Requirement for the Rac GTPase in Chlamydia trachomatis invasion of non-phagocytic cells

Chlamydiae are gram-negative obligate intracellular pathogens to which access to an intracellular environment is paramount to their survival and replication. To this end, chlamydiae have evolved extremely efficient means of invading nonphagocytic cells. To elucidate the host cell machinery utilized by Chlamydia trachomatis in invasion, we examined the roles of the Rho GTPase family members in the internalization of chlamydial elementary bodies. Upon binding of elementary bodies on the cell surface, actin is rapidly recruited to the sites of internalization. Members of the Rho GTPase family are frequently involved in localized recruitment of actin. Clostridial Toxin B, which is a known enzymatic inhibitor of Rac, Cdc42 and Rho GTPases, significantly reduced chlamydial invasion of HeLa cells. Expression of dominant negative constructs in HeLa cells revealed that chlamydial uptake was dependent on Rac, but not on Cdc42 or RhoA. Rac but not Cdc42 was found to be activated by chlamydial attachment. The effect of dominant negative Rac expression on chlamydial uptake is manifested through the inhibition of actin recruitment to the sites of chlamydial entry. Studies utilizing Green Fluorescent Protein fusion constructs of Rac, Cdc42 and RhoA, showed Rac to be the sole member of the Rho GTPase family recruited to the site of chlamydial entry.     

IQGAP1 regulates Salmonella invasion through interactions with actin, Rac1, and Cdc42

To infect host cells, Salmonella utilizes an intricate system to manipulate the actin cytoskeleton and promote bacterial uptake. Proteins injected into the host cell by Salmonella activate the Rho GTPases, Rac1 and Cdc42, to induce actin polymerization. Following uptake, a different set of proteins inactivates Rac1 and Cdc42, returning the cytoskeleton to normal. Although the signaling pathways allowing Salmonella to invade host cells are beginning to be understood, many of the contributing factors remain to be elucidated. IQGAP1 is a multidomain protein that influences numerous cellular functions, including modulation of Rac1/Cdc42 signaling and actin polymerization. Here, we report that IQGAP1 regulates Salmonella invasion. Through its interaction with actin, IQGAP1 co-localizes with Rac1, Cdc42, and actin at sites of bacterial uptake, whereas infection promotes the interaction of IQGAP1 with both Rac1 and Cdc42. Knockdown of IQGAP1 significantly reduces Salmonella invasion and abrogates activation of Cdc42 and Rac1 by Salmonella. Overexpression of IQGAP1 significantly increases the ability of Salmonella to enter host cells and required interaction with both actin and Cdc42/Rac1. Together, these data identify IQGAP1 as a novel regulator of Salmonella invasion.     

Beyond DNA binding - a review of the potential mechanisms mediating quinacrine's therapeutic activities in parasitic infections,inflammation, and cancers

Background

Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin^-/-, integrin-beta1^-/-, focal adhesion kinase (FAK)^-/- and Src/Yes/Fyn^-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake.

Results

Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2^-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry.

Conclusion

Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.     

Antagonistic cross-talk between Rac and Cdc42 GTPases regulates generation of reactive oxygen species

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b(558) and p67(phox) oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b(558) that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b(558) in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.     

Burkholderia cenocepacia infection: Disruption of phagocyte immune functions through Rho GTPase inactivation

Phagocytosis is an important component of innate immunity that contributes to the eradication of infectious microorganisms; however, successful bacterial pathogens often evade different aspects of host immune responses. A common bacterial evasion strategy entails the production of toxins and/or effectors that disrupt normal host cell processes and because of their importance Rho-family GTPases are often targeted. Burkholderia cenocepacia, an opportunistic pathogen that has a propensity to infect cystic fibrosis patients, is an example of a pathogenic bacterium that has only recently been shown to disrupt Rho GTPase function in professional phagocytes. More specifically, B. cenocepacia disrupts Rac and Cdc42 seemingly through perturbation of guanine nucleotide exchange factor function. Inactivation of Rac, Cdc42 and conceivably other Rho GTPases seriously compromises phagocyte function.     

Rho GTPase signaling in the development of colorectal cancer

The involvement of Rho GTPases in major aspects of cancer development, such as cell proliferation, apoptosis, cell polarity, adhesion, migration, and invasion, have recently been attracting increasing attention. In this review, we have summarized the current findings in the literature, and we discuss the participation of the Rho GTPase members RhoA, Rac1, and Cdc42 in the development of colorectal cancer, the second most lethal neoplasia worldwide. First, we present an overview of the mechanisms of Rho GTPase regulation and the impact that regulator proteins exert on GTPase signaling. Second, we focus on the participation of Rho GTPases as modulators of colorectal cancer development. Third, we emphasize the involvement of activation and expression alterations of Rho GTPases in events associated with cancer progression, such as loss of cell-cell adhesion, proliferation, migration, and invasion. Finally, we highlight the potential use of novel anticancer drugs targeting specific components of the Rho GTPase signaling pathway with antineoplastic activity in this cancer type.     

Differential role of Rho GTPases in intestinal epithelial barrier regulation in vitro

Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho-family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF-1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF-1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC-dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF-1 after 3 h. In contrast, after 24 h of incubation with CNF-1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions.     

CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion

CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.     

IpgB1 is a novel Shigella effector protein involved in bacterial invasion of host cells. Its activity to promote membrane ruffling via Rac1 and Cdc42 activation

Shigella, the causative agent of bacillary dysentery, is capable of inducing the large scale membrane ruffling required for the bacterial invasion of host cells. Shigella secrete a subset of effectors via the type III secretion system (TTSS) into the host cells to induce membrane ruffling. Here, we show that IpgB1 is secreted via the TTSS into epithelial cells and plays a major role in producing membrane ruffles via stimulation of Rac1 and Cdc42 activities, thus promoting bacterial invasion of epithelial cells. The invasiveness of the ipgB1 mutant was decreased to less than 50% of the wild-type level (100%) in a gentamicin protection or plaque forming assay. HeLa cells infected with the wild-type or a IpgB1-hyperproducing strain developed membrane ruffles, with the invasiveness and the scale of membrane ruffles being comparable with the level of IpgB1 production in bacteria. Upon expression of EGFP-IpgB1 in HeLa cells, large membrane ruffles are extended, where the EGFP-IpgB1 was predominantly associated with the cytoplasmic membrane. The IpgB1-mediated formation of ruffles was significantly diminished by expressing Rac1 small interfering RNA and Cdc42 small interfering RNA or by treatment with GGTI-298, an inhibitor of the geranylgeranylation of Rho GTPases. When IpgB1 was expressed in host cells or wild-type Shigella-infected host cells, Rac1 and Cdc42 were activated. The results thus indicate that IpgB1 is a novel Shigella effector involved in bacterial invasion of epithelial cells via the activation of Rho GTPases.     

Targeting and activation of Rac1 are mediated by the exchange factor beta-Pix

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator beta-Pix (p21-activated kinase [Pak]-interacting exchange factor). The interaction with beta-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1-beta-Pix interaction is required for Rac1 activation by beta-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the beta-Pix-binding kinase Pak1, we show that Pak1 regulates the Rac1-beta-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor beta-Pix.     

Burkholderia cenocepacia infection

Phagocytosis is an important component of innate immunity that contributes to the eradication of infectious microorganisms; however, successful bacterial pathogens often evade different aspects of host immune responses. A common bacterial evasion strategy entails the production of toxins and/or effectors that disrupt normal host cell processes and because of their importance Rho-family GTPases are often targeted. Burkholderia cenocepacia, an opportunistic pathogen that has a propensity to infect cystic fibrosis patients, is an example of a pathogenic bacterium that has only recently been shown to disrupt Rho GTPase function in professional phagocytes. More specifically, B. cenocepacia disrupts Rac and Cdc42 seemingly through perturbation of guanine nucleotide exchange factor function. Inactivation of Rac, Cdc42 and conceivably other Rho GTPases seriously compromises phagocyte function.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

Cell migration and signaling specificity is determined by the phosphatidylserine recognition motif of Rac1

The Rho guanosine triphosphatases (GTPases) control cell shape and motility and are frequently overexpressed during malignant growth. These proteins act as molecular switches cycling between active GTP- and inactive GDP-bound forms. Despite being membrane anchored via their isoprenylated C termini, Rho GTPases rapidly translocate between membrane and cytosolic compartments. Here, we show that the Rho GTPase Rac1 preferentially interacts with phosphatidylserine (PS)-containing bilayers through its polybasic motif (PBM). Rac1 isoprenylation contributes to membrane avidity but is not critical for PS recognition. The similar protein Cdc42 (cell division cycle 42), however, only associates with PS when prenylated. Conversely, other Rho GTPases such as Rac2, Rac3, and RhoA do not bind to PS even when they are prenylated. Cell stimulation with PS induces translocation of Rac1 toward the plasma membrane and stimulates GTP loading, membrane ruffling, and filopodia formation. This stimulation also promotes Cdc42 activation and phosphorylation of mitogen-activated protein kinase through Rac1/PS signaling. Consequently, the PBM specifically directs Rac1 to effect cytoskeletal rearrangement and cell migration by selective membrane phospholipid targeting.     

An essential role for Rac/Cdc42 GTPases in cerebellar granule neuron survival

Rho family GTPases are critical molecular switches that regulate the actin cytoskeleton and cell function. In the current study, we investigated the involvement of Rho GTPases in regulating neuronal survival using primary cerebellar granule neurons. Clostridium difficile toxin B, a specific inhibitor of Rho, Rac, and Cdc42, induced apoptosis of granule neurons characterized by c-Jun phosphorylation, caspase-3 activation, and nuclear condensation. Serum and depolarization-dependent survival signals could not compensate for the loss of GTPase function. Unlike trophic factor withdrawal, toxin B did not affect the antiapoptotic kinase Akt or its target glycogen synthase kinase-3beta. The proapoptotic effects of toxin B were mimicked by Clostridium sordellii lethal toxin, a selective inhibitor of Rac/Cdc42. Although Rac/Cdc42 GTPase inhibition led to F-actin disruption, direct cytoskeletal disassembly with Clostridium botulinum C2 toxin was insufficient to induce c-Jun phosphorylation or apoptosis. Granule neurons expressed high basal JNK and low p38 mitogen-activated protein kinase activities that were unaffected by toxin B. However, pyridyl imidazole inhibitors of JNK/p38 attenuated c-Jun phosphorylation. Moreover, both pyridyl imidazoles and adenoviral dominant-negative c-Jun attenuated apoptosis, suggesting that JNK/c-Jun signaling was required for cell death. The results indicate that Rac/Cdc42 GTPases, in addition to trophic factors, are critical for survival of cerebellar granule neurons.     

Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.     

A New Look at Rho GTPases in the Cell Cycle: Their Role in Kinetochore-Microtubule Attachment

Rho GTPases including Rho, Rac and Cdc42 are involved in cell morphogenesis by inducing specific types of actin cytoskeleton and alignment and stabilization of microtubules. Previous studies suggest that they also regulate cell cycle progression; Rho, Rac and Cdc42 regulate the G1-S progression and Rho controls cytokinesis. However, a role of Rho GTPases in nuclear division has not been definitely shown. We have recently found that Cdc42 and its downstream effector mDia3 are involved in bi-orientation and stabilization of spindle microtubules attachment to kinetochores and regulate chromosome alignment and segregation. Here, we discuss how this is coordinated with other events in mitosis, particularly, with the action of Rho in cytokinesis and how attachment of microtubules to kinetochores is achieved and stabilized. We also discuss redundancy of Cdc42 and Cdc42-related GTPase(s) and potential mechanisms of chromosome instability in cancer     

The cooperative action of bacterial fibronectin-binding proteins and secreted proteins promote maximal Campylobacter jejuni invasion of host cells by stimulating membrane ruffling

This study was performed to elucidate the host cell scaffolding and signalling molecules that Campylobacter jejuni utilizes to invade epithelial cells. We hypothesized that the C. jejuni fibronectin‐binding proteins and secreted proteins are required for cell signalling and maximal invasion of host cells. C. jejuni binding to host cells via the CadF and FlpA fibronectin‐binding proteins activated the epidermal growth factor (EGF) pathway, as evidenced by inhibitor studies and immunoprecipitation coupled with immunoblot analysis using antibodies reactive against total and active EGF receptor. Inhibitor studies revealed maximal C. jejuni host cell invasion was dependent upon PI3‐Kinase, c‐Src and focal adhesion kinase (FAK), all of which are known to participate in cytoskeletal rearrangements. Knockdown of endogenous Dock180, which is a Rac1‐specific guanine nucleotide exchange factor, using siRNA revealed that C. jejuni invasion was significantly reduced compared with cells treated with scrambled siRNA. We further demonstrated that the C. jejuni Cia proteins are, in part, responsible for Rho GTPase Rac1 recruitment and activation, as judged by immunofluorescence microscopy and Rac1 activation. Based on these data, we present a model that illustrates that C. jejuni utilizes a coordinated mechanism involving both adhesins and secreted proteins to promote membrane ruffling and host cell invasion.