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Prediction of T4SS Effector Proteins for Anaplasma phagocytophilum Using OPT4e, A New Software Tool.

ABSTRACT: Type IV secretion systems (T4SS) are used by a number of bacterial pathogens to attack the host cell. The complex protein structure of the T4SS is used to directly translocate effector proteins into host cells, often causing fatal diseases in humans and animals. Identification of effector proteins is the first step in understanding how they function to cause virulence and pathogenicity. Accurate prediction of effector proteins via a machine learning approach can assist in the process of their identification. The main goal of this study is to predict a set of candidate effectors for the tick-borne pathogen Anaplasma phagocytophilum, the causative agent of anaplasmosis in humans. To our knowledge, we present the first computational study for effector prediction with a focus on A. phagocytophilum. In a previous study, we systematically selected a set of optimal features from more than 1,000 possible protein characteristics for predicting T4SS effector candidates. This was followed by a study of the features using the proteome of Legionella pneumophila strain Philadelphia deduced from its complete genome. In this manuscript we introduce the OPT4e software package for Optimal-features Predictor for T4SS Effector proteins. An earlier version of OPT4e was verified using cross-validation tests, accuracy tests, and comparison with previous results for L. pneumophila. We use OPT4e to predict candidate effectors from the proteomes of A. phagocytophilum strains HZ and HGE-1 and predict 48 and 46 candidates, respectively, with 16 and 18 deemed most probable as effectors. These latter include the three known validated effectors for A. phagocytophilum.

SUBMITTER: Esna Ashari Z

PROVIDER: S-EPMC6598457 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Prediction of T4SS Effector Proteins for <i>Anaplasma phagocytophilum</i> Using OPT4e, A New Software Tool.

Esna Ashari Zhila Z Brayton Kelly A KA Broschat Shira L SL

Frontiers in microbiology 20190621

Type IV secretion systems (T4SS) are used by a number of bacterial pathogens to attack the host cell. The complex protein structure of the T4SS is used to directly translocate effector proteins into host cells, often causing fatal diseases in humans and animals. Identification of effector proteins is the first step in understanding how they function to cause virulence and pathogenicity. Accurate prediction of effector proteins via a machine learning approach can assist in the process of their id ...[more]

PMID: 31293540

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Project description:Pathogens must adapt to disparate environments in permissive host species, a feat that is especially pronounced for vector-borne microbes, which transition between vertebrate hosts and arthropod vectors to complete their lifecycles. Most knowledge about arthropod-vectored bacterial pathogens centers on their life in the mammalian host, where disease occurs. However, disease outbreaks are driven by the arthropod vectors. Adapting to the arthropod is critical for obligate intracellular rickettsial pathogens, as they depend on eukaryotic cells for survival. To manipulate the intracellular environment, these bacteria use type IV secretion systems (T4SS) to deliver effectors into the host cell. To date, few rickettsial T4SS translocated effectors have been identified and have only been examined in the context of mammalian infection. We identified an effector from the tick-borne rickettsial pathogen Anaplasma phagocytophilum, HGE1_02492, as critical for survival in tick cells and acquisition by ticks in vivo. Conversely, HGE1_02492 was dispensable during mammalian cell culture and murine infection. We show that HGE1_02492 is translocatable in a T4SS-dependent manner to the host cell cytosol. In eukaryotic cells, the HGE1_02492 localized with cortical actin filaments, which is dependent on multiple sub-domains of the protein. HGE1_02492 is the first arthropod-vector specific T4SS translocated effector identified from a rickettsial pathogen. Moreover, the subcellular target of HGE1_02492 suggests that A. phagocytophilum is manipulating actin to enable arthropod colonization. Based on these findings, we propose the name AteA for Anaplasma (phagocytophilum) tick effector A. Altogether, we show that A. phagocytophilum uses distinct strategies to cycle between mammals and arthropods.IMPORTANCETicks are the number one vector of pathogens for livestock worldwide and for humans in the United States. The biology of tick transmission is an understudied area. Understanding this critical interaction could provide opportunities to affect the course of disease spread. In this study, we examined the zoonotic tick-borne agent Anaplasma phagocytophilum and identified a secreted protein, AteA, which is expressed in a tick-specific manner. These secreted proteins, termed effectors, are the first proteins to interact with the host environment. AteA is essential for survival in ticks and appears to interact with cortical actin. Most effector proteins are studied in the context of the mammalian host; however, understanding how this unique set of proteins affects tick transmission is critical to developing interventions.

Project description:SUMOylation, the covalent attachment of a member of the small ubiquitin-like modifier (SUMO) family of proteins to lysines in target substrates, is an essential post-translational modification in eukaryotes. Microbial manipulation of SUMOylation recently emerged as a key virulence strategy for viruses and facultative intracellular bacteria, the latter of which have only been shown to deploy effectors that negatively regulate SUMOylation. Here, we demonstrate that the obligate intracellular bacterium, Anaplasma phagocytophilum, utilizes an effector, AmpA (A. phagocytophilum post-translationally modified protein A) that becomes SUMOylated in host cells and this is important for the pathogen's survival. We previously discovered that AmpA (formerly APH1387) localizes to the A. phagocytophilum-occupied vacuolar membrane (AVM). Algorithmic prediction analyses denoted AmpA as a candidate for SUMOylation. We verified this phenomenon using a SUMO affinity matrix to precipitate both native AmpA and ectopically expressed green fluorescent protein (GFP)-tagged AmpA. SUMOylation of AmpA was lysine dependent, as SUMO affinity beads failed to precipitate a GFP-AmpA protein when its lysine residues were substituted with arginine. Ectopically expressed and endogenous AmpA were poly-SUMOylated, which was consistent with the observation that AmpA colocalizes with SUMO2/3 at the AVM. Only late during the infection cycle did AmpA colocalize with SUMO1, which terminally caps poly-SUMO2/3 chains. AmpA was also detected in the cytosol of infected host cells, further supporting its secretion and likely participation in interactions that aid pathogen survival. Indeed, whereas siRNA-mediated knockdown of Ubc9 - a necessary enzyme for SUMOylation - slightly bolstered A. phagocytophilum infection, pharmacologically inhibiting SUMOylation in infected cells significantly reduced the bacterial load. Ectopically expressed GFP-AmpA served as a competitive agonist against native AmpA in infected cells, while lysine-deficient GFP-AmpA was less effective, implying that modification of AmpA lysines is important for infection. Collectively, these data show that AmpA becomes directly SUMOylated during infection, representing a novel tactic for A. phagocytophilum survival.

Project description:BACKGROUND: Anaplasma phagocytophilum is the etiological agent of granulocytic anaplasmosis in humans and animals. Wild animals and ticks play key roles in the enzootic cycles of the pathogen. Potential ecotypes of A. phagocytophilum have been characterized genetically, but their host range, zoonotic potential and transmission dynamics has only incompletely been resolved. METHODS: The presence of A. phagocytophilum DNA was determined in more than 6000 ixodid ticks collected from the vegetation and wildlife, in 289 tissue samples from wild and domestic animals, and 69 keds collected from deer, originating from various geographic locations in The Netherlands and Belgium. From the qPCR-positive lysates, a fragment of the groEL-gene was amplified and sequenced. Additional groEL sequences from ticks and animals from Europe were obtained from GenBank, and sequences from human cases were obtained through literature searches. Statistical analyses were performed to identify A. phagocytophilum ecotypes, to assess their host range and their zoonotic potential. The population dynamics of A. phagocytophilum ecotypes was investigated using population genetic analyses. RESULTS: DNA of A. phagocytophilum was present in all stages of questing and feeding Ixodes ricinus, feeding I. hexagonus, I. frontalis, I. trianguliceps, and deer keds, but was absent in questing I. arboricola and Dermacentor reticulatus. DNA of A. phagocytophilum was present in feeding ticks and tissues from many vertebrates, including roe deer, mouflon, red foxes, wild boar, sheep and hedgehogs but was rarely found in rodents and birds and was absent in badgers and lizards. Four geographically dispersed A. phagocytophilum ecotypes were identified, that had significantly different host ranges. All sequences from human cases belonged to only one of these ecotypes. Based on population genetic parameters, the potentially zoonotic ecotype showed significant expansion. CONCLUSION: Four ecotypes of A. phagocytophilum with differential enzootic cycles were identified. So far, all human cases clustered in only one of these ecotypes. The zoonotic ecotype has the broadest range of wildlife hosts. The expansion of the zoonotic A. phagocytophilum ecotype indicates a recent increase of the acarological risk of exposure of humans and animals.

Dataset Information

Prediction of T4SS Effector Proteins for Anaplasma phagocytophilum Using OPT4e, A New Software Tool.

Publications

Prediction of T4SS Effector Proteins for <i>Anaplasma phagocytophilum</i> Using OPT4e, A New Software Tool.

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