Type 1 pilus-mediated bacterial invasion of bladder epithelial cells

Most strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili. We have determined that the type 1 pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells. In contrast, adherence mediated by another pilus adhesin, PapG, did not initiate bacterial internalization. FimH-mediated invasion required localized host actin reorganization, phosphoinositide 3-kinase (PI 3-kinase) activation and host protein tyrosine phosphorylation, but not activation of Src-family tyrosine kinases. Phosphorylation of focal adhesin kinase (FAK) at Tyr397 and the formation of complexes between FAK and PI 3-kinase and between alpha-actinin and vinculin were found to correlate with type 1 pilus-mediated bacterial invasion. Inhibitors that prevented bacterial invasion also blocked the formation of these complexes. Our results demonstrate that UPEC strains are not strictly extracellular pathogens and that the type 1 pilus adhesin FimH can directly trigger host cell signaling cascades that lead to bacterial internalization.     

Integrin-mediated host cell invasion by type 1-piliated uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, typically express filamentous adhesive organelles called type 1 pili that mediate both bacterial attachment to and invasion of bladder urothelial cells. Several host proteins have previously been identified as receptors for type 1 pili, but none have been conclusively shown to promote UPEC entry into host bladder cells. Using overlay assays with FimH, the purified type 1 pilus adhesin, and mass spectroscopy, we have identified beta1 and alpha3 integrins as key host receptors for UPEC. FimH recognizes N-linked oligosaccharides on these receptors, which are expressed throughout the urothelium. In a bladder cell culture system, beta1 and alpha3 integrin receptors co-localize with invading type 1-piliated bacteria and F-actin. FimH-mediated bacterial invasion of host bladder cells is inhibited by beta1 and alpha3 integrin-specific antibodies and by disruption of the beta1 integrin gene in the GD25 fibroblast cell line. Phosphorylation site mutations within the cytoplasmic tail of beta1 integrin that alter integrin signaling also variably affect UPEC entry into host cells, by either attenuating or boosting invasion frequencies. Furthermore, focal adhesion and Src family kinases, which propagate integrin-linked signaling and downstream cytoskeletal rearrangements, are shown to be required for FimH-dependent bacterial invasion of target host cells. Cumulatively, these results indicate that beta1 and alpha3 integrins are functionally important receptors for type 1 pili-expressing bacteria within the urinary tract and possibly at other sites within the host.     

Clathrin, AP-2, and the NPXY-binding subset of alternate endocytic adaptors facilitate FimH-mediated bacterial invasion of host cells

The FimH adhesin, localized at the distal tips of type 1 pili, binds mannose-containing glycoprotein receptors like alpha3beta1 integrins and stimulates bacterial entry into target host cells. Strains of uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections, utilize FimH to invade bladder epithelial cells. Here we set out to define the mechanism by which UPEC enters host cells by investigating four of the major entry routes known to be exploited by invasive pathogens: caveolae, clathrin, macropinocytosis and secretory lysosomes. Using pharmacological inhibitors in combination with RNA interference against specific endocytic pathway components, mutant host cell lines and a mouse infection model system, we found that type 1 pili-dependent bacterial invasion of host cells occurs via a cholesterol- and dynamin-dependent phagocytosis-like mechanism. This process did not require caveolae or secretory lysosomes, but was modulated by calcium levels, clathrin, and cooperative input from the primary clathrin adaptor AP-2 and a subset of alternate adaptors comprised of Numb, ARH and Dab2. These alternate clathrin adaptors recognize NPXY motifs, as found within the cytosolic tail of beta1 integrin, suggesting a functional link between the engagement of integrin receptors by FimH and the clathrin-dependent uptake of type 1-piliated bacteria.     

Dynamin2- and endothelial nitric oxide synthase-regulated invasion of bladder epithelial cells by uropathogenic Escherichia coli

Invasion of bladder epithelial cells by uropathogenic Escherichia coli (UPEC) contributes to antibiotic-resistant and recurrent urinary tract infections (UTIs), but this process is incompletely understood. In this paper, we provide evidence that the large guanosine triphosphatase dynamin2 and its partner, endothelial nitric oxide (NO) synthase (NOS [eNOS]), mediate bacterial entry. Overexpression of dynamin2 or treatment with the NO donor S-nitrosothiols increases, whereas targeted reduction of endogenous dynamin2 or eNOS expression with ribonucleic acid interference impairs, bacterial invasion. Exposure of mouse bladder to small molecule NOS inhibitors abrogates infection of the uroepithelium by E. coli, and, concordantly, bacteria more efficiently invade uroepithelia isolated from wild-type compared with eNOS(-/-) mice. E. coli internalization promotes rapid phosphorylation of host cell eNOS and NO generation, and dynamin2 S-nitrosylation, a posttranslational modification required for the bacterial entry, also increases during E. coli invasion. These findings suggest that UPEC escape urinary flushing and immune cell surveillance by means of eNOS-dependent dynamin2 S-nitrosylation and invasion of host cells to cause recurrent UTIs.     

QseC-mediated dephosphorylation of QseB is required for expression of genes associated with virulence in uropathogenic Escherichia coli

Bacteria sense environmental cues and regulate gene expression accordingly so as to persist in diverse niches. QseC is a membrane sensor kinase shown in enterohemorrhagic Escherichia coli to respond to host and bacterial signals by phosphorylating the QseB response regulator at residue D51, resulting in QseB activation and presumably upregulation of virulence genes. We studied QseBC in uropathogenic E. coli (UPEC). UPEC establish infection by colonizing and invading bladder cells. After invasion, UPEC can escape into the cytoplasm where they can form intracellular bacterial communities. Deletion of qseC significantly attenuated intracellular bacterial community formation and virulence, whereas paradoxically qseB deletion did not impact pathogenesis. We found that QseB upregulates its own expression in the qseC mutant, arguing that it is activated even in the absence of QseC. However, expression of QseB, but not a QseB_D51A mutant, in the absence of QseC resulted in downregulation of type 1 pili, curli and flagella. We observed similar phenotypes with enterohemorrhagic E. coli , showing that this is not a UPEC-specific phenomenon. Target gene expression is restored when QseC is present. We discovered that QseC has phosphatase activity required for QseB dephosphorylation. Thus, the QseC phosphatase capacity is critical for modulating QseB activity and subsequent gene expression.     

G-CSF induction early in uropathogenic Escherichia coli infection of the urinary tract modulates host immunity

Uropathogenic Escherichia coli (UPEC), the causative agent of approximately 85% of urinary tract infections (UTI), is a major health concern primarily affecting women. During infection, neutrophils infiltrate the bladder, but the mechanism of recruitment is not well understood. Here, we investigated the role of UPEC-induced cytokine production in neutrophil recruitment and UTI progression. We first examined the kinetics of cytokine expression during UPEC infection of the bladder, and their contribution to neutrophil recruitment. We found that UPEC infection induces expression of several pro-inflammatory cytokines including granulocyte colony-stimulating factor (G-CSF, CSF-3), not previously known to be involved in the host response to UTI. G-CSF induces neutrophil emigration from the bone marrow; these cells are thought to be critical for bacterial clearance during infection. Upon neutralization of G-CSF during UPEC infection, we found fewer circulating neutrophils, decreased neutrophil infiltration into the bladder and, paradoxically, a decreased bacterial burden in the bladder. However, depletion of G-CSF resulted in a corresponding increase in macrophage-activating cytokines, such as monocyte chemotactic protein-1 (MCP-1, CCL-2) and Il-1beta, which may be key in host response to UPEC infection, potentially resolving the paradoxical decreased bacterial burden. Thus, G-CSF acts in a previously unrecognized role to modulate the host inflammatory response during UPEC infection.     

Covert operations of uropathogenic Escherichia coli within the urinary tract

Entry into host cells is required for many bacterial pathogens to effectively disseminate within a host, avoid immune detection and cause disease. In recent years, many ostensibly extracellular bacteria have been shown to act as opportunistic intracellular pathogens. Among these are strains of uropathogenic Escherichia coli (UPEC), the primary causative agents of urinary tract infections (UTIs). UPEC are able to transiently invade, survive and multiply within the host cells and tissues constituting the urinary tract. Invasion of host cells by UPEC is promoted independently by distinct virulence factors, including cytotoxic necrotizing factor, Afa/Dr adhesins, and type 1 pili. Here we review the diverse mechanisms and consequences of host cell invasion by UPEC, focusing also on the impact of these processes on the persistence and recurrence of UTIs.     

Transposon Mutagenesis Identifies Uropathogenic Escherichia coli Biofilm Factors

Uropathogenic Escherichia coli (UPEC), which accounts for 85% of urinary tract infections (UTI), assembles biofilms in diverse environments, including the host. Besides forming biofilms on biotic surfaces and catheters, UPEC has evolved an intracellular pathogenic cascade that culminates in the formation of biofilm-like intracellular bacterial communities (IBCs) within bladder epithelial cells. Rapid bacterial replication during IBC formation augments a build-up in bacterial numbers and persistence within the host. Relatively little is known about factors mediating UPEC biofilm formation and how these overlap with IBC formation. To address this gap, we screened a UPEC transposon mutant library in three in vitro biofilm conditions: Luria broth (LB)-polyvinyl chloride (PVC), YESCA (yeast extract-Casamino Acids)-PVC, and YESCA-pellicle that are dependent on type 1 pili (LB) and curli (YESCA), respectively. Flagella are important in all three conditions. Mutants were identified that had biofilm defects in all three conditions but had no significant effects on the expression of type 1 pili, curli, or flagella. Thus, this approach uncovered a comprehensive inventory of novel effectors and regulators that are involved in UPEC biofilm formation under multiple conditions. A subset of these mutants was found to be dramatically attenuated and unable to form IBCs in a murine model of UTI. Collectively, this study expands our insights into UPEC multicellular behavior that may provide insights into IBC formation and virulence.     

LeuX tRNA-dependent and -independent mechanisms of Escherichia coli pathogenesis in acute cystitis

Uropathogenic Escherichia coli (UPEC) contain multiple horizontally acquired pathogenicity-associated islands (PAI) implicated in the pathogenesis of urinary tract infection. In a murine model of cystitis, type 1 pili-mediated bladder epithelial invasion and intracellular proliferation are key events associated with UPEC virulence. In this study, we examined the mechanisms by which a conserved PAI contributes to UPEC pathogenesis in acute cystitis. In the human UPEC strain UTI89, spontaneous excision of PAI II(UTI89) disrupts the adjacent leuX tRNA locus. Loss of wild-type leuX-encoded tRNA(5)(Leu) significantly delayed, but did not eliminate, FimB recombinase-mediated phase variation of type 1 pili. FimX, an additional FimB-like, leuX-independent recombinase, was also found to mediate type 1 pili phase variation. However, whereas FimX activity is relatively slow in vitro, it is rapid in vivo as a non-piliated strain lacking the other fim recombinases rapidly expressed type 1 pili upon experimental infection. Finally, we found that disruption of leuX, but not loss of PAI II(UTI89) genes, reduced bladder epithelial invasion and intracellular proliferation, independent of type 1 piliation. These findings indicate that the predominant mechanism for preservation of PAI II(UTI89) during the establishment of acute cystitis is maintenance of wild-type leuX, and not PAI II(UTI89) gene content.     

Escherichia coli producing CNF1 toxin hijacks Tollip to trigger Rac1-dependent cell invasion

Rho GTPases, which are master regulators of both the actin cytoskeleton and membrane trafficking, are often hijacked by pathogens to enable their invasion of host cells. Here we report that the cytotoxic necrotizing factor-1 (CNF1) toxin of uropathogenic Escherichia coli (UPEC) promotes Rac1-dependent entry of bacteria into host cells. Our screen for proteins involved in Rac1-dependent UPEC entry identifies the Toll-interacting protein (Tollip) as a new interacting protein of Rac1 and its ubiquitinated forms. We show that knockdown of Tollip reduces CNF1-induced Rac1-dependent UPEC entry. Tollip depletion also reduces the Rac1-dependent entry of Listeria monocytogenes expressing InlB invasion protein. Moreover, knockdown of Tollip, Tom1 and clathrin, decreases CNF1 and Rac1-dependent internalization of UPEC. Finally, we show that Tollip, Tom1 and clathrin associate with Rac1 and localize at the site of bacterial entry. Collectively, these findings reveal a new link between Rac1 and Tollip, Tom1 and clathrin membrane trafficking components hijacked by pathogenic bacteria to allow their efficient invasion of host cells.     

Uropathogenic Escherichia coli induces serum amyloid a in mice following urinary tract and systemic inoculation

Serum amyloid A (SAA) is an acute phase protein involved in the homeostasis of inflammatory responses and appears to be a vital host defense component with protective anti-infective properties. SAA expression remains poorly defined in many tissues, including the urinary tract which often faces bacterial challenge. Urinary tract infections (UTIs) are usually caused by strains of uropathogenic Escherichia coli (UPEC) and frequently occur among otherwise healthy individuals, many of whom experience bouts of recurrent and relapsing infections despite the use of antibiotics. To date, whether SAA is present in the infected urothelium and whether or not the induction of SAA can protect the host against UPEC is unclear. Here we show, using mouse models coupled with immunofluorescence microscopy and quantitative RT-PCR, that delivery of UPEC either directly into the urinary tract via catheterization or systemically via intraperitoneal injection triggers the expression of SAA. As measured by ELISA, serum levels of SAA1/2 were also transiently elevated in response to UTI, but circulating SAA3 levels were only up-regulated substantially following intraperitoneal inoculation of UPEC. In in vitro assays, physiological relevant levels of SAA1/2 did not affect the growth or viability of UPEC, but were able to block biofilm formation by the uropathogens. We suggest that SAA functions as a critical host defense against UTIs, preventing the formation of biofilms both upon and within the urothelium and possibly providing clinicians with a sensitive serological marker for UTI.     

Actin-gated intracellular growth and resurgence of uropathogenic Escherichia coli

Strains of uropathogenic Escherichia coli (UPEC) can invade terminally differentiated superficial bladder epithelial cells and subsequently multiply, forming large biofilm-like inclusions referred to as pods. In contrast, within immature bladder cells UPEC enter a more quiescent state and often fail to replicate appreciably. As immature bladder epithelial cells undergo terminal differentiation the actin cytoskeleton is radically diminished, a phenomenon that we reasoned could influence the intracellular fate of UPEC. Here we show that UPEC within undifferentiated bladder cells is trafficked into acidic compartments having key features of late endosomes and lysosomes. These UPEC-containing vacuoles are often enmeshed within a network of actin filaments, the disruption of which stimulates intravacuolar growth and efflux of UPEC in cell culture-based studies. In this in vitro model system, release of UPEC into the host cytosol further stimulates intracellular bacterial growth and the rapid development of pod-like inclusions. These inclusions, as well as those observed using an in vivo mouse model, develop in association with cytokeratin intermediate filaments that may act as scaffolding for intracellular biofilm formation. Our data suggest an aetiological basis for recurrent urinary tract infections, linking bladder cell differentiation and the accompanying redistribution of actin microfilaments with the resurgence of UPEC from quiescent intravacuolar reservoirs within the bladder epithelium.     

Ferritinophagy drives uropathogenic Escherichia coli persistence in bladder epithelial cells

Autophagy is a cellular recycling pathway, which in many cases, protects host cells from infections by degrading pathogens. However, uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections (UTIs), persist within the urinary tract epithelium (urothelium) by forming reservoirs within autophagosomes. Iron is a critical nutrient for both host and pathogen, and regulation of iron availability is a key host defense against pathogens. Iron homeostasis depends on the shuttling of iron-bound ferritin to the lysosome for recycling, a process termed ferritinophagy (a form of selective autophagy). Here, we demonstrate for the first time that UPEC shuttles with ferritin-bound iron into the autophagosomal and lysosomal compartments within the urothelium. Iron overload in urothelial cells induces ferritinophagy in an NCOA4-dependent manner causing increased iron availability for UPEC, triggering bacterial overproliferation and host cell death. Addition of even moderate levels of iron is sufficient to increase and prolong bacterial burden. Furthermore, we show that lysosomal damage due to iron overload is the specific mechanism causing host cell death. Significantly, we demonstrate that host cell death and bacterial burden can be reversed by inhibition of autophagy or inhibition of iron-regulatory proteins, or chelation of iron. Together, our findings suggest that UPEC persist in host cells by taking advantage of ferritinophagy. Thus, modulation of iron levels in the bladder may provide a therapeutic avenue to controlling UPEC persistence, epithelial cell death, and recurrent UTIs.     

Matrix stiffness regulates endosomal escape of uropathogenic E. coli

Uropathogenic E. coli (UPEC) infection in vivo is characterized by invasion of bladder umbrella epithelial cells followed by endosomal escape and proliferation in the cytoplasm to form intracellular bacterial communities. By contrast, UPEC infection in tissue culture models results in bacteria being trapped within Lamp1‐positive endosomes where proliferation is limited. Pharmacological disruption of the actin cytoskeleton has been shown to facilitate UPEC endosomal escape in vitro and extracellular matrix stiffness is a well‐characterized physiological regulator of actin dynamics; therefore, we hypothesized that substrate stiffness may play a role in UPEC endosomal escape. Using functionalized polyacrylamide substrates, we found that at physiological stiffness, UPEC escaped the endosome and proliferated rapidly in the cytoplasm of bladder epithelial cells. Dissection of the cytoskeletal signaling pathway demonstrated that inhibition of the Rho GTPase RhoB or its effector PRK1 was sufficient to increase cytoplasmic bacterial growth and that RhoB protein level was significantly reduced at physiological stiffness. Our data suggest that tissue stiffness is a critical regulator of intracellular bacterial growth. Due to the ease of doing genetic and pharmacological manipulations in cell culture, this model system may provide a useful tool for performing mechanistic studies on the intracellular life cycle of uropathogens.     

Surfactant Protein D Inhibits Adherence of Uropathogenic Escherichia coli to the Bladder Epithelial Cells and the Bacterium-induced Cytotoxicity: A POSSIBLE FUNCTION IN URINARY TRACT*

The adherence of uropathogenic Escherichia coli (UPEC) to the host urothelial surface is the first step for establishing UPEC infection. Uroplakin Ia (UPIa), a glycoprotein expressed on bladder urothelium, serves as a receptor for FimH, a lectin located at bacterial pili, and their interaction initiates UPEC infection. Surfactant protein D (SP-D) is known to be expressed on mucosal surfaces in various tissues besides the lung. However, the functions of SP-D in the non-pulmonary tissues are poorly understood. The purposes of this study were to investigate the possible function of SP-D expressed in the bladder urothelium and the mechanisms by which SP-D functions. SP-D was expressed in human bladder mucosa, and its mRNA was increased in the bladder of the UPEC infection model in mice. SP-D directly bound to UPEC and strongly agglutinated them in a Ca²⁺-dependent manner. Co-incubation of SP-D with UPEC decreased the bacterial adherence to 5637 cells, the human bladder cell line, and the UPEC-induced cytotoxicity. In addition, preincubation of SP-D with 5637 cells resulted in the decreased adherence of UPEC to the cells and in a reduced number of cells injured by UPEC. SP-D directly bound to UPIa and competed with FimH for UPIa binding. Consistent with the in vitro data, the exogenous administration of SP-D inhibited UPEC adherence to the bladder and dampened UPEC-induced inflammation in mice. These results support the conclusion that SP-D can protect the bladder urothelium against UPEC infection and suggest a possible function of SP-D in urinary tract.     

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.     

Regulation of cyclooxygenase-2 expression in human bladder epithelial cells infected with type I fimbriated uropathogenic E. coli

The type 1 fimbriae of uropathogenic Escherichia coli (UPEC) have been described as important for the establishment of bladder infections and urinary tract infections (UTI). Urinary prostaglandin (PG) levels and cyclooxygenase (COX)-2 expression in urine particulates may increase with infectious and inflammatory processes, including UTIs. We investigated the mechanisms underlying the modulation of COX-2 expression through the invasion of type 1 fimbriated UPEC strain J96 (J96-1) in human bladder 5637 cells. Bladder 5637 cells infected with J96-1 induced increases in the expression of COX-2 and secretion of PGE(2) . By using specific inhibitors and short hairpin RNA (shRNA), we have demonstrated that the activation of extracellular signal-related kinase (ERK), c-Jun-NH(2) -terminal kinase (JNK) and p38 MAPK pathways is critical for J96-1-induced COX-2 expression. Luciferase reporters and chromatin immunoprecipitation assays suggest that J96-1 invasion increases NF-κB- and AP-1-DNA-binding activities in 5637 cells. Inhibition of NF-κB and AP-1 activations blocked the J96-1-induced COX-2 promoter activity and expression. The effect of J96-1 on 5637 cell signalling and COX-2 expression is mediated by Toll-like receptor (TLR)-4. In summary, our findings provide the molecular pathways underlying type 1 fimbriated J96-dependent COX-2 expression in 5637 cells, providing insight into the function of UPEC invasion in bladder epithelial cells.     

Expression and Antimicrobial Function of Beta-Defensin 1 in the Lower Urinary Tract

Beta defensins (BDs) are cationic peptides with antimicrobial activity that defend epithelial surfaces including the skin, gastrointestinal, and respiratory tracts. However, BD expression and function in the urinary tract are incompletely characterized. The purpose of this study was to describe Beta Defensin-1 (BD-1) expression in the lower urinary tract, regulation by cystitis, and antimicrobial activity toward uropathogenic Escherichia coli (UPEC) in vivo. Human DEFB1 and orthologous mouse Defb1 mRNA are detectable in bladder and ureter homogenates, and human BD-1 protein localizes to the urothelium. To determine the relevance of BD-1 to lower urinary tract defense in vivo, we evaluated clearance of UPEC by Defb1 knockout (Defb1 ^-/-) mice. At 6, 18, and 48 hours following transurethral UPEC inoculation, no significant differences were observed in bacterial burden in bladders or kidneys of Defb1 ^-/- and wild type C57BL/6 mice. In wild type mice, bladder Defb1 mRNA levels decreased as early as two hours post-infection and reached a nadir by six hours. RT-PCR profiling of BDs identified expression of Defb3 and Defb14 mRNA in murine bladder and ureter, which encode for mBD-3 and mBD-14 protein, respectively. MBD-14 protein expression was observed in bladder urothelium following UPEC infection, and both mBD-3 and mBD-14 displayed dose-dependent bactericidal activity toward UPEC in vitro. Thus, whereas mBD-1 deficiency does not alter bladder UPEC burden in vivo, we have identified mBD-3 and mBD-14 as potential mediators of mucosal immunity in the lower urinary tract.     

Type 1 pili-mediated adherence of Escherichia coli strain LF82 isolated from Crohn's disease is involved in bacterial invasion of intestinal epithelial cells

We previously characterized the invasive ability of Escherichia coli strain LF82, isolated from an ileal biopsy of a patient with Crohn's disease. In the present study, we performed TnphoA insertion mutagenesis to identify genes involved in LF82 invasion of intestinal epithelial cells. Most of the non-invasive mutants had an insertion mutation within the type 1 pili-encoding operon. Two non-invasive fim mutants, which harboured an insertion within the fimI and fimF genes, still adhered but had lost the ability to induce host cell membrane elongations at the sites of contact with the epithelial cells. Transcomplementation experiments with a fim operon cloned from E. coli K-12 restored both invasive ability and the ability to induce host cell membrane elongations. Expression of the cloned LF82 or K-12 fim operon into the non-invasive laboratory strain JM109 did not confer invasive properties. Thus, these findings showed that: (i) type 1 pili-mediated adherence is involved in LF82-induced perturbation of host cell signalling responsible for membrane elongations; (ii) native shafts are required for type 1 pilus-mediated induction of membrane elongations; (iii) this active phenomenon is a key step in the establishment of the invasive process; and (iv) type 1 pili alone are not sufficient to trigger bacterial internalization.