Why put yourself on a pedestal? The pathogenic role of the A/E pedestal

ABSTRACT

Certain Escherichia coli (E. coli) strains are attaching and effacing (A/E) lesion pathogens that primarily infect intestinal epithelial cells. They cause actin restructuring and polymerization within the host cell to create an actin-rich protrusion below the site of adherence, termed the pedestal. Although there is clarity on the pathways initiating pedestal formation, the underlying purpose(s) of the pedestal remains ambiguous. The conservation of pedestal-forming activity across multiple pathogens and redundancy in formation pathways indicate a pathogenic advantage. However, few decisive conclusions have been drawn, given that the results vary between model systems. Some research argues that the pedestal increases the colonization capability of the bacterium. These studies utilize A/E pathogens specifically deficient in pedestal formation to evaluate adhesion and intestinal colonization following infection. There have been many proposed mechanisms for the colonization benefit conferred by the pedestal. One suggested benefit is that the pedestal allows for direct cytosolic anchoring through incorporation of the established host cortical actin, causing a stable link between the pathogen and cell structure. The pedestal may confer enhanced motility, as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are better able to migrate on the surface of host cells and infect neighboring cells in the presence of the pedestal. Additionally, some research suggests that the pedestal improves effector delivery. This review will investigate the purpose of pedestal formation using evidence from recent literature and will critically evaluate the methodology and model systems. Most importantly, we will contextualize the proposed functions to reconcile potential synergistic effects.

A/E PATHOGENS

The study of enterobacterial pathogens is of critical importance, given the nearly 63,000 annual diarrheagenic Escherichia coli (E. coli)-related deaths worldwide (1, 2). In particular, enteropathogenic E. coli (EPEC) is a leading cause of infantile diarrhea, often contracted through the fecal-oral route via food or water (3). Attaching and effacing (A/E) pathogens, like EPEC, are named for their signature lesion on the intestinal epithelium during infection. The characteristics of these attaching and effacing lesions are effacement of the intestinal brush border, intimate attachment to the host epithelium, and the formation of actin-rich pedestals below the site of bacterial adherence. These pedestals are derived from bacterial-driven actin nucleation inside the host cell, creating a protrusion in the plasma membrane. Enterohemorrhagic E. coli (EHEC) is another human A/E pathogen that is similar to EPEC but can be distinguished from EPEC by its pathogenic and geographic features. Although EPEC is prevalent in low- and middle-income countries, EHEC is pervasive in developed countries. EPEC preferentially infects the small intestine, and EHEC preferentially infects the colon. Additionally, only EHEC produces Shiga toxin (Stx) that can inhibit protein synthesis in eukaryotic cells and is a cause of many of the systemic symptoms experienced, including long-term renal damage and hemolytic uremic syndrome (4, 5). Regardless of their clinical differences, both EPEC and EHEC are often studied using the murine pathogen Citrobacter rodentium (C. rodentium) as a proxy (6–8). All three pathogens encode the Locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system and is required for virulence. C. rodentium possesses the same LEE pathogenicity island-encoded effectors that are required for EPEC and EHEC virulence (9–11). However, the three AE pathogens have other effectors that are not a part of the LEE pathogenicity island, contributing to distinct characteristics in each pathogen. These A/E lesion pathogens are a continuous object of study due, in part, to their unique ability to form pedestals and the unknown role the pedestals play in infection.

MECHANISM OF PEDESTAL FORMATION

The study of early signaling cascades during the adherence stage of A/E pathogenic infection has led to an established understanding of the pedestal formation pathways. These pathways have been previously reviewed in detail by references 12 and 13 and, therefore, will only be touched on briefly for essential context . Upon contact with the host cell, the bacterial type three secretion system (T3SS) is used to puncture the host membrane and release a multitude of effectors. One of these effectors is the translocated intimin receptor (Tir). Tir is embedded into the plasma membrane with both extracellular and intracellular domains (14). The extracellular domain of Tir binds to the bacterial outer membrane protein intimin, clustering Tir, initiating pedestal formation, and facilitating intimate attachment (15, 16). In each of these three A/E pathogens, there are similar pathways leading to actin nucleation, yet different dependencies on each pathway, as described below. The earliest discovery was the Tir-Nck-NWASP pathway, which utilizes the host adaptor protein Nck. Once a specific tyrosine residue in a Tir cytoplasmic domain (Tir tyrosine residue 471 in C. rodentium or 474 in EPEC) is phosphorylated, Nck will bind via its SH2 domains (17, 18). Upon binding, Nck can activate N-WASP, either directly or through WASP-interacting protein, which then recruits Arp2/3 (19–21). Arp2/3 is responsible for the de novo nucleation of actin at the pedestal site. Mice lacking N-WASP do not create pedestals following C. rodentium infection, indicating a reliance on this pathway in vivo (22).

Another pathway to actin polymerization utilized primarily by EHEC is mediated by EspF_u also known as TccP (23–25). EspF_u is another pathogenic effector secreted by T3SS during initial infection. The NPY458 sequence of a cytoplasmic domain of EHEC Tir binds host membrane-deforming proteins IRTKS and IRSp53 (26–28). EspF_u has a higher affinity to IRTKS and IRSp53 than even the host adaptor protein EPS8, allowing it to outcompete native interactions (29). EspF_u can then bind the autoinhibitory region of WASP, which causes the activation of N-WASP (30, 31). It has been shown that clustering of EspF_u repeats will more efficiently initiate actin assembly (32). Additionally, it is thought that although most EPEC strains do not utilize EspF_u, they can nucleate actin via the NPY454 sequence. However, this process has low efficiency and is secondary to the Tir-Nck mediated pathway (13). Certain EPEC strains have acquired EspF_u, thereby enhancing colonization efficiency during in vitro infection (33). In contrast, the ectopic expression of EspF_u in C. rodentium did not enhance virulence in vivo (34). In vitro studies of EHEC with HeLa cells or fibroblast-like cells have shown that there is still actin assembly in the absence of N-WASP, alluding to a N-WASP-independent pathway that contributes to pedestal formation (23, 35). These studies highlight important differences between A/E pathogens and possibly between model systems.

Many other proteins, both bacterial- and host-derived, have been revealed to play roles in pedestal formation. On the host side, endocytic proteins like CD2AP and Dynamin 2 have been associated with pedestal formation due to their cortactin associations (36, 37). Cytoskeleton proteins, such as spectrin and adducin, are thought to structurally stabilize the pedestal (38). Additionally, mitotic cyclin-dependent kinase 1 and diaphanous-related formin 1 are important mediators of microvillar effacement during EHEC infection (39). Similarly, mammalian diaphanous1, a formin protein, has been shown to contribute specifically to EPEC pedestal formation (40). To accommodate the actin build-up, membrane deforming protein TOCA1 is hijacked by EHEC (41). An extensive number of bacterial proteins have also been implicated in this formation process. Some of these proteins have well-illustrated functions that include more than one process leading to pedestal formation, whereas others have simply been shown to localize to the pedestal or have a debated function. For instance, one study determined that NleL downregulates the formation of pedestals (42). However, another study found that NleL promotes EHEC A/E lesions through inactivating c-Jun N-terminal kinase (43). Similarly, A/E pathogenic effector EspJ targets Src kinase, making it a potential candidate for the dynamics needed for the pedestal, but the true mechanism has yet to be revealed (44). A description of the bacterial effectors and their proposed role in pedestal formation is shown in Table 1.

TABLE 1.

A/E pathogen effectors implicated in A/E lesion formation and pedestal biology

Effector	Purpose	Source
Tir	Double transmembrane protein that binds intimin and links the bacterium to the plasma membrane of the host cell. In EPEC and C. rodentium, phosphorylation of the C-terminus of the cytoplasmic domain of Tir initiates actin nucleation, whereas EHEC Tir binds I-BAR proteins for the actin nucleation cascade.	Kenny et al., Kenny et al., Deibel et al. (15, 45, 46)
EspFu/TccP	Responsible for the cascade of nucleating actin in EHEC through interactions with Tir-binding proteins.	Vingadassalom et al., Campellone et al., Garmendia et al., Garber et al. (23–25, 47)
EspH	Secreted by EPEC, causes accumulation of WASP Interacting Protein at the tip of actin pedestals. Disassembly of focal adhesions. Modulate pedestal length during EHEC infection.	Wong et al., Wong et al., Tu et al., Dong et al. (48–51)
NleL	Modulates EHEC A/E lesions. Exact role remains unclear.	Sheng et al., Piscatelli et al. (42, 43)
Map	Guanine nucleotide exchange factor that causes formation of filopodia, membrane extensions, early in infection.	Lai et al., Huang et al., Alto et al. (13, 52, 53,53)
EspB	Assists with microvilli effacing through inhibition of myosin. Causes extensive changes in the morphology of actin and stress fibers after transfection into HeLa cells.	Lizumi et al., Taylor et al. (54, 55,55)
EspG	Plays a role in pedestal development, disassembly of microtubules, tight junction, and endosome disruption.	Cepeda-molero et al., Glotfelty et al., Singh et al. (56–58,58)
EspY3	Localizes to the pedestal. Role remains unclear.	Larzabal et al. (59)

THE ROLE OF THE PEDESTAL IN PATHOGENESIS

Bacterial-driven actin rearrangement is not only a characteristic of A/E pathogens but is a feature of many enteric pathogens including Salmonella enterica serovar Typhimurium and Shigella flexneri. Actin rearrangement is used for internalization (S. Typhimurium) and migration to neighboring cells (S. flexneri) (60, 61). The widespread use of actin polymerization by bacteria for infectivity suggests that the pedestals could confer an advantage for A/E pathogens. These pedestals are conserved between the three A/E pathogens and are not restricted to the intestinal epithelium as they have been identified in the urinary tract epithelium during enterohemorrhagic E. coli infection (62). Bacteria capable of actin rearrangement cause an increase in the host inflammatory response; however, the reason remains uncertain. For example, during C. rodentium infection, the mutation of the Tir phosphorylation sites (Y451A/Y471A) reduces the recruited neutrophils and hyperplasia in the colon compared with WT (63). In vitro, the particular pedestal formation pathway utilized dictates the upregulated gene profile of the host (33). Given the potential interplay actin polymerization has with the host, it is pertinent to understand the possible reason, or reasons, that a pedestal would be initiated by a pathogen. This research may better direct our efforts to combat these pathogens. For example, nanobody technology that targets the Tir extracellular loop reduces EHEC burden and therefore could be a promising therapy technique (64).

Although this review will focus on the most recent publications, identifying the purpose of the pedestal has been a focus of the field since the early 2000s. Some of these works may have to be revisited, considering inconsistencies in the model systems as described above. Furthermore, distinguishing the benefit of intimin attachment from the benefit of actin polymerization was difficult, considering that both require Tir transmembrane embedding. Tir deficiency experiments investigating the purpose of the pedestal are confounded by the possibility that the results are explained by the lack of intimin attachment. To address this, researchers use directed tyrosine mutation to investigate only the actin nucleation capability of the Tir protein. In the following sections, we review theories on the various roles of pedestals that have been proposed with an emphasis on the most recent work.

LIMITATIONS AND NEW AVENUES OF TISSUE CULTURE MODELS FOR PEDESTAL FORMATION

Lack of polarization

Studies of the pathogenic role of pedestal formation by A/E pathogens have generated contradictory data for decades. One of the factors in these contradictions is the discrepancies between manifestations of pedestals in different model systems. HeLa cells, an immortalized human cervical carcinoma cell line, became a prominent way to study pedestals as they can still undergo actin generation and are susceptible to pedestal formation (65, 66). However, this model has shortcomings. HeLa cells lack microvilli as well as the apical-basal polarity of intestinal epithelial cells unless grown on Transwells filters. Therefore, they do not recapitulate the polarized signaling and trafficking that happens in the intestinal epithelium (67). The epithelium relies on this polarization for absorbing nutrients and maintaining intestinal barrier integrity. The apical side of the cell is the site of interaction between bacteria and host and is therefore the site where bacteria specifically attempt to manipulate host processes (68). The lack of polarized signaling in HeLa cells grown in regular culture may explain why many infections of cell lines by enteropathogens do not mimic primary cell results. Corroborating these concerns, the phosphorylation of EPEC Tir was essential for actin assembly on HEp-2 cells and HeLa cell derivatives, but not during in vitro organ culture due to poorer activity of the Nck-independent pathways in cultured cells (69, 70).

In vitro culturing

In vitro culturing of EHEC displayed reduced pedestal formation compared with mammalian infection of EHEC, which exhibited profuse A/E lesions (71, 72). One study reported that some EHEC isolates that form pedestals on nonpolarized cells are incapable of forming a pedestal on a polarized intestinal epithelial cell line (73). Some of these phenomena hint at the functional changes A/E pathogens undergo during mammalian in vivo adaptation. When EHEC was adapted in a mammalian host prior to in vitro infection, cell attachment, pedestal formation, and translocation increased in efficiency. EHEC was isolated from gnotobiotic piglets after infection and compared with an in vitro-grown EHEC O157:H7 strain. There was a significant increase in pedestals of bound bacteria. This host adaptation phenotype was explained by an increase in translocated effectors (71).

Effect of the microbiome

The host microbiome is also crucial in promoting productive C. rodentium infection. When C. rodentium is administered to germ-free mice, the bacteria will not produce virulence factors such as those transcribed from the LEE pathogenicity island and will, instead, act as an avirulent “commensal” (74, 75). Additionally, when EHEC infection was coupled with certain commensals in in vitro culture, more bacterial pedestals formed (76). A recent study has identified that tryptophan metabolites I3A, IPγA, and IEt, all produced by intestinal microbiota, protect mice from C. rodentium infection by downregulating the expression of N-WASP, subsequently decreasing pedestal formation (77). This defines an important dichotomy between pathogens studied in the host and pathogens studied in vitro.

Intestinal organoids as potential improved models

The variability in the abovementioned models demonstrates the need for critical analysis of data acquisition and reproducibility in other models. Recently, newer models of primary epithelial cells derived from organoids have been established. These cells are much closer to an in vivo scenario as they are polarized and can include all epithelial cell types while also producing mucus (76, 78–81). Ultimately, updated in vitro model systems will help distinguish true physiology from in vitro artifacts, building on the important insights gathered from earlier models.

COLONIZATION FUNCTION OF PEDESTAL FORMATION

The most well-recognized pathogenic advantage of the pedestal is enhancing colonization. It is well established that a total deletion of Tir decreases colonization compared with WT, thereby making the distinction between the role of Tir for intimate attachment and for actin nucleation more difficult to decipher.

Interestingly, early studies demonstrated no appreciable difference in bacterial burden with or without pedestal creation. The Tir phosphorylation site, and therefore pedestal formation, was shown to be dispensable for colonization of the murine colon. Additionally, A/E lesions and colonic hyperplasia were still seen in mice with the Tir phosphorylation mutation (82). This indicated that actin polymerization and pedestal formation were not part of the pathogenesis of A/E lesions. Corroborating this work was a study which showed that fecal shedding in calves and lambs was equivalent, regardless of the pedestal status during EHEC infection (83). A later study showed the same results with C. rodentium in mice. At 14 days postinfection, both animals infected with Tir Y471A mutant C. rodentium and WT C. rodentium strains had equivalent bacterial shedding in the feces (63). However, during EHEC infection in rabbits, the EHEC EspF_u mutants had reduced bacterial burden on the cecum and midcolon at 7 days postinfection (84).

For investigations of the systemic manifestations of EHEC infection, C. rodentium is not ideal, as only EHEC can produce a Stx that is required for systemic disease. C. rodentium, lysogenized with a phage that produces Stx, can be used to circumvent this issue and provide a toxigenic model of infection in mice (85). C. rodentium PhiStx expressing phosphorylation mutant Tir exhibits less severe intestinal pathology, including necrosis and inflammatory colitis, 10 days postinfection (22). This was a definitive confirmation that Tir-mediated actin polymerization plays an advantageous role in disease manifestation, causing pathology and unfavorable outcomes. These findings indicate that the severity of EHEC in humans is partially reliant on pedestal formation for virulence.

None of the above studies specifically evaluated intestinal tissue colonization.

Many more recent studies suggest that the pedestal functions to specifically enhance epithelial colonization of the pathogen throughout the course of infection but does affect luminal growth less. One of the most comprehensive analyses, published in 2014, investigated colonization capacity over a long duration of infection through the use of C. rodentium strains incapable of actin nucleation. The actin nucleation capability of Tir was decidedly necessary for efficient colonization by C. rodentium (22). Mice infected with C. rodentium Δtir + pTir_Y471F shed lower levels of bacteria than C. rodentium Δtir + pTir_WT at peak shedding day. Additionally, the burden of bacteria on the cecum and colon tissue was decreased; however, only the decrease in colon bacterial burden was significant (22). One postulation for this tissue-specific bacterial burden is that the cecum experiences more limited intestinal flow than the colon; therefore, pedestal formation may contribute more to the colonization of the colon than the cecum. The previously cited publication (82), which showed no difference, evaluated colonization by quantifying the entire colon and feces combined, which may have disguised a phenotype that is specific to tissue adherence in the colon, but not important for luminal growth (82). This is an example of the discrepancies that may reflect varying methods of data collection.

Collectively, these data highlight the intricacies of pedestal formation and function. It underscores the challenges of pinpointing actin nucleation’s role at the site of infection while also considering the physiological relevance of the chosen model system. After decades of research, experiments in several model systems suggest that there is an epithelial colonization advantage conferred by the pedestal. There is speculation that multiple mechanisms could contribute to this colonization advantage. In the following sections, we describe evidence for several of these suggested mechanisms (Fig. 1).

Fig 1.

Potentially overlapping roles of the A/E pedestal in pathogenesis. There may be numerous roles for pedestal function, and many of the proposed functions may be complementary. An A/E pathogen pedestal on an intestinal epithelial cell is depicted here with Tir and T3SS spanning the host membrane. Translocation and colonization may mutually benefit from pedestal formation. Anchoring of polymerized actin to host cortical actin provides for intimate adherence that could contribute to colonization and antiphagocytic function or allow for motility without the bacterium detaching from the host membrane. This anchoring may create a linkage to the host cytoskeleton, stabilizing a separate compartment that could contribute to the expansion of a signaling niche. Finally, the motility of an A/E pathogen upon the pedestal facilitates macrocolonies while still being tethered to the host cell. It is difficult to discern the exact contribution that any of these proposed roles provide to pathogenesis; however, it is evident that there is a substantial advantage to the creation of the pedestal itself.

HYPOTHESES FOR COLONIZATION FUNCTION OF PEDESTAL FORMATION

Actin-based motility

Some studies suggested that the pedestal confers actin-based motility for A/E pathogens as it does for Shigella or Listeria. Actin-dependent migration of bacteria on the surface of infected cells termed “pathogen surfing” was appreciated early on during the study of EPEC infection. The pedestal would grow in length and wave back and forth (86). Later, FRAP was used to visualize the pedestal undulation and translocation of the pathogen across the cell surface (87). This discovery has made way for the theory that the pedestal is more flexible and fluid than previously thought, setting the stage for a pedestal-specific role in migration and pathogen movement. A subsequent study revealed that this motility leads to further bacterial dissemination and larger colonized area (88). Fibroblasts were infected with EPEC, and bacterial movement was evaluated across the cell surface. EPEC utilized pedestals to create macrocolonies, thereby encompassing the surface of the cells. The EPEC strain proficient for pedestal formation traveled twice the distance and with double the speed than the pedestal-deficient strains. The use of a TirY474F EPEC mutant capable of mediating cellular adherence, but not pedestal formation, specifically implicated the pedestal in these actions and not the intimate adherence driven by the Tir intimin binding (88). This model also provided evidence that the creation of pedestals significantly increases the breadth of these macrocolonies on polarized human intestinal epithelial cell lines. EPEC preferentially “surfs” to sites of epithelial junctions and creates additional pedestal structures on a neighboring cell. The authors imaged attached EPEC to demonstrate that a bacterium could replicate atop a pedestal, and the pedestal could “split” to leave a new daughter cell on the new pedestal on a new cell; however, quantifiable evidence of enhanced bacterial replication due to this pedestal action is lacking (88). The findings in this study implicate the pedestal in the expanse of colonized tissue area. This is corroborated by a finding that rabbits infected with EHEC strains had the same colonization capacity at day 2 of infection, but EspF_u mutants neglected to expand in the cecum and colon (84).

There are multiple implications for the hypothesis of motility, the first being that the formation of the pedestal provides a dynamic structure with which to move across the host surface to expand the infection while still maintaining strong adherence to the host cell. Additionally, this activity demonstrates an active interplay between the host and the bacteria. This motility mechanism could be an alternative to flagella for A/E pathogens. Although these works were limited to in vitro methodologies, and only in some cases polarized cells, the results align well with the evidence of increased colonization capacity in WT vs Tir mutant strains (22).

Anchoring

The actin polymerization induced during pedestal formation may create a sturdy cup for the bacteria to adhere to, which may help it to maintain structural stability atop the plasma membrane of the host cell. Given the early stage of infection at which pedestals are formed, actin of the pedestal may connect the bacteria stably to the actin cortex of the host cell. In doing so, the pathogens may be able to resist shear flow that naturally occurs in the intestines and can cause bacteria to slough off and be excreted. This could lead to better attachment and therefore greater bacterial burden across the intestinal epithelium.

Enhanced attachment was shown to be the direct result of Tir-mediated actin assembly. In vitro, EHEC Tir Y458A, a strain specifically deficient in Tir-mediated actin assembly, displayed a decrease in intimin binding, indicating that Tir-mediated actin polymerization is required for optimal bacterial binding (89). EHEC that entirely lacked Tir had an even greater reduction in binding efficiency compared with WT. The differential in the binding efficiencies of these two mutants demonstrates that the Tir-Intimin interaction is essential for efficient bacterial adherence, but Tir-mediated actin nucleation contributes as well (89). This contextualizes prior studies suggesting that colonization is fully dependent on Tir-intimin binding. Instead, it appears that EHEC Tir contributes to adherence through more than one mechanism (89). Contrary to EHEC, C. rodentium Tir mutant infection did not show a difference in adherence in vitro; however, in vivo infection increased the numbers of attached bacteria upon pedestal actin nucleation, as described above (22).

Other works have incidentally proposed a mechanism for this observed bacterial adherence by demonstrating that the dynamics of actin could modulate the interactions between the host and the pathogen. Not only do pedestals form from de novo actin nucleation, but host microvillar actin is redirected to the pedestal. Microvillar actin is an important part of the main actin cortex of the host cell. Therefore, incorporation of the microvillar actin into the pedestal could anchor the pedestal to the actin cortex of the cell. It was demonstrated that EPEC will recruit parallel actin from surrounding microvilli to the pedestal during its formation. Infected Caco-2 cells, which are a colorectal adenocarcinoma cell line that can be grown in a polarized fashion, were observed with elongated microvilli that leaned toward the location of the adherent bacteria. Following this directed motion, the actin from these microvilli was incorporated into the pedestal, which was evidenced by the presence of parallel microvillar actin-bundling protein, Espin (90). This may provide the bacteria with stronger adherence to the host cell by deliberately interacting with the host actin cortex. Additional research on the physical properties of the actin involved in the pedestal formation showed that host cortical actin becomes thicker and stiffer during EHEC infection. The formation of the actin pedestal specifically causes the mechanical properties of the apical side of the infected cell to become twice as stiff as uninfected cells. Cellular stiffness has been correlated with an increase in cell adhesion, potentially implicating a change in the bacterial-host interaction (91). These findings demonstrate that pedestal formation engages extensive manipulation of host mechanics. However, it is important to note that neither of these studies compare anchoring specifically through investigating Tir or Tir mutant bacteria. Therefore, it is unclear if increased anchoring would be valuable or inhibitory for required actin modifications. Alternatively, these effects may be outcomes of pedestal formation and bear no significance on adherence.

Although the idea of anchoring seems to contradict the theory of mobility, it is possible that they work in concert. Host microvilli have dedicated pathways for movement (92). Therefore, incorporation of these structures in the pedestal may allow bacteria to harness microvillar actin to assist in motility while remaining closely attached to the host membrane.

Translocation efficiency

There is an extensive body of work which has indicated that actin polymerization can induce greater translocation of pathogenic effectors. For instance, during infection with Shigella, a non-A/E pathogen, actin polymerization is required for the translocation of effectors through the T3SS system. Shigella effectors, IpaA and OspB, were demonstrated to translocate less in the presence of actin inhibitors (93, 94). Similarly, Pseudomonas aeruginosa and Yersinia pseudotuberculosis T3SS translocation is facilitated by actin (95, 96). Therefore, it has been proposed that the purpose of actin nucleation at the site of the pedestal could be to enhance the translocation of A/E effectors during infection.

Although Tir- and EspF_u-mediated actin polymerization can enhance translocation, translocation can still occur in the absence of the actin polymerization pathway. In murine fibroblasts, the activity of N-WASP increases T3SS translocation of Tir and EspF_u into the host cytosol, which was determined using N-WASP knockout cells (23). Tir and EspF_u utilize N-WASP to nucleate actin; therefore, the translocation of Tir induces the nucleation of actin through N-WASP and, as described in this study, ensures more translocation of Tir in a feed-forward mechanism. Without N-WASP, and therefore pedestal formation, the boost in Tir that is necessary for enhanced colonization is lost. Previous colonization research corroborates these results, demonstrating that there is a translocation decrease in Tir mutant infections. However, the phenotype disappears when normalizing to the number of colonized bacteria, showing that in this study, the translocation defect could be entirely explained by the decreased adherence (89). This suggested that optimal translocation is dependent on the Tir phosphorylation site and thus pedestal formation. It is also important to note that the aforementioned studies have been performed in HeLa cells and MEFs, and these data have not yet been recapitulated in polarized epithelial cells. Additionally, A/E pathogens have many other effectors that are secreted during early infection which could play an undetermined role in translocation efficiency and colonization.

Deciphering translocation analyses can create multiple different interpretations depending on the perspective and experimental setup. Translocation can lead to better colonization due to the secretion of Tir; however, better adherence and therefore colonization can then increase translocation. If actin polymerization is necessary for only one of these events, then is the other complemented simply by a circuitous relationship between the two?

Antiphagocytic function

One theory provided the rationale for pedestal functioning in a different cell type altogether. Although EPEC and EHEC primarily infect intestinal epithelial cells, they also can infect or be phagocytosed by immune cells, namely macrophages, during systemic infection. If an A/E pathogen breaches the epithelial barrier, either through cell death or M cell phagocytosis, it will contact macrophages and build pedestals. Some literature has pointed to an antiphagocytic function for the pedestal-building machinery during macrophage interactions with A/E pathogens. For instance, both EPEC and EHEC recruit host factors to the pedestal that are used to remodel the plasma membrane during phagocytosis including Clathrin, PtdIns3K, and Esp15 (97, 98). In addition to this recruitment, pathogenic effector EspH, which induces pedestal elongation and disrupts RhoGEF signaling, has been shown to attenuate phagocytosis of EPEC into murine macrophages (49, 50). Another A/E pathogenic effector, EspB, involved in pedestal creation, decreased phagocytosis through the inhibition of myosin activity (54). Infection of bone marrow-derived macrophages with EPEC deficient in EspB led to increased internalization. The recruitment of these effectors that are important for pedestal creation may indirectly demonstrate a purposeful disruption in phagocytosis that allows for more bacterial survival. Intestinal epithelial cells can also phagocytose under certain circumstances; however, it has not been examined whether pedestal formation interferes with this process (99, 100).

MISLOCALIZATION OF HOST PROTEINS

Other possibilities for the function of the pedestal have been suggested but remain to be explored. A variety of proteins are aberrantly localized to the pedestal, and their role in pedestal formation or other host-pathogen activities has not been revealed. Several tight junction proteins, including ZO-1 and focal adhesion proteins like talin, vinculin, and ezrin, are localized to pedestals on cultured cells. However, only ZO-1 and vinculin have a dependence on Tir phosphorylation (101–104). Ezrin may assist pathogens with motility or colonization (104). In rabbit kidney cells infected with rabbit EPEC, many transmembrane junctional proteins, like occludin, are relocated to the pedestal (105). This induced paracellular permeability, suggesting that the sequestration of host proteins may also be a purpose for the pedestal. Other aberrantly recruited proteins have been reviewed previously (101). Despite all these recruited proteins, it remains unclear how the proteins are related to the pedestal or what role they play.

With the sheer number of host proteins recruited to the site of adherence, it is tempting to speculate that the pedestal forms a signaling niche or a location for the sequestration of vital host proteins. This may allow for the pathogens to harness these proteins for their own use and simultaneously sequester them from the location of their host functional role. In fact, linker proteins that bind Tir proteins to each other could organize connections between bacteria (13, 106). The greater surface area at the pedestal may create a location for recruited proteins and create a signaling niche for subsequent infectivity steps.

It is also important to consider the importance of host defensive functions beyond phagocytosis during infection and how those may apply to the findings regarding pedestal purpose. In macrophages, EPEC can cause the activation of Caspase 4 and subsequently NLRP3 to trigger inflammatory cell death, termed pyroptosis (107). Similarly, it has been reported that EPEC triggers pyroptosis through Tir clustering in intestinal epithelial cells, which leads to the uptake of LPS into the cytosol where it can be sensed by Caspase-4 (108). Recently, it has been demonstrated that in vivo infection with Salmonella induces activation of the NAIP-NLRC4 inflammasome in intestinal epithelial cells. This host protein complex is a cytosolic sensor that triggers pyroptosis and, in primary epithelial cells, a cascade of host actin rearrangement that leads to extrusion of the infected cell into the lumen of the intestines before further infection can ensue (109, 110). Extrusion is an important innate immune defense that maintains the intestinal barrier integrity during infection. However, this process could mislead the interpretation of in vivo colonization phenotypes demonstrated if the Tir mutant and WT bacteria lead to differences in cell extrusion. If any actin-modifying bacterial effectors target this extrusion pathway to reduce its efficiency, then blocking the effector function would lead to more cell extrusion, and colonization would appear diminished. Given the extensive actin nucleation and manipulation during pedestal formation, it is conceivable that Tir may purposely nucleate actin to interfere with extrusion. Ultimately, it is tempting to speculate that the pedestal is largely a byproduct of the disruption of host cell actin that, in turn, impacts colonization.

CONCLUSIONS AND FUTURE PERSPECTIVES

It remains difficult to elucidate a singular definitive purpose of the pedestal during bacterial infection. It is highly likely that many of the functions of the pedestal demonstrated by work to date function in concert to create an advantage for the pathogen. Given the conservation of the structure of the pedestal, it seems plausible that its function may be versatile (Fig. 1). For example, the ability to be motile while still maintaining strong anchoring capacity on the cell would provide an outlet for bacterial spread and macrocolony formation while preventing removal due to weakened host-bacterial interaction. The heightened level of epithelial colonization may be examined through the perspective that physical adherence is increased. The impact of the pedestal on translocation efficiency and colonization creates difficulty in distinguishing their value independently of each other, as they may be inextricably linked. Given that even the interpretation of these experiments can be confounded by numerous factors, it is critical that these topics are counter investigated through multiple experimental models. The suggestions of mobility, anchoring, and signaling niche are ripe for further investigation as they have profound implications for how we understand A/E pathogenesis. As investigations continue in this field, the commitment to verifying results in appropriate model systems and acknowledging compounding factors will be vital to resolving the importance of the pedestal formation during A/E pathogenesis. Understanding the interplay between host actin activity and bacterial infection may expose important context about the actions of individual signaling pathways. Linking A/E pathogenic activities to their infection outcomes provides us with a holistic picture of the evolutionary arms race between the pathogen and the host.

Biographies

M. V. Miner is a Ph.D. candidate in the lab of Dr. Isabella Rauch pursuing her degree in Biomedical Sciences at Oregon Health & Science University. She received her Bachelor of Science degree in Molecular Biology from the University of Denver where she worked under the guidance of Dr. Robert Dores exploring phylogenetic relationships between ray finned fish in the Neopterygii subclass. Her focus was on melanocortin receptor evolution and conservation. Now, under the guidance of Dr. Rauch, Marin has spent three years studying innate immunity during intestinal infections. In 2022, she won the Ruth L. Kirschstein T32 training grant to pursue her research on dynamics of A/E pathogenic interactions with host defense mechanisms, namely the NAIP-NLRC4 inflammasome. Marin strives to use all her previous studies to inform her work on the host-pathogen interface.

I. Rauch is an Assistant Professor at the Department of Molecular Microbiology and Immunology at Oregon Health and Science University. She received her Doctorate (PhD) from the University of Salzburg in Austria, where she studied antimicrobial neuropeptides in the skin. She worked on interferons in intestinal inflammation and inflammasome mediated gastrointestinal pathogen defense in her postdoctoral research at the University of Vienna and during an Erwin-Schrödinger postdoctoral fellowship at the University of California, Berkeley. She received the Austrian scientists and scholars in the northern Americas award and the UC Berkeley outstanding postdoc award for her work showing rapid epithelial cell extrusion upon cytosolic pathogen detection by inflammasomes. Dr. Rauch started her own independent lab focusing on epithelial responses to pathogen infection in 2019. Their research uses genetic mouse models of in vivo infection as well as stem cell derived organoids as primary epithelial cell in vitro models.