EPEC Recruits a Cdc42-Specific GEF, Frabin, To Facilitate PAK Activation and Host Cell Colonization

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is an extracellular pathogen that tightly adheres to host cells by forming “actin pedestals” beneath the bacteria, a critical step in pathogenesis. EPEC injects effector proteins that manipulate host cell signaling cascades to trigger pedestal assembly. We have recently shown that one such effector, EspG, hijacks p21-activated kinase (PAK) and sustains its activated state to drive the cytoskeletal changes necessary for attachment of the pathogen to target cells. This EspG subversion of PAK required active Rho family small GTPases in the host cell. Here we show that EPEC itself promotes the activation of Rho GTPases by recruiting Frabin, a host guanine nucleotide exchange factor (GEF) for the Rho GTPase Cdc42. Cells devoid of Frabin showed significantly lower EPEC-induced PAK activation, pedestal formation, and bacterial attachment. Frabin recruitment to sites of EPEC attachment was driven by EspG and required localized enrichment of phosphatidylinositol 4,5-bisphosphate (PIP2) and host Arf6. Our findings identify Frabin as a key target for EPEC to ensure the activation status of cellular GTPases required for actin pedestal formation. IMPORTANCE Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea in children, especially in the developing world. EPEC initiates infection by attaching to cells in the host intestine, triggering the formation of actin-rich “pedestal” structures directly beneath the adherent pathogen. These bacteria inject their own receptor into host cells, which upon binding to a protein on the pathogen surface triggers pedestal formation. Multiple other proteins are also delivered into the cells of the host intestine, which work together to hijack host signaling pathways to drive pedestal production. Here we show how EPEC hijacks a host protein, Frabin, which creates the conditions in the cell necessary for the pathogen to manipulate a specific pathway that promotes pedestal formation. This provides new insights into this essential early stage in disease caused by EPEC.

Formin FHOD1 regulates the size of EPEC pedestals

SummaryEnteropathogenic E. coli (EPEC) is an extracellular pathogen that causes polymerization of actin filaments at the site of bacterial attachment, referred to as ‘actin pedestals’. Actin polymerization in the pedestal was believed to be solely regulated via the Nck-WASp-Arp2/3 pathway before formins were recently discovered to be associated with pedestals. Herein, we explored the collaborative role of formins in contributing to EPEC pedestal formation. In particular, we discovered that the formin FHOD1 preferentially localized to the pedestal base and its knockdown drastically reduced pedestal surface area. The pedestal localization of formin FHOD1 was found to be dependent on Tir phosphorylation at Y474, and on FHOD1 phosphorylation at Y99 from host Src family kinases (SFKs). Interestingly, differences in Arp2/3 and FHOD1 dynamics were observed. In large pedestals, Arp3 was nearly absent, but FHOD1 levels were high, suggesting that Arp2/3 and formins were segregated temporally. In line with this observation, as the pedestals grew in size, FHOD1 localization increased, while Arp3 localization decreased along the pedestals. Together, our results suggest that EPEC employs multiple actin nucleators that act at different stages of pedestal formation.Graphical abstract

Is diaphyseal fixation of short neck-retaining stem prostheses related to the size of the implant?

Introduction: Short-stem hip prostheses present variable proximal femoral bone radiological findings. The aims of this study were to analyse, in our patients with implanted collum femoris-preserving (CFP) stems, cancellous bone remodelling, cortical distal hypertrophy and pedestal formation, and the relationship between those radiological changes that suggest distal fixation with the size of the stem. Methods: From October 2001 to December 2012 a total of 199 consecutive primary total hip arthroplasties in 180 patients were performed at our department using the CFP stem and followed up for a minimum of 5 years until December 2017. Results: Stress shielding was present in 74% of oversized stems cases, but in normal or undersized stems, stress shielding was present in 8.5%. Cortical hyperthrophy was observed in 49% of the oversized stems and in 6% of the normal or undersized ones. Finally, non-statistically significant differences ( p = 0.089) in pedestal formation were found, present in 16.3% of the oversized stems and in 6% of normal or undersized ones. Conclusions: Oversized stems cause more stress shielding and distal cortical hypertrophy in the distal part of the stem, which indicates distal fixation in bigger sizes of stem.

EspFu-Mediated Actin Assembly Enhances Enteropathogenic Escherichia coli Adherence and Activates Host Cell Inflammatory Signaling Pathways

ABSTRACT The translocation of effectors into the host cell through type 3 secretion systems (T3SS) is a sophisticated strategy employed by pathogenic bacteria to subvert host responses and facilitate colonization. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) utilize the Tir and EspFu (also known as TccP) effectors to remodel the host cytoskeleton, culminating in the formation of attaching and effacing (AE) lesions on enterocytes. While some EPEC strains require tyrosine phosphorylation of Tir and recruitment of the host Nck to trigger actin polymerization, EHEC and certain EPEC strains, whose Tir is not phosphorylated, rely on the effector EspFu for efficient actin remodeling. Here, we investigated the role played by Tir-Nck and Tir-EspFu actin polymerization pathways during the infection of epithelial cells, as well as the host transcriptional response to the AE lesion formation induced by EPEC. We found that EspFu-mediated actin assembly promotes bacterial attachment and epithelial colonization more efficiently than Tir-Nck. Moreover, we showed that both actin polymerization mechanisms can activate inflammatory pathways and reverse the anti-inflammatory response induced by EPEC in epithelial cells. However, this activity is remarkably more evident in infections with EspFu-expressing EPEC strains. This study demonstrates the complex interactions between effector-mediated actin remodeling and inflammation. Different strains carry different combinations of these two effectors, highlighting the plasticity of pathogenic E. coli enteric infections. IMPORTANCE EPEC is among the leading causes of diarrheal disease worldwide. The colonization of the gut mucosa by EPEC results in actin pedestal formation at the site of bacterial attachment. These pedestals are referred to as attaching and effacing (AE) lesions. Here, we exploit the different molecular mechanisms used by EPEC to induce AE lesions on epithelial cells, showing that the effector EspFu is associated with increased bacterial attachment and enhanced epithelial colonization compared to the Tir-Nck pathway. Moreover, we also showed that actin pedestal formation can counterbalance the anti-inflammatory activity induced by EPEC, especially when driven by EspFu. Collectively, our findings provide new insights into virulence mechanisms employed by EPEC to colonize epithelial cells, as well as the host response to this enteric pathogen.

Pathogenic Escherichia coli Hijacks GTPase-Activated p21-Activated Kinase for Actin Pedestal Formation

ABSTRACT Enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC, respectively) are extracellular pathogens that reorganize the host cell cytoskeleton to form “actin pedestals” beneath the tightly adherent bacteria, a critical step in pathogenesis. EPEC and EHEC inject effector proteins that manipulate host cell signaling cascades to trigger pedestal assembly. One such effector, EspG, has been reported to bind and activate p21-activated kinase (PAK), a key cytoskeletal regulator, but the function of this interaction and whether it impacts pedestal assembly are unknown. Here, we demonstrate that deletion of espG significantly impairs pedestal formation and attachment by both EPEC and EHEC. This role of EspG is shown to be dependent on its interaction with PAK. Unexpectedly, EspG was able to subvert PAK only in the presence of Rho family small GTPases, which function to both concentrate PAK at the membrane and stimulate PAK activation. Our findings reveal a novel mechanism by which EspG hijacks PAK and sustains its active state to drive bacterial attachment to host cells. IMPORTANCE Enteropathogenic E. coli and enterohemorrhagic E. coli (EPEC and EHEC, respectively) remain a significant global health problem. Both EPEC and EHEC initiate infection by attaching to cells in the host intestine, triggering the formation of actin-rich “pedestal” structures directly beneath the adherent pathogen. These bacteria inject their own receptor into host cells, which upon binding to a protein on the pathogen surface triggers pedestal formation. Multiple other proteins are also delivered into the cells of the host intestine, but how they contribute to disease is often less clear. Here, we show how one of these injected proteins, EspG, hijacks a host signaling pathway for pedestal production. This provides new insights into this essential early stage in EPEC and EHEC disease.

Mid-Term Outcome of Total Hip Arthroplasty Using a Short Stem

Purpose To review the outcome of total hip arthroplasty (THA) using a short femoral stem in 33 hips. Methods Records of 33 hips in 20 men and 10 women aged 25 to 40 (mean, 30) years who underwent cementless THA using a short femoral stem by a single senior surgeon were reviewed. The diagnosis included avascular necrosis (n=9), ankylosing spondylitis (n=12), rheumatoid arthritis (n=7), post-traumatic arthritis (n=4), and Hurler syndrome (n=1). Clinical outcome was assessed using the Harris Hip Score. Radiological outcome was assessed according to a modified Gruen zoning system. Stem positioning (neutral, varus, valgus) and bone contact were evaluated, as were fixation and early host response as well as subsidence and changes in the calcar region (zone 5). Trabecular response (trabecular attachment), spot welds, cortical hypertrophy, and pedestal formation were determined. Heterotopic ossification was graded by the Brooker classification. Results The mean follow-up period was 6.5 years. The mean Harris Hip Score improved from 40 to 90. All hips achieved immediate postoperative stability. No patient had thigh pain. Four hips had varus placement (5°–7°) of the stem; all were asymptomatic and remained stable without any migration. Evidence of proximal load transfer (endosteal spot welds) between the endosteum and the stem in zones 2 and/or 4 was noted in 12 hips on both sides and in 8 hips on the lateral side only. At one year, all stems showed evidence of osseointegration. None had subsidence or progressive varus migration. There was no radiolucent line or osteolysis around the stem, pedestal formation or buttressing at the prosthesis tip, or cortical hypertrophy. One patient had grade I heterotopic ossification that was not clinically significant. One patient had a 1.5 cm leg lengthening. One patient had a discharging sinus, a loosened acetabular component, and intrapelvic migration at 2 years and underwent implant removal and debridement. One patient developed a crack in the proximal femur even with the smallest stem. The stem was fixed with cerclage wiring and remained stable with no migration. Conclusion A short femoral stem design that transfers load proximally through a prominent lateral flare achieved good short-term outcome in younger patients. Nonetheless, the ease of removal and preservation of bone at the time of revision should guide the choice of the design of the short stem.

Actin Pedestal Formation by Enterohemorrhagic Escherichia coli Enhances Bacterial Host Cell Attachment and Concomitant Type III Translocation

ABSTRACTAttachment of enterohemorrhagicEscherichia coli(EHEC) to intestinal epithelial cells is critical for colonization and is associated with localized actin assembly beneath bound bacteria. The formation of these actin “pedestals” is dependent on the translocation of effectors into mammalian cells via a type III secretion system (T3SS). Tir, an effector required for pedestal formation, localizes in the host cell plasma membrane and promotes attachment of bacteria to mammalian cells by binding to the EHEC outer surface protein Intimin. Actin pedestal formation has been shown to foster intestinal colonization by EHEC in some animal models, but the mechanisms responsible for this remain undefined. Investigation of the role of Tir-mediated actin assembly promoting host cell binding is complicated by other, potentially redundant EHEC-encoded binding pathways, so we utilized cell binding assays that specifically detect binding mediated by Tir-Intimin interaction. We also assessed the role of Tir-mediated actin assembly in two-step assays that temporally segregated initial translocation of Tir from subsequent Tir-Intimin interaction, thereby permitting the distinction of effects on translocation from effects on cell attachment. In these experimental systems, we compromised Tir-mediated actin assembly by chemically inhibiting actin assembly or by infecting mammalian cells with EHEC mutants that translocate Tir but are specifically defective in Tir-mediated pedestal formation. We found that an inability of Tir to promote actin assembly resulted in a significant and striking decrease in bacterial binding mediated by Tir and Intimin. Bacterial mutants defective for pedestal formation translocated type III effectors to mammalian cells with reduced efficiency, but the decrease in translocation could be entirely accounted for by the decrease in host cell attachment.

Posttranscriptional Control of Microbe-Induced Rearrangement of Host Cell Actin

ABSTRACTRemodeling of the host cytoskeleton is a common strategy employed by bacterial pathogens. Although there is vigorous investigation of the cell biology underlying these bacterially mediated cytoskeleton modifications, knowledge of the plasticity and dynamics of the bacterial signaling networks that regulate the expression of genes necessary for these phenotypes is lacking. EnterohemorrhagicEscherichia coliattaches to enterocytes, forming pedestal-like structures. Pedestal formation requires the expression of the locus-of-enterocyte-effacement (LEE) andespFugenes. The LEE encodes a molecular syringe, a type III secretion system (T3SS) used by pathogens to translocate effectors such as EspFu into the host cell. By using a combination of genetic, biochemical, and cell biology approaches, we show that pedestal formation relies on posttranscriptional regulation by two small RNAs (sRNAs), GlmY and GlmZ. The GlmY and GlmZ sRNAs are unique; they have extensive secondary structures and work in concert. Although these sRNAs may offer unique insights into RNA and posttranscriptional biology, thus far, only one target and one mechanism of action (exposure of the ribosome binding site from theglmSgene to promote its translation) has been described. Here we uncovered new targets and two different molecular mechanisms of action of these sRNAs. In the case of EspFu expression, they promote translation by cleavage of the transcript, while in regard to the LEE, they promote destabilization of the mRNA. Our findings reveal that two unique sRNAs act in concert through different molecular mechanisms to coordinate bacterial attachment to mammalian cells.IMPORTANCEPathogens evolve by horizontal acquisition of pathogenicity islands. We describe here how two sRNAs, GlmY and GlmZ, involved in cellular metabolism and cellular architecture, through the posttranscriptional control of GlmS (the previously only known target of GlmY and GlmZ), which controls amino sugar synthesis, have been coopted to modulate the expression of virulence. These sRNAs quickly allow for plasticity in gene expression in order for enterohemorrhagicEscherichia colito fine-tune the expression of its complex type III secretion machinery and its effectors to promote bacterial attachment and subsequent actin rearrangement on host cells. Pedestal formation is a very dynamic process. Many of the genes necessary for pedestal formation are located within the same operon to evolutionarily guarantee that they are inherited together. However, it is worth noting that within these operons, several genes need to yield more proteins than others and that these differences cannot be efficiently regulated at the transcriptional level.