Group B Streptococcus (GBS) has evolved several strategies to avoid host defences where macrophages are one of main targets. Since pathogens frequently target the cytoskeleton to evade immune defences, we investigated if GBS manipulates macrophage cytoskeleton. GBS-III-COH31 in a time- and infection ratio-dependent manner induces great macrophage cytoskeleton alterations, causing degradation of several structural and regulatory cytoskeletal proteins. GBS β-haemolysin is involved in cytoskeleton alterations causing plasma membrane permeability defects which allow calcium influx and calpain activation. In fact, cytoskeleton alterations are not induced by GBS-III-COH31 in conditions that suppress β-haemolysin expression/activity and in presence of dipalmitoylphosphatidylcholine (β-haemolysin inhibitor). Calpains, particularly m-calpain, are responsible for GBS-III-COH31-induced cytoskeleton disruption. In fact, the calpain inhibitor PD150606, m-calpain small-interfering-RNA and EGTA which inhibit calpain activation prevented cytoskeleton degradation whereas µ-calpain and other protease inhibitors did not. Finally, calpain inhibition strongly increased the number of viable intracellular GBS-III-COH31, showing that cytoskeleton alterations reduced macrophage phagocytosis. Marked macrophage cytoskeleton alterations are also induced by GBS-III-NEM316 and GBS-V-10/84 through β-haemolysin-mediated plasma membrane permeability defects which allow calpain activation. This study suggests a new GBS strategy to evade macrophage antimicrobial responses based on cytoskeleton disruption by an unusual mechanism mediated by calcium influx and calpain activation.
Streptococcus agalactiae (Group B Streptococcus, GBS) is a pathogen that causes serious invasive infections in human neonates and adults, particularly the elderly and individuals with underlying chronic disease (Baker and Edwards, 1995; Maisey et al., 2008). The physiopathology of GBS infection implies that this pathogen can escape the first line of host innate immune defence mechanisms especially macrophages (MΦ) which can engulf and kill bacteria. In fact, it has been shown that GBS has evolved several strategies to overcome MΦ defences. In particular, it resists phagocytosis by MΦ through an antiphagocytic capsule which prevents opsonic complement-mediated and non-opsonic scavenger receptor A mediated phagocytosis (Rubens et al., 1987; Areschoug et al., 2008). GBS, like intracellular microorganisms, can survive inside MΦ (Valentin-Weigand et al., 1996; Cornacchione et al., 1998) and it also delays MΦ pro-inflammatory cytokine responses (Rosati et al., 1998). GBS, through β-haemolysin, can also kill murine MΦ and human monocytes by apoptosis, triggered by a strong extracellular calcium (Ca2+) influx and consequently calpain activation which initiate a caspase-independent pathway (Fettucciari et al., 2000; 2006). Ulett et al. (2003; 2005) showed that also phagocytosed GBS induce apoptosis in a MΦ-like cell line J774 and that nitric oxide is a key determinant in apoptosis which occurs by a caspase-dependent pathway (Ulett and Adderson, 2005).
The recent discovery that GBS through activation of Rho GTPases (Rho A, Rac1 and Cdc42), FAK or PI3K/Akt manipulates the cytoskeleton of non-phagocytic epithelial and endothelial cells to promote its internalization in thehost (Tyrrell et al., 2002; Shin and Kim, 2006; Shin et al., 2006; Burnham et al., 2007), raises the question if GBS may also manipulate the MΦ cytoskeleton as another strategy to evade immune defence mechanisms.
Switching off or altering actin and microtubule dynamics of phagocytes will help pathogens to avoid the immune response by subversion of several antimicrobial MΦ responses (May and Machesky, 2001; Gruenheid and Finlay, 2003; Yoshida and Sasakawa, 2003; Rottner et al., 2005). Because understanding the cytoskeletal manipulation mechanisms induced by bacteria in phagocytes could be important for the management of infectious diseases we investigated if GBS manipulates the cytoskeleton of MΦ and the mechanisms involved.
Our results show that the more haemolytic GBS strains, GBS type III strain COH31 r/s (GBS-III-COH31), GBS type III strain NEM316 (GBS-III-NEM316) and GBS type V strain NCTC10/84 (GBS-V-10/84), alter the MΦ cytoskeleton by inducing an early and strong degradation of several structural and regulatory cytoskeletal proteins and this degradation depended on plasma membrane permeability defects and calpain activation which, as demonstrated for GBS-III-COH31, is due to Ca2+ influx. Furthermore the demonstration that infection with weakly haemolytic GBS type Ia A909 (GBS-Ia-A909), weakly haemolytic GBS type Ib H36B (GBS-Ib-H36B), in presence of dipalmitoylphosphatidylcholine (DPPC, an inhibitor of β-haemolytic activity) and with GBS-III-COH31 in conditions that suppress haemolytic activity does not induce cytoskeleton alterations, suggests that β-haemolysin is involved in MΦ cytoskeleton alterations inducing MΦ plasma membrane permeability defects which can allow Ca2+ influx and consequently calpain activation. In fact, inhibition of Ca2+ influx and calpain activation prevents such proteolysis. Finally, inhibition of cytoskeletal protein degradation, by preventing calpain activation, increases also the number of intracellular viable GBS-III-COH31 in inhibitor-treated MΦ. Therefore, disruption of the actin and microtubule MΦ cytoskeleton by GBS could also contribute to its overall strategy to counteract antimicrobial MΦ functions.
GBS-III-COH31 induces alterations in MΦ cytoskeleton
The results of immunoblot analysis showed that GBS-III-COH31 induces marked changes in some relevant MΦ cytoskeletal proteins and the effect was time- and MΦ:GBS infection ratio-dependent. At 1 h after GBS-III-COH31 infection no significant changes in the expression of α-actinin, Vinculin, β-tubulin, Pyk2/Cakβ (Pyk2), Paxillin, were found at all ratios used (Fig. 1) and the expression of Talin was only slightly reduced with the MΦ:GBS ratio 1:100 (Fig. 1A). In contrast, at 2 h after infection, in response to GBS-III-COH31 at 1:100 we found about 68–75% inhibition of expression of α-actinin, Vinculin (Fig. 1A) and Paxillin (Fig. 1B), 85–95% for Talin, β-tubulin (Fig. 1A) and Pyk2 (Fig. 1B). In response to GBS-III-COH31 at a 1:50 ratio there was a 35–44% decrease in α-actinin, Vinculin, β-tubulin expression levels and 85% for Talin (Fig. 1A) while Pyk2 and Paxillin expression was not affected (Fig. 1B). GBS-III-COH31 at a 1:10 ratio did not alter expression levels in any of the cytoskeletal proteins examined (Fig. 1).
These results indicate that GBS-III-COH31 can induce marked alterations in the MΦ cytoskeleton, causing a great reduction of Talin, Vinculin, α-actinin, Paxillin, Pyk2 and β-tubulin expression and that this early effect was time- and infection ratio-dependent.
Calpains are activated during GBS-III-COH31 infection of MΦ
Since calpains have been reported to act as regulators of the actin cytoskeleton and FA turnover, by cleaving FA proteins such as Vinculin, Talin, α-actinin and Paxillin and several cytoskeletal proteins such as Cortactin, Pyk2 and β-tubulin (Glading et al., 2002; Franco and Huttenlocher, 2005) and since we have previously demonstrated that GBS-III-COH31 activates calpain in MΦ (Fettucciari et al., 2006) the above results led us to evaluate calpain activation during infection of MΦ with GBS-III-COH31 at different MΦ:GBS ratios and if calpains could be involved in the cleavage of cytoskeletal proteins.
To this end we first analysed calpain activation by monitoring at 1 and 2 h MΦ infection with GBS-III-COH31 at different MΦ:GBS ratios, both the cleavage of α-spectrin into 150 kDa and 145 kDa breakdown products (BP), a characteristic sign of calpain activity (Wang et al., 1996; Goll et al., 2003; Fettucciari et al., 2006) and the cleavage of the fluorogenic-calpain substrate Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) in live cells, as a marker of changes in intracellular calpain activity (Wang et al., 1996; Fettucciari et al., 2006).
The results obtained, like those previously demonstrated (Fettucciari et al., 2006), indicate that GBS-III-COH31 activates calpains in MΦ in a time- and infection ratio-dependent manner (Fig. 2A and B). The immunoblot analyses (Fig. 2A) show that the calpain specific 145 kDa BP appeared strongly at 2 h in response to GBS-III-COH31 at a 1:100 ratio, while it did not appear in response to GBS-III-COH31 at a 1:10 ratio. Fluorogenic-substrate assay (Fig. 2B) confirmed the results of immunoblot indicating that GBS-III-COH31, at 2 h after infection at a 1:100 MΦ:GBS ratio, elicited in MΦ about threefold induction of fluorescence over the basal level, while no increase of fluorescence was observed with a 1:10 MΦ:GBS ratio. Bacterial interference in the enzymatic calpain assay was excluded since no enzymatic activity was detected in the bacterial preparation itself (results not shown). Since GBS-III-COH31 at a 1:100 MΦ:GBS ratio at 2 h after infection induced the greatest α-spectrin and Suc-LLVY-AMC cleavage (Fig. 2A and B) and greatest reduction in cytoskeletal protein expression levels (Fig. 1) in the following experiments only the 1:100 MΦ:GBS infection ratio and the 2 h infection were used.
To confirm that calpains were responsible for the cleavage of α-spectrin and Suc-LLVY-AMC we used the highly selective µ- and m-calpain inhibitors which target the Ca2+-binding [3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, PD150606] and the active (Carbobenzoxy valyl phenylalanial, MDL28170) site (Wang et al., 1996; Fettucciari et al., 2006; Niapour and Berger, 2007). Pre-treating MΦ with 100 µM PD150606, before GBS-III-COH31 infection, strongly inhibited the formation of 145 kDa BP of α-spectrin (Fig. 2C) and the increase of fluorescence by Suc-LLVY-AMC cleavage (Fig. 2D). This demonstrates that calpains are responsible for α-spectrin and Suc-LLVY-AMC cleavage. Similar results were obtained with the MDL28170 inhibitor (results not shown).
Since calpain can be activated mainly by an increase in intracellular Ca2+ levels, to determine the possible involvement of extracellular Ca2+ in calpain activation, we analysed the effect of the Ca2+ chelator, EGTA, on the appearance of calpain specific α-spectrin BP and Suc-LLVY-AMC cleavage. To this purpose MΦ monolayers at 18 h were infected or not with GBS in the presence of 1 mM EGTA in RPMI-1640 medium (containing Ca2+ and Mg2+) with 10% fetal bovine serum at pH 7.3 for 2 h. Addition of EGTA in these conditions and in particular in the presence of Ca2+ did not affect MΦ adherence, morphology and viability which were like to control MΦ, then EGTA treatment in our model did not lead to detachment of MΦ from plastic dish. EGTA at 1 mM strongly inhibited the formation of 145 kDa BP α-spectrin (Fig. 2C) and Suc-LLVY-AMC cleavage (Fig. 2D). The effect of EGTA can be reversed by addition of an excess of CaCl2 but not MgCl2 during incubation with EGTA (results not shown). Therefore these results demonstrate that calpains in the presence of EGTA are not activated and then their activation is mediated by extracellular Ca2+ influx.
Since there are two isoforms of ubiquitously expressed Ca2+-dependent calpains, the µ- and m-calpain (Goll et al., 2003) and since PD150606 and MDL28170 can inhibit both isoforms, to determine if both are involved in α-spectrin and Suc-LLVY-AMC cleavage in our experimental model, we also evaluated the effect of the highly selective µ-calpain inhibitor, 3-(5-fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (PD151746) (Wang et al., 1996; Fettucciari et al., 2006). Pre-treating MΦ with 50 µM PD151746 before GBS-III-COH31 infection did not inhibit α-spectrin (Fig. 2E) and Suc-LLVY-AMC cleavage (Fig. 2D).
To exclude the involvement of other proteases in α-spectrin and Suc-LLVY-AMC cleavage we performed experiments with: (i) the cathepsin B inhibitor IV (CA-074 Me), (ii) the inhibitor of all lysosomal cathepsins, ammonium chloride (NH4Cl), (iii) the cathepsin D inhibitor (Pepstatin A), (iv) the caspase inhibitor, boc-Asp(OMe)-fluoromethylketone (BAF) and (v) the proteasome inhibitor, clasto-Lactacystin β-Lactone (clasto-Lactacystin). The results obtained show that Suc-LLVY-AMC (Fig. 2D) and α-spectrin (Fig. 2F) cleavage were not affected by CA-074 Me (10 µM; 50 µM) (Fig. 2D and F, left panel), NH4Cl (10 mM) (Fig. 2D and F, left panel), Pepstatin A (1 µM; 10 µM) (Fig. 2D and F, right panel), BAF (25 µM) (results not shown) and clasto-Lactacystin (2 µM) (results not shown). This indicates that α-spectrin and Suc-LLVY-AMC cleavage is not due to the proteolytic activity of other proteases.
Overall, the results indicate that GBS-III-COH31, by an influx of extracellular Ca2+, activates the calpains, particularly m-calpain in MΦ.
Calpains are responsible for MΦ cytoskeletal protein degradation induced by GBS-III-COH31
To link the above demonstrated calpain activation with MΦ cytoskeleton alterations, we determined whether calpains could be involved in GBS-III-COH31-induced MΦ cytoskeletal protein degradation by analysing: (i) the effect of the calpain inhibitors (PD150606, MDL28170) and the Ca2+ chelator (EGTA) on the cleavage of Vinculin, Talin, α-actinin, Paxillin, Pyk2 and β-tubulin, and (ii) the effect of the cathepsin, caspase and proteasome inhibitors on the expression of β-tubulin, as a representative microtubule component, and the expression of Vinculin, Talin and Paxillin, as representative proteins of the actin cytoskeleton and FA.
Treating MΦ with 100 µM PD150606 or 1 mM EGTA before GBS-III-COH31 infection, strongly reduced the degradation of Vinculin, Talin, β-tubulin (Fig. 3A), Pyk2, Paxillin (Fig. 3B) and prevented that of α-actinin (Fig. 3C). Similar results were obtained with the MDL28170 inhibitor (results not shown). On the contrary, no inhibition of cytoskeletal protein degradation was observed with CA-074 Me (10 µM; 50 µM) (Fig. 3D, left panel), NH4Cl (10 mM) (Fig. 3D, left panel), Pepstatin A (1 µM; 10 µM) (Fig. 3D, right panel), BAF (25 µM) (results not shown) and clasto-Lactacystin (2 µM) (results not shown).
These data taken together indicate that calpains are specifically involved in MΦ cytoskeletal degradations induced by GBS-III-COH31.
To assess the respective role of µ- and m-calpain on MΦ cytoskeleton alterations, we first used PD151746, a µ-calpain inhibitor. PD151746 did not affect GBS-III-COH31-induced MΦ cytoskeletal protein degradation (Fig. 4A and B), suggesting that m-calpains are involved in MΦ cytoskeletal alterations.
To provide more direct evidence about the role of m-calpain in the cleavage of representative FA proteins, actin and microtubule cytoskeleton (e.g. Talin, Vinculin, Paxillin and β-tubulin) we specifically downregulated m-calpain expression by the small-interfering-RNA (siRNA)-mediated gene silencing technique. MΦ were transfected for 72 h with 100 nM of siRNA specific for m-calpain. Then the cells were: (i) harvested for analysis of m-calpain expression by immunoblotting, and (ii) infected with GBS-III-COH31 at a 1:100 ratio for immunoblot analysis of α-spectrin and cytoskeletal proteins. Transfection with m-calpain siRNA gave an optimum reduction in protein level (Fig. 4C and D).
Downregulation of m-calpain by m-calpain siRNA, which significantly reduced α-spectrin cleavage (results not shown), inhibited by about 50% the GBS-III-COH31-induced Talin and Vinculin cleavage (Fig. 4E) but did not significantly prevent the GBS-III-COH31-induced cleavage of β-tubulin and Paxillin (Fig. 4E and F).
Overall, the results demonstrate that calpains, particularly m-calpain, as a consequence of extracellular Ca2+ influx, are activated and mainly responsible for the cleavage of cytoskeletal proteins in GBS-III-COH31-infected MΦ.
GBS-III-COH31 induces quantitative and qualitative alterations in MΦ cytoskeleton
Confocal microscopy analysis performed on control MΦ and MΦ infected with GBS-III-COH31 at a ratio of 1:100 and 1:10 for 2 h, double-labelled using Alexa Fluor 488 phalloidin to detect β-actin and an Alexa Fluor 546-labelled secondary antibody (Ab) to detect Vinculin, Talin, Paxillin or β-tubulin by indirect immunofluorescence, confirmed that GBS-III-COH31 infection induces extensive quantitative alterations in the MΦ cytoskeleton.
As illustrated in Fig. 5, control MΦ as well as MΦ infected with GBS-III-COH31 at a 1:10 ratio showed an intense cytoplasmic immunostaining for Vinculin, Talin, Paxillin and β-tubulin whereas in MΦ infected with GBS-III-COH31 at a 1:100 ratio, Vinculin, Paxillin and Talin were no longer detectable and only a few cells remained positive for β-tubulin. These alterations were prevented by pre-treatment with 100 µM PD150606 (Fig. 5) in agreement with the results obtained by immunoblot analysis.
Although we observed no significant quantitative alterations in β-actin expression by immunoblot analysis, Alexa Fluor 488 phalloidin staining of GBS-III-COH31-infected MΦ revealed significant changes in β-actin localization (Fig. 5). In fact, in control MΦ, β-actin was mainly localized below the plasma membrane while most MΦ infected with GBS-III-COH31 at a 1:100 ratio were characterized by a marked increase in intracytoplasmic β-actin.
These findings show that by inducing the degradation of Vinculin, Talin, Paxillin and β-tubulin and the redistribution of β-actin, GBS-III-COH31 induces quantitative and qualitative alterations in the MΦ cytoskeleton.
Microbial factors responsible for GBS-III-COH31 alterations in MΦ cytoskeleton
In a further series of experiments we examined the possible microbial factor/s involved in GBS-III-COH31-induced MΦ cytoskeleton alterations. GBS has a pluripotent virulence factor the β-haemolysin, strictly bound to the cell surface, highly unstable when released in culture supernatants, requiring metabolic activity for its production and like pore-forming proteins is active against the membrane of several eukaryotic cells (Fettucciari et al., 2000; Nizet, 2002; Maisey et al., 2008). Because it is generally assumed that pore-forming proteins can induce Ca2+ influx which can lead to several effects (Almeida-Campos et al., 2002; Tran Van Nhieu et al., 2004) and since in our model, as reported above, the cytoskeleton alterations were mediated by calpains activated by extracellular Ca2+ influx we examined the possibility that GBS-III-COH31 could cause MΦ cytoskeleton alterations by β-haemolysin. Therefore, immunoblot analysis for Talin, Vinculin, Paxillin, Pyk2, β-tubulin degradation, to show cytoskeleton alterations, immunoblot analysis for α-spectrin cleavage, to show calpain activation and propidium iodide (PI) uptake assay, to evaluate plasma membrane permeability alterations, were performed at 2 h in MΦ incubated, at an infection ratio of 1:100 with: (i) supernatant of GBS-III-COH31 growth in culture medium for 2 h, (ii) GBS-III-COH31, in contiguous medium separated by a 0.45 µm pore size membrane of cell culture insert, (iii) hiGBS-III-COH31, GBS-III-COH31 killed at 60°C for 30 min, a condition that causes GBS β-haemolysin inactivation, and (iv) gGBS-III-COH31, GBS-III-COH31 grown for 18 h in the presence of 10 mg ml−1 glucose, a condition that abolishes GBS β-haemolysin synthesis.
Under these conditions, we observed no cleavage of α-spectrin to calpain specific 145 kDa BP (Fig. 6A, left panel) or degradation of all cytoskeletal proteins examined (Fig. 6B–D, left panels). Under these conditions we found no alterations in MΦ plasma membrane permeability that are induced only by haemolytic GBS-III-COH31 (Fig. 6E). In fact, as shown in Fig. 6E, the MΦ infected with haemolytic GBS-III-COH31 showed 77% PI+ cells, while in the MΦ infected in all other conditions the percentage of PI+ cells was 19–22% similar to that of control MΦ (19%) (Fig. 6E). In spite of 77% PI+ cells the total number of MΦ infected with haemolytic GBS-III-COH31 at 2 h after infection, evaluated by Trypan blue exclusion assay, was the same as control MΦ and MΦ infected under all other conditions (results not shown).
To provide further evidence about the role of β-haemolysin in cytoskeletal alterations, since it has been reported that some phospholipids such as DPPC inhibited GBS β-haemolytic activity (Fettucciari et al., 2000; Nizet, 2002), we also analysed the effect of DPPC. We found that DPPC in MΦ infected with GBS-III-COH31 at a 1:100 ratio strongly inhibited degradation of Vinculin, Talin, Paxillin, Pyk2 and β-tubulin (Fig. 6B–D, right panels) and prevented both α-spectrin cleavage (Fig. 6A, right panel) and alterations in membrane permeability (Fig. 6E).
Loss of β-haemolysin synthesis, due to growing GBS-III-COH31 in the presence of glucose, and inhibition of GBS β-haemolytic activity by heat-inactivation of GBS-III-COH31 or by DPPC, was confirmed by evaluating the haemolytic activity of gGBS-III-COH31, hiGBS-III-COH31 and GBS-III-COH31 in the presence of DPPC or not against sheep red blood cells (results not shown) with the haemolytic activity assay (Marchlewicz and Duncan, 1980) performed as described previously (Fettucciari et al., 2000).
Overall, these results suggest that β-haemolysin is involved in MΦ cytoskeleton alterations causing MΦ plasma membrane permeability defects which lead to Ca2+ influx and calpain activation which mediate cytoskeletal protein degradation.
GBS-III-COH31 induces degradation of the Rho GTPase family members Rho A, Rac1 and Cdc42 and of their effectors
Therefore, in an attempt to characterize the mechanisms used by GBS to subvert MΦ cytoskeleton, we examined if the GBS-III-COH31 effect against MΦ cytoskeleton was specific to structural cytoskeletal proteins or, like many other pathogens, the GBS effect was also addressed against the key regulatory cytoskeletal proteins, Rho GTPases. To this end immunoblot analysis for Rho A, Rac1 and Cdc42 was performed in MΦ infected with GBS-III-COH31, at different infection ratios.
The results show that Rho A, Rac1 and Cdc42 expression was markedly reduced at 2 h after infection in MΦ infected with GBS at a 1:100 ratio and that the effect depended on the MΦ:GBS ratio (Fig. 7A–C).
Since it is known that in several experimental models the calpains can be an intermediate signal upstream of the Rho family GTPases which are also calpain substrates (Glading et al., 2002; Franco and Huttenlocher, 2005) we investigated whether calpains could cleave Rho GTPase family members in GBS-III-COH31-infected MΦ. Immunoblot analysis shows that the decrease of f.l. Rho A, Rac1 and Cdc42 induced by GBS-III-COH31 at a 1:100 ratio was prevented by pre-treating MΦ with PD150606 (Fig. 7H–J), indicating that in our infection model, calpains are upstream of Rho GTPases and responsible of their degradation.
We also found that GBS-III-COH31-induced Rho A, Rac1 and Cdc42 degradation, like Talin, Vinculin, Paxillin, Pyk2 and β-tubulin degradation, was directly correlated to β-haemolysin expression. In fact, no reduction in the expression of Rho A, Rac1 and Cdc42 was detected with GBS-III-COH31 supernatant, GBS-III-COH31 infection, in contiguous medium separated by a 0.45 µm pore size membrane of cell culture insert, hiGBS-III-COH31, gGBS-III-COH31 and DPPC (Fig. S1).
Since Rho GTPases can be upstream regulators of actin reorganization (Hall and Nobes, 2000; Ahmadian et al., 2002; Ridley et al., 2003) and since they interact with downstream effectors, e.g. ROCK1, PAK1 and Cortactin, which converge on proteins directly regulating the actin cytoskeleton such as Filamin A and Cofilin, we determined if GBS-III-COH31 also manipulates the expression of these proteins. The results show that Cortactin, PAK1, ROCK1, Cofilin and Filamin A expression was markedly reduced at 2 h after infection in MΦ infected with GBS-III-COH31 at a 1:100 ratio (Fig. 7D–G). The effect depended on MΦ:GBS ratio (Fig. 7D–G) and this degradation was strongly inhibited by PD150606 (Fig. 7K–N).
Overall, the results indicate that GBS-III-COH31 through calpains could alter the actin cytoskeleton in MΦ not only by inducing the degradation of structural cytoskeletal proteins but also of some Rho GTPase family members and their effectors.
Effect of cytoskeletal alterations on internalization and survival of GBS-III-COH31 in MΦ
It is known that microfilament and/or microtubule elements of the eukaryotic cell cytoskeleton are required for the efficient uptake of GBS within endocytic vacuoles. In fact, disruption of actin filaments by Cytochalasin D (CytD) inhibited GBS invasion of HUVEC, HBMEC and HeLa cells (Shin et al., 2006; Maisey et al., 2008) and phagocytosis by MΦ (Fettucciari et al., 2000, and results not shown). Also disruption of microtubules by Nocodazole or colchicine inhibited GBS invasion of HEp-2, J774 and HeLa cells (Valentin-Weigand et al., 1996; 1997; Tyrrell et al., 2002) and greatly reduced the uptake of GBS-III-COH31 by MΦ (results not shown).
Since the results reported above show that inhibition of calpain activity prevents GBS-induced cytoskeletal protein degradation, to evaluate whether cytoskeletal disruption affects uptake and/or intracellular survival of GBS in MΦ we performed a GBS intracellular survival assay with calpain inhibitor, PD150606. MΦ pre-treated or not with 100 µM PD150606, were infected with GBS-III-COH31 at 1:100 and 1:10 ratios and the number of viable GBS-III-COH31 in MΦ evaluated both at 2 h after infection and at 4 h after infection (2 h infection plus 2 h incubation with antibiotics) by quantitative plating (Cornacchione et al., 1998; Fettucciari et al., 2006). The data obtained show that at 4 h after infection, at a 1:100 infection ratio, the number of intracellular GBS-III-COH31 recovered from PD150606-treated MΦ had increased about 50-fold compared with untreated MΦ (Table 1) while at a 1:10 infection ratio, the number of viable GBS-III-COH31 was the same in inhibitor-treated and untreated MΦ (Table 1). Remarkably, the number of viable GBS-III-COH31 at 2 h after infection was the same in inhibitor-treated and untreated MΦ, and the addition of PD150606 or vehicle to GBS cultures did not affect GBS-III-COH31 viability and growth (results not shown). Moreover, the total number of MΦ at 2 and 4 h after infection, evaluated by Trypan blue exclusion assay, was the same in PD150606-treated or untreated samples (results not shown). These results suggest that the increase in the number of intracellular GBS-III-COH31 in PD150606-treated MΦ is not due to an effect of the inhibitor by increasing GBS-III-COH31 growth and adhesion to MΦ or by affecting MΦ survival but to inhibition of cytoskeletal protein degradation.
Table 1. Effect of the calpain inhibitor, PD150606, on the number of intracellular GBS-III-COH31 in MΦ.
MΦ(3 × 106), pre-treated or not for 1 h with 100 µM PD150606, were infected for 2 h with GBS-III-COH31 at a ratio of 1:100(3 × 108 cfu) and 1:10(3 × 107 cfu). The inhibitor was kept all during the course of experiments.
Number of GBS-III-COH31 was evaluated by viable plate count of cfu. Data are presented as the means ± SD of three individual experiments performed in triplicate.
MΦ were recovered after 2 h GBS-III-COH31 infection as described in Experimental procedures.
MΦ infected with GBS-III-COH31 for 2 h were treated with antibiotics to kill extracellular bacteria and recovered after 2 h incubation with antibiotics(4 h) as described in Experimental procedures.
P < 0.01(GBS-infected MΦ treated with PD150606 versus untreated GBS-infected MΦ) according to Student's t-test.
To provide further evidence and depict the different number of intracellular GBS-III-COH31 in MΦ pre-treated or not with PD150606 we performed confocal microscopy analysis labelling GBS-III-COH31 with anti-GBS Ab and Alexa Fluor 568-labelled secondary Ab, and cytoskeleton with anti-Vinculin and Alexa Fluor 488-labelled secondary Ab in MΦ pre-treated or not with PD150606 infected with GBS-III-COH31 at a 1:100 ratio. To discriminate between intracellular and extracellular GBS-III-COH31 localization we also reconstructed cell volume through the ‘Cell’ module of the Imaris software, using the fluorescence of Vinculin as the protein representative of the cytoskeleton and identified each single GBS-III-COH31 as a single spot of red fluorescence. We identified the dots located in the cell volume and those located outside and automatically calculated the number and the percentage of intracellular and extracellular dots (GBS-III-COH31). As illustrated in Fig. 8 MΦ pre-treated with PD150606 had an increased number of intracellular GBS-III-COH31 with respect to MΦ infected but not treated with the calpain inhibitor, confirming the data obtained by GBS intracellular assay. Therefore the increase in the number of intracellular GBS-III-COH31 in PD150606-treated MΦ is due to an increase of GBS-III-COH31 phagocytosis by inhibition of cytoskeletal degradation.
Overall, our data suggest that cytoskeletal protein degradation is involved in GBS subversion of MΦ functions.
Effect of different GBS strains on MΦ cytoskeleton
To determine if the ability to induce marked MΦ cytoskeleton alterations is a general mechanism of GBS or is specific for the clinical GBS strain COH31 used in this study we employed the clinically isolated highly haemolytic GBS type V strain NCTC10/84 (GBS-V-10/84) (Wilkinson, 1977; Liu et al., 2004) and three different GBS strains clinically isolated and fully sequenced: GBS type III strain NEM316 (GBS-III-NEM316), GBS type Ia strain A909 (GBS-Ia-A909) and GBS type Ib strain H36B (GBS-Ib-H36B) (Glaser et al., 2002; Liu et al., 2004; Tettelin et al., 2005) for which previously a different serotype and haemolytic activity was demonstrated (Marchlewicz and Duncan, 1980; Nizet et al., 1996; Liu et al., 2004) and recently multilocus sequence type (ST) and a dispensable genome also (Glaser et al., 2002; Tettelin et al., 2005; Brochet et al., 2006). We analysed by immunoblot at 2 h of MΦ infection with GBS-III-NEM316, GBS-V-10/84, GBS-Ib-H36B, GBS-Ia-A909 at different MΦ:GBS ratios the expression of: (i) Vinculin, Talin and Paxillin, as representative proteins of the actin cytoskeleton and FA, (ii) β-tubulin as a representative microtubule component, (iii) Rho A as a representative of Rho GTPase proteins, and (iv) Cofilin as a representative downstream effector of Rho GTPases.
The results showed that at 2 h after infection GBS-III-NEM316 at MΦ:GBS ratios of 1:50 and 1:20 induces more than 90% decrease of Talin, Vinculin, Paxillin, β-tubulin, Rho A and Cofilin expression (Fig. 9A, left panels) and at a ratio of 1:10 causes 87% decrease of Rho A expression levels, 50–64% decrease for Talin and Cofilin expression, and 24% decrease of Vinculin, β-tubulin, Paxillin expression (Fig. 9A, left panels). GBS-III-NEM316 at 1:5 ratio did not alter significantly the expression levels of Vinculin, β-tubulin, Paxillin while decrease of about 40% the expression levels of Rho A, Talin and Cofilin (Fig. 9A, left panels). Similar results were found after infection with GBS-V-10/84. At 2 h infection of MΦ with GBS-V-10/84 at a MΦ:GBS ratios of 1:50 and 1:20, we found 58–70% decrease in Talin and Vinculin expression levels and more than 85% for Paxillin, β-tubulin, Rho A and Cofilin (Fig. 9A, middle panels). In response to GBS-V-10/84 at 1:10 ratio we found about 24–34% decrease in Talin, Vinculin, β-tubulin, Paxillin, Rho A and Cofilin expression while no significant alteration in the expression levels of all cytoskeletal proteins examined was observed at a 1:5 ratio (Fig. 9A, middle panels). In contrast GBS-Ib-H36B and GBS-Ia-A909 did not significantly alter the expression levels in all cytoskeletal proteins examined at all MΦ:GBS ratios used (Fig. 9A, right panels).
The above results demonstrating that marked MΦ cytoskeletal alterations are induced by GBS-III-NEM316 and GBS-V-10/84 that have a haemolytic titre of 8 and 64, respectively, but not by GBS-Ib-H36B and GBS-Ia-A909 that have a haemolytic titre of 4 (Marchlewicz and Duncan, 1980; Nizet et al., 1996; Liu et al., 2004) suggest that like what was demonstrated for GBS-III-COH31, β-haemolysin could be involved in MΦ cytoskeletal alterations inducing MΦ membrane permeability defects and consequently calpain activation. Therefore we first analysed the effect of GBS-III-NEM316, GBS-V-10/84, GBS-Ib-H36B and GBS Ia-A909 on MΦ plasma membrane permeability by PI uptake assay.
GBS-III-NEM316 and GBS-V-10/84 were able to induce marked alterations in MΦ plasma membrane permeability and the effect depended on the infection ratio (Fig. 9B). At 2 h after infection, the percentage of MΦ with permeabilized membrane increased significantly at a MΦ:GBS ratio of 1:10 to about 70% PI+ cells with GBS-III-NEM316 and to about 80% PI+ cells with GBS-V-10/84 (Fig. 9B), then at 1:20 and 1:50 ratios the percentage of PI+ cells reached 85% for GBS-III-NEM316-infected MΦ and 90% for GBS-V-10/84-infected MΦ (Fig. 9B). Instead only a weak increase in the percentage of PI+ cells was found with MΦ infected with GBS-Ib-H36B and GBS-Ia-A909 at a 1:50 ratio (Fig. 9B).
On the basis of these results we investigated if these GBS strains activate calpains evaluating by immunoblot the cleavage of α-spectrin in MΦ at 2 h after infection with GBS-III-NEM316, GBS-V-10/84, GBS-Ib-H36B and GBS-Ia-A909 at different MΦ:GBS ratios. Only GBS-III-NEM316 and GBS-V-10/84 activated calpains in MΦ and the effect was infection ratio-dependent (Fig. 9C). In fact, at 2 h infection a strong appearance of the calpain specific 145 kDa BP was found with GBS-III-NEM316 and GBS-V-10/84 at 1:50 and 1:20 ratios (Fig. 9C, left and middle panel) but not with GBS-III-NEM316 and GBS-V-10/84 at a 1:5 ratio (Fig. 9C, left and middle panel), and with GBS-Ib-H36B and GBS-Ia-A909 at all ratios used (Fig. 9C, right panel).
To confirm that calpains were also responsible for α-spectrin cleavage induced by GBS-III-NEM316 and GBS-V-10/84 we used the calpain inhibitor PD150606. Since at 2 h after infection the 1:20 MΦ:GBS ratio had already induced the greatest α-spectrin cleavage (Fig. 9C) and cytoskeletal protein degradation (Fig. 9A, left and middle panels) in these experiments and those following only the 1:20 MΦ:GBS infection ratio was used. Pre-treating MΦ with 100 µM PD150606, before infection with GBS-III-NEM316 and GBS-V-10/84, abolished the formation of 145 kDa BP of α-spectrin (Fig. 10A), demonstrating so that these two strains activate calpains.
In an attempt to determine if calpains could be involved also in the cleavage of cytoskeletal proteins induced by GBS-III-NEM316 and GBS-V-10/84 we analysed the effect of PD150606 on the expression of Vinculin, Talin, Paxillin, β-tubulin, Rho A and Cofilin. The results obtained showed that pre-treating MΦ with 100 µM PD150606 strongly inhibited cytoskeletal protein degradation induced by GBS-V-10/84 and prevented that induced by GBS-III-NEM316 (Fig. 10B and C).
Since the above reported results with GBS strains suggest a key role of β-haemolysin in MΦ cytoskeletal alterations induced by GBS-III-NEM316 and GBS-V-10/84, to provide more evidence of β-haemolysin involvement we examined the effect of DPPC. DPPC in MΦ infected with GBS-III-NEM316 and GBS-V-10/84 at a 1:20 ratio inhibited Talin, Vinculin (Fig. 11A), β-tubulin, Paxillin, Rho A and Cofilin degradation (Fig. 11B), prevent α-spectrin cleavage (results not shown) and alterations in MΦ plasma membrane permeability (results not shown). These results indicate that β-haemolysin is also involved in MΦ cytoskeletal alterations induced by GBS-III-NEM316 and GBS-V-10/84.
All together these results indicate that, like GBS-III-COH31, marked MΦ cytoskeleton alterations are also induced by other GBS strains through calpains, and the effect is correlated with β-haemolysin-induced MΦ plasma membrane permeability defects.
This study demonstrates that GBS-III-COH31 during infection induces early marked alterations of murine MΦ cytoskeleton. In particular GBS-III-COH31 affects two major classes of cytoskeletal fibres, the microtubules and the actin microfilaments. By immunoblot analysis we demonstrated that at 2 h after infection GBS-III-COH31, at a 1:100 ratio, induced extensive alterations in actin and microtubule cytoskeleton, characterized by a marked reduction in Talin, Vinculin, α-actinin, Paxillin, Pyk2 and β-tubulin expression although there was no change in β-actin expression. By confocal microscopy we confirmed that GBS-III-COH31 caused disruption of MΦ microtubules, loss of FA-associated proteins (Talin, Vinculin and Paxillin) and also showed that such disruption is associated with the redistribution of β-actin from the plasma membrane to the cytoplasm. The GBS-III-COH31-induced MΦ cytoskeleton alterations were time- and infection ratio-dependent. In fact, no significant changes in expression of all the cytoskeletal proteins examined was observed both at 1 h after GBS-III-COH31 infection with all MΦ:GBS ratios used and at 2 h after infection with GBS-III-COH31 at a MΦ:GBS ratio of 1:10.
It is well known that proteolysis and activity of many structural and regulatory cytoskeletal proteins is mediated by calpain activation (Glading et al., 2002; Goll et al., 2003; Franco and Huttenlocher, 2005) and the increase of intracellular Ca2+ concentrations (TranVan Nhieu et al., 2004; Melendez and Tay, 2008) play a crucial role both in calpain activation and in the activity of many cytoskeletal proteins. Since we have previously demonstrated that GBS-III-COH31 at a 1:100 ratio induces a strong Ca2+ influx and activates calpain in MΦ (Fettucciari et al., 2006) we investigated calpain activation during GBS-III-COH31 infection of MΦ at different infection ratios and the role of calpains and Ca2+ in the cleavage of MΦ cytoskeletal proteins, in an attempt to define the mechanisms by which GBS-III-COH31 induces marked MΦ cytoskeleton alterations.
This study shows that GBS-III-COH31 at a 1:100 ratio activates in MΦ the Ca2+-dependent protease calpains, demonstrated by: (i) the cleavage of calpain substrate α-spectrin into the calpain specific 145 kDa BP, (ii) the increase in total levels of calpain activity measured by the fluorogenic calpain substrate (Suc-LLVY-AMC) cleavage in situ, and (iii) the inhibition of α-spectrin and Suc-LLVY-AMC cleavage by the µ- and m-calpain inhibitor PD150606 but not by PD151746 (µ-calpain inhibitor) and by cathepsin, caspase and proteasome inhibitors. Furthermore, the demonstration that EGTA (extracellular Ca2+ chelator) inhibited α-spectrin and Suc-LLVY-AMC cleavage indicates that in GBS-III-COH31-infected MΦ the calpains are activated as a consequence of extracellular Ca2+ influx and also suggests that calpain activation, unlike what occurs in other experimental models, does not require other factors (Glading et al., 2002; Goll et al., 2003; Franco and Huttenlocher, 2005; Fettucciari et al., 2006). The GBS-III-COH31-induced calpain activation was time- and infection ratio-dependent. In fact, no cleavage of α-spectrin or Suc-LLVY-AMC was observed both at 1 h after GBS-III-COH31 infection with all MΦ:GBS ratios used and at 2 h after infection with GBS-III-COH31 at a MΦ:GBS ratio of 1:10.
Interestingly, calpains, particularly m-calpain, and Ca2+ play a major role in the MΦ cytoskeleton alterations as demonstrated by inhibition of cytoskeletal protein cleavage by PD150606, siRNA for m-calpain and EGTA but not by PD151746 (µ-calpain inhibitor) and other protease inhibitors. The apparent discrepancies between the effect of pharmacological inhibition (PD150606) and m-calpain siRNA in the cleavage of some cytoskeletal proteins (β-tubulin and Paxillin) could be explained taking into account that the regulation of calpains is complex and their activity is poorly correlated with protein levels (Niapour and Berger, 2007) so that in GBS-III-COH31-infected MΦ the residual m-calpain activity after knock-down by siRNA could suffice for the proteolysis of some cytoskeletal proteins. Moreover, PD150606 acts through a mixed form of inhibition affecting both catalytic activity and substrate binding (Wang et al., 1996; Niapour and Berger, 2007).
It is known that Rho GTPases, a protein family that in several cellular processes including antimicrobial MΦ defences act as upstream regulators of actin reorganization since they mediate this function by interacting with downstream effectors which converge on proteins that directly regulate the behaviour of actin cytoskeleton (Hall and Nobes, 2000; Ahmadian et al., 2002; Ridley et al., 2003), are a common target of pathogens used to manipulate cytoskeletal and subvert host defences (Fiorentini et al., 2003; Aktories and Barbieri, 2005). Recently Shin and Kim (2006) and Burnham et al. (2007) have shown that GBS activates Rho family GTPase members (Rho A, Rac1 and Cdc42) to manipulate the cytoskeleton of non-phagocytic epithelial and endothelial cells and promote its internalization in the host. Our results indicate that GBS-III-COH31 induces degradation of the Rho GTPase family members, Rho A, Rac1, Cdc42, but also of downstream Rho GTPase effectors, ROCK1, PAK1, Cortactin and of some proteins that directly regulate the actin cytoskeleton such as Filamin A and Cofilin. The degradation of all these proteins, as demonstrated for the FA and cytoskeletal proteins reported above, is time- and infection ratio-dependent and is mediated by calpains as shown by protection against proteolysis by PD150606. Therefore GBS seems to use several targets to disarm the MΦ cytoskeleton functions.
Two main putative mechanisms of calpain action in cytoskeletal rearrangements have been described in literature (Glading et al., 2002; Franco and Huttenlocher, 2005). The first is based on the effects of calpains on focal structure and disassembly. In fact, several FA components, including Paxillin, Talin, α-actinin, Vinculin, Cortactin and Pyk2, have been identified in vitro as potential calpain substrates. The second is based on the role of calpains as a signalling intermediate upstream of the Rho family GTPases. In this latter mechanism the calpains by activation, inhibition or cleavage of Rho GTPase members might arrest their functional activities and then GTPase-mediated cytoskeletal reorganization. Our results, showing that GBS-III-COH31 through calpain activation induced loss of FA-associated proteins and Rho GTPases (Rho A, Rac1, Cdc42), suggest that GBS can use the two putative calpain action mechanisms for cytoskeleton manipulation.
Our results and the report by Shin and Kim (2006) and Burnham et al. (2007) show that GBS to survive in the host can act on Rho GTPase family members in different ways inducing opposite effects. In fact, GBS induces Rho GTPase degradation in MΦ or their activation in endothelial and epithelial cells leading to reduction of uptake by MΦ or invasion by endothelial and epithelial cells. This difference is very interesting and suggests that GBS, like other microorganisms, could have evolved different mechanisms for Rho GTPase utilization, depending on the cell type (Fiorentini et al., 2003; Aktories and Barbieri, 2005; Rottner et al., 2005). However, further studies are necessary to provide more information about GBS manipulation of Rho GTPases in MΦ.
The mechanism and strategy by which GBS-III-COH31 inhibits the activity of the MΦ cytoskeleton differs from that used by intracellular and extracellular pathogens such as Clostridium, Yersinia, Salmonella, Pseudomonas and Escherichia. In fact, inhibition of actin cytoskeleton activity induced by these pathogens is mediated in two main ways (Rottner et al., 2005): one mediated by interaction of microorganisms with eukaryotic receptors, which initiates actin rearrangements. Another way is mediated by directly introducing bacterial effector molecules into the cytoplasm of a target cell which can modify actin directly (e.g. actin-ADP ribosylation activity) or indirectly (e.g. dephosphorylation of cytoskeletal signalling proteins; activation/inactivation of the Rho GTPases) by several mechanisms (Fiorentini et al., 2003; Gruenheid and Finlay, 2003; TranVan Nhieu et al., 2004; Aktories and Barbieri, 2005; Rottner et al., 2005; Bhavsar et al., 2007). Instead, inhibition of cytoskeleton activity by GBS-III-COH31 is mediated by influx of extracellular Ca2+ and activation of calpains which cleave several structural and regulatory cytoskeletal proteins.
In an attempt to define the GBS-III-COH31 virulence factor/s involved in MΦ cytoskeleton alterations we tested the role of GBS β-haemolysin. GBS β-haemolysin contributes to GBS pathogenicity by virtue of different ability against various targets including its activity, like pore-forming proteins, against the membrane of several eukaryotic cells that lead to necrosis in epithelial/endothelial cells and to apoptosis in macrophages (Nizet et al., 1996; Fettucciari et al., 2000; Nizet, 2002; Liu et al., 2004; Maisey et al., 2008; Rajagopal, 2009). The crucial role of haemolysin in GBS pathogenicity is also supported by the observation that the virulence of haemolysin-deficient GBS mutants is attenuated in various animal models of GBS infection and that non-haemolytic strains rarely cause infections (Liu et al., 2004; Sigge et al., 2008; Rajagopal, 2009).
Our data show that the expression of GBS β-haemolytic activity is directly correlated with MΦ cytoskeleton alterations. In fact, infection in different conditions that suppress the haemolytic activity of GBS-III-COH31 (Fettucciari et al., 2000; Nizet, 2002) did not induce MΦ cytoskeletal protein degradation. Moreover, the demonstration that in these conditions the alterations of plasma membrane permeability and calpain activation did not occur suggests that GBS β-haemolysin is responsible for generating small pores and in these conditions the MΦ plasma membrane allows the influx of Ca2+ which directly activates calpains and these mediate cytoskeletal protein degradation (Fig. 12).
We previously demonstrated that GBS by β-haemolysin induces murine MΦ apoptosis at 24 h (Fettucciari et al., 2000; 2006) and although the time frame of MΦ cytoskeletal disruption look to indicate that it will lead to apoptosis, preliminary results showing that GBS-induced apoptosis occurs also in the absence/reduction of MΦ cytoskeletal disruption suggest that MΦ cytoskeletal disruption and apoptosis are two independent phenomena linked only by the necessity of Ca2+ influx and calpains (K. Fettucciari et al., unpubl. obs.). However further comprehensive studies are necessary to provide definitive finding on the relation that exists between the two phenomena.
Interestingly, since the microtubule and microfilaments play a pivot role in GBS phagocytosis, as demonstrated by the fact that depolymerizing them, respectively, with nocodazole and CytD blocked GBS phagocytosis, the demonstration (by intracellular survival assay and confocal microscopy analysis) that at 4 h after infection the number of live GBS-III-COH31 within MΦ had increased after treatment with PD150606, which inhibited the degradation of all cytoskeletal proteins and Rho GTPase members examined, suggests that GBS through early MΦ cytoskeleton alterations could escape cytoskeleton dependent MΦ defence mechanisms.
Recently it was discover that GBS can be defined by a ‘pan genome’ including a core genome that represents genes present in all GBS strains and a dispensable genome constituted by genes absent in one or more GBS strains and genes that are unique to each GBS strains (Tettelin et al., 2005; Brochet et al., 2006). Therefore, to determine if the ability of GBS-III-COH31 to induce marked alterations of cytoskeleton is unique for this strain or a general mechanism used by GBS, we analysed whether other clinically isolated GBS strains and some fully sequenced strains could alter the MΦ cytoskeleton. Our data indicate that marked cytoskeleton alterations are also induced, by GBS-III-NEM316 and GBS-V-10/84, even at low MΦ:GBS ratios, through calpain activation as demonstrated by inhibition of MΦ cytoskeleton alterations by PD150606. Whereas MΦ cytoskeleton alterations were not appreciable, even at a 1:50 ratio, with GBS-Ia-A909 and GBS-Ib-H36B, which did not induce MΦ plasma membrane permeability defects and calpain activation. Our results also demonstrated that DPPC, a β-haemolysin inhibitor, prevents MΦ cytoskeleton alterations, calpain activation and plasma membrane permeability defects induced by GBS-III-NEM316 and GBS-V-10/84. Therefore, these results, together with the knowledge that these strains showed different haemolytic activity, indeed GBS-V-10/84 had a haemolytic titre of 64 units, GBS-III-NEM316 had a haemolytic titre of 8 units while GBS-Ia-A909 and GBS-Ib-H36B had a haemolytic titre of 4 units (Marchlewicz and Duncan, 1980; Nizet et al., 1996; Liu et al., 2004), strongly suggest that the ability of GBS to cause MΦ cytoskeleton disruption depends on GBS β-haemolysin activity. It seems that a threshold of β-haemolytic activity is required to trigger the effect likely to induce a significant intracellular Ca2+ increase.
However, in the light of the genetic diversity of GBS strains and the recent evidence that various genes in the operon cyl other than cylE (cylA, cylB, cylJ, cylK) are associated with post-translation modification of β-haemolysin expression (Rajagopal, 2009), further comprehensive studies also with other GBS strains are necessary to full understand the role of β-haemolysin on MΦ cytoskeleton alterations, the possible involvement of other virulence factors and how the expression of these is regulated during infection with the different GBS strains.
Altogether the results obtained with the different GBS strains and with GBS-III-COH31 showing that β-haemolysin is involved in MΦ cytoskeleton alterations in virtue of its pore-forming-like activity against eukaryotic cell membrane add another effect of β-haemolysin to those previously demonstrated based on alterations of MΦ cytoskeleton that could contribute to evasion of antimicrobial MΦ functions.
These in vitro results mean that this pathogen has evolved a multiple and more complex strategy than previous thought by which it can escape the first line of innate host defence mechanisms, played in particular by MΦ. In fact, while until now it was known that GBS can evade MΦ phagocytosis by an antiphagocytic capsule, survive in MΦ like an intracellular microorganism and induce apoptosis, this study adds evidence of a further mechanism by which GBS can avoid MΦ antimicrobial responses based on induction of a precocious MΦ cytoskeleton disruption by GBS β-haemolysin. It is very interesting that only the more haemolytic GBS strains are able to degrade MΦ cytoskeleton at infection ratios in the range of 1:20 to 1:100. This evidence could be an additional feature of major pathogenicity. It is possible to speculate that GBS regarding MΦ have evolved a complex strategy to enhance their chance of survival so exemplified: GBS non-haemolytic, GBS weakly haemolytic and GBS haemolytic at low multiplicity of infection, can survive inside MΦ. Whereas GBS with more haemolytic activity when the infection load progressively increases can cause early MΦ cytoskeleton degradation and then apoptosis.
In conclusion, this study provides the first evidence that GBS (GBS-III-COH31, GBS-III-NEM316 and GBS-V-10/84) causes an early marked disruption of the actin and microtubule MΦ cytoskeleton through calpain activation mediated by extracellular Ca2+ influx (demonstrated for GBS-III-COH31) following induction of MΦ plasma membrane permeability defects. This effect is directly correlated with the expression of GBS β-haemolytic activity (Fig. 12). The ability of some GBS strains to manipulate the MΦ cytoskeleton by degradation mediated by calpains activated by the influx of extracellular Ca2+ seems unusual among the mechanisms of bacteria-induced cytoskeletal rearrangements where, with only a few exceptions, they subvert and control the assembly/disassembly of actin filaments and microtubules by modulating activation of the cellular regulators of this process through the activity of delivered effectors, toxins or the engagement of host cell receptors. Furthermore, the demonstration that prevention of cytoskeletal alterations by inhibition of calpain activity increases the number of viable intracellular GBS-III-COH31 in MΦ suggests that early alterations in MΦ cytoskeleton induced by GBS with a greater haemolytic activity could be another important pathogenic mechanism that could contribute to GBS overall strategy to counteract antimicrobial MΦ functions.
PD150606 (a selective inhibitor for µ- and m-calpain directed to the Ca2+ binding sites; IC50: 210 nM for µ-calpain; IC50: 370 nM for m-calpain), PD151746 (a 20-fold selective inhibitor for µ-calpain directed to the Ca2+ binding sites; IC50: 260 nM for µ-calpain; IC50: 5.8 µM for m-calpain), MDL28170 (a potent cell-permeable inhibitor of µ- and m-calpain directed to the active site; IC50: 8 nM), CA-074 Me (an inhibitor of cathepsin B; IC50: 2.24 nM), Pepstatin A (an inhibitor of cathepsin D; IC50: 10 nM), NH4Cl (an inhibitor of all lysosomal proteases; IC50: 5 mM), BAF (a broad-spectrum caspase inhibitor; IC50: 1.2 nM), clasto-Lactacystin (a proteasome inhibitor; IC50: 1 µM) were obtained from Calbiochem (San Diego, CA). BSA, Triton X-100, Nocodazole, CytD, DPPC (an inhibitor of β-haemolytic activity), Suc-LLVY-AMC (a calpain-specific and membrane-permeable fluorogenic substrate), PI, rabbit polyclonal Ab anti-m-Calpain raised against Domain III, mouse monoclonal Abs anti-β-actin (clone AC-15), anti-β-tubulin (clone TUB 2.1), anti-Talin (clone 8D4), anti-Vinculin (clone hVIN-1), anti-α-actinin (clone BM-75.2) were obtained from Sigma (St. Louis, MO). Rabbit polyclonal Ab anti-Cdc42 (P1) and mouse monoclonal Ab anti-Rho A (26C4) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal Ab anti-Rac1 (clone 23A8) and anti-Cortactin (clone 4F11) were obtained from Upstate (Lake Placid, NY). Mouse Ab anti-Paxillin (clone 349) and Pyk2 (clone 11) were obtained from BD Trasduction Laboratories (Lexington, KY). Mouse α-spectrin (nonerythroid; MAB1622) mAb was purchased from Chemicon International (Temecula, CA). Rabbit polyclonal Abs anti-Filamin A, anti-Cofilin, anti-PAK1 and rabbit mAb anti-ROCK1 were obtained from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase (HRP)-conjugated secondary anti-mouse IgG and ECL system were obtained from GE Healthcare (Arlington Heights, IL), HRP-linked anti-rabbit IgG Abs were obtained from Cell Signaling Technology. Rabbit polyclonal Ab anti-GBS was purchased from AbD serotec (Langford lane, Kidlington, Oxford, UK). 4′,6′-Diamidino-2-phenylindole,dilactate (DAPI), ProLong Gold antifade reagent, Alexa Fluor 546-labelled goat anti-mouse IgG (H + L) Ab, Alexa Fluor 488-labelled goat anti-mouse IgG (H + L) Ab, Alexa Fluor 568-labelled goat anti-rabbit IgG (H + L) Ab and Alexa Fluor 488 phalloidin were obtained from Molecular Probes, Invitrogen detection technologies (Eugene, Oregon). Paraformaldehyde extra pure was obtained from Merck (Darmstadt, Germany). The siRNA duplexes specific for mouse m-calpain (Catalogue No. M-043027) were obtained from Dharmacon RNA Technologies (Boulder, CO). The siRNA for m-calpain contained four RNA sequences in a pool SmartPool selected from the NCBI RefSeq Database by a proprietary algorithm.
Group B Streptococcus, type III, strain COH31 r/s (GBS-III-COH31), clinically isolated from a foot ulcer of a diabetic adult, rendered resistant to rifampicin and streptomycin (Gibson et al., 1989) was kindly provided by Dr M. Wessel (Channing Laboratory, Boston, MA). GBS-III-COH31 was grown in Todd Hewitt Broth (THB; Difco Laboratories, Detroit, MI) at 37°C and aliquots stored at −70°C until used.
Group B Streptococcus, type V, strain NCTC10/84 (S. agalactiae ATCC®49447) (GBS-V-10/84) freshly purchased from American Type Culture Collection (Manassas, USA) (Wilkinson, 1977; Nizet et al., 1996; Liu et al., 2004), Group B Streptococcus, type III, strain NEM316 (GBS-III-NEM316; CIP 82.45), Group B Streptococcus, type Ia strain A909 (GBS-Ia-A909; CIP 82.43), Group B Streptococcus, type Ib strain H36B (GBS-Ib-H36B; CIP 82.42), freshly purchased from the Collection of the Institute Pasteur (Paris, France) (Glaser et al., 2002; Liu et al., 2004; Tettelin et al., 2005; Brochet et al., 2006), were propagated according to their respective product information sheets, then aliquots and stored at −70°C until used.
For assays, all the GBS strains were grown in THB overnight, back diluted in fresh THB and grown to late-log phase, washed three times with PBS (3000 g, 10 min, 22°C) and resuspended in RPMI-1640 with glutamine to the desired number of cfu ml−1 (Cornacchione et al., 1998). Bacterial numbers were determined photometrically (600 nm) and confirmed in each experiment by quantitative culture on Islam agar (Difco Laboratories) plates containing 5% heat-inactivated horse serum (Cornacchione et al., 1998) and on Todd Hewitt agar (THA, Difco Laboratories).
For some experiments, GBS-III-COH31, resuspended to the desired number of cfu ml−1 as described above (Cornacchione et al., 1998), was heat-inactivated (0.5 h 80°C; hiGBS-III-COH31) then extensively washed in PBS (3000 g, 10 min, 22°C), aliquoted and stored at −70°C until used. Sterility was confirmed by culture on THA plates.
For some experiments GBS-III-COH31 was grown for 18 h in THB in the presence of 10 mg ml−1 glucose (gGBS-III-COH31), conditions that do not allow haemolytic activity expression (Fettucciari et al., 2000; Nizet, 2002), and then treated as described above.
For experiments with GBS-III-COH31 supernatant, GBS-III-COH31 was grown for 2 h in RPMI-1640 medium with 10% fetal bovine serum at a concentration equivalent to the 1:100 MΦ:GBS ratio (3 × 108 cfu of GBS-III-COH31 for each 2 ml medium). The bacteria were then pelleted by centrifugation at 3000 g for 10 min at 22°C and the resulting supernatant was filter sterilized with a 0.2 µm nitrocellulose membrane.
Preparation of peritoneal MΦ
Outbred female CD-1 8- to 10-week-old mice were obtained from Charles River Breeding Laboratories, Calco, Milan, Italy.
Murine peritoneal MΦ were elicited by intraperitoneal injection of 1 ml of 10% Thioglycollate broth solution (Difco Laboratories) and cells were recovered 4 days later as previously described (Fettucciari et al., 2000). Cells were resuspended in cold antibiotic-free RPMI-1640 medium with 10% fetal bovine serum (complete medium) and cell viability evaluated by Trypan blue exclusion method.
The purity of Thioglycollate-elicited MΦ obtained by washing the peritoneal cavity was more than 80%, as determined by binding of monoclonal Ab to CD14 (Fettucciari et al., 2006). When the MΦ were allowed to adhere for 90 min in six-well tissue culture plates and the non-adherent cells removed by washing, the resulting MΦ population was 98% pure as determined by non-specific esterase staining and by binding of monoclonal Ab to CD14 (Fettucciari et al., 2006).
For PI uptake assay and Trypan blue exclusion assay, MΦ were harvested from monolayers by gently pipetting repeatedly after incubation with EDTA 0.5 mM in PBS without Ca2+ and Mg2+ pH 8 at 37°C for 15 min which, rather than MΦ monolayer scraping, allows the recovery of MΦ without significantly altering plasma membrane permeability and cell viability.
MΦ (3 × 106 in 2 ml of complete medium) were allowed to adhere for 90 min at 37°C, 5% CO2 in six-well plates and then non-adherent cells were removed. After 18 h, MΦ monolayers were infected with GBS-III-COH31, at a cell:microorganism ratio of 1:100, 1:50 or 1:10 for 1 or 2 h. For some experiments, MΦ, seeded 18 h before in a six-well plate, infected for 2 h washed and reincubated for 2 h in complete medium containing 100 U ml−1 penicillin and 100 µg ml−1 gentamicin, were also used. Control MΦ were incubated in medium without GBS-III-COH31 for the same times.
Infection of MΦ monolayers with hiGBS-III-COH31 or gGBS-III-COH31 at a cell:microorganism ratio of 1:100 was as described above. For some experiments MΦ monolayers were also incubated for 2 h with GBS-III-COH31 supernatant.
Infection of MΦ monolayers with GBS-III-NEM316, GBS-V-10/84, GBS-Ia-A909, GBS-Ib-H36B, at a cell:microorganism ratio of 1:50, 1:20, 1:10 and 1:5, was as described above.
For experiments with calpain inhibitors we added PD150606 (100 µM), MDL28170 (10 µM), PD151746 (50 µM) to MΦ 1 h before GBS infection and kept during the 2 h infection at the same concentration. During the 2 h reincubation PD150606 was added at 50 µM.
For EGTA experiments, 1 mM was added to MΦ during the 2 h infection.
For experiments with other protease inhibitors, CA-074 Me (10 and 50 µM), NH4Cl (10 mM), BAF (25 µM) or clasto-Lactacystin (2 µM) was added to MΦ 1.5 h before GBS infection and kept during the 2 h of infection.
For Pepstatin A experiments, 1 and 10 µM was added to MΦ 18 h before GBS infection and kept during the 2 h of infection.
For experiments with CytD (1 µg ml−1) and Nocodazole (30 µM), the inhibitors were added to MΦ 1 h before GBS infection and kept during the 2 h of infection.
Controls were MΦ treated with each inhibitor under the same conditions (time and concentration) as GBS-infected MΦ, but not infected.
For DPPC experiments, 2 mg ml−1 in PBS of sonicated DPPC (1 min, 30 W) was added to GBS for 5 min before infection with MΦ. Controls were MΦ treated with DPPC in the same condition as infected MΦ, but not infected.
Dose–response experiments evaluating calpain activity and cytoskeletal degradation in GBS-infected MΦ, and cell viability in control MΦ showed that in our cellular model the IC50 for PD150606 is 40 µM, and the maximal concentration of inhibitor tolerated is 125 µM, while for MDL28170 the IC50 is 5 µM and the maximal concentration of inhibitor tolerated is 12.5 µM. As regards PD151746, CA-074 Me, Pepstatin A since these inhibitors did not affect cytoskeletal degradation dose–response experiments were performed in control MΦ, evaluating cell viability by the Trypan blue exclusion method after incubation with serial dilutions of each inhibitor for different times. The results obtained showed that 50 µM for PD151746, 50 µM for CA-074 Me, 10 µM for Pepstatin A, 10 mM for NH4Cl, 25 µM for BAF and 2 µM for clasto-Lactacystin are the maximal concentrations of inhibitor tolerated and can be used in MΦ without affecting cell viability, which remained at 98% as evaluated by Trypan blue assay. The effectiveness of the dose of BAF (25 µM) and clasto-Lactacystin (2 µM) used in MΦ was previously demonstrated evaluating, respectively, caspase activity and proteosome activity (Fettucciari et al., 2006). Therefore the doses were chosen taking account of inhibitor specificity and cytotoxicity.
PD150606, MDL28170, PD151746, CA-074 Me, Pepstatin A, CytD and Nocodazole were dissolved in dimethylsulfoxide at a concentration 400 times greater than the final concentration used. This dilution (1:400) in medium showed no vehicle toxic effect.
The different times of MΦ pre-incubation with different inhibitors were necessary to achieve the maximum inhibitor effect.
At different times, infected and control MΦ monolayers (3 × 106 cells per well; 12 × 106 cells per sample) were scraped into 100 µl well of a modified Radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 2 mM EDTA, 0.1% SDS, 20 mM β-glycerophosphate, 1 mM PMSF, 10 µg ml−1 leupeptin, 10 µg ml−1 aprotinin, 1 mM sodium orthovanadate, 1 µg ml−1 Pepstatin A, 10 mM benzamidine and 10 mM sodium fluoride, Sigma) on ice and clarified by centrifuging at 16 000 g for 15 min at 4°C. Protein concentrations were determined by a standard Bradford protein assay (Bio-Rad Laboratories, Hercules, CA).
Proteins, 20 µg, were boiled for 5 min at 95°C, separated on 10% or 12% SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. The blots were blocked with 5% milk in Tris-buffered saline plus 0.1% Tween-20 (TBST) for 1 h at room temperature, and then incubated overnight at 4°C with an appropriate dilution of the primary specific Abs. The blots were then washed four times with TBST and incubated for 1 h with the appropriate HRP-conjugated secondary Ab. Immunoreactive bands were developed using ECL. Autoradiography was performed for variable times. Stripping the blots was performed in stripping buffer (0.2 M NaOH in bidistilled water) for 15 min at room temperature and after four washes with TBST the blots were reprobed as described above. β-actin was used as loading control.
Each blot was subjected to densitometric analysis and the density of the bands corresponding to the respective protein was quantified after scanning by Quantity One software (Bio-Rad) and expressed as arbitrary units relative to the densitometric units of β-actin.
Measurement of calpain activity by a fluorogenic-substrate assay in intact cells
A Fluorogenic-substrate assay, using the calpain-specific and membrane-permeable fluorogenic substrate Suc-LLVY-AMC, was performed as previously described (Fettucciari et al., 2006) to detect calpain activity in intact cells (Wang et al., 1996; Niapour and Berger, 2007). Proteolytic hydrolysis of the peptidyl-7-amino bond liberates the highly fluorescent AMC moiety.
MΦ monolayers in 96-well black plates (Costar, Cambridge, MA) at 105 cells per well in 100 µl of assay buffer (115 mM NaCl, 1 mM KH2PO4, 5 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, 25 mM Hepes, pH 7.4) were pre-incubated with or without calpain and protease inhibitors and then 80 µM Suc-LLVY-AMC was added.
After incubation for 30 min at 37°C 5% CO2, cells were infected with GBS-III-COH31 and the plates incubated at 37°C in the fluorometer.
Controls were: MΦ monolayers not infected pre-treated or not with the inhibitors and incubated with and without Suc-LLVY-AMC, GBS-III-COH31, without MΦ, pre-treated or not with the inhibitors incubated with and without Suc-LLVY-AMC.
Fluorescence was measured at 30 min intervals starting immediately after addition of GBS-III-COH31 for up to 2 h with a Titertek Fluoroskan II platereader fluorometer with 355 nm excitation and 460 nm emission filters (Flow Laboratories, Mc Lean, VA). Standard curves were generated with free AMC (Sigma) for each experiment. Calpain activity was calculated and gave fold increases over basal activity.
Immunofluorescence and phalloidin staining
MΦ (2 × 106) were allowed to adhere on coverslips immersed in six-well plates for 90 min and then non-adherent cells were removed. After 18 h, MΦ monolayers pre-treated or not with 100 µM PD150606 were infected for 2 h with GBS-III-COH31, as described above, in presence or absence of the calpain inhibitor. After PBS washes, the cells were fixed for 20 min with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 10 min, and after washing in PBS containing 0.05% Triton X-100 (PBSTr) incubated in blocking buffer (PBSTr containing 2.5% BSA) for 30 min. The primary Ab (anti-β-tubulin, anti-Talin, anti-Vinculin, anti-Paxillin), diluted 1:100 in blocking buffer, was added and incubated for 1 h. Then Alexa Fluor 546-labelled goat anti-mouse IgG Ab was added at a 1:200 dilution. Alexa Fluor 488 phalloidin was used to detect F-actin. DAPI was added at 1 µg ml−1 to counterstain nuclei. The coverslips were mounted on microscopic glass slides with ProLong Gold antifade medium. All steps were performed at room temperature.
Fluorescence was evaluated by confocal microscopy (Nikon, C1 on Eclipse Ti-5; EZC1 software) fitted with an argon laser (488 nm excitation), a He/Ne laser (542 nm excitation) and UV excitation at 405 nm (DAPI staining) from a blue diode.
Assay for GBS intracellular survival in MΦ
The MΦ monolayers pre-treated or not with 100 µM PD150606 were infected for 2 h with GBS-III-COH31 at a 1:100 and 1:10 ratio, as described above, in the presence or absence of the calpain inhibitor, PD150606. The culture supernatants of infected MΦ were then removed by aspiration. MΦ monolayers were washed three times with antibiotic-free medium. To quantify the number of intracellular and adherent GBS-III-COH31, the cells were washed and then lysed with Triton X-100 at a final concentration of 0.1% (v/v) in sterile distilled water. Serial dilutions of lysate from each well were prepared and 0.1 ml of each dilution was plated on Islam agar. The number of cfu was determined after 24 h incubation under anaerobic conditions.
To kill extracellular bacteria, the cultures of MΦ infected for 2 h were incubated for a further 2 h (time 4 h after infection: 2 h infection plus 2 h incubation with antibiotics) in complete medium containing 100 U ml−1 penicillin and 100 µg ml−1 gentamicin. At 4 h the supernatants containing antibiotics were removed, the cells washed and the number of intracellular GBS-III-COH31 quantified as described above and in our previous reports (Cornacchione et al., 1998; Fettucciari et al., 2006).
Exposure of GBS-III-COH31 to 100 U ml−1 penicillin and 100 µg ml−1 gentamicin for 2 h was sufficient to kill 100% of extracellular microorganisms.
Assay for GBS-III-COH31 intracellular survival in MΦ with CytD and Nocodazole was performed as described above.
siRNA transfection was performed as previously described (Fettucciari et al., 2006) and according to the manufacturer's protocol for Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Briefly, 100 nM siRNA specific for the catalytic subunit of mouse m-calpain was solubilized in the appropriate amount of RPMI-1640 without serum and mixed gently. Lipofectamine 2000 was mixed gently, then diluted in the appropriate amount of RPMI-1640 without serum, mixed gently and incubated at room temperature. After 5 min incubation, the diluted siRNA was combined with the diluted Lipofectamine 2000, mixed gently and incubated for 20 min to allow complex formation. The siRNA–Lipofectamine complexes were added to the cultured MΦ seeded 18 h before in a six-well plate at 70–80% cell confluence. The plates were mixed gently by rocking back and forth and incubated for 72 h at 37°C 5% CO2. Cell viability was monitored continuously throughout the experiments. Ninety-eight per cent cells were vital during the course of all experiments as determined by Trypan blue exclusion assay.
At 72 h post transfection the effect of siRNA was evaluated analysing the m-calpain knockdown by immunoblotting using specific Ab, and β-actin as loading control. Furthermore, at 72 h post transfection the cells were washed, placed in fresh RPMI and infected with GBS-III-COH31 as described above. Immunoblot analysis was performed as described above.
PI uptake assay
At 2 h after infection, infected and control MΦ were recovered from monolayer as described above, washed, adjusted to 1 × 106 ml−1 in PBS containing PI (5 µg ml−1), incubated at 23°C for 5 min and analysed on an EPICS XL-MCL flow cytometer (Instrumentation Laboratory, Beckman Coulter, Miami, FL). Data were processed by an Intercomp computer and analysed with SYSTEM II software (Instrumentation Laboratory, Beckman Coulter).
Immunofluorescence staining of GBS-III-COH31
MΦ monolayers on coverslips, prepared as described above, pre-treated or not with 100 µM PD150606, were infected with GBS-III-COH31, as described above. After 2 h the culture supernatants of infected MΦ were removed by aspiration and the MΦ-infected cultures were incubated for a further 2 h in complete medium containing antibiotics (Cornacchione et al., 1998; Fettucciari et al., 2006). At 4 h (2 h infection plus 2 h incubation with antibiotics) the supernatants containing antibiotics were removed by washing and the MΦ fixed, permeabilized and blocked as described above. Next the MΦ were incubated for 1 h with polyclonal anti-rabbit anti-GBS Ab (1:100) and with monoclonal anti-Vinculin Ab (1:100). After washes, Alexa Fluor 568-labelled goat anti-rabbit IgG Ab (1:200), to detect GBS, and Alexa Fluor 488 goat anti-mouse IgG Ab (1:200), to detect Vinculin as a marker of cytoskeleton alteration, were added for 1 h. After washing, the coverslips were mounted on microscopic glass slides with ProLong Gold antifade medium. All steps were performed at room temperature.
The images were acquired with a confocal microscope (Zeiss, LSM 510). The files were transferred to a graphic work station and analysed with the ‘Imaris’ software (Bitplane, Zurich, Switzerland). For identification and quantification of intracellular GBS-III-COH31 the ‘Cell’ module of the software was used. Cell volume was reconstructed using fluorescence of Vinculin as the protein representative of the cytoskeleton. Each single GBS was identified as a single spot of red fluorescence (dot). The dots located in the cell volume and external to it were identified by the software, and the number and the percentage were automatically calculated.
Data of calpain activity assays in absence and presence of inhibitors, PI uptake assay and GBS intracellular survival in MΦ are presented as the means ± standard deviation (SD) of six independent experiments performed in triplicate. The data were analysed by Student's t-test. Immunoblot analysis was repeated six times in four independent experiments and representative blots are shown. Immunoblot analysis in absence and presence of inhibitors with the other GBS strains was repeated four times in three independent experiments. Confocal microscopy was repeated four times and representative images are shown.
The authors thank Catherine Bennett Gillies for the excellent assistance in preparing the manuscript.
This work was supported by a grant from Fondazione Cassa di Risparmio di Perugia, Italy (bando 2010-2010.020.0131 Ricerca Scientifica e Tecnologica; K.F.), PRIN (Anno 2008 – prot. 2008L57JXW_003; K.F.) and Polo Didattico e Scientifico di Terni, Italy (2007-09; P.M.).