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.

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.

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.

Quantitative mass spectrometry catalogues Salmonella pathogenicity island-2 effectors and identifies their cognate host binding partners.

Gram-negative bacterial pathogens have developed specialized secretion systems to transfer bacterial proteins directly into host cells. These bacterial effectors are central to virulence and reprogram host cell processes to favor bacterial survival, colonization, and proliferation. Knowing the complete set of effectors encoded by a particular pathogen is the key to understanding bacterial disease. In addition, the identification of the molecular assemblies that these effectors engage once inside the host cell is critical to determining the mechanism of action of each effector. In this work we used stable isotope labeling of amino acids in cell culture (SILAC), a powerful quantitative proteomics technique, to identify the proteins secreted by the Salmonella pathogenicity island-2 type three secretion system (SPI-2 T3SS) and to characterize the host interaction partners of SPI-2 effectors. We confirmed many of the known SPI-2 effectors and were able to identify several novel substrate candidates of this secretion system. We verified previously published host protein-effector binding pairs and obtained 11 novel interactions, three of which were investigated further and confirmed by reciprocal co-immunoprecipitation. The host cell interaction partners identified here suggest that Salmonella SPI-2 effectors target, in a concerted fashion, cellular processes such as cell attachment and cell cycle control that are underappreciated in the context of infection. The technology outlined in this study is specific and sensitive and serves as a robust tool for the identification of effectors and their host targets that is readily amenable to the study of other bacterial pathogens.

The pathogenic E. coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling.

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are diarrhoeal pathogens that cause the formation of attaching and effacing (A/E) lesions on infected host cells. These pathogens encode a type III secretion system (T3SS) used to inject effector proteins directly into host cells, an essential requirement for virulence. In this study, we identified a function for the type III secreted effector EspZ. Infection with EPEC DeltaespZ caused increased cytotoxicity in HeLa and MDCK cells compared with wild-type EPEC, and expressing espZ in cells abrogated this effect. Using yeast two-hybrid, proteomics, immunofluorescence and co-immunoprecipitation, it was demonstrated that EspZ interacts with the host protein CD98, which contributes to protection against EPEC-mediated cytotoxicity. EspZ enhanced phosphorylation of focal adhesion kinase (FAK) and AKT during infection with EPEC, but CD98 only appeared to facilitate FAK phosphorylation. This study provides evidence that EspZ and CD98 promote host cell survival mechanisms involving FAK during A/E pathogen infection.

A comprehensive proteomic analysis of the type III secretome of Citrobacter rodentium.

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.

A mammalian organelle map by protein correlation profiling.

Protein localization to membrane-enclosed organelles is a central feature of cellular organization. Using protein correlation profiling, we have mapped 1,404 proteins to ten subcellular locations in mouse liver, and these correspond with enzymatic assays, marker protein profiles, and confocal microscopy. These localizations allowed assessment of the specificity in published organellar proteomic inventories and demonstrate multiple locations for 39% of all organellar proteins. Integration of proteomic and genomic data enabled us to identify networks of coexpressed genes, cis-regulatory motifs, and putative transcriptional regulators involved in organelle biogenesis. Our analysis ties biochemistry, cell biology, and genomics into a common framework for organelle analysis.

Proteomics.

The genome sequences of important model systems are available and the focus is now shifting to large-scale experiments enabled by this data. Following in the footsteps of genomics, we have functional genomics, proteomics, and even metabolomics, roughly paralleling the biological hierarchy of the transcription, translation, and production of small molecules. Proteomics is initially concerned with determining the structure, expression, localization, biochemical activity, interactions, and cellular roles of as many proteins as possible. There has been great progress owing to novel instrumentation, experimental strategies, and bioinformatics methods. The area of protein-protein interactions has been especially fruitful. First pass interaction maps of some model organisms exist, and the proteins in many important organelles are about to be determined. Researchers are also beginning to integrate large-scale data sets from various "omics" disciplines in targeted investigations of specific biomedical areas and in pursuit of a general framework for systems biology.

RNA and RNA binding proteins participate in early stages of cell spreading through spreading initiation centers.

Focal adhesions are specialized attachment and signaling centers that form at sites of cell-matrix contacts. We employed a quantitative mass spectrometry-based method called SILAC to identify and quantify proteins interacting in an attachment-dependent manner with focal adhesion proteins. Subsequent confocal microscopy revealed a previously undescribed structure, which we have termed a spreading initiation center (SIC), existing only in early stages of cell spreading. SICs contain focal adhesion markers, appear to be surrounded by an actin sheath, and, surprisingly, contain numerous RNA binding proteins, ribosomal RNA, and perhaps other RNAs. Interfering with the function of FUS/TLS, hnRNP K, and hnRNP E1 results in increased spreading. Spreading initiation centers are ribonucleoprotein complexes distinct from focal adhesions and demonstrate a role for RNA and RNA binding proteins in the initiation of cell spreading.

Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors.

Membrane lipids were once thought to be homogenously distributed in the 2D surface of a membrane, but the lipid raft theory suggests that cholesterol and sphingolipids partition away from other membrane lipids. Lipid raft theory further implicates these cholesterol-rich domains in many processes such as signaling and vesicle traffic. However, direct characterization of rafts has been difficult, because they cannot be isolated in pure form. In the first functional proteomic analysis of rafts, we use quantitative high-resolution MS to specifically detect proteins depleted from rafts by cholesterol-disrupting drugs, resulting in a set of 241 authentic lipid raft components. We detect a large proportion of signaling molecules, highly enriched versus total membranes and detergent-resistant fractions, which thus far biochemically defined rafts. Our results provide the first large-scale and unbiased evidence, to our knowledge, for the connection of rafts with signaling and place limits on the fraction of plasma membrane composed by rafts.