The super repertoire of type IV effectors in the pangenome of Ehrlichia spp. provides insights into host-specificity and pathogenesis

The identification of bacterial effectors is essential to understand how obligatory intracellular bacteria such as Ehrlichia spp. manipulate the host cell for survival and replication. Infection of mammals–including humans–by the intracellular pathogenic bacteria Ehrlichia spp. depends largely on the injection of virulence proteins that hijack host cell processes. Several hypothetical virulence proteins have been identified in Ehrlichia spp., but one so far has been experimentally shown to translocate into host cells via the type IV secretion system. However, the current challenge is to identify most of the type IV effectors (T4Es) to fully understand their role in Ehrlichia spp. virulence and host adaptation. Here, we predict the T4E repertoires of four sequenced Ehrlichia spp. and four other Anaplasmataceae as comparative models (pathogenic Anaplasma spp. and Wolbachia endosymbiont) using previously developed S4TE 2.0 software. This analysis identified 579 predicted T4Es (228 pT4Es for Ehrlichia spp. only). The effector repertoires of Ehrlichia spp. overlapped, thereby defining a conserved core effectome of 92 predicted effectors shared by all strains. In addition, 69 species-specific T4Es were predicted with non-canonical GC% mostly in gene sparse regions of the genomes and we observed a bias in pT4Es according to host-specificity. We also identified new protein domain combinations, suggesting novel effector functions. This work presenting the predicted effector collection of Ehrlichia spp. can serve as a guide for future functional characterisation of effectors and design of alternative control strategies against these bacteria.     

Secretion of type III effectors into host cells in real time

Type III secretion (T3S) systems are key features of many gram-negative bacteria that translocate T3S effector proteins directly into eukaryotic cells. There, T3S effectors exert many effects, such as cellular invasion or modulation of host immune responses. Studying spatiotemporal orchestrated secretion of various effectors has been difficult without disrupting their functions. Here we developed a new approach using Shigella flexneri T3S as a model to investigate bacterial translocation of individual effectors via multidimensional time-lapse microscopy. We demonstrate that direct fluorescent labeling of tetracysteine motif-tagged effectors IpaB and IpaC is possible in situ without loss of function. Studying the T3S kinetics of IpaB and IpaC ejection from individual bacteria, we found that the entire pools of IpaB and IpaC were released concurrently upon host cell contact, and that 50% of each effector was secreted in 240 s. This method allows an unprecedented analysis of the spatiotemporal events during T3S.     

青枯劳尔氏菌多基因家族III型效应蛋白在植物病害发展及防御系统中的作用

青枯劳尔氏菌是导致多种重要经济作物毁灭性枯萎（bacterial wilt）的一种土传病害,严重危害热带和亚热带地区食品安全。该细菌通过III型分泌系统（T3SS）向寄主细胞注射大量效应蛋白（T3Es）。效应蛋白是把双刃剑,既可诱导植物感病,又能激活植物防御系统。具有特殊重复结构的效应蛋白被归类成多基因家族,各家族成员协同致病,但其分子机制尚不清楚。本文围绕近年来有关多基因家族效应蛋白结构、功能和致病性等方面最新进展进行综述,为青枯菌致病机理和病害防治提供新思路。     

Ehrlichia type IV secretion effector ECH0825 is translocated to mitochondria and curbs ROS and apoptosis by upregulating host MnSOD

Ehrlichia chaffeensis infects monocytes/macrophages and causes human monocytic ehrlichiosis. To determine the role of type IV secretion (T4S) system in infection, candidates for T4S effectors were identified by bacterial two‐hybrid screening of E. chaffeensis hypothetical proteins with positively charged C‐terminus using E. chaffeensis VirD4 as bait. Of three potential T4S effectors, ECH0825 was highly upregulated early during exponential growth in a human monocytic cell line. ECH0825 was translocated from the bacterium into the host‐cell cytoplasm and localized to mitochondria. Delivery of anti‐ECH0825 into infected host cells significantly reduced bacterial infection. Ectopically expressed ECH0825 also localized to mitochondria and inhibited apoptosis of transfected cells in response to etoposide treatment. In double transformed yeast, ECH0825 localized to mitochondria and inhibited human Bax‐induced apoptosis. Mitochondrial manganese superoxide dismutase (MnSOD) was increased over ninefold in E. chaffeensis‐infected cells, and the amount of reactive oxygen species (ROS) in infected cells was significantly lower than that in uninfected cells. Similarly, MnSOD was upregulated and the ROS level was reduced in ECH0825‐transfected cells. These data suggest that, by upregulating MnSOD, ECH0825 prevents ROS‐induced cellular damage and apoptosis to allow intracellular infection. This is the first example of host ROS levels linked to a bacterial T4S effector.     

Type IV secretion systems and their effectors in bacterial pathogenesis

Type IV secretion systems (T4SSs) are membrane-associated transporter complexes used by various bacteria to deliver substrate molecules to a wide range of target cells. T4SSs are involved in horizontal DNA transfer to other bacteria and eukaryotic cells, in DNA uptake from or release into the extracellular milieu, in toxin secretion and in the injection of virulence factors into eukaryotic host target cells by several mammalian pathogens. Rapid progress has been made towards defining the structures and functions of T4SSs, identifying the translocated effector molecules and elucidating the mechanisms by which the effectors subvert eukaryotic cellular processes during infection. These findings have had an important impact on our understanding of how these pathogens manipulate host cell functions to trigger bacterial uptake, facilitate intracellular growth and suppress defence mechanisms, thus facilitating bacterial colonization and disease development.     

Diverse toxic effectors are harbored by vgrG islands for interbacterial antagonism in type VI secretion system

The type VI secretion system (T6SS) is considered as one of the key competition strategies by injecting toxic effectors for intestinal pathogens to acquire optimal colonization in host gut, a microenviroment with high-density polymicrobial community where bacteria compete for niches and resources. Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea in human and animals, widely encode T6SS clusters in their genomes. In this report, we first identified VT1, a novel amidase effector in ETEC, significantly hydrolyzed D-lactyl-L-Ala crosslinks between N-acetylmuramoyl and L-Ala in peptidoglycan. Further study showed that the VT1/VTI1 effector/immunity pair is encoded within a typical vgrG island, and plays a critical role for the successful establishment of ETEC in host gut. Numerous putative effectors with diverse toxin domains were found by retrieving vgrG islands in pathogenic E. coli, and designated as VT modules. Therein, VT5, a lysozyme-like effector widely encoded in ETEC, was confirmed to effectively kill adjacent cells, suggesting that VT toxin modules may be critical for pathogenic E. coli to seize a significantly competitive advantage for optimal intestinal colonization. To expand our analyses for large-scale search of VT antibacterial effectors based on vgrG island, >200 predicted effectors from 20 bacterial species were found and classified into 11 predicted toxins. This work reports a new retrieval strategy for screening T6SS effectors, and provides an example how pathogenic bacteria antagonize and displace commensal microbiome to successfully colonize in the host niches through a T6SS-dependent manner.     

Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae

Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.     

Prime time for minor subunits of the type II secretion and type IV pilus systems

The type II secretion system (T2SS) exports folded proteins from the periplasms of Gram‐negative bacteria. The type IV pilus system (T4PS) is a multifunctional machine used for adherence, motility and DNA transfer in bacteria and archaea. Partial sequence identity between the two systems suggests that they are related and might function via a similar mechanism, the dynamic assembly and disassembly of pseudopilus (T2SS) or pilus (T4PS) filaments. The major subunit in each system is thought to form the bulk of the (pseudo)pilus, while minor (low‐abundance) subunits have proposed roles in assembly initiation, antagonism of disassembly, or modulation of (pseudo)pilus functional properties. In this issue, Cisneros et al. ( 2012 ) extend their previous finding that pseudopilus assembly is primed by the minor pseudopilins, showing that the same proteins can initiate assembly of Escherichia coli T4P. Similarly, they show that the E. coli minor pilins prime the polymerization of T2S pseudopili, although unlike genuine pseudopili, the chimeric filaments did not support secretion. This work reinforces the notion of a common assembly mechanism for the T2S and T4P systems.     

Salmonella effectors: important players modulating host cell function during infection

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative facultative food-borne pathogen that causes gastroenteritis in humans. This bacterium has evolved a sophisticated machinery to alter host cell function critical to its virulence capabilities. Central to S. Typhimurium pathogenesis are two Type III secretion systems (T3SS) encoded within pathogenicity islands SPI-1 and SPI-2 that are responsible for the secretion and translocation of a set of bacterial proteins termed effectors into host cells with the intention of altering host cell physiology for bacterial entry and survival. Thus, once delivered by the T3SS, the secreted effectors play critical roles in manipulating the host cell to allow for bacteria invasion, induction of inflammatory responses, and the assembly of an intracellular protective niche created for bacterial survival and replication. Emerging evidence indicates that these effectors are modular proteins consisting of distinct functional domains/motifs that are utilized by the bacteria to activate intracellular signalling pathways modifying host cell function. Also, recently reported are the dual functionality of secreted effectors and the concept of 'terminal reassortment'. Herein, we highlight some of the nascent concepts regarding Salmonella effectors in the context of infection.     

The large,diverse, and robust arsenal of Ralstonia solanacearum type III effectors and their in planta functions

The type III secretion system with its delivered type III effectors (T3Es) is one of the main virulence determinants of Ralstonia solanacearum, a worldwide devastating plant pathogenic bacterium affecting many crop species. The pan-effectome of the R. solanacearum species complex has been exhaustively identified and is composed of more than 100 different T3Es. Among the reported strains, their content ranges from 45 to 76 T3Es. This considerably large and varied effectome could be considered one of the factors contributing to the wide host range of R. solanacearum. In order to understand how R. solanacearum uses its T3Es to subvert the host cellular processes, many functional studies have been conducted over the last three decades. It has been shown that R. solanacearum effectors, as those from other plant pathogens, can suppress plant defence mechanisms, modulate the host metabolism, or avoid bacterial recognition through a wide variety of molecular mechanisms. R. solanacearum T3Es can also be perceived by the plant and trigger immune responses. To date, the molecular mechanisms employed by R. solanacearum T3Es to modulate these host processes have been described for a growing number of T3Es, although they remain unknown for the majority of them. In this microreview, we summarize and discuss the current knowledge on the characterized R. solanacearum species complex T3Es.     

Identification of Anaplasma marginale type IV secretion system effector proteins

Background

Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.

Results

By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141) of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system.

Conclusions

The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work.     

The type III effectors of Xanthomonas

A review of type III effectors (T3 effectors) from strains of Xanthomonas reveals a growing list of candidate and known effectors based on functional assays and sequence and structural similarity searches of genomic data. We propose that the effectors and suspected effectors should be distributed into 39 so-called Xop groups reflecting sequence similarity. Some groups have structural motifs for putative enzymatic functions, and recent studies have provided considerable insight into the interaction with host factors in their function as mediators of virulence and elicitors of resistance for a few specific T3 effectors. Many groups are related to T3 effectors of plant and animal pathogenic bacteria, and several groups appear to have been exploited primarily by Xanthomonas species based on available data. At the same time, a relatively large number of candidate effectors remain to be examined in more detail with regard to their function within host cells.     

WEDeepT3: predicting type III secreted effectors based on word embedding and deep learning

Background: The type III secreted effectors (T3SEs) are one of the indispensable proteins in the growth and reproduction of Gram-negative bacteria. In particular, the pathogenesis of Gram-negative bacteria depends on the type III secreted effectors, and by injecting T3SEs into a host cell, the host cell’s immunity can be destroyed. The high diversity of T3SE sequences and the lack of defined secretion signals make it difficult to identify and predict. Moreover, the related study of the pathological system associated with T3SE remains a hot topic in bioinformatics. Some computational tools have been developed to meet the growing demand for the recognition of T3SEs and the studies of type III secretion systems (T3SS). Although these tools can help biological experiments in certain procedures, there is still room for improvement, even for the current best model, as the existing methods adopt hand-designed feature and traditional machine learning methods. Methods: In this study, we propose a powerful predictor based on deep learning methods, called WEDeepT3. Our work consists mainly of three key steps. First, we train word embedding vectors for protein sequences in a large-scale amino acid sequence database. Second, we combine the word vectors with traditional features extracted from protein sequences, like PSSM, to construct a more comprehensive feature representation. Finally, we construct a deep neural network model in the prediction of type III secreted effectors. Results: The feature representation of WEDeepT3 consists of both word embedding and position-specific features. Working together with convolutional neural networks, the new model achieves superior performance to the state-of-the-art methods, demonstrating the effectiveness of the new feature representation and the powerful learning ability of deep models. Conclusion: WEDeepT3 exploits both semantic information of k-mer fragments and evolutional information of protein sequences to accurately differentiate between T3SEs and non-T3SEs. WEDeepT3 is available at bcmi.sjtu.edu.cn/~yangyang/WEDeepT3.html.     

A human pathogenic bacterium Shigella proliferates in plants through adoption of type III effectors for shigellosis

Shigella, which infects primates, can be transmitted via fresh vegetables; however, its molecular interactions with plants have not been elucidated. Here, we show that four Shigella strains, Shigella boydii, Shigella sonnei, Shigella flexneri 2a, and S. flexneri 5a, proliferate at different levels in Arabidopsis thaliana. Microscopic studies revealed that these bacteria were present inside leaves and damaged plant cells. Green fluorescent protein (GFP)‐tagged S. boydii and S. flexneri 5a colonized leaves only, whereas S. flexneri 2a colonized both leaves and roots. Using Shigella mutants lacking type III secretion systems (T3SSs), we found that T3SSs that regulate the pathogenesis of shigellosis in humans also play a central role in bacterial proliferation in Arabidopsis. Strikingly, the immunosuppressive activity of two T3S effectors, OspF and OspG, was required for proliferation of Shigella in Arabidopsis. Of note, delivery of OspF or OspG effectors inside plant cells upon Shigella inoculation was confirmed using a split GFP system. These findings demonstrate that the human pathogen Shigella can proliferate in plants by adapting immunosuppressive machinery used in the original host human.     

Modulation of innate immune responses by Yersinia type III secretion system translocators and effectors

The innate immune system of mammals responds to microbial infection through detection of conserved molecular determinants called ‘pathogen‐associated molecular patterns’ (PAMPs). Pathogens use virulence factors to counteract PAMP‐directed responses. The innate immune system can in turn recognize signals generated by virulence factors, allowing for a heightened response to dangerous pathogens. Many Gram‐negative bacterial pathogens encode type III secretion systems (T3SSs) that translocate effector proteins, subvert PAMP‐directed responses and are critical for infection. A plasmid‐encoded T3SS in the human‐pathogenic Yersinia species translocates seven effectors into infected host cells. Delivery of effectors by the T3SS requires plasma membrane insertion of two translocators, which are thought to form a channel called a translocon. Studies of the Yersinia T3SS have provided key advances in our understanding of how innate immune responses are generated by perturbations in plasma membrane and other signals that result from translocon insertion. Additionally, studies in this system revealed that effectors function to inhibit innateimmune responses resulting from insertion of translocons into plasma membrane. Here, we review these advances with the goal of providing insight into how a T3SS can activate and inhibit innate immune responses, allowing a virulent pathogen to bypass host defences.     

Genomic mining type III secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors

Many bacterial pathogens of plants and animals use a type III secretion system (TTSS) to deliver virulence effector proteins into host cells. Because effectors are heterogeneous in sequence and function, there has not been a systematic way to identify the genes encoding them in pathogen genomes, and our current inventories are probably incomplete. A pre-closure draft sequence of Pseudomonas syringae pv. tomato DC3000, a pathogen of tomato and Arabidopsis, has recently supported five complementary studies which, collectively, identify 36 TTSS-secreted proteins and many more candidate effectors in this strain. These studies demonstrate the advantages of combining experimental and computational approaches, and they yield new insights into TTSS effectors and virulence regulation in P. syringae, potential effector targeting signals in all TTSS-dependent pathogens, and strategies for finding TTSS effectors in other bacteria that have sequenced genomes.