Host Cell Signaling Pathways Research Articles

Epithelial Cells Are Sensitive Detectors of Bacterial Pore-forming Toxins

Epithelial cells act as an interface between human mucosal surfaces and the surrounding environment. As a result, they are responsible for the initiation of local immune responses, which may be crucial for prevention of invasive infection. Here we show that epithelial cells detect the presence of bacterial pore-forming toxins (including pneumolysin from Streptococcus pneumoniae, alpha-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and anthrolysin O from Bacillus anthracis) at nanomolar concentrations, far below those required to cause cytolysis. Phosphorylation of p38 MAPK appears to be a conserved response of epithelial cells to subcytolytic concentrations of bacterial poreforming toxins, and this activity is inhibited by the addition of high molecular weight osmolytes to the extracellular medium. By sensing osmotic stress caused by the insertion of a sublethal number of pores into their membranes, epithelial cells may act as an early warning system to commence an immune response, while the local density of toxin-producing bacteria remains low. Osmosensing may thus represent a novel innate immune response to a common bacterial virulence strategy.

Differential Regulation of Src Family Kinases by HIV‐1 Nef

The HIV-1 virulence factor, Nef, promotes viral pathogenicity by altering host cell signaling pathways. Nef is known to bind several members of the Src kinase family, implicating these non-receptor protein-tyrosine kinases in the pathogenesis of HIV/AIDS. However, the direct effect of Nef binding on Src family kinase (SFK) activity has not been systematically investigated. We explored this issue using S. cerevisiae, a well-defined system for the study of SFK regulation. Previous studies have shown that ectopic expression of c-Src arrests yeast cell growth in a kinase-dependent manner. We expressed Fgr, Fyn, Hck, Lck, Lyn, and Yes as well as c-Src in yeast and found that each kinase was active and induced growth suppression. Co-expression of the negative regulatory kinase Csk reversed the growth-inhibitory effect. We then co-expressed each SFK with HIV-1 Nef in the presence of Csk. Nef strongly activated Hck, Lyn, and Src, partially activated Fgr, but did not affect Fyn, Lck, or Yes. Mutagenesis of the Nef PxxP motif essential for SH3 binding greatly reduced the effect of Nef on Hck, Lyn, and Src, suggesting that SFK SH3 domain engagement and linker displacement is a common feature of Nef-induced SFK activation. Our data show that Nef selectively activates Hck, Lyn and Src among SFKs, identifying these kinases as mediators of Nef signaling and as potential targets for anti-HIV drug discovery. Research supported by NIH Grant R01 AI057083.

Proteomic and functional analysis of the suite of Ysp proteins exported by the Ysa type III secretion system of Yersinia enterocolitica Biovar 1B

Full virulence of Yersinia enterocolitica Biovar 1B requires two distinct and distantly related contact-dependent type III secretion (T3S) systems. The plasmid-encoded Ysc T3S system is essential for systemic stages of infection and the Yop effector proteins it translocates have been extensively studied. The chromosome-encoded Ysa T3S system contributes to gastrointestinal stages of infection, but the suite of Ysp effectors proteins it translocates into host cells remains obscure. Using a proteomics-based approach, the Ysa T3S system was analysed revealing a complex set of 15 secreted Ysp proteins. Seven of these proteins were previously described (YspA, YspB, YspC, YspD, YopE, YopN and YopP). Eight of these Ysps (YspK, YspI, YspE, YspF, YspP, YspY, YspN and YspL) had not previously been characterized. Several of the new Ysps are homologous to other virulence factors, including YspP with similarity to the Yersinia protein tyrosine phosphatase YopH and YspK with similarity to the Shigella serine/threonine kinase OspG. Biochemical analysis of purified hexa-histidine tagged YspK and YspP established that these proteins have kinase and phosphatase activity respectively. Infection of eukaryotic cells with Y. enterocolitica strains expressing a Ysp-CyaA chimeric protein resulted in Ysa T3S system-dependent increases in cytosolic levels of cAMP for six Ysps (YspK, YspI, YspE, YspF, YspP and YspL), but not two others (YspY and YspN). YspN, however, was required for translocation of effector proteins into eukaryotic cells by the Ysa T3S system. Competition assays in BALB/c mice revealed that mutants defective for the production of an individual Ysp are affected for colonization of gastrointestinal tissues. Collectively, the results of this study support the hypothesis that the Ysa T3S system targets a complex suite of effector proteins into host cells to affect the outcome of an infection. Identification of the suite of effectors delivered by the Ysa T3S system reveals that host cell signalling pathways are the probable targets of several Ysp effectors.

A Humble Bacterium Sweeps This Year's Nobel Prize

Earlier this month, the Nobel Prize in Physiology or Medicine was awarded to the Australians Barry Marshall and Robin Warren for their discovery of the bacterium Helicobacter pylori and its role in peptic ulcer disease and gastric cancer. It is the first time since 1928 that the Nobel Prize has been awarded for the discovery of a bacterium, and H. pylori is the first bacterium to be associated with cancer.

Helicobacter pylori Activates Myosin Light-Chain Kinase To Disrupt Claudin-4 and Claudin-5 and Increase Epithelial Permeability

Helicobacter pylori is a spiral, gram-negative bacterium that specifically and persistently infects the human stomach. In some individuals, H. pylori-induced chronic gastritis may progress to gastroduodenal ulcers and gastric cancer. Currently, the host-microbe interactions that determine the clinical outcome of infection are not well defined. H. pylori strains capable of disrupting the gastric epithelial barrier may increase the likelihood of developing serious disease. In this study, H. pylori strain SS1 increased gastric, but not small intestinal, permeability in C57BL/6 mice. H. pylori strain SS1 was able to directly increase paracellular permeability, in the absence of host inflammatory cells, by disrupting the tight-junctional proteins occludin, claudin-4, and claudin-5 in confluent nontransformed epithelial cells. H. pylori SS1 also reduced claudin-4 protein levels in human gastric AGS cells. The ability of H. pylori SS1 to increase permeability appeared to be independent of the well-characterized virulence factors vacuolating cytotoxin and CagA protein. H. pylori activated myosin light-chain kinase in epithelial cells to phosphorylate myosin light chain and increase permeability by disrupting claudin-4 and claudin-5. The bacterial factor responsible for increasing epithelial permeability was heat sensitive, membrane bound, and required apical contact with monolayers. In conclusion, disruptions of the tight junctions observed in this study implicate host cell signaling pathways, including the phosphorylation of myosin light chain and the regulation of tight-junctional proteins claudin-4 and claudin-5, in the pathogenesis of H. pylori infection.

Genome-wide analysis of host responses to the Pseudomonas aeruginosa type III secretion system yields synergistic effects

The type III secretion system (TTSS) is a dedicated bacterial pathogen protein targeting system that directly affects host cell signalling and response pathways. Our goal was to identify host responses to the Pseudomonas aeruginosa effectors, introduced into target cells utilizing the TTSS. We carried out expression profiling of a human lung pneumocyte cell line A549 exposed to isogenic mutants of P. aeruginosa PAK lacking individual or a combination of TTSS components. We then devised a data analysis method to isolate the key responses to specific secreted bacterial effector proteins as well as components of the TTSS machinery. Individually, the effector proteins elicited host responses consistent with their known functions, many of which were cell cycle-related. However, our analysis has shown that the effector proteins elicit a distinct host transcriptional response when present in combination, suggesting a synergistic effect. Furthermore, the pattern of host transcriptional responses is consistent with the pore forming ability of the TTSS needle complex. This study shows that the individual components of the TTSS define an integrated system and that a systems biology approach is required to fully understand the complex interplay between pathogen and host.

WAVE2 Signaling Mediates Invasion of Polarized Epithelial Cells by Salmonella typhimurium

The bacterial pathogen Salmonella penetrates the intestinal epithelium by inducing its own phagocytosis into epithelial cells. The dramatic reorganization of the actin cytoskeleton required for internalization is driven by bacterial manipulation of host signaling pathways, including activation of the Rho family GTPase Rac1 and subsequent activation of the Arp2/3 complex. However, the mechanisms linking these two events remain poorly understood. Rac1 is thought to promote activation of the Arp2/3 complex through its interaction with suppressor of cAMP receptor/WASP family verprolin-homologous (SCAR/WAVE) family proteins, but this interaction is apparently indirect. Two different Rac1 effectors have been shown to bind WAVE2: IRSp53, the SH3 domain of which binds the WAVE2 proline-rich domain, and PIR121/Sra-1, which forms a pentameric complex containing WAVE, Abi1, Nap1, and HSPC300. However, the extent to which each of these complexes contributes to Arp2/3 complex activation in the context of Salmonella infection is unclear. Here, we show that WAVE2 is necessary for efficient invasion of epithelial cells by Salmonella typhimurium. We found that although Salmonella infection strongly promotes the formation of an IRSp53/WAVE2 complex, IRSp53 is not necessary for bacterial internalization. In contrast, disruption of the PIR121/Nap1/Abi1/WAVE2/HSPC300 complex potently inhibits bacterial uptake. These results indicate that WAVE2 is an important component in signaling pathways leading to Salmonella invasion. Although infection leads to the formation of an IRSp53/WAVE2 complex, it is the association of WAVE2 with the Abi1/Nap1/PIR121/HSPC300 complex that regulates bacterial internalization.

Host Cell Signaling and Trypanosoma cruzi Invasion: Do All Roads Lead to Lysosomes?

Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease in humans, is capable of invading and replicating within a wide variety of nucleated mammalian cell types. Host cell invasion by infective T. cruzi trypomastigotes is governed by parasite-triggered activation of host cell signaling pathways. Recent studies highlighting a role for host cell phosphatidylinositol 3-kinases (PI3Ks) in the T. cruzi invasion process have revealed surprising new insights into the mechanism of host cell invasion by this pathogen. In this Perspective, we discuss these findings and propose alternative models of T. cruzi invasion that incorporate this new information.

Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus

Human herpesvirus 8 (HHV-8; also called Kaposi's sarcoma-associated herpesvirus), which is implicated in the pathogenesis of Kaposi's sarcoma (KS) and lymphoproliferative disorders, infects a variety of target cells both in vivo and in vitro. HHV-8 binds to several in vitro target cells via cell surface heparan sulfate and utilizes the alpha3beta1 integrin as one of its entry receptors. Interactions with cell surface molecules induce the activation of host cell signaling cascades and cytoskeletal changes (P. P. Naranatt, S. M. Akula, C. A. Zien, H. H. Krishnan, and B. Chandran, J. Virol. 77:1524-1539, 2003). However, the mechanism by which the HHV-8-induced signaling pathway facilitates the complex events associated with the internalization and nuclear trafficking of internalized viral DNA is as yet undefined. Here we examined the role of HHV-8-induced cytoskeletal dynamics in the infectious process and their interlinkage with signaling pathways. The depolymerization of microtubules did not affect HHV-8 binding and internalization, but it inhibited the nuclear delivery of viral DNA and infection. In contrast, the depolymerization of actin microfilaments did not have any effect on virus binding, entry, nuclear delivery, or infection. Early during infection, HHV-8 induced the acetylation of microtubules and the activation of the RhoA and Rac1 GTPases. The inactivation of Rho GTPases by Clostridium difficile toxin B significantly reduced microtubular acetylation and the delivery of viral DNA to the nucleus. In contrast, the activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor significantly augmented the nuclear delivery of viral DNA. Among the Rho GTPase-induced downstream effector molecules known to stabilize the microtubules, the activation of RhoA-GTP-dependent diaphanous 2 was observed, with no significant activation in the Rac- and Cdc42-dependent PAK1/2 and stathmin molecules. The nuclear delivery of viral DNA increased in cells expressing a constitutively active RhoA mutant and decreased in cells expressing a dominant-negative mutant of RhoA. HHV-8 capsids colocalized with the microtubules, as observed by confocal microscopic examination, and the colocalization was abolished by the destabilization of microtubules with nocodazole and by the phosphatidylinositol 3-kinase inhibitor affecting the Rho GTPases. These results suggest that HHV-8 induces Rho GTPases, and in doing so, modulates microtubules and promotes the trafficking of viral capsids and the establishment of infection. This is the first demonstration of virus-induced host cell signaling pathways in the modulation of microtubule dynamics and in the trafficking of viral DNA to the infected cell nucleus. These results further support our hypothesis that HHV-8 manipulates the host cell signaling pathway to create an appropriate intracellular environment that is conducive to the establishment of a successful infection.

Inhibition of host connective tissue growth factor expression: a novel Trypanosoma cruzi‐mediated response

Connective tissue growth factor (CTGF) is a secreted cytokine that plays a fundamental role in the development of tissue fibrosis by mediating many of the profibrotic effects of TGF-beta. We present the novel finding that the intracellular pathogen Trypanosoma cruzi elicits immediate and sustained repression of basal CTGF expression in dermal fibroblasts, followed by down-regulation of the extracellular matrix proteins, fibronectin, and collagen I alpha1. To address mechanisms underlying this response, the major CTGF-regulating pathways were investigated. We report that both T. cruzi trypomastigotes and secreted parasite factor(s) antagonize TGF-beta-dependent induction of CTGF in fibroblasts. Of the TGF-beta-dependent signaling pathways required for CTGF expression, we demonstrate that T. cruzi interferes with cellular Erk1/2 phosphorylation but not Smad3 signaling. While increased stimulation of Erk phosphorylation alone was insufficient to override the parasite-mediated repression of CTGF, stimulation of fibroblasts with increased concentrations of TGF-beta, which activates both Smad3 and Erk1/2, completely abrogated this inhibition. Together with the finding that T. cruzi-mediated down-regulation of CTGF expression requires de novo host cell protein synthesis, our data indicate that the unique ability of T. cruzi to interfere with the host fibrogenic response is a complex process requiring input from multiple host cell signaling pathways.

Analysis of the mechanisms of Salmonella-induced actin assembly during invasion of host cells and intracellular replication.

Salmonella enterica serovar Typhimurium (S. typhimurium) induces actin assembly both during invasion of host cells and during the course of intracellular bacterial replication. In this study, we investigated the involvement in these processes of host cell signalling pathways that are frequently utilized by bacterial pathogens to manipulate the eukaryotic actin cytoskeleton. We confirmed that Cdc42, Rac, and Arp3 are involved in S. typhimurium invasion of HeLa cells, and found that N-WASP and Scar/WAVE also play a role in this process. However, we found no evidence for the involvement of these proteins in actin assembly during intracellular replication. Cortactin was recruited by Salmonella during both invasion and intracellular replication. However, RNA interference directed against cortactin did not inhibit either invasion or intracellular actin assembly, although it resulted in increased cell spreading and a greater number of lamellipodia. We also found no role for either the GTPase dynamin or the formin family member mDia1 in actin assembly by intracellular bacteria. Collectively, these data provide evidence that signalling pathways leading to Arp2/3-dependent actin nucleation play an important role in S. typhimurium invasion, but are not involved in intracellular Salmonella-induced actin assembly, and suggest that actin assembly by intracellular S. typhimurium may proceed by a novel mechanism.

The pivotal role of phosphatidylinositol 3-kinase-Akt signal transduction in virus survival

Over the course of evolution, viruses have developed the ability to modulate a variety of host cell signalling pathways. Inhibition of apoptosis, in particular, has become recognized as an important contributory factor in virus survival. Apoptotic inhibition contributes to the establishment of latent and chronic infections and has been implicated in viral oncogenesis. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is utilized by many cell types for inhibition of apoptosis and cellular survival. Virus modulation of this pathway provides an alternative to the expression of viral oncogenes or the direct inhibition of pro-apoptotic proteins. It has become evident that many viruses require up-regulation of this pathway to sustain long-term infections and it is modulated, in some cases, by specific viral products to create an environment favourable for cellular transformation. In other cases, PI3K-Akt signalling simply helps to create an environment favourable for virus replication and virion assembly. This review details the modulation and function of PI3K-Akt signalling for virus survival.

Interplay between mycobacteria and host signalling pathways.

Pathogenesis by mycobacteria requires the exploitation of host-cell signalling pathways to enhance the intracellular survival and persistence of the pathogen. The disruption of these pathways by mycobacteria causes impaired maturation of phagosomes into phagolysosomes, modulates host-cell apoptotic pathways and suppresses the host immune response. This review highlights the strategies employed by mycobacteria to subvert host-cell signalling and identifies key molecules involved in these processes that might serve as potential targets for new antimycobacterial therapies.

Reciprocal regulation of the nuclear factor of activated T cells and HIV-1

The human immunodeficiency virus type 1 (HIV-1) has evolved to coordinate its replication with the activation state of the host CD4T cell. To this end, it taps into major host cell signaling pathways and their associated transcription factors. Of these, T-cell activation and the transcription factor NF-kappaB, respectively, have become the best-studied examples. The past several years have revealed compelling evidence that another transcription factor family involved in T-cell activation, the nuclear factor of activated T cells (NFAT), plays an important role in the regulation of HIV-1. Major advances have been made in our understanding of the interaction of HIV-1 with this intriguing transcription factor. The duplicated NF-kappaB binding sites in the HIV-1 enhancer surprisingly also bind NFAT proteins and appear to be the most important targets for NFAT transactivation of the HIV-1 long terminal repeat. The crystal structure of NFAT1 bound to one of these duplicated sites was solved recently. Interestingly, it showed that NFAT1 binds to this site as a homodimer and occupies the core of the NF-kappaB site, suggesting mutually exclusive binding and alternate transactivation by these two factors. NFAT also regulates HIV-1 infection indirectly, as it can relieve a block to reverse transcription in quiescent T cells. In turn, HIV-1, and particularly its Tat and Nef gene products, can upregulate NFAT expression and activity. This reciprocal regulation between virus and transcription factor potentially creates a positive feedback loop, which may facilitate the establishment of early HIV-1 infection and, later, the transition from latent to productive infection. The immunosuppressive drug cyclosporin A (CsA) inhibits NFAT activity and thus represents a potential treatment for HIV-1 infection. Recent small-scale clinical trials have yielded optimistic results, suggesting roles for CsA after organ transplantation in HIV-1+ individuals and as adjunct treatment in stable early HIV-1 infection.

Bacterial responses to neutrophil phagocytosis.

This review focuses on adaptive bacterial interactions with neutrophils, emphasizing information communicated within the past year about bacterial factors that respond to contact with or phagocytosis by PMN. Since the discovery of type III and IV secretion, progress in the analysis of bacterial interactions with host phagocytes has been extensive but largely focused on the macrophage. The remarkable growth of information about bacterial subversion of macrophage metabolism has been summarized in several excellent reviews. The scope of progress on neutrophil-bacteria interactions is more limited and dominated by recent studies of the granulocyte pathogen, Anaplasma phagocytophilum, the agent of granulocytic ehrlichiosis. For many pathogens, contact with or ingestion by phagocytes elicits a vigorous but varied microbial response. The response repertoire includes activation of type III and type IV secretion systems that inject effector molecules into the host cell. Effectors modify host cell signaling and metabolic pathways to favor survival of the microbe. Whereas microbial secretory structures are few in kind and relatively conserved, effector molecules are numerous and variable. Effectors may promote phagocytosis by nonphagocytic cells or suppress phagocytosis by macrophages and neutrophils. They may suppress assembly or misdirect localization of the phagocyte NADPH oxidase that is responsible for generating toxic oxidants, and they may suppress phagosome-lysosome fusion. Phagocytosed bacteria may also up-regulate the expression of defensive proteins that attenuate the effects of phagocyte-derived antimicrobial toxins. These pathogenic stratagems probably have their origins in the competition among single-celled organisms, eukaryotes versus prokaryotes, that arose early in evolution.

Abl tyrosine kinases are required for infection by Shigella flexneri.

Infection by the opportunistic bacterial pathogen Shigella flexneri stimulates tyrosine phosphorylation of host cell proteins, but the kinases involved and their effects on the regulation of cell signaling pathways during bacterial entry remain largely undefined. Here, we demonstrate a requirement for the Abl family of tyrosine kinases during Shigella internalization. Family members Abl and Arg are catalytically activated upon Shigella infection, accumulate at the site of bacterial entry, and are required for efficient bacterial uptake, as internalization is blocked upon targeted deletion of these kinases or treatment with a specific pharmacological inhibitor. We identify the adapter protein Crk as a target for Abl kinases during Shigella uptake, and show that a phosphorylation-deficient Crk mutant significantly inhibits bacterial uptake. Moreover, we define a novel signaling pathway activated during Shigella entry that links Abl kinase phosphorylation of Crk to activation of the Rho family GTPases Rac and Cdc42. Together, these findings reveal a new role for the Abl kinases, and suggest a novel approach to treatment of Shigella infections through inhibition of host cell signaling pathways.

Functional Insights Revealed by the Crystal Structures of Escherichia coli Glucose-1-phosphatase

The Escherichia coli periplasmic glucose-1-phosphatase is a member of the histidine acid phosphatase family and acts primarily as a glucose scavenger. Previous substrate profiling studies have demonstrated some of the intriguing properties of the enzyme, including its unique and highly selective inositol phosphatase activity. The enzyme is also potentially involved in pathogenic inositol phosphate signal transduction pathways via type III secretion into the host cell. We have determined the crystal structure of E. coli glucose-1-phosphatase in an effort to unveil the structural mechanism underlying such unique substrate specificity. The structure was determined by the method of multiwavelength anomalous dispersion using a tungstate derivative together with the H18A inactive mutant complex structure with glucose 1-phosphate at 2.4-A resolution. In the active site of glucose-1-phosphatase, there are two unique gating residues, Glu-196 and Leu-24, in addition to the conserved features of histidine acid phosphatases. Together they create steric and electrostatic constraints responsible for the unique selectivity of the enzyme toward phytate and glucose-1-phosphate as well as its unusually high pH optimum for the latter. Based on the structural characterization, we were able to derive simple structural principles that not only precisely explains the substrate specificity of glucose-1-phosphatase and the hydrolysis products of various inositol phosphate substrates but also rationalizes similar general characteristics across the histidine acid phosphatase family.

Pathogenesis of Salmonella-induced enteritis.

Infections with Salmonella serotypes are a major cause of food-borne diseases worldwide. Animal models other than the mouse have been employed for the study of nontyphoidal Salmonella infections because the murine model is not suitable for the study of Salmonella-induced diarrhea. The microbe has developed mechanisms to exploit the host cell machinery to its own purpose. Bacterial proteins delivered directly into the host cell cytosol cause cytoskeletal changes and interfere with host cell signaling pathways, which ultimately enhance disease manifestation. Recently, marked advances have been made in our understanding of the molecular interactions between Salmonella serotypes and their hosts. Here, we discuss the molecular basis of the pathogenesis of Salmonella-induced enteritis.

Chlamydiae Host Cell Interactions Revealed Using DNA Microarrays

Chlamydiae are obligate intracellular bacterial parasites that infect eukaryotic cells and live their entire life cycle within a cytoplasmic vacuole or inclusion. We have employed cDNA microarray and conventional biological approaches to study the pathogen-host cell interaction during C. pneumoniae infection of eukaryotic cells. Two host cell signaling pathways, MEK/ERK and PI 3-kinase/Akt, were activated within 5 and 20 minutes, respectively, following infection with chlamydiae. Pharmacological inhibition of these pathways blocked invasion of HEp2 cells indicating that activation of these pathways was required for infection. Rho family GTPase activity was essential for invasion, since the pan-Rho GTPase inhibitor, compactin, blocked infection of HEp2 cells. cDNA microarrays and reverse transcriptase PCR were used to study host cell and chlamydial gene expression during the replication cycle. Analysis of host cell gene expression following infection with C. pneumoniae indicated that genes coding for cytokines, growth factors, and signaling molecules were upregulated, as early as 2 hours postinfection. Analysis of chlamydial gene expression indicated a temporal regulation of transcription with distinct early-, mid-, and late-cycle classes of RNA transcripts. Newly discovered genes encoding three Ser/Thr protein kinases and one protein phosphatase were upregulated 6-12 hours postinfection. One protein kinase, designated CpnPK1, was first detected at 12 hours postinfection, accumulated in the inclusion throughout the replication cycle, and may be a type III effector molecule. An increased understanding of chlamydial host cell interactions, in particular the role of various chlamydial proteins in infection and identification of essential virulence factors should provide novel targets for the development of new antimicrobials.

Cdc42 and Rac1 Regulate Late Events in Salmonella typhimurium-induced Interleukin-8 Secretion from Polarized Epithelial Cells

Salmonella typhimurium colonization of the intestinal epithelium initiates biochemical cross-talk between pathogen and host that results in the secretion of chemokines, such as interleukin (IL)-8, that direct neutrophil migration to the site of infection. In nonpolarized cells, Rac1 and Cdc42 have been shown to regulate both bacterial invasion and signaling events leading to nuclear responses and IL-8 secretion. However, because the underlying actin cytoskeleton and the associated signaling machinery are distributed much differently in polarized epithelial cells, we used polarized Madin-Darby canine kidney monolayers to investigate the role of Rac1 and Cdc42 in S. typhimurium-induced pro-inflammatory responses in the more physiologically relevant polarized state. In Madin-Darby canine kidney monolayers expressing dominant-negative Rac1 or Cdc42, both Salmonella- and tumor necrosis factor alpha-induced activation of NFkappaB and mitogen-activated protein kinase signaling cascades proceeded normally, but IL-8 secretion was inhibited. We found that Rac1 and Cdc42 were not involved in early pro-inflammatory signaling events, as in nonpolarized cells, but rather regulated the basolateral exocytosis and secretion of IL-8. In contrast, dominant-negative Rac1 inhibited apical actin pedestal formation, indicating that pedestal formation and nuclear signaling for pro-inflammatory activation are not linked. These findings indicate that there are significant differences in the requirements of pathogen-induced host cell signaling pathways in polarized and nonpolarized cells.