion of strain 90-226 by epithelial cells was significantly prevented by the inhibitor in a dose-dependent manner (Fig. 2A). The inhibitor did not affect viability of either epithelial cells or streptococci when evaluated by trypan blue exclusion or colony counts respectively (data not shown). To confirm previous reports that KP-392-inhibited ILK kinase activity (D’Amico et al., 2000), HEp-2 cells were plated on poly- l-lysine-coated plates to reduce the basal level of ILK and pretreated with the inhibitor, then infected with Fn-coated streptococci. ILK isolated by immunoprecipitation (Experimental procedures) was assessed for kinase activity. As shown in Fig. 2B, Akt-GST was phosphorylated by ILK from non-infected cells (lane 1) and cells infected with strain 90-226 without KP-392 (lane 2); however, much less kinase activity was associated with ILK isolated from KP-392-treated cells (lane 3). Although total ILK protein in immunoprecipitates from KP-392-treated cells (lane 3) equalled that from untreated cells (lane 2), the kinase activity in immunoprecipitates from 90-226-treated cells was as low as that in mock precipitates without anti-ILK antibody (lane 4). The weak band in lane 4 is likely background, non-specific phosphorylation of Akt-GST in the presence of ATP or non-specific binding of the anti-phospho-Akt antibody to non-phosphorylated Akt-GST. This result confirmed that KP-392 effectively inhibited the ILK kinase activity of cells used for invasion assays.

image

Figure 2. ILK Specific Chemical inhibitor reduced GAS invasion. A. HEp-2 cells were pretreated with inhibitors KP-392 and Wortmannin (WM) or solvent (DMSO) as a control, overnight. Invasion assays were performed with GAS strain 90-226 (M+) or 90-226Δemm1 (M1) with Fn present as described in Experimental procedures. B. Kinase assay. HEp-2 cells were pretreated with KP-392 (50 µ M) or DMSO overnight and plated on poly- l-lysine-coated plates without serum for 2 h. Bacteria were preincubated with Fn (300 µg ml−1 in DMEM) 30 min at room temperature, washed and incubated with HEp-2 cells for 2 h. ILK was isolated from cell lysates by immunoprecipitation with excess ILK- specific antibody and kinase assays were performed as described in Experimental procedures. The reaction shown in lane 4 was the same as that in lane 2 but omitted anti-ILK antibody in immunoprecipitation. IP is immunoprecipitate and P-Akt-GST is phosphorylated Akt-GST protein. Ctrl is ILK IP from uninfected cells treated with DMSO.

Download figure to PowerPoint

Integrin-linked kinase is known to be activated upon integrin engagement in a PI3K-dependent manner (Delcommenne et al., 1998). Kinase activity of ILK requires the ILK pleckstrin homology-like domain to be bound by phosphatidylinositol 3, 4, 5-triphosphate, a product of PI3K (Persad and Dedhar, 2003). A specific PI3K inhibitor, Wortmannin, substantially blocks ILK kinase activity (Delcommenne et al., 1998). We previously showed that the same inhibitor efficiently prevented ingestion of strain 90-226 by epithelial cells (Purushothaman et al., 2003), a finding repeated here (Fig. 2A). The inhibitory effects of KP-392 and Wortmannin on GAS invasion indicated that either direct or indirect inhibition of ILK kinase activity efficiently prevents GAS entry.

To confirm the requirement for ILK kinase activity in GAS invasion, epithelial cells were transiently transfected with a kinase-inactive form of the ILK gene (ILK KI), in which serine at position 343 was changed to an alanine (Persad et al., 2001). Under the conditions used, fluorescence microscopy with an antibody against the V5 epitope, which is a fusion partner with ILK in this construct and used as indication of ILK expression, showed that 20–30% of the cells had acquired the ILK KI DNA (data not shown). Transfection efficiency was also assessed by Western blot with anti-V5 antibody (top of lower panel in Fig. 3). Ingestion of GAS by cells transfected with ILK KI was significantly reduced compared to cells transfected with control vector (Fig. 3). To confirm that ILK kinase activity was inhibited by the kinase-inactive form of ILK (ILK KI), kinase assays were performed on extracts from transfected cells. As expected, less Akt-GST was phosphorylated by ILK isolated from ILK KI-transfected cells than ILK from cells transfected with the control vector (bottom, lower panel of Fig. 3). Therefore, ILK kinase activity is required for efficient invasion of epithelial cells by GAS.

image

Figure 3. Genetic inhibition of ILK kinase activity inhibited GAS invasion. HeLa cells were transiently transfected with equal amounts of mutant ILK KI or control vector DNAs. Invasion assays were performed 48 h post transfection. The V5-ILK fusion protein from transfected cell lysates was immunoprecipitated with anti-V5 epitope and displayed by Western blot. ILK kinase assays were performed as described in Fig. 1. Asterisk (*) indicates significant differences between the treatments (P ≤ 0.05). Data are presented as means ± standard deviations (n = 3) for a representative from three separate experiments.

Download figure to PowerPoint

M1 protein is required to trigger ILK-dependent signaling that leads to GAS internalization

Expression of full-length M1 protein by L. lactis strain (pLM1) was previously shown to confer to this organism the ability to invade host cells efficiently (Cue et al., 2001) and ingestion of M+ lactococci was significantly inhibited by PI3K inhibitors (Purushothaman et al., 2003). Therefore, it was reasonable to postulate that ILK kinase activity is required for M1-mediated L. lactis invasion of epithelial cells. This was tested by observing the effect of KP-392 on invasion by this bacterium. As shown in Fig. 4, L. lactis efficiently invaded host cells when expressing M1 protein, and as predicted, invasion of epithelial cells was significantly impaired by pretreatment with KP-392 or Wortmannin. Thus, M1 protein is required to trigger ILK-dependent signalling that leads to GAS internalization. The possibility that other GAS molecules also initiate signalling through ILK has not been excluded.

image

Figure 4. M1 protein is required to trigger ILK-dependent signalling that leads to GAS internalization. HEp-2 cells were pretreated with ILK inhibitor KP-392 (100 µ M), Wortmannin (200 nM) (WM) and DMSO overnight. Invasion assays were performed with M1+ and M1L. lactis. Ctrl cells were treated with DMSO and infected with M+ lactococci. Asterisk (*) indicates significant differences between the treatments (P ≤ 0.05). Data are presented as means ± standard deviations (n = 3) for a representative from three separate experiments.

Download figure to PowerPoint

Integrin-mediated invasion of epithelial cells by another serotype of GAS and other bacterial pathogens is dependent on ILK

A variety of Fn-binding proteins are produced by very diverse bacterial pathogens (Joh et al., 1999) and in many cases bound Fn was shown to mediate invasion of human cells by interacting with appropriate integrins (Byrd et al., 1993; Watarai et al., 1996; Dehio et al., 1998; Fowler et al., 2000; Isberg et al., 2000; Yilmaz et al., 2002). ILK is known to interact with cytoplasmic domains of integrins β1, and β3, which are isoforms in a majority of integrin heterodimers, identified as receptors for many bacterial invasions. Therefore, it is likely that ILK is a common component in the internalization mechanism. Another streptococcal invasin, designated PrtF1/SfbI, was previously shown to bind Fn and to promote internalization of streptococcus in a Fn-, α5β1- or αVβ3-dependent manner (Ozeri et al., 1998). PrtF1/SfbI protein has a higher affinity for Fn, and invasion mediated by this protein is more sensitive to a tyrosine inhibitor genistein than M1 protein-Fn promoted invasion (Purushothaman, Wang and Cleary, 2003). Therefore, the ILK requirement for invasion of epithelial cells by the PrtF1+ M6+ strain JRS4 was investigated. Invasion of HEp2 cell by this strain was highly efficient (50–60% of total inoculum was internalized) (Fig. 5A). As anticipated both ILK and PI3K inhibitors significantly reduced ingestion of these streptococci. This strain also expresses the M6 invasin, though, less important than PrtF1 for this strain. To separate PrtF1-mediated ingestion of streptococci from that of M6 protein, an M PrtF1+ variant of strain 90-226 was constructed. The plasmid pPTF8 was introduced into an M1 mutant strain with a  complete  in  frame  deletion  of  the emm1  to  produce  an M1 PrtF1+-derivative strain (90-226Δemm1(pPTF8) (Experimental procedures). Western blots showed that surface extracts of the strain contained PrtF protein (data not shown). Uptake of this recombinant by HEp-2 cells was as efficient as that of JRS4 and was significantly inhibited by the ILK inhibitor (Fig. 5B) indicating that ILK is also the integrin-proximal signal transducer required by PrtF1–Fn complexes.

image

Figure 5. PrtF1/Sfb1-mediated invasion of epithelial cells is dependent on ILK. HEp-2 cells were pretreated with inhibitors overnight or 1 h (KP-392, 100 µ M and Wortmannin, 200 nM). Invasion assays were performed with (A) M6+ PrtF1+ strain JRS4, (B) an M1 PrtF1+ recombinant of strain 90-226. PrtF1 is encoded by the plasmid pPTF8 in this recombinant. Asterisk (*) indicates significant differences between the treatments (P ≤ 0.05). M1 and PrtF1 phenotypes are shown in square brackets. Data are presented as means ± standard deviations (n = 3) for a representative from two or three separate experiments.

Download figure to PowerPoint

To further test the universality of involvement of ILK in Fn-integrin mediated invasion of epithelial cells by bacterial pathogens, we investigated another important pathogenic genus, Staphylococcus aureus, strain RN6390. The efficient entry of this bacterium is dependent on expression of Fn-binding protein FnBPA or FnBPB (Fowler et al., 2000). Ingestion of wild-type staphylococci by HEp-2 cells was significantly inhibited when cells were pretreated with KP-392 (Fig. 6A).

image

Figure 6. Staphylococcus aureus and Yersinia invasins require ILK to promote invasion of epithelial cells. HEp-2 cells were pretreated with inhibitors as described in Fig. 5 before they were infected with (A) FnBPA+B+S. aureus NR6930 with Fn in wells, (B) with Inv+E. coli HB101 pVM101(Inv) or InvE. coli HB101 without Fn and (C) with Salmonella typhimurium without Fn in the wells. Asterisk (*) indicates significant differences between the treatments (P ≤ 0.05). Data are presented as means ± standard deviations (n = 3) for a representative from two or three separate experiments.

Download figure to PowerPoint

Enteropathogenic Yersinia, Y. pseudoberculosis and Y. enterocolitica have multiple invasion systems. The most efficient entry into host cells is promoted by the protein-termed invasin. Invasin has high affinity for integrin α5β1 to compete for Fn binding, and directly induces integrin signalling that leads to bacterial entry. We postulated that interruption of integrin signalling would also prevent entry of these bacteria. To avoid complications of multiple invasion systems, the effect of KP-392 on ingestion of an Escherichia coli strain carrying the Inv invasion gene from Y. enterocolitica (E. coli HB101 pVM101) (Rosenshine et al., 1992) was tested. As shown in Fig. 6B, the parental strain E. coli HB101 was not able to invade HEp-2 cells. As observed before, introduction of the Inv gene into the bacterium efficiently promoted its uptake by HEp-2 cells (> 50% of the inoculum). As predicted, both KP-392 and Wortmannin significantly reduced invasion by E. coli carrying the Inv gene (6.6% and 13.3% of inoculum respectively).

To eliminate the possibility that the ILK inhibitor KP-392 deranged cellular responses in some non-specific way that disrupted internalization of the above pathogens, we tested its impact on internalization of S. typhimurium, a mechanism known to be independent of Fn–integrin interactions (Brumell et al., 1999). As shown in Fig. 6C, the inhibitor did not prevent entry of this bacterium into epithelial cells; instead, uptake was enhanced somewhat. A potential explanation for this increase will be discussed later. Overall these results confirm that the inhibitory effect of KP392 specifically prevents integrin-mediated events.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Intracellular streptococci are known to persist in tonsils of children who experience recurrent tonsillitis and pharyngitis, and likely contribute to a carrier state, following antibiotic therapy (Osterlund et al., 1997; Podbielski et al., 2003). Our earlier studies demonstrated a requirement for engagement of α5β1 integrin and Fn bound to M1 protein (Cue et al., 2001) and for the PI3K signalling pathway for efficient ingestion of streptococci (Purushothaman et al., 2003). In the present study, M1 type GAS strain 90-226 was used as a model to dissect the connection between integrins and downstream cellular events. Results showed that disruption of ILK kinase activity by various approaches significantly inhibited M1-Fn mediated GAS entry. In addition, we found that inhibition of ILK kinase activity significantly prevented invasion mediated by different invasins of two other important pathogens.

Another high-affinity GAS FnBP protein PrtF1/SfbI is produced by 70% of human GAS isolates and mediates invasion of epithelial cells by GAS (Valentin-Weigand et al., 1994). Invasion of epithelial cells was significantly reduced when PtrF1/SfbI protein was not expressed (Molinari et al., 1997). Ozeri et al. (2001) demonstrated a requirement for Src and FAK when PrtF1 protein mediates epithelial cell ingestion of GAS. Their experiments did not implicate involvement of PI3K or ILK. As PtrF1-mediated streptococcal invasion is integrin α5β1- or αVβ3-dependent (Ozeri et al., 1998), it is not surprising that ILK is also required for PrtF1-mediated entry. Indeed, invasion assays with PrtF1-expressing strains JRS4 or 90226 M1/PrtF1+ showed that ILK and PI3K inhibitors significantly prevented PrtF1-mediated invasion of epithelial cells. Thus, ILK is required for invasion mediated by two distinct major streptococcal Fn-binding proteins. Because expression of PrtF1 is enhanced in an O2-rich environment while expression of M1 protein is greater at a higher partial pressure of CO2 (Caparon et al., 1992; Podbielski et al., 1992), uptake of GAS may be mediated by alternative Fn-binding proteins in different tissue environments: a potential advantageous trick for survival at various locations in a host.

Besides streptococci, a variety of other bacterial pathogens are known to interact directly or indirectly with different integrin receptors to promote internalization by epithelial cells, including major human pathogens S. aureus, Y. pseudotuberculosis, Y. enterocolitica, Shigella flexneri, Porphryomonas gingivalis, Neisseria meningitidis and Mycobacterium leprae (Byrd et al., 1993; Watarai et al., 1996; Dehio et al., 1998; Fowler et al., 2000; Isberg et al., 2000; Yilmaz et al., 2002). But like streptococci, links between cellular receptors and downstream signalling and focal adhesion components had not been uncovered. Connection of ILK with most integrin heterodimers (Reddy and Mangale, 2003) endows the possibility of universal participation of ILK in bacterial invasion. We show here that disruption of ILK kinase activity efficiently prevented  bacterial  invasion  mediated  by  two  different  Fn-binding proteins of GAS and by different invasins expressed by staphylococci and yersinia. Although contribution of ILK to invasion of these other bacterial pathogens remains to be further defined, our results suggest that ILK is shared by different bacterial pathogens although different invasin are used to exploit the integrin signal pathway for their entry.

As we predicted, inhibition of ILK activity did not impair integrin-independent ingestion of S. typhimurium, confirming that the effect of the chemical inhibitor KP-392 is specific for integrin-dependent invasion. Intriguingly, inhibition of ILK substantially enhanced uptake of this bacterium. E-cadherin is a transmembrane glycoprotein for formation of cell-to-cell adherent junctions and colocalizes with adherent S. typhimurium (Jepson et al., 1995). E-cadherin is downregulated by overexpression of ILK and upregulated by inhibition of ILK activity (Oloumi et al., 2004), which could account for the increased S. typhimurium entry.

A model based on our findings is shown in Fig. 7. M1 proteins function as a platform on the bacterial surface for deposition of soluble Fn. M1 proteins bound to Fn induce α5β1 integrins clustering (Schwarz-Linek et al., 2004), which leads to ILK activation in a PI3K-dependent manner. Activated ILK is both a signal transducer and adapter protein for recruitment of focal adhesion proteins, such as paxillin and FAK, and is followed by rearrangement of the cytoskeleton and ingestion of GAS by epithelial cells. The cellular events induced by GAS are similar to those by which cells recognize the ECM. However, due to the small size of bacterial cells, integrin interaction with M1 proteins bound to Fn may result in internalization rather than just adhesion to the bacterium (Joh et al., 1999). This idea is supported by the observations that Fn or Fn-binding protein-coated latex beads are efficiently internalized by epithelial cells (Molinari et al., 1997; Ozeri et al., 1998; Dombek et al., 1999). In one sense, bacteria disguised themselves as ECM by expression of FnBPs or integrin-binding proteins for successful survival in tissue.

image

Figure 7. Schematic diagram of postulated signalling pathways that lead to GAS invasion of epithelial cells. FnBP (fibronectin-binding protein) M1 or PrtF proteins (•) on the surface of GAS bind to soluble Fn molecules, which in turn bind to α5β1 integrin. This causes clustering that triggers signalling through ILK activation. ILK activation is dependent on PIP3, products of PI3K, in the membrane. This subsequently recruits focal adhesion proteins, including paxillin and FAK, which ultimately leads to actin cytoskeleton rearrangement and uptake of GAS. GAS invasion of epithelial cells can be prevented by inhibition of ILK kinase activity directly with inhibitor KP-392 (KP) or indirectly by PI3K inhibitor, Wortmannin (WM), or by inhibition of paxillin phosphorylation with PP2 (data not shown). inline image indicates inhibition.

Download figure to PowerPoint

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Reagents

Integrin-linked kinase inhibitor KP-392 was obtained from Kinetek Pharmaceuticaks, Vancouver, BC Canada (Yoganathan et al., 2002). Wortmannin was purchased from Calbiochem (La Jolla, CA). Poly- l-Lysine was from Sigma (St. Louis, MO).

Bacterial strains and culture conditions

Group A streptococcus strain 90-226 is a serotype of M1 strain (Cue et al., 1998). The M1 deletion variant of strain 90-226 (90-226Δemm1) carries a complete non-polar deletion of emm1, is sensitive to phagocytosis and produces no M1 antigen or peptide fragment (Zimmerlein et al., 2005). The PrtF1/SfbI-expressing variant (90-226 M1 PrtF1+) of strain 90-226 was constructed by introducing plasmid pPTF8, which contains prtF1 gene (Hanski and Caparon, 1992), into strain 90-226Δemm1. It was cultured in medium containing Kanamycin 500 µg ml−1. GAS strain JRS4 (emm6.1) (PrtF+ and M6+) was a gifts from Dr M. Caparon (Hanski and Caparon, 1992; Jadoun et al., 1997), and S. aureus strain RN 6390 (FnBPA+B+) was a gift from Dr P. Schlievert (Department of Microbiology, University of Minnesota). GAS and S. aureus strains were maintained on sheep blood agar and grown in THY-Neo medium for invasion assays. E. coli HB101 pVM101 was a gift from Dr Brett Finlay (Rosenshine et al., 1992). Plasmid pVM101 contains the inv gene from Y. enterocolitica which constitutively expresses invasin protein when E. coli is grown in Luria–Bertani (LB) broth containing ampicillin 50 µg ml−1. M+Lactococcus lactis (pLM1) is a derivative of strain VELL122 containing plasmid pLM1 which encodes full-length M1 protein. L. lactis (pP59) carries the empty vector and is used as an M control (Cue et al., 2001). L. lactis strains were cultured in M17 medium containing erythromycin (5 µg ml−1). Salmonella enterica serovar Typhimurium (S. typhimurium) strain SL1344 was obtained from Brett Finlay (Steele-Mortimer et al., 2002) and was grown in LB broth at 37°C.

Cell culture

The HEp-2 (human larynx epithelial) and HeLa (human cervix epithelial) cell lines were cultured in minimal essential medium (MEM) or Dulbecco's modified Eagle's medium (DMEM) at a humidified atmosphere of 5% CO2 and 95% air. The media contained 10% fetal bovine serum (Invitrogen), penicillin (100 units ml−1) and streptomycin (0.1 mg ml−1).

Invasion assay

Invasion assays were performed as previously described (Cue and Cleary, 1997) with some modifications. PBS was used instead of HBSS for washing monolayers. MEM or DMEM was used for resuspending streptococci. 0.025% Triton X-100 was used instead of H2O for better cell lysis. Bacterial invasion per well is CFU that survived antibiotic treatment and expressed as percentage of CFU of inoculum (Cue et al., 1998). Controls were pretreated with medium alone (DMEM) or solvent used for inhibitors (DMSO) and considered 100%. Invasion was then normalized against controls.

siRNA transfection

siRNA duplex DNA (ILK-H) was synthesized by Xeragon, Germantown, MD. Twenty-one-base sequences of the human ILK gene (GenBank accession number gi 3150001) specifically targeting the pH domain was chosen (Troussard et al., 2003). An inverted ILK-H was used as a control siRNA. Transfections of HEp-2 cells with siRNA were promoted with the Lipofectin reagent (Invitrogen) according to the manufacturer's guidelines. ILK silencing was examined by Western blot with an anti-ILK antibody.

Western blot analysis

Epithelial cells were lysed with lysis buffer (Cell Signaling) after infection. Proteins of cell lysates were measured and equal amount of proteins was analysed by antibodies against ILK, phospho-paxillin (Tyr118), phospho-Akt (Ser473) (Cell Signaling), phospho-FAK (Try397) (Upstate) and Actin (Sigma) as described by the manufacturer's instructions.

Transient transfection of cells with ILK kinase-inactive DNA construct

HeLa cells were transiently transfected with V5/His-tagged-ILK kinase mutant cDNA encoded on plasmid pcDNA3.1 (Troussard et al., 2003). The negative control was plasmid pCDNA3.1 (Invitrogen) without an insert. Transfection with plasmid DNAs was promoted with Lipofactamine™ 2000 reagent (Invitrogen) according to the manufacturer's guidelines. 48 h post-transfection invasion assays were performed and transfection efficiency was examined by fluorescence microscopy with anti-V5 antibody (Invitrogen) and a secondary antibody conjugated with TRITC (Sigma) and by Western blot with anti-V5 antibody according to the manufacturer's instructions after immunoprecipitation with anti-V5.

Immunoprecipitation and ILK kinase assay

HeLa cells were lysed in NP-40 lysis buffer (Persad et al., 2000). Cell lysates were precleaned with protein A/G-sepharose (Amersham Pharmacia Biotech UK, Buckinghamshire, UK) 4°C for 1 h and incubated with monoclonal anti-ILK antibody (Clone 65.1.9, Upstate Biotechnology, Lake Placid, NY) at 4°C for 16 h. After incubation, immune complexes were collected with protein A/G-sepharose. The immunoprecipitated ILK was incubated with unactivated Akt1/PKBα-GST (Upstate Biotechnology) for 30 min at 30°C in a total volume of 50 µl of kinase reaction buffer (50 mM Hepes, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 2 mM NaF, 1 mM Na3VO4, 200 µM ATP). Phosphorylation of Akt1/PKB was detected using anti-phospho-PKB/Akt (Ser 473), secondary antibody coupled to horseradish peroxidase, and LumiGLO (Cell Signaling Technology) by Western blot.

Statistical analysis

The significance of differences in invasion between samples was determined using the two-tailed Student t-test. P < 0.05 was defined as significant. Results are expressed as the averages and standard deviation (n = 3) for a representative experiment performed at least two or three times.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Dr M. Rezcallah for making M1 ProtF1+ variant of strain 90-226. This work was supported by National Institutes of Health Grant RO1-AI34503 (to P.P. Cleary).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Bisno, A.L., Brito, M.O., and Collins, C.M. (2003) Molecular basis of group A streptococcal virulence. Lancet Infect Dis 3: 191200.
  • Brumell, J.H., Steele-Mortimer, O., and Finlay, B.B. (1999) Bacterial invasion: force feeding by Salmonella. Curr Biol 9: R277R280.
  • Byrd, S.R., Gelber, R., and Bermudez, L.E. (1993) Roles of soluble fibronectin and beta 1 integrin receptors in the binding of Mycobacterium leprae to nasal epithelial cells. Clin Immunol Immunopathol 69: 266271.
  • Caparon, M.G., Geist, R.T., Perez-Casal, J., and Scott, J.R. (1992) Environmental regulation of virulence in group A streptococci: transcription of the gene encoding M protein is stimulated by carbon dioxide. J Bacteriol 174: 56935701.
  • Cleary, P.P., Kaplan, E.L., Handley, J.P., Wlazlo, A., Kim, M.H., Hauser, A.R., and Schlievert, P.M. (1992) Clonal basis for resurgence of serious Streptococcus pyogenes disease in the 1980s. Lancet 339: 518521.
  • Cue, D.R., and Cleary, P.P. (1997) High-frequency invasion of epithelial cells by Streptococcus pyogenes can be activated by fibrinogen and peptides containing the sequence RGD. Infect Immun 65: 27592764.
  • Cue, D., Dombek, P.E., Lam, H., and Cleary, P.P. (1998) Streptococcus pyogenes serotype M1 encodes multiple pathways for entry into human epithelial cells. Infect Immun 66: 45934601.
  • Cue, D., Lam, H., and Cleary, P.P. (2001) Genetic dissection of the Streptococcus pyogenes M1 protein: regions involved in fibronectin binding and intracellular invasion. Microb Pathog 31: 231242.
  • D’Amico, M., Hulit, J., Amanatullah, D.F., Zafonte, B.T., Albanese, C., Bouzahzah, B., et al. (2000) The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways. J Biol Chem 275: 3264932657.
  • Dehio, M., Gomez-Duarte, O.G., Dehio, C., and Meyer, T.F. (1998) Vitronectin-dependent invasion of epithelial cells by Neisseria gonorrhoeae involves alpha (v) integrin receptors. FEBS Lett 424: 8488.
  • Delcommenne, M., Tan, C., Gray, V., Rue, L., Woodgett, J., and Dedhar, S. (1998) Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc Natl Acad Sci USA 95: 1121111216.
  • Dombek, P.E., Cue, D., Sedgewick, J., Lam, H., Ruschkowski, S., Finlay, B.B., and Cleary, P.P. (1999) High-frequency intracellular invasion of epithelial cells by serotype M1 group A streptococci: M1 protein-mediated invasion and cytoskeletal rearrangements. Mol Microbiol 31: 859870.
  • Fowler, T., Wann, E.R., Joh, D., Johansson, S., Foster, T.J., and Hook, M. (2000) Cellular invasion by Staphylococcus aureus involves a fibronectin bridge between the bacterial fibronectin-binding MSCRAMMs and host cell beta1 integrins. Eur J Cell Biol 79: 672679.
  • Hannigan, G.E., Leung-Hagesteijn, C., Fitz-Gibbon, L., Coppolino, M.G., Radeva, G., Filmus, J., et al. (1996) Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase. Nature 379: 9196.
  • Hanski, E., and Caparon, M. (1992) Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes. Proc Natl Acad Sci USA 89: 61726176.
  • Isberg, R.R., Hamburger, Z., and Dersch, P. (2000) Signaling and invasin-promoted uptake via integrin receptors. Microbes Infect 2: 793801.
  • Jadoun, J., Burstein, E., Hanski, E., and Sela, S. (1997) Proteins M6 and F1 are required for efficient invasion of group A streptococci into cultured epithelial cells. Adv Exp Med Biol 418: 511515.
  • Jepson, M.A., Collares-Buzato, C.B., Clark, M.A., Hirst, B.H., and Simmons, N.L. (1995) Rapid disruption of epithelial barrier function by Salmonella typhimurium is associated with structural modification of intercellular junctions. Infect Immun 63: 356359.
  • Joh, D., Wann, E.R., Kreikemeyer, B., Speziale, P., and Hook, M. (1999) Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells. Matrix Biol 18: 211223.
  • Lancefield, R.C. (1969) Current problems in studies of streptococci. J Gen Microbiol 55: 161163.
  • Molinari, G., Talay, S.R., Valentin-Weigand, P., Rohde, M., and Chhatwal, G.S. (1997) The fibronectin-binding protein of Streptococcus pyogenes, SfbI, is involved in the internalization of group A streptococci by epithelial cells. Infect Immun 65: 13571363.
  • Oloumi, A., McPhee, T., and Dedhar, S. (2004) Regulation of E-cadherin expression and beta-catenin/Tcf transcriptional activity by the integrin-linked kinase. Biochim Biophys Acta 1691: 115.
  • Osterlund, A., Popa, R., Nikkila, T., Scheynius, A., and Engstrand, L. (1997) Intracellular reservoir of Streptococcus pyogenes in vivo: a possible explanation for recurrent pharyngotonsillitis. Laryngoscope 107: 640647.
  • Ozeri, V., Rosenshine, I., Mosher, D.F., Fassler, R., and Hanski, E. (1998) Roles of integrins and fibronectin in the entry of Streptococcus pyogenes into cells via protein F1. Mol Microbiol 30: 625637.
  • Ozeri, V., Rosenshine, I., Ben-Ze’Ev, A., Bokoch, G.M., Jou, T.S., and Hanski, E. (2001) De novo formation of focal complex-like structures in host cells by invading Streptococci. Mol Microbiol 41: 561573.
  • Pankov, R., and Yamada, K.M. (2002) Fibronectin at a glance. J Cell Sci 115: 38613863.
  • Persad, S., and Dedhar, S. (2003) The role of integrin-linked kinase (ILK) in cancer progression. Cancer Metastasis Rev 22: 375384.
  • Persad, S., Attwell, S., Gray, V., Delcommenne, M., Troussard, A., Sanghera, J., and Dedhar, S. (2000) Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells. Proc Natl Acad Sci USA 97: 32073212.
  • Persad, S., Attwell, S., Gray, V., Mawji, N., Deng, J.T., Leung, D., et al. (2001) Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343. J Biol Chem 276: 2746227469.
  • Podbielski, A., Peterson, J.A., and Cleary, P. (1992) Surface protein-CAT reporter fusions demonstrate differential gene expression in the vir regulon of Streptococcus pyogenes. Mol Microbiol 6: 22532265.
  • Podbielski, A., Beckert, S., Schattke, R., Leithauser, F., Lestin, F., Gossler, B., and Kreikemeyer, B. (2003) Epidemiology and virulence gene expression of intracellular group A streptococci in tonsils of recurrently infected adults. Int J Med Microbiol 293: 179190.
  • Purushothaman, S.S., Wang, B., and Cleary, P.P. (2003) M1 protein triggers a phosphoinositide cascade for group A Streptococcus invasion of epithelial cells. Infect Immun 71: 58235830.
  • Reddy, K.V., and Mangale, S.S. (2003) Integrin receptors: the dynamic modulators of endometrial function. Tissue Cell 35: 260273.
  • Rosenshine, I., Duronio, V., and Finlay, B.B. (1992) Tyrosine protein kinase inhibitors block invasin-promoted bacterial uptake by epithelial cells. Infect Immun 60: 22112217.
  • Schwarz-Linek, U., Hook, M., and Potts, J.R. (2004) The molecular basis of fibronectin-mediated bacterial adherence to host cells. Mol Microbiol 52: 631641.
  • Steele-Mortimer, O., Brumell, J.H., Knodler, L.A., Meresse, S., Lopez, A., and Finlay, B.B. (2002) The invasion-associated type III secretion system of Salmonella enterica serovar Typhimurium is necessary for intracellular proliferation and vacuole biogenesis in epithelial cells. Cell Microbiol 4: 4354.
  • Troussard, A.A., Mawji, N.M., Ong, C., Mui, A., St-Arnaud, R., and Dedhar, S. (2003) Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation. J Biol Chem 278: 2237422378.
  • Valentin-Weigand, P., Talay, S.R., Kaufhold, A., Timmis, K.N., and Chhatwal, G.S. (1994) The fibronectin binding domain of the Sfb protein adhesin of Streptococcus pyogenes occurs in many group A streptococci and does not cross-react with heart myosin. Microb Pathog 17: 111120.
  • Watarai, M., Funato, S., and Sasakawa, C. (1996) Interaction of Ipa proteins of Shigella flexneri with alpha5beta1 integrin promotes entry of the bacteria into mammalian cells. J Exp Med 183: 991999.
  • Yilmaz, O., Watanabe, K., and Lamont, R.J. (2002) Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis. Cell Microbiol 4: 305314.
  • Yoganathan, N., Yee, A., Zhang, Z., Leung, D., Yan, J., Fazli, L., et al. (2002) Integrin-linked kinase, a promising cancer therapeutic target: biochemical and biological properties. Pharmacol Ther 93: 233242.
  • Zimmerlein, B., Park, H.S., Li, S., Podbielski, A., and Cleary, P.P. (2005) The M protein is dispensable for maturation of streptococcal cysteine protease SpeB. Infect Immun 73: 859864.