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      Advances and Challenges in Studying Type III Secretion Effectors of Attaching and Effacing Pathogens

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          Introduction Outbreaks of the diarrhoeal disease caused by enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) present a significant burden to public health in countries with low and high human development indices (HDIs) alike. Over the course of evolution, horizontal gene transfer events have expanded the 4.1 Mb core genome of the human colonic commensal E. coli by 1 Mb, resulting in the emergence of pathogens (Croxen and Finlay, 2010; Clements et al., 2012). Each pathogenic strain is characterized by their unique virulence factor repertoire and clinical epidemiology (Nataro and Kaper, 1998; Gomes et al., 2016). EPEC causes infantile diarrhea in countries with a low HDI. Indeed, from 2007 to 2015 the World Health Organization (WHO) estimate 230,000 cases of death from diarrhoeal disease, of which EPEC was responsible for 16%, disproportionately affecting children under five (WHO, 2015). EHEC, on the other hand, is defined by its ability to produce Shiga toxins (Stx) (Melton-Celsa, 2014; Krause et al., 2018). Distinguishing between EPEC and EHEC is clinically important, as treatment of EHEC with antibiotics can incite Stx expression (Zhang et al., 2000) and consequently acute kidney failure, a sequela of haemolytic uremic syndrome (HUS) (Pacheco and Sperandio, 2012). EPEC and EHEC are united in their ability to intimately adhere to human enterocytes, causing elongation and loss of microvilli, and the formation of actin-rich pedestals at the site of bacterial attachment (Finlay et al., 1992; Frankel and Phillips, 2008) As such, EPEC and EHEC are members of the attaching and effacing (A/E) family of pathogens, which also include E. albertii (Bhatt et al., 2019), the murine-restricted Citrobacter rodentium (Mullineaux-Sanders et al., 2019) and rabbit enteropathogenic E. coli (REPEC) (Milon et al., 1999). The formation of A/E lesions is facilitated by proteins encoded on the locus of enterocyte effacement (LEE), a largely conserved 35.6 kb pathogenicity island that encodes components of the Type 3 Secretion System (T3SS). The T3SS of A/E pathogens is ~3.5 MDa and includes several elements. Its cytoplasmic complex is equipped with an ATPase (EscN), secretion regulators (SepL and SepD) and chaperones (such as CesT). A basal body spans the inner and outer membranes, tethering the sheathed extracellular EscF needle to EspA filaments (Knutton et al., 1998) and culminates in the translocation pore (comprising EspB and EspD) in the host membrane (reviewed by Slater et al., 2018). The integration of signals from the gut environment, the microbiome and chaperones facilitate T3SS assembly, and translocation of effector proteins from the bacterium directly into the host cytosol (McDaniel et al., 1995; Connolly et al., 2015; Furniss and Clements, 2017; Katsowich et al., 2017; Serapio-Palacios and Finlay, 2020). The effectors of A/E pathogens are encoded on either the LEE, prophages or insertion elements. While all the effectors rely on an N-terminal translocation sequence and specific chaperone-binding motifs to guide secretion (Deng et al., 2017; Slater et al., 2018; Wagner et al., 2018), their sequences are otherwise highly adapted to intercept specific host processes. Additionally, effector functions can be antagonistic or cooperative (Shenoy et al., 2018), which taken together with their low abundance and continuous acquisition, underpins the challenge of identifying and studying effectors in a meaningful context. Current research into the effector biology of A/E pathogens can be considered in three phases: discovery, in vitro functional characterization, and defining the contribution of each effector to the pathogen's infection strategy in vivo. To date, 30 families of effectors have been identified in A/E pathogens (Table 1). However, as with many pathogens, the rate of effector discovery has surpassed their biological characterization, and the contribution of many effectors to pathogenesis remains unknown. Here we highlight recent advances in technical and conceptual approaches to characterize effectors in A/E pathogens in the context of human disease. Table 1 Reported repertoire of A/E effectors, their method of discovery and inferred role during infection. Effector Discovery method Inferred function during infection Cif Transposon mutagenesis in rabbit ileal loop model Promotes bacterial survival/ cell cycle arrest Tir/EspE Anti-phosphotyrosine blotting Host adherence, actin polymerisation and pyroptosis EspF LEE inspection Induces apoptosis and disrupts tissue architecture EspG LEE inspection/ effector homology Manipulates small GTPases EspH LEE inspection Cytoskeletal remodeling, inhibition of phagocytosis EspI/NleA Coomassie of secreted proteins Inhibits inflammasome activation and protein secretion EspJ Transcriptome analysis of adhered bacteria Inhibits phagocytosis EspK Transposon mutagenesis in calf model Unknown EspL Mass spectrometry (MS) of secreted proteins Inhibits necroptosis EspM1/2 MS of secreted proteins Cytoskeletal remodeling EspN MS of secreted proteins Unknown EspO MS of secreted proteins Promotes tissue integrity by promoting IL-22 secretion EspR MS of secreted proteins Unknown EspS/Ibe/OspB Effector homology Suppresses colonic crypt hyperplasia EspT Effector homology Cytoskeletal remodeling, NF-κB modulation EspW MS of secreted proteins Cytoskeletal remodeling EspX/NleL MS of secreted proteins Ligates ubiquitin EspY MS of secreted proteins Unknown EspZ/SepZ Transposon mutagenesis in cell culture Limits effector translocation Map LEE inspection Cytoskeletal remodeling, mitochondrial disruption and colonic oxygenisation NleB MS of secreted proteins Inhibits pro-inflammatory signaling and necroptosis NleC MS of secreted proteins NF-κB inhibition NleD Transposon mutagenesis in bovine gastrointestinal tract model MAPK signaling inhibition NleE MS of secreted proteins NF-κB inhibition NleF MS of secreted proteins Inhibits caspase-4/8/9 activity to limit cell death NleG MS of secreted proteins Ligates ubiquitin NleH/OspG Proximity to known effector Inhibits cell death and NF-κB signaling NleJ MS of secreted proteins Unknown NleK MS of secreted proteins Unknown TccP/EspFU Transcriptome analysis of adhered bacteria Actin polymerisation Predicting and Verifying Translocation Substrates Several resources have been developed to identify new effectors. Effector-encoding genes can be predicted in silico to varying degrees of accuracy (McDermott et al., 2011; Hobbs et al., 2016; Xue et al., 2019). These algorithms harness experimental knowledge of typical type III effector features, such as N-terminal enrichment of small polar amino acids (e.g., serine and threonine; Arnold et al., 2009), conservation of regulatory motifs upstream of the gene, a differing GC content to the rest of the genome, lack of gene homology to non-T3SS-encoding strains, and gene proximity to known effectors (Teper et al., 2016). Indeed, many novel type III effectors have been identified and validated using algorithm-based approaches, including Pseudomonas syringae and P. fluorescens (Vinatzer et al., 2005; Samudrala et al., 2009), Ralstonia spp. (Sabbagh et al., 2019), S. Typhimurium (Samudrala et al., 2009), Chlamydia trachomatis and C. psittaci (Hovis et al., 2013), Xanthomonas euvesicatoria (Teper et al., 2016) and Pantoea agglomerans (Nissan et al., 2018). Despite these successes, algorithm-based approaches have yet to be applied to A/E genomes. Instead, effectors in A/E pathogens were historically discovered through manually curating pathogenicity island genes and mutagenesis screening (Dziva et al., 2004; Mundy et al., 2004; Kanack et al., 2005), homology searches to other T3SS effectors in different species (Bulgin et al., 2009; Petty et al., 2010), mRNA profiling during infection (Dahan et al., 2005), and peptide discovery mass spectrometry (MS) of secreted proteins, notably in combination with 2D gel electrophoresis and effector hypersecretion mutants (Kresse et al., 2000; Creasey et al., 2003; Deng et al., 2004, 2010, 2012; Gruenheid et al., 2004; O'Connell et al., 2004; Tobe et al., 2006; Orton et al., 2013). Moving forward, the employment of techniques that do not rely on homology offer less bias and are therefore preferable. Additionally, A/E pathogens that encode a second functional T3SS (named ETT2) may also secrete its own cognate effectors (Fox et al., 2020), and secretion substrates could be shared between the two T3SSs, as there is evidence for regulatory crossover (Zhang et al., 2004; Luzader et al., 2016). Once identified, the T3SS-dependent translocation of candidate effectors must be experimentally confirmed. A common approach, developed in 2004 by Charpentier and Oswald, is to C-terminally tag the effector with the TEM-1 β-lactamase and infect CCF2-loaded cells (Charpentier and Oswald, 2004); alternative and refined protocols have since been developed that decrease the tag size, minimize cell toxicity and offer single cell resolution. Collectively, these approaches benefit from their capacity to support different modes of analysis depending on the infection setup, such as enzymatic assays, optical readouts in a 96-well plate, flow cytometry and immunofluorescence microscopy (Mills et al., 2008; Miyake et al., 2008; Gawthorne et al., 2016; O'Boyle et al., 2018). In vitro Characterization of the Effectors Approaches to characterize the role of each new effector during infection are ever-developing. At its most fundamental, effector functionality can be investigated under overexpression conditions in vitro, where amenable cells are transfected for ectopic effector expression or microinjected with purified protein. Overexpression protocols can provide readouts for drastic visual phenotypes, such as the radical cytoskeletal rearrangements resulting from the transfection of EspV (Arbeloa et al., 2011). Non-mammalian eukaryotic systems such as Saccharomyces cerevisiae have also been instrumental in the definition of eukaryotic interaction partners for A/E effectors (Hardwidge et al., 2004; Popa et al., 2016), as well as delineating interfaces for substrate interaction and catalytic residues (Blasche et al., 2013, 2014; Sandu et al., 2017). These approaches, however, share a common weakness: effectors localize differently when not natively translocated through the injectisome. As such, the infection of appropriate mammalian cells (i.e., colonic epithelial cells) with bacteria translocating a tagged effector protein can provide a more physiologically relevant readout for effector localization. Indeed, native expression of a tagged effector is readily achievable by introducing a C-terminal tag onto the chromosome for effector visualization by immunofluorescence, or for use in tandem with co-immunoprecipitation and/or MS to probe for host protein interactors upon infection. Chromosomal manipulation by triparental conjugation works efficiently in A/E pathogens and other enteric pathogens (Mullineaux-Sanders et al., 2017; Watson et al., 2019; Wong et al., 2019). This conjugation protocol can similarly be used to generate scarless isogenic effector mutants in lieu of traditional gene disruption with antibiotic resistance cassettes or transposon elements (Cepeda-Molero et al., 2017). Defining Roles for the Effectors During Infection Despite the ease of culturing, transfection and microscopy offered by non-polarized and non-colonic cells, the integrated use of more relevant models circumvents cell-line-specific phenotypes. A shift in practice toward more native models for infection is therefore evolving, using differentiated, polarized colonic cell lines, explants and organoids, primary tissues and laboratory animals (Carvalho et al., 2005; Law et al., 2013; Lewis et al., 2016; Cepeda-Molero et al., 2017). Increasingly, infections with A/E pathogens are also modeled in immune-associated cells, whose distinctive protein expression profiles allows researchers to probe the impact of effector delivery on immune-specific pathways (Pearson et al., 2017; Goddard et al., 2019), providing an alternative insight into the impact of effectors in human infection. One particularly useful model for probing effector function is the infection of mice with C. rodentium. C. rodentium is a natural murine pathogen which causes transmissible colonic crypt hyperplasia (CCH) and A/E lesions that are indistinguishable from those cause by EPEC and EHEC in humans (Barthold et al., 1978). Critically, C. rodentium shares 67% homology with EPEC and EHEC genomes, most notably in the LEE (Petty et al., 2010, 2011), making it an invaluable tool for the study of the role of the T3SS and its cognate effectors in vivo (Mundy et al., 2005; Borenshtein et al., 2008; Collins et al., 2014; Mullineaux-Sanders et al., 2019). The contribution of single or multiple effectors to pathogenesis can be assayed via infection with C. rodentium deletion or point mutants (Crepin et al., 2016). Key to revealing novel and physiological phenotypes is the selection of an appropriate mouse strain, or knock-out mice (Simmons et al., 2002; Zheng et al., 2008; Carson et al., 2019). To highlight some examples, the mouse model has delineated Tir, NleA and NleB as essential effectors for efficient colonization (Deng et al., 2003; Mundy et al., 2004; Kelly et al., 2006), demonstrated the impact of individual effectors deletions on host physiology, such as EspO and EspS impacting CCH (Berger et al., 2018; Connolly et al., 2018), and substantiated in vitro data indicating Map impacts colonic oxygen availability through mitochondrial disruption (Berger et al., 2017). Recently, mouse-specific differences in infection signatures have been identified through RNAseq and proteomics (Kang et al., 2018; Carson et al., 2019); it remains to be seen whether these differences are fine-tuned by the synergistic action of the effectors. There are important genetic differences between C. rodentium and human A/E pathogens. While C. rodentium encodes a type IV pilus named colonization factor Citrobacter (CFC), which is related to the EPEC bundle forming pilus (BFP) (Mundy et al., 2003), it does not encode some strain-specific effectors, nor does it express the Shiga toxin or a flagellum, and it likely benefits from mouse-specific host adaptations. Nevertheless, modifications to this model can be implemented to investigate specific aspects of infection by human A/E pathogens, such as HUS and diarrhea (Vallance et al., 2003; Mallick et al., 2012), and drawing parallels from C. rodentium studies offers invaluable insight into human infections. At the other end of the spectrum, the use of minimal effector models should prove instrumental for delineating the complex interplay between effectors, where the creation and complementation of isogenic strains cannot. As has been employed for Yersinia pestis (Palace et al., 2018), all known effectors were recently removed from the EPEC E2348/69 genome to investigate the contribution of select effectors and intact pathogenicity islands to A/E lesion formation on the human intestinal mucosa ex vivo, confirming that while Tir is essential, it is not sufficient and other elusive effectors are required (Cepeda-Molero et al., 2017). Undoubtedly the same approach would be of great use for other T3SS-encoding pathogens. Building on the decades of fundamental biochemical research into individual effector proteins, the mutation of clusters of effectors with similar functions in vitro should also be considered. Despite the wealth of technical and biological knowledge unearthed over the last two decades, the synergies and redundancies in the function of the effectors hamper the comparison of in vitro research to in vivo scenarios. However, large-scale sequencing efforts of human pathogenic E. coli isolates have revealed the correlation between the presence of effectors, and other virulence factors, and the severity of human pathology through comparative genomics (Donnenberg et al., 2015; Hazen et al., 2016). This can be extended to assaying the prevalence of effectors in strains from different environments (Xu et al., 2017). Although challenging to integrate, these datasets offer unparalleled insight into the relative importance of effector proteins during human infection. Finally, following translocation, effectors form tight interaction networks. As clinical EPEC and EHEC isolates encode strain-specific effector gene combinations, it would be interesting to test the robustness of these networks in vivo. This will offer insight into the virulence potential of alternative effector combinations present in clinical isolates. C. rodentium provides an ideal model to address this. Conclusions and Future Perspectives With technological advances, our conceptual approaches must keep up. For example, moving beyond the idea that effectors act exclusively once translocated has illuminated greater functionality for C. rodentium NleB, which was recently demonstrated to GlcNAcylate an intrabacterial glutathione synthase GshB to promote bacterial survival (El Qaidi et al., 2020); other effectors with enzymatic functions may also have intrabacterial targets. In the same vein, addressing effector functionality in a top-down fashion (from mammalian phenotype to bacterial genotype) has recently unveiled both a unique mode of EPEC-induced inflammatory cell death and a further role for Tir during infection (Goddard et al., 2019). Effector proteins exhibit fascinating diversity and specificity. Consequently, their study is warranted not only by their importance in human disease. Effectors can demonstrate first-in-class functions, the characterization of which broadens our understanding of protein biochemistry. Additionally, as effectors have witnessed the complexity of eukaryotic signaling cascades for considerably longer than modern researchers, they offer a unique insight into the breadth of function our own cells. Author Contributions SS and GF wrote the manuscript. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past collaboration with the author GF.

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          Most cited references 85

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          A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens.

          Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 are intestinal pathogens that profoundly damage the microvilli and subapical cytoskeleton of epithelial cells. Here we report finding in EPEC a 35-kbp locus containing several regions implicated in formation of these lesions. DNA probes throughout this locus hybridize to E. coli O157:H7 and other pathogens of three genera that cause similar lesions but do not hybridize to avirulent members of the same species. The EPEC locus and a different virulence locus of uropathogenic E. coli insert into the E. coli chromosome at the identical site and share highly similar sequences near the point of insertion.
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            Citrobacter rodentium: infection, inflammation and the microbiota.

            Citrobacter rodentium is a mucosal pathogen of mice that shares several pathogenic mechanisms with enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), which are two clinically important human gastrointestinal pathogens. Thus, C. rodentium has long been used as a model to understand the molecular basis of EPEC and EHEC infection in vivo. In this Review, we discuss recent studies in which C. rodentium has been used to study mucosal immunology, including the deregulation of intestinal inflammatory responses during bacteria-induced colitis and the role of the intestinal microbiota in mediating resistance to colonization by enteric pathogens. These insights should help to elucidate the roles of mucosal inflammatory responses and the microbiota in the virulence of enteric pathogens.
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              Citrobacter rodentium of mice and man.

              The major classes of enteric bacteria harbour a conserved core genomic structure, common to both commensal and pathogenic strains, that is most likely optimized to a life style involving colonization of the host intestine and transmission via the environment. In pathogenic bacteria this core genome framework is decorated with novel genetic islands that are often associated with adaptive phenotypes such as virulence. This classical genome organization is well illustrated by a group of extracellular enteric pathogens, which includes enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium, all of which use attaching and effacing (A/E) lesion formation as a major mechanism of tissue targeting and infection. Both EHEC and EPEC are poorly pathogenic in mice but infect humans and domestic animals. In contrast, C. rodentium is a natural mouse pathogen that is related to E. coli, hence providing an excellent in vivo model for A/E lesion forming pathogens. C. rodentium also provides a model of infections that are mainly restricted to the lumen of the intestine. The mechanism's by which the immune system deals with such infections has become a topic of great interest in recent years. Here we review the literature of C. rodentium from its emergence in the mid-1960s to the most contemporary reports of colonization, pathogenesis, transmission and immunity.

                Author and article information

                Front Cell Infect Microbiol
                Front Cell Infect Microbiol
                Front. Cell. Infect. Microbiol.
                Frontiers in Cellular and Infection Microbiology
                Frontiers Media S.A.
                07 July 2020
                : 10
                MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London , London, United Kingdom
                Author notes

                Edited by: Gunnar Neels Schroeder, Queen's University Belfast, United Kingdom

                Reviewed by: V. K. Viswanathan, University of Arizona, United States

                *Correspondence: Gad Frankel g.frankel@ 123456imperial.ac.uk

                This article was submitted to Bacteria and Host, a section of the journal Frontiers in Cellular and Infection Microbiology

                Copyright © 2020 Slater and Frankel.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 0, Tables: 1, Equations: 0, References: 95, Pages: 7, Words: 6035
                Funded by: Biotechnology and Biological Sciences Research Council 10.13039/501100000268
                Funded by: Wellcome Trust 10.13039/100004440
                Funded by: Medical Research Council 10.13039/501100000265
                Cellular and Infection Microbiology


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