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CELLULAR AND MOLECULAR
Ball State University (M.P.H., S.M.K., F.L.R., J.B., C.P.S., C.M.J., T.N.A., A.B., C.B.V., K.G., J.L.M., S.A.M.), Indiana School of Medicine (D.L.B.), Muncie, Indiana
Received February 13, 2008; accepted April 2, 2008.
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Abstract |
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). The most prevalent etiologic agent, Staphylococcus aureus (Diekema et al., 2001), is a leading hospital-acquired pathogen, and community-acquired infection is becoming increasingly widespread (Lowy, 1998). The incidence of complicated infection has increased with the development of invasive procedures and with increased aging, immunocompromised patient populations. Bacterial sepsis is complicated further by invasive endocarditis and osteomyelitis, with persistent infections commonly requiring lengthy hospital stays and treatment. The emergence of antibiotic-resistant strains, including methicillin-resistant S. aureus (MRSA), has limited treatment options. We were surprised to find that statins, a class of drugs generally prescribed for lowering cholesterol, may provide a new adjunctive therapy for bacterial sepsis (Liappis et al., 2001; Krüger and Merx, 2007).
The pharmacology of statins includes effects caused by the lowering of plasma cholesterol and the depletion of intermediates within the cholesterol biosynthesis pathway. Statins lower cholesterol through inhibition of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis (Tobert, 2003). In addition to the reduction in cholesterol levels, inhibition diminishes the synthesis of intermediates within the pathway, including mevalonate and the hydrophobic isoprenoids geranylgeranyl pyrophosphate (GGpp) and farnesyl pyrophosphate (Fpp) (Goldstein and Brown, 1990). Pleiotropic benefits independent of cholesterol lowering have been associated with this depletion of isoprenoid intermediates. Isoprenoids function as membrane anchors and in protein-protein interactions. During post-translational prenylation, 10-carbon GGpp or 15-carbon Fpp is covalently added at the cysteine residue of the conserved carboxyl-terminal motif CaaX, where "C" designates the prenylated cysteine residue, "a" designates aliphatic residues, and "X" determines whether the target is recognized by geranylgeranyl or farnesyl transferases (Zhang and Casey, 1996). With statin treatment, the pool of GGpp and Fpp is reduced, diminishing prenylation, sequestering CaaX-containing proteins within the cytosol, and thereby impairing functions that require membrane localization.
At the cell membrane, the interaction between the CaaX-containing proteins Rac and CDC42 and the phosphoinositide 3-kinase (PI3K) isoform p85 (Zheng et al., 1994; Bokoch et al., 1996) may facilitate actin dynamics and endocytic trafficking. p85 is a regulatory subunit within the PI3K family of proteins (Vanhaesebroeck and Waterfield, 1999). The regulatory subunits, which include p85
and -β, p55 and -
, and p50, possess domains for interaction with membrane-bound proteins and for heterodimerization with catalytic subunits p110,-β, and -
. Regulatory subunits provide a linkage between membrane-bound proteins and the catalytic subunits. These catalytic subunits phosphorylate membrane-bound phosphoinositides, including phosphoinositide 4,5-bisphosphate, forming phosphoinositide 3,4,5-trisphosphate. By coupling through the Rho-GTPase-activating protein (GAP) domain of p85, a domain missing in the truncated p55 and p50 isoforms, prenylated small GTPases would be well positioned to bring the catalytic p110 domains in proximity with membrane-bound phosphoinositides. PI3K may function in this manner as an intermediary to regulate actin dynamics and initiate endocytosis (Johnson, 1999) as membrane-bound phosphoinositide 3,4,5-trisphosphate binds to -actinin, dislodging this bundling protein from actin filaments and disassembling actin stress fibers (Fraley et al., 2005). Thus, prenylation of a subset of CaaX-containing proteins coupled to p85 possibly localizes PI3K at the cell membrane to facilitate the reordering of actin stress fibers.
Actin dynamics can drive both clathrin- and nonclathrin-mediated endocytosis, cellular processes that are exploited by pathogenic bacteria for host invasion (Sinha and Herrmann, 2005; Nitsche-Schmitz et al., 2007). To initiate invasion, surface adhesins of pathogenic S. aureus bind to extracellular matrix proteins. As matrix-bound S. aureus engages host receptors, the pathogen enters the cell during endocytosis of matrix/receptor complexes. Although S. aureus has been primarily considered as an extracellular pathogen, it is increasingly clear that invasiveness contributes to pathogenesis (Lowy, 1998; Alexander and Hudson, 2001; Foster, 2005; Que et al., 2005; Sinha and Herrmann, 2005; Hauck and Ohlsen, 2006; Proctor et al., 2006), and blocking receptor engagement is an emerging immunotherapeutic target (Rivas et al., 2004).
We explored whether statins inhibit endocytic invasion by S. aureus. Our hypothesis is that by diminishing protein prenylation, simvastatin sequesters CaaX-containing proteins coupled to PI3K within the cytosol, inhibiting actin dynamics required for S. aureus endocytic invasion. We have examined whether pretreatment of host cells with simvastatin at therapeutic concentrations prevents S. aureus invasion and the mechanism of this inhibition.
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Materials and Methods |
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). Endothelial Cell Culture. Human umbilical vein endothelial cells (HUVEC; Cascade Biologics, Portland, OR) were grown in M200 medium supplemented with low serum growth supplement (Cascade Biologics; 5% CO2, 37°C, 75-cm2-vented cap flasks) before plating. For invasion assays and flow cytometry, 105 cells were plated on 35-mm tissue culture dishes (Thermo Fisher Scientific) coated with Attachment Factor (a gelatin that contains no extracellular matrix proteins; Cascade Biologics). For confocal imaging, cells were plated on 35-mm glass-bottom dishes (MatTek, Ashland, MA) and coated, as described above. Simvastatin treatments were initiated on day 3 of plating. LY294002 and secramine A treatments were performed on day 4.
Invasion Assay. S. aureus (29213; American Type Culture Collection, Manassas, VA) were subcultured daily in tryptic soy broth (200 rpm, 37°C). Bacteria were harvested by centrifugation (10,000 rpm, 3 min, 37°C), washed once, and resuspended at 3 x 108 cells/ml in saline. HUVEC were washed once with 1x PBS and incubated with S. aureus (1.2 x 108) in 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA)/PBS (1 h, 5% CO2, 37°C). Invasion was terminated by incubation with lysostaphin (20 µg/ml) and gentamicin (50 µg/ml) in 10% FBS/PBS (45 min, 5% CO2, 37°C). Intracellular bacteria were released into the medium using 1% saponin/PBS (20 min, 5% CO2, 37°C). Serial dilutions of the medium were plated on tryptic soy agar, and colony counts were performed (16 h, 37°C). All invasion assays were performed on day 4 of plating. To assess whether compounds were bactericidal, S. aureus (1.2 x 108 cfu) were incubated with compounds at the concentrations indicated in 10% FBS (1 h), and serial dilutions were plated on tryptic soy agar.
Generation of CDC42 C507V/V5. Site-directed mutagenesis (QuikChange; Agilent Technologies, Santa Clara, CA) of human CDC42 cDNA in pUSE (Millipore, Billerica, MA) was performed to substitute the cysteine required for prenylation at position 507 with a valine, remove the stop codon, and fuse to the V5 tag (Invitrogen). The following primer sequence was used: ccg aag aag agc cgc agg gta gtg ctg cta cta ggt aag cct atc cct aac cct ctc ctc ggt cta gat tct acg tga ctc gag tct aga ggg ccc g. Human embryonic kidney (HEK) 293A cells (Invitrogen) were transfected with CDC42 C507V/V5/pUSE using Lipofectamine (Invitrogen), and a stable cell line was created from a geneticin-resistant clone. Invasion assays were performed as described above, with the exception that after the gentamicin/lysostaphin treatment, cells were suspended using trypsin and pelleted before the saponin addition.
Cytotoxicity Assay. HUVEC or HEK 293A were pretreated with compounds at the concentrations indicated in the figure legends, and cytotoxicity was assessed using flow cytometry. Propidium iodide uptake was measured in 1 x 104 cells/treatment.
Immunofluorescence. After infection, HUVEC were washed with 1x PBS, fixed (4% paraformaldehyde/PBS, 30 min; Electron Microscopy Sciences, Hatfield, PA), permeabilized, blocked (0.1% Triton X-100, 1% bovine serum albumin, 30 min), and incubated with Alexa Fluor 488 phalloidin for actin (1:40; Invitrogen). Confocal images were acquired using an inverted Zeiss Axiovert200 microscope equipped with a plan-apochromat 40x, 1.2 NA water immersion lens with correction collar and LSM 5 pascal scan head. Alexa 488 was excited by the 488-nm argon laser line and detected by using a 505- to 530-nm bandpass filter. Z-sectioning and frame size were set to Nyquist sampling. Maximal pixel projections from the Z-stacks were generated and analyzed for actin morphology. Differential interference contrast images were acquired simultaneously with fluorescence images.
Adenoviral Constructs. Wild-type human PI3Kp110 in pDONR201 was kindly provided by Dr. Jim Thomas (Eli Lilly and Co.). By site-directed mutagenesis (QuikChange), the catalytic domain was truncated at amino acid 915 to remove the region that putatively interacts with the phosphoinositide polar head group for lipid kinase activity (Yart et al., 2002). A point mutation was generated to substitute lysine with arginine at amino acid 805, the site required for protein kinase activity (Bondeva et al., 1998). Mutated p110 was cloned into pAD/CMV/V5 through LR recombination (Gateway System; Invitrogen). Kinase-dead p110/pAD/CMV/V5 and LacZ/pAD/CMV/V5 (Invitrogen) were transfected into HEK 293A using Lipofectamine, viral particles were harvested and purified (BD Biosciences, Franklin Lakes, NJ), and titer was determined. Optimal multiplicity of infection and duration of infection were determined for both constructs by infecting HEK 293A and assessing expression by Western blot analysis. Invasion assays were performed as described above for assessing CDC42 C507V/V5.
Cell Fractionation. HEK 293A cells were plated in Dulbecco's modified Eagle's medium/10% FBS at a density of 7.5 x 105 cells on 100-mm tissue culture dishes (Thermo Fisher Scientific) coated with Attachment Factor. At day 3, cells were pretreated with DMSO or simvastatin. On day 4, cells were washed once with ice-cold 1x PBS, incubated on ice in buffer (15 min; 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, and 10 mM PIPES, pH 7.3; 1x Mini-Tab), and harvested using cell scrapers. After sonication (10 s, 40TL probe; Ultrasonic Power Corporation, Freeport, Illinois), lysates were pelleted (500g, 5 min, 4°C), and supernatants were centrifuged (1 h, 110,000g, 4°C, Beckman SW41). The supernatant was designated as the cytosolic fraction. Total protein concentration was determined using the Bio-Rad Protein Assay (Hercules, CA).
Western Blot Analysis. Cytosolic and membrane fractions were subjected to electrophoresis using NU-PAGE (1x MES buffer) and transferred to polyvinylidene difluoride (Invitrogen). Membranes were blocked using Odyssey Blocking Buffer (LI-COR, Lincoln, NE; room temperature, 1 h) and then incubated in blocking buffer/0.1% Tween 20 (4°C, 16 h) with anti-CDC42 (no. 610928; BD Biosciences), with anti-Rac (no. 05-389; Millipore), or anti-RhoB (sc-180; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and subsequently with anti-flotillin-1 (no. 610820; BD Biosciences) and anti-actin (Millipore Bioscience Research Reagents, Temecula, CA). Membranes were washed in 1x PBS/0.1% Tween 20 and incubated with the following: anti-mouse IRDye 800CW (Rockland Immunochemicals, Gilbertsville, PA) for CDC42, Rac, and actin; anti-rabbit IRDye 800CW (Rockland Immunochemicals) for RhoB; and anti-mouse Alexa Fluor 680 for flotillin and/or actin (1:40,000, room temperature, 1 h, in blocking buffer/0.1% Tween 20). Membranes were washed in 1x PBS/0.1% Tween 20, and fluorescent signals (680 and 800) were detected using the Odyssey Infrared Imaging System (LI-COR). Integrated intensity values were acquired using Odyssey software (LI-COR).
Immunoprecipitation. To assess coupling between PI3K p85 and CDC42, Rac, or RhoB, cytosolic fractions (500–1000 µg) were incubated overnight with anti-CDC42, anti-Rac, or anti-RhoB, and immunocomplexes were recovered using Protein A Agarose (Invitrogen). After extensive washing with ice-cold 1x PBS and subsequently with ice-cold LiCl buffer (500 mM LiCl/100 mM Tris-HCl, pH 7.5), immunocomplexes were retrieved by boiling in LDS-Sample buffer containing reducing agent (Invitrogen). Immunocomplexes were assessed by Western blot analysis as described above, probed with anti-PI3K p85 (no. 06-497; Millipore; 16 h, 4°C) followed by anti-rabbit IRDye 800CW (1:40,000, room temperature, 1 h), and detected using the Odyssey Infrared Imaging System. Integrated intensity values were acquired for p85 bands and background using Odyssey software.
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2 test of association (SPSS, Inc., Chicago, IL). Differences between groups were considered statistically significant at p 
0.05. |
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) and to mediate actin dynamics (Johnson, 1999). To determine whether membrane localization by prenylated CDC42 is required for S. aureus invasion, HEK 293A cells were pretreated with secramine A (1 h), an antagonist of prenylated CDC42 membrane association (Xu et al., 2006). Secramine A inhibited S. aureus invasion (Fig. 3A) at concentrations that were neither cytotoxic (Fig. 3B) nor bactericidal (data not shown). To directly assess the role of prenylated CDC42 in S. aureus invasion, a mutated construct was generated in which the cysteine residue required for prenylation was substituted with a valine and expressed as a stable clone. Expression of CDC42 C507V/V5 inhibited S. aureus invasion (Fig. 3C).
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Pretreatment with Simvastatin (20 h) Inhibited Actin Stress Fiber Disassembly during S. aureus Invasion. At day 4 of plating, HUVEC displayed extensive stress fiber formations (Fig. 4A). During invasion, stress fibers disassembled (Fig. 4A), and disassembly persisted throughout 2 h of invasion (Fig. 4B). In contrast, stress fibers remained intact in control cells over time (Fig. 4B). Stress fibers depolymerized in the presence of heat-killed S. aureus (data not shown). Simvastatin reduced actin stress fiber disassembly (Fig. 4C).
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inhibited S. aureus invasion by 70%, more than the 45% decrease in response to adenoviral expression of the control LacZ (Fig. 5C).
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), membrane and cytosolic fractions from cells pretreated with simvastatin or DMSO were assessed upon initial infection (2 min). In the presence of simvastatin, CDC42, Rac, and RhoB remained sequestered in the cytosol with infection (Fig. 6B).
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Discussion |
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Complex actin dynamics mediate the endocytosis of S. aureus (Alexander and Hudson, 2001; Schröder et al., 2006), possibly generating the pulling forces necessary for inward movement and vesicular trafficking (Agerer et al., 2005). Inhibition of actin stress fiber depolymerization by LY294002 (Fig. 5A) indicated that these actin dynamics are in part regulated via a PI3K p110 catalytic domain, because the inhibitory action of this compound is within the ATP-binding site of the p110 domain (Walker et al., 2000). Sequestering the regulatory subunit p85 within the cytosol could impair p110 catalytic activity by restricting access to membrane-bound phosphoinositide. p85 could also affect actin dynamics independently of the p110 subunits because p85 is functional as an unbound monomer (Brachmann et al., 2005). Our data indicate a continuous loss of fibers during the invasion by S. aureus (Fig. 4B), which corresponds temporally with the recent report that bridging between S. aureus and the integrin receptor 5β1 results in sustained actin dynamics (Schröder et al., 2006). Bacteria were found to spend, on average, 45 min on the endothelial cell surface with stimulation of actin waves, formation of actin cups, and generation of comet tails before endocytosis of the bacterium. The current findings indicate that depletion of isoprenoid intermediates by simvastatin and inhibition of PI3K catalytic activity attenuates the cortical actin dynamics required for invasion by the bacterium.
An early event in S. aureus invasion is the activation of CDC42 (Arbibe et al., 2000), a CaaX-containing small GTPase known to facilitate actin dynamics (Johnson, 1999). CDC42 stimulates actin dynamics through a number of effectors. One potential effector is PI3K. CDC42 couples to PI3K through the Rho-GAP domain of PI3Kp85, and it has been postulated that the interaction determines appropriate cellular localization for the lipid kinase (Zheng et al., 1994). Therefore, we examined the possibility that simvastatin inhibits S. aureus invasion by impeding the access of PI3K to membrane-bound phosphoinositide. Nanomolar concentrations of simvastatin were sufficient to significantly inhibit invasion, suggesting that the depletion of isoprenoid intermediates impairs the translocation of multiple prenylated proteins, possibly coupled to distinct PI3K isoforms. In addition to CDC42, Rac had previously been found to couple with PI3K within the Rho-GAP domain of p85 (Bokoch et al., 1996), and, more recently, both Rac and RhoB were found to localize within the cytosol in response to simvastatin (Stamatakis et al., 2002; Cordle et al., 2005). Interaction between p85 and Rac or CDC42 requires GTP loading of these small GTPases. It is interesting to note that Cordle et al. (2005) found that simvastatin stimulates GTP loading of Rac, whereas the small GTPase remained functionally inactivated and translocation to the cell membrane was inhibited. Likewise, S. aureus stimulates an immediate and transient GTP loading of CDC42 (Arbibe et al., 2000). Therefore, in response to either simvastatin or S. aureus, the requirement for GTP loading of small GTPases for interaction with p85 seems to be met, and our findings indicate that this association sequesters p85 within the cytosol in response to simvastatin.
In addition to the small GTPases examined in the current study, additional candidates that may be mediating inhibition include the -subunit of heterotrimeric G proteins that couple to the PI3K isoforms p110 and -β and which require prenylation for membrane insertion (Zhang and Casey, 1996) and small GTPases capable of coupling with p85 through the Rho-GAP domain that have yet to be identified. Our findings also reveal a role for the p110 catalytic domain because treatment with LY294002 or adenoviral expression of a kinase-dead form of p110 decreased invasion more than DMSO or LacZ controls, respectively (Fig. 5B). Adenoviral expression of LacZ diminished infection relative to DMSO control by 45% (Fig. 5B), supporting the concept that the endocytic pathway used for adenoviral uptake is shared by S. aureus for invasion (Sinha and Herrmann, 2005). Previous work has clearly demonstrated that the acute effect of simvastatin on the PI3K signaling pathway is the activation of the downstream mediator Akt (Kureishi et al., 2000). Our study reveals that the pharmacology of long-term (20 h) statin treatment includes sequestration of PI3K through coupling with CaaX-containing proteins.
Although a number of clinical studies indicate an improved prognosis for individuals on a statin regimen, the safety and efficacy of statin therapy during sepsis has been questioned (Mahboobi et al., 2006; Vincent and Miller, 2006; Bromilow and Schuster Bruce, 2007). Concerns include the altered pharmacokinetics of statins in critically ill patients that result in high levels of circulating, nonmetabolized drug, predisposing this patient population to myopathy and rhabdomyolysis. Large, clinical studies will begin to address these concerns. In addition, developing an understanding of the underlying mechanism of statin pharmacology will aid in determining whether statins are an efficacious adjunctive therapy in infectious disease and may provide the rationale for the development of more targeted therapeutics.
Invasion by S. aureus facilitates persistent, relapsing infection (Clement et al., 2005; Proctor et al., 2006) and, in the case of endocarditis, is central to pathogenesis (Yeaman and Bayer, 2000; Que et al., 2005). Immunotherapeutics directed against bacterial adhesin molecules have shown clinical promise, and it is postulated that the protective effect is due in part to impaired endothelial invasion (Rivas et al., 2004). The current study raises the possibility that inhibition of invasion by simvastatin could improve extracellular antibiotic efficacy, reduce hematogenous spread, and impede intracellular pathogenicity including persistent infection. Previous work has shown that statin treatment during sepsis also improves cardiovascular function and modulates the immune response (Kwak et al., 2000; Merx et al., 2004; Jacobson et al., 2005). Taken together, these findings reveal that the pharmacology of simvastatin extends to the inhibition of host invasion and provide a basis for further studies into the usefulness and efficacy of simvastatin, as well as therapeutics targeting distinct PI3K isoforms, as adjunctive approaches in the treatment of invasive infection.
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Acknowledgements |
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that was used to generate a mutated form for these studies; and Xiaoling Liu for generation of adenoviral constructs during his thesis studies at Ball State. Secramine A came jointly from the Kirchhausen laboratory (Harvard Medical School) and the Hammond laboratory (University of Louisville) and was synthesized by Bo Xu and G. B. Hammond (University of Louisville). |
Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: MRSA, methicillin-resistant S. aureus; GGpp, geranylgeranyl pyrophosphate; Fpp, farnesyl pyrophosphate; PI3K, phosphoinositide 3-kinase; GAP, GTPase-activating protein; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; HUVEC, human umbilical vein endothelial cells; FBS, fetal bovine serum; HEK, human embryonic kidney; ANOVA, analysis of variance; GGOH, dephosphorylated form of GGpp; PAGE, polyacrylamide gel electrophoresis; GGTI-2147, 4-{[N-(imidazol-4-yl)methyleneamino]-2-(1-naphthyl)benzoyl}leucine methyl ester; FTI-277, methyl {N-[2-phenyl-4-N[2(R)-amino-3-mercaptopropylamino]benzoyl]}-methionate.
Address correspondence to: Dr. Susan A. McDowell, Cooper Science Complex, CL 171C, Ball State University, Muncie, IN 47306. E-mail: samcdowell{at}bsu.edu
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, less than GGOH/Fpp in the presence of simvastatin; p 
