Quantitative Mass Spectrometry Identifies Novel Host Binding Partners for Pathogenic Escherichia coli Type III Secretion System Effectors.

Enteropathogenic and enterohemorrhagic Escherichia coli cause enteric diseases resulting in significant morbidity and mortality worldwide. These pathogens remain extracellular and translocate a set of type III secreted effector proteins into host cells to promote bacterial virulence. Effectors manipulate host cell pathways to facilitate infection by interacting with a variety of host targets, yet the binding partners and mechanism of action of many effectors remain elusive. We performed a mass spectrometry screen to identify host targets for a library of effectors. We found five known effector targets and discovered four novel interactions. Interestingly, we identified multiple effectors that interacted with the microtubule associated protein, ensconsin. Using co-immunoprecipitations, we confirmed that NleB1 and EspL interacted with ensconsin in a region that corresponded to its microtubule binding domain. Ensconsin is an essential cofactor of kinesin-1 that is required for intracellular trafficking, and we demonstrated that intracellular trafficking was severely disrupted during wild type EPEC infections but not during infections with ΔnleB1 or ΔespL mutants. Our findings demonstrate the efficacy of quantitative proteomics for identifying effector-host protein interactions and suggest that vesicular trafficking is a crucial cellular process that may be targeted by NleB1 and EspL through their interaction with ensconsin.

Structural analysis of a specialized type III secretion system peptidoglycan-cleaving enzyme.

The Gram-negative bacterium enteropathogenic Escherichia coli uses a syringe-like type III secretion system (T3SS) to inject virulence or "effector" proteins into the cytoplasm of host intestinal epithelial cells. To assemble, the T3SS must traverse both bacterial membranes, as well as the peptidoglycan layer. Peptidoglycan is made of repeating N-acetylmuramic acid and N-acetylglucosamine disaccharides cross-linked by pentapeptides to form a tight mesh barrier. Assembly of many macromolecular machines requires a dedicated peptidoglycan lytic enzyme (PG-lytic enzyme) to locally clear peptidoglycan. Here we have solved the first structure of a T3SS-associated PG-lytic enzyme, EtgA from enteropathogenic E. coli. Unexpectedly, the active site of EtgA has features in common with both lytic transglycosylases and hen egg white lysozyme. Most notably, the ß-hairpin region resembles that of lysozyme and contains an aspartate that aligns with lysozyme Asp-52 (a residue critical for catalysis), a conservation not observed in other previously characterized lytic transglycosylase families to which the conserved T3SS enzymes had been presumed to belong. Mutation of the EtgA catalytic glutamate, Glu-42, conserved across lytic transglycosylases and hen egg white lysozyme, and this differentiating aspartate diminishes type III secretion in vivo, supporting its essential role in clearing the peptidoglycan for T3SS assembly. Finally, we show that EtgA forms a 1:1 complex with the building block of the polymerized T3SS inner rod component, EscI, and that this interaction enhances PG-lytic activity of EtgA in vitro, collectively providing the necessary strict localization and regulation of the lytic activity to prevent overall cell lysis.

SepD/SepL-dependent secretion signals of the type III secretion system translocator proteins in enteropathogenic Escherichia coli.

UNLABELLED: The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by the sepD and sepL mutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals. IMPORTANCE: Many bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into the host cells to cause disease. The secreted proteins perform different functions at various stages during infection and are classified into three substrate categories (T3SS components, translocators, and effectors). They all contain secretion signals at their N termini, but how their secretion hierarchy is determined is poorly understood. Here, we show that the N-terminal secretion signals from different substrate categories all behave the same and do not confer substrate specificity. We further characterize the secretion signals of the translocators and identify a translocator-specific signal, demonstrating that substrate-specific secretion signals are required in regulating T3SS substrate hierarchy.

Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS.

During infection by Gram-negative pathogenic bacteria, the type III secretion system (T3SS) is assembled to allow for the direct transmission of bacterial virulence effectors into the host cell. The T3SS system is characterized by a series of prominent multi-component rings in the inner and outer bacterial membranes, as well as a translocation pore in the host cell membrane. These are all connected by a series of polymerized tubes that act as the direct conduit for the T3SS proteins to pass through to the host cell. During assembly of the T3SS, as well as the evolutionarily related flagellar apparatus, a post-translational cleavage event within the inner membrane proteins EscU/FlhB is required to promote a secretion-competent state. These proteins have long been proposed to act as a part of a molecular switch, which would regulate the appropriate chronological secretion of the various T3SS apparatus components during assembly and subsequently the transported virulence effectors. Here we show that a surface type II beta-turn in the Escherichia coli protein EscU undergoes auto-cleavage by a mechanism involving cyclization of a strictly conserved asparagine residue. Structural and in vivo analysis of point and deletion mutations illustrates the subtle conformational effects of auto-cleavage in modulating the molecular features of a highly conserved surface region of EscU, a potential point of interaction with other T3SS components at the inner membrane. In addition, this work provides new structural insight into the distinct conformational requirements for a large class of self-cleaving reactions involving asparagine cyclization.

Quantitative proteomic analysis of type III secretome of enteropathogenic Escherichia coli reveals an expanded effector repertoire for attaching/effacing bacterial pathogens.

Type III secretion systems are central to the pathogenesis and virulence of many important Gram-negative bacterial pathogens, and elucidation of the secretion mechanism and identification of the secreted substrates are critical to our understanding of their pathogenic mechanisms and developing potential therapeutics. Stable isotope labeling with amino acids in cell culture-based mass spectrometry is a quantitative and highly sensitive proteomics tool that we have previously used to successfully analyze the type III secretomes of Citrobacter rodentium and Salmonella enterica serovar Typhimurium. In this report, stable isotope labeling with amino acids in cell culture was used to analyze the type III secretome of enteropathogenic Escherichia coli (EPEC), an important human pathogen, which, together with enterohemorrhagic E. coli and C. rodentium, represents the family of attaching and effacing bacterial pathogens. We not only confirmed all 25 known EPEC type III-secreted proteins and effectors previously identified by conventional molecular and bioinformatical techniques but also identified several new type III-secreted proteins, including two novel effectors, C_0814/NleJ and LifA, that were shown to be translocated into host cells. LifA is a known virulence factor believed to act as a toxin as well as an adhesin, but its mechanism of secretion and function is not understood. With a predicted molecular mass of 366 kDa, LifA is the largest type III effector identified thus far in any pathogen. We further demonstrated that Efa1, ToxB, and Z4332 (homologs of LifA in enterohemorrhagic E. coli) are also type III effectors. This study has comprehensively characterized the type III secretome of EPEC, expanded the repertoire of type III-secreted effectors for the attaching and effacing pathogens, and provided new insights into the mode of function for LifA/Efa1/ToxB/Z4332, an important family of virulence factors.

Global, regional, and national trends in metabolic risk factor-associated mortality among the working-age population from 1990-2019: An age-period-cohort analysis of the Global Burden of Disease 2019 study.

BACKGROUND: Metabolic diseases contribute significantly to premature mortality worldwide, with increasing burdens observed among the working-age population (WAP). This study assessed global, regional, and national trends in metabolic disorders and associated mortality over three decades in WAP. METHODS: Data from the Global Burden of Disease 2019 study were leveraged to assess global metabolism-associated mortality and six key metabolic risk factors in WAP from 1990-2019. An age-period-cohort model was employed to determine the overall percentage change in mortality. RESULTS: The 2019 global metabolic risk-related mortality rate in WAP rose significantly by 50.73%, while the age-standardized mortality rate declined by 21.5%. India, China, Indonesia, the USA, and the Russian Federation were the top contributing countries to mortality in WAP, accounting for 51.01% of the total. High systolic blood pressure (HSBP), high body mass index (HBMI), and high fasting plasma glucose (HFPG) were the top metabolic risk factors for the highest mortality rates. Adverse trends in HBMI-associated mortality were observed, particularly in lower sociodemographic index (SDI) regions. HFPG-related mortality declined globally but increased in older age groups in lower SDI countries. CONCLUSIONS: Despite a general decline in metabolic risk-related deaths in WAP, increasing HBMI- and HFPG-related mortality in lower SDI areas poses ongoing public health challenges. Developing nations should prioritize interventions addressing HBMI and HFPG to mitigate mortality risks in WAP.

Multi-step validation of a deep learning-based system with visual explanations for optical diagnosis of polyps with advanced features.

Artificial intelligence (AI) has been found to assist in optical differentiation of hyperplastic and adenomatous colorectal polyps. We investigated whether AI can improve the accuracy of endoscopists' optical diagnosis of polyps with advanced features. We introduced our AI system distinguishing polyps with advanced features with more than 0.870 of accuracy in the internal and external validation datasets. All 19 endoscopists with different levels showed significantly lower diagnostic accuracy (0.410-0.580) than the AI. Prospective randomized controlled study involving 120 endoscopists into optical diagnosis of polyps with advanced features with or without AI demonstration identified that AI improved endoscopists' proportion of polyps with advanced features correctly sent for histological examination (0.960 versus 0.840, p < 0.001), and the proportion of polyps without advanced features resected and discarded (0.490 versus 0.380, p = 0.007). We thus developed an AI technique that significantly increases the accuracy of colorectal polyps with advanced features.

EscE and EscG are cochaperones for the type III needle protein EscF of enteropathogenic Escherichia coli.

Type III secretion systems (T3SSs) are central virulence mechanisms used by a variety of Gram-negative bacteria to inject effector proteins into host cells. The needle polymer is an essential part of the T3SS that provides the effector proteins a continuous channel into the host cytoplasm. It has been shown for a few T3SSs that two chaperones stabilize the needle protein within the bacterial cytosol to prevent its premature polymerization. In this study, we characterized the chaperones of the enteropathogenic Escherichia coli (EPEC) needle protein EscF. We found that Orf2 and Orf29, two poorly characterized proteins encoded within the EPEC locus of enterocyte effacement (LEE), function as the needle protein cochaperones. Our finding demonstrated that both Orf2 and Orf29 are essential for type III secretion (T3S). In addition, we found that Orf2 and Orf29 associate with the bacterial membrane and form a complex with EscF. Orf2 and Orf29 were also shown to disrupt the polymerization of EscF in vitro. Prediction of the tertiary structures of Orf2 and Orf29 showed high structural homology to chaperones of other T3SS needle proteins. Overall, our data suggest that Orf2 and Orf29 function as the chaperones of the needle protein, and therefore, they have been renamed EscE and EscG.

Role of inflammasomes in host defense against Citrobacter rodentium infection.

Citrobacter rodentium is an enteric bacterial pathogen of the mouse intestinal tract that triggers inflammatory responses resembling those of humans infected with enteropathogenic and enterohemorrhagic Escherichia coli. Inflammasome signaling is emerging as a central regulator of inflammatory and host responses to several pathogens, but the in vivo role of inflammasome signaling in host defense against C. rodentium has not been characterized. Here, we show that mice lacking the inflammasome components Nlrp3, Nlrc4, and caspase-1 were hypersusceptible to C. rodentium-induced gastrointestinal inflammation. This was due to defective interleukin (IL)-1ß and IL-18 production given that il-1ß(-/-) and il-18(-/-) mice also suffered from increased bacterial burdens and exacerbated histopathology. C. rodentium specifically activated the Nlrp3 inflammasome in in vitro-infected macrophages independently of a functional bacterial type III secretion system. Thus, production of IL-1ß and IL-18 downstream of the Nlrp3 and Nlrc4 inflammasomes plays a critical role in host defense against enteric infections caused by C. rodentium.

A novel subcellular machine contributes to basal junction remodeling in the seminiferous epithelium.

Tubulobulbar complexes are cytoskeleton-related membrane structures that develop at sites of intercellular attachment in mammalian seminiferous epithelium. At apical junctions between Sertoli cells and spermatids, the structures internalize adhesion junctions and are a component of the sperm release mechanism. Here we explore the possibility that tubulobulbar complexes that form at the blood-testis barrier are subcellular machines that internalize basal junction complexes. Using electron microscopy, we confirmed that morphologically identifiable tight and gap junctions are present in basal tubulobulbar complexes in rats. In addition, immunological probes for claudin-11 (CLDN11), connexin-43 (GJA1), and nectin-2 (PVRL2) react with linear structures at the light level that we interpret as tubulobulbar complexes, and probes for early endosome antigen 1 (EEA1) and Rab5 (RAB5A) react in similar locations. Significantly, fluorescence patterns for actin and claudin-11 indicate that the amount of junction present is dramatically reduced over the time period that tubulobulbar complexes are known to be most prevalent during spermatogenesis. We also demonstrated, using electron microscopy and fluorescence microscopy, that tubulobulbar complexes develop at basal junctions in primary cultures of Sertoli cells and that like their in vivo counterparts, the structures contain junction proteins. We use this culture system together with transfection techniques to show that junction proteins from one transfected cell occur in structures that project into adjacent nontransfected cells as predicted by the junction internalization hypothesis. On the basis of our findings, we present a new model for basal junction remodeling as it relates to spermatocyte translocation in the seminiferous epithelium.

EscI: a crucial component of the type III secretion system forms the inner rod structure in enteropathogenic Escherichia coli.

The T3SS (type III secretion system) is a multi-protein complex that plays a central role in the virulence of many gram-negative bacterial pathogens. This apparatus spans both bacterial membranes and transports virulence factors from the bacterial cytoplasm into eukaryotic host cells. The T3SS exports substrates in a hierarchical and temporal manner. The first secreted substrates are the rod/needle proteins which are incorporated into the T3SS apparatus and are required for the secretion of later substrates, the translocators and effectors. In the present study, we provide evidence that rOrf8/EscI, a poorly characterized locus of enterocyte effacement-encoded protein, functions as the inner rod protein of the T3SS of EPEC (enteropathogenic Escherichia coli). We demonstrate that EscI is essential for type III secretion and is also secreted as an early substrate of the T3SS. We found that EscI interacts with EscU, the integral membrane protein that is linked to substrate specificity switching, implicating EscI in the substrate-switching event. Furthermore, we showed that EscI self-associates and interacts with the outer membrane secretin EscC, further supporting its function as an inner rod protein. Overall, the results of the present study suggest that EscI is the YscI/PrgJ/MxiI homologue in the T3SS of attaching and effacing pathogens.

GrlR, a negative regulator in enteropathogenic E. coli, also represses the expression of LEE virulence genes independently of its interaction with its cognate partner GrlA.

Introduction: Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) belong to a group of pathogens that share the ability to form "attaching and effacing" (A/E) lesions on the intestinal epithelia. A pathogenicity island known as the locus of enterocyte effacement (LEE) contains the genes required for A/E lesion formation. The specific regulation of LEE genes relies on three LEE-encoded regulators: Ler activates the expression of the LEE operons by antagonizing the silencing effect mediated by the global regulator H-NS, GrlA activates ler expression and GrlR represses the expression of the LEE by interacting with GrlA. However, despite the existing knowledge of LEE regulation, the interplay between GrlR and GrlA and their independent roles in gene regulation in A/E pathogens are still not fully understood. Methods: To further explore the role that GrlR and GrlA in the regulation of the LEE, we used different EPEC regulatory mutants and cat transcriptional fusions, and performed protein secretion and expression assays, western blotting and native polyacrylamide gel electrophoresis. Results and discussion: We showed that the transcriptional activity of LEE operons increased under LEE-repressing growth conditions in the absence of GrlR. Interestingly, GrlR overexpression exerted a strong repression effect over LEE genes in wild-type EPEC and, unexpectedly, even in the absence of H-NS, suggesting that GrlR plays an alternative repressor role. Moreover, GrlR repressed the expression of LEE promoters in a non-EPEC background. Experiments with single and double mutants showed that GrlR and H-NS negatively regulate the expression of LEE operons at two cooperative yet independent levels. In addition to the notion that GrlR acts as a repressor by inactivating GrlA through protein-protein interactions, here we showed that a DNA-binding defective GrlA mutant that still interacts with GrlR prevented GrlR-mediated repression, suggesting that GrlA has a dual role as a positive regulator by antagonizing GrlR's alternative repressor role. In line with the importance of the GrlR-GrlA complex in modulating LEE gene expression, we showed that GrlR and GrlA are expressed and interact under both inducing and repressing conditions. Further studies will be required to determine whether the GrlR alternative repressor function depends on its interaction with DNA, RNA, or another protein. These findings provide insight into an alternative regulatory pathway that GrlR employs to function as a negative regulator of LEE genes.

Quorum sensing modulates bacterial virulence and colonization dynamics of the gastrointestinal pathogen Citrobacter rodentium.

Quorum Sensing (QS) is a form of cell-to-cell communication that enables bacteria to modify behavior according to their population density. While QS has been proposed as a potential intervention against pathogen infection, QS-mediated communication within the mammalian digestive tract remains understudied. Using an LC-MS/MS approach, we discovered that Citrobacter rodentium, a natural murine pathogen used to model human infection by pathogenic Escherichia coli, utilizes the CroIR system to produce three QS-molecules. We then profiled their accumulation both in vitro and across different gastrointestinal sites over the course of infection. Importantly, we found that in the absence of QS capabilities the virulence of C. rodentium is enhanced. This highlights the role of QS as an effective mechanism to regulate virulence according to the pathogen's spatio-temporal context to optimize colonization and transmission success. These results also demonstrate that inhibiting QS may not always be an effective strategy for the control of virulence.

The pathogenic Escherichia coli type III secreted protease NleC degrades the host acetyltransferase p300.

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively) are attaching and effacing bacterial pathogens that cause devastating diarrhoeal disease worldwide. These pathogens depend on a type III secretion system, which functions as a molecular syringe to translocate bacterial effector proteins directly into infected host cells. One of these effectors, NleC, was recently described as a zinc metalloprotease that targets NF-κB Rel-A (p65) and thus contributes to dampening of inflammatory signalling during EPEC and EHEC infection. We have identified the acetyltransferase p300 as an additional target of NleC. Several biochemical techniques were employed to demonstrate specific binding of p300 by NleC. We also show that NleC causes decreased abundance of p300 in cellular nuclei and that the metalloprotease domain of NleC is responsible for this phenotype. Furthermore, we demonstrate that overexpression of p300 can antagonize repression of IL-8 secretion by EPEC and that siRNA knock-down of p300 dampens IL-8 secretion by EPEC ΔnleC-infected cells. We have therefore identified a second target of NleC and provided the first example of a bacterial virulence factor targeting the acetyltransferase p300.

Structural analysis of a prototypical ATPase from the type III secretion system.

The type III secretion system (T3SS) ATPase is the conserved and essential inner-membrane component involved in the initial stages of selective secretion of specialized T3SS virulence effector proteins from the bacterial cytoplasm through to the infected host cell, a process crucial to subsequent pathogenicity. Here we present the 1.8-A-resolution crystal structure of the catalytic domain of the prototypical T3SS ATPase EscN from enteropathogenic Escherichia coli (EPEC). Along with in vitro and in vivo mutational analysis, our data show that the T3SS ATPases share similarity with the F1 ATPases but have important structural and sequence differences that dictate their unique secretory role. We also show that T3SS ATPase activity is dependent on EscN oligomerization and describe the molecular features and possible functional implications of a hexameric ring model.