Application of a C. trachomatis expression system to identify C. pneumoniae proteins translocated into host cells

Chlamydia pneumoniae is a Gram-negative, obligate intracellular pathogen that causes community-acquired respiratory infections. C. pneumoniae uses a cell contact-dependent type-III secretion (T3S) system to translocate pathogen effector proteins that manipulate host cellular functions. While several C. pneumoniae T3S effectors have been proposed, few have been experimentally confirmed in Chlamydia. In this study, we expressed 382 C. pneumoniae genes in C. trachomatis as fusion proteins to an epitope tag derived from glycogen synthase kinase 3β (GSK3β) which is the target of phosphorylation by mammalian kinases. Based on the detection of the tagged C. pneumoniae protein with anti-phospho GSK3β antibodies, we identified 49 novel C. pneumoniae-specific proteins that are translocated by C. trachomatis into the host cytoplasm and thus likely play a role as modifiers of host cellular functions. In this manner, we identified and characterized a new C. pneumoniae effector CPj0678 that recruits the host cell protein PACSIN2 to the plasma membrane. These findings indicate that C. trachomatis provides a powerful screening system to detect candidate effector proteins encoded by other pathogenic and endosymbiotic Chlamydia species. Importance Chlamydia injects numerous effector proteins into host cells to manipulate cellular functions important for bacterial survival. Based on findings in C. trachomatis, it has been proposed that between 5-10% of the C. pneumoniae genome, a related respiratory pathogen, encodes secreted effectors. However only a few C. pneumoniae effectors have been identified and experimentally validated. With the development of methods for the stable genetic transformation of C. trachomatis, it is now possible to use C. trachomatis shuttle plasmids to express and explore the function of proteins from other Chlamydia in a more relevant bacterial system. In this study, we experimentally determined that 49 C. pneumoniae-specific proteins are translocated into the host cytoplasm by Chlamydia secretion systems, and identify a novel effector required to recruit the host factor PACSIN2 to the plasma membrane during infection.

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Bacterial Effector HopF2 Suppresses Arabidopsis Innate Immunity at the Plasma Membrane

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-10-0150 ◽

2011 ◽

Vol 24 (5) ◽

pp. 585-593 ◽

Cited By ~ 39

Author(s):

Shujing Wu ◽

Dongping Lu ◽

Mehdi Kabbage ◽

Hai-Lei Wei ◽

Bryan Swingle ◽

...

Keyword(s):

Innate Immunity ◽

Plasma Membrane ◽

Pseudomonas Syringae ◽

Plant Immunity ◽

Mapk Signaling ◽

Effector Proteins ◽

Host Cells ◽

Mitogen Activated Protein Kinase ◽

Immune Response Gene ◽

Secretion Systems

Many bacterial pathogens inject a cocktail of effector proteins into host cells through type III secretion systems. These effectors act in concert to modulate host physiology and immune signaling, thereby promoting pathogenicity. In a search for additional Pseudomonas syringae effectors in suppressing plant innate immunity triggered by pathogen or microbe-associated molecular patterns (PAMPs or MAMPs), we identified P. syringae tomato DC3000 effector HopF2 as a potent suppressor of early immune-response gene transcription and mitogen-activated protein kinase (MAPK) signaling activated by multiple MAMPs, including bacterial flagellin, elongation factor Tu, peptidoglycan, lipopolysaccharide and HrpZ1 harpin, and fungal chitin. The conserved surface-exposed residues of HopF2 are essential for its MAMP suppression activity. HopF2 is targeted to the plant plasma membrane through a putative myristoylation site, and the membrane association appears to be required for its MAMP-suppression function. Expression of HopF2 in plants potently diminished the flagellin-induced phosphorylation of BIK1, a plasma membrane–associated cytoplasmic kinase that is rapidly phosphorylated within one minute upon flagellin perception. Thus, HopF2 likely intercepts MAMP signaling at the plasma membrane immediately of signal perception. Consistent with the potent suppression function of multiple MAMP signaling, expression of HopF2 in transgenic plants compromised plant nonhost immunity to bacteria P. syringae pv. Phaseolicola and plant immunity to the necrotrophic fungal pathogen Botrytis cinerea.

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Small-Molecule Type III Secretion System Inhibitors Block Assembly of the Shigella Type III Secreton

Journal of Bacteriology ◽

10.1128/jb.01004-08 ◽

2008 ◽

Vol 191 (2) ◽

pp. 563-570 ◽

Cited By ~ 71

Author(s):

Andreas K. J. Veenendaal ◽

Charlotta Sundin ◽

Ariel J. Blocker

Keyword(s):

Type Iii Secretion ◽

Bacterial Infections ◽

Shigella Flexneri ◽

Effector Proteins ◽

Host Cells ◽

Incomplete Penetrance ◽

Secretion Systems ◽

Bacterial Surface ◽

Type Iii ◽

Type Iii Secretion Systems

ABSTRACT Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.

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A Salmonella type III effector, PipA, works in a different manner than the PipA family effectors GogA and GtgA

PLoS ONE ◽

10.1371/journal.pone.0248975 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0248975

Author(s):

Momo Takemura ◽

Takeshi Haneda ◽

Hikari Idei ◽

Tsuyoshi Miki ◽

Nobuhiko Okada

Keyword(s):

Hela Cells ◽

Critical Role ◽

Effector Proteins ◽

Host Cells ◽

Microbial Pathogens ◽

Secretion Systems ◽

Type Iii ◽

Zinc Metalloprotease ◽

Type Iii Secretion Systems ◽

Serovar Typhimurium

Nuclear factor-kappa B (NF-κB) plays a critical role in the host defense against microbial pathogens. Many pathogens modulate NF-κB signaling to establish infection in their host. Salmonella enterica serovar Typhimurium (S. Typhimurium) possesses two type III secretion systems (T3SS-1 and T3SS-2) and directly injects many effector proteins into host cells. It has been reported that some effectors block NF-κB signaling, but the molecular mechanism of the inactivation of NF-κB signaling in S. Typhimurium is poorly understood. Here, we identified seven type III effectors—GogA, GtgA, PipA, SseK1, SseK2, SseK3, and SteE—that inhibited NF-κB activation in HeLa cells stimulated with TNF-α. We also determined that only GogA and GtgA are involved in regulation of the activation of NF-κB in HeLa cells infected with S. Typhimurium. GogA, GtgA, and PipA are highly homologous to one another and have the consensus zinc metalloprotease HEXXH motif. Our experiments demonstrated that GogA, GtgA, and PipA each directly cleaved NF-κB p65, whereas GogA and GtgA, but not PipA, inhibited the NF-κB activation in HeLa cells infected with S. Typhimurium. Further, expressions of the gogA or gtgA gene were induced under the SPI-1-and SPI-2-inducing conditions, but expression of the pipA gene was induced only under the SPI-2-inducing condition. We also showed that PipA was secreted into RAW264.7 cells through T3SS-2. Finally, we indicated that PipA elicits bacterial dissemination in the systemic stage of infection of S. Typhimurium via a T3SS-1-independent mechanism. Collectively, our results suggest that PipA, GogA and GtgA contribute to S. Typhimurium pathogenesis in different ways.

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Cooperative Immune Suppression by Escherichia coli and Shigella Effector Proteins

Infection and Immunity ◽

10.1128/iai.00560-17 ◽

2018 ◽

Vol 86 (4) ◽

Cited By ~ 6

Author(s):

Maarten F. de Jong ◽

Neal M. Alto

Keyword(s):

Escherichia Coli ◽

Defense Mechanisms ◽

Immune Defense ◽

Effector Proteins ◽

Innate Immune ◽

Host Cells ◽

Secretion Systems ◽

Content Type ◽

E Coli ◽

Type Iii Secretion Systems

ABSTRACT The enteric attaching and effacing (A/E) pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) and the invasive pathogens enteroinvasive E. coli (EIEC) and Shigella encode type III secretion systems (T3SS) used to inject effector proteins into human host cells during infection. Among these are a group of effectors required for NF-κB-mediated host immune evasion. Recent studies have identified several effector proteins from A/E pathogens and EIEC/ Shigella that are involved in suppression of NF-κB and have uncovered their cellular and molecular functions. A novel mechanism among these effectors from both groups of pathogens is to coordinate effector function during infection. This cooperativity among effector proteins explains how bacterial pathogens are able to effectively suppress innate immune defense mechanisms in response to diverse classes of immune receptor signaling complexes (RSCs) stimulated during infection.

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Salmonella in Australia: understanding and controlling infection

Microbiology Australia ◽

10.1071/ma17044 ◽

2017 ◽

Vol 38 (3) ◽

pp. 112

Author(s):

Joshua PM Newson

Keyword(s):

Host Cell ◽

Cell Biology ◽

Protein Translocation ◽

Cell Signalling ◽

Effector Proteins ◽

Host Cells ◽

Secretion Systems ◽

Significant Burden ◽

Systemic Infections ◽

Bacterial Effectors

The bacterium Salmonella causes a spectrum of foodborne diseases ranging from acute gastroenteritis to systemic infections, and represents a significant burden of disease globally. In Australia, Salmonella is frequently associated with outbreaks and is a leading cause of foodborne illness, which results in a significant medical and economic burden. Salmonella infection involves colonisation of the small intestine, where the bacteria invades host cells and establishes an intracellular infection. To survive within host cells, Salmonella employs type-three secretion systems to deliver bacterial effector proteins into the cytoplasm of host cells. These bacterial effectors seek out and modify specific host proteins, disrupting host processes such as cell signalling, intracellular trafficking, and programmed cell death. This strategy of impairing host cells allows Salmonella to establish a replicative niche within the cell, where they can replicate to high numbers before escaping to infect neighbouring cells, or be transmitted to new hosts. While the importance of effector protein translocation to infection is well established, our understanding of many effector proteins remains incomplete. Many Salmonella effectors have unknown function and unknown roles during infection. A greater understanding of how Salmonella manipulates host cells during infection will lead to improved strategies to prevent, control, and eliminate disease. Further, studying effector proteins can be a useful means for exploring host cell biology and elucidating the details of host cell signalling.

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Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals

Microbiology ◽

10.1099/mic.0.28368-0 ◽

2006 ◽

Vol 152 (1) ◽

pp. 143-152 ◽

Cited By ~ 12

Author(s):

Ciara M. Shaver ◽

Alan R. Hauser

Keyword(s):

Pseudomonas Aeruginosa ◽

Disease Severity ◽

Type Iii Secretion ◽

Disease Process ◽

Effector Proteins ◽

Host Cells ◽

Secreted Proteins ◽

Secretion Systems ◽

Type Iii

The effector proteins of the type III secretion systems of many bacterial pathogens act in a coordinated manner to subvert host cells and facilitate the development and progression of disease. It is unclear whether interactions between the type-III-secreted proteins of Pseudomonas aeruginosa result in similar effects on the disease process. We have previously characterized the contributions to pathogenesis of the type-III-secreted proteins ExoS, ExoT and ExoU when secreted individually. In this study, we extend our prior work to determine whether these proteins have greater than expected effects on virulence when secreted in combination. In vitro cytotoxicity and anti-internalization activities were not enhanced when effector proteins were secreted in combinations rather than alone. Likewise in a mouse model of pneumonia, bacterial burden in the lungs, dissemination and mortality attributable to ExoS, ExoT and ExoU were not synergistically increased when combinations of these effector proteins were secreted. Because of the absence of an appreciable synergistic increase in virulence when multiple effector proteins were secreted in combination, we conclude that any cooperation between ExoS, ExoT and ExoU does not translate into a synergistically significant enhancement of disease severity as measured by these assays.

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Dynamics of the Type III Secretion System Activity of Enteropathogenic Escherichia coli

mBio ◽

10.1128/mbio.00303-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 37

Author(s):

Erez Mills ◽

Kobi Baruch ◽

Gili Aviv ◽

Mor Nitzan ◽

Ilan Rosenshine

Keyword(s):

Escherichia Coli ◽

Type Iii Secretion ◽

Public Health Problem ◽

Effector Proteins ◽

Host Cells ◽

Global Public Health ◽

Secretion Systems ◽

Comprehensive Description ◽

Content Type ◽

Type Iii

ABSTRACT Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics. IMPORTANCE EPEC and the closely related enterohemorrhagic Escherichia coli (EHEC) represent a global public health problem. New strategies to combat EPEC and EHEC infections are needed, and development of such strategies requires better understanding of their virulence machinery. The TTSS is a critical virulence mechanism employed by these pathogens, and by others, including Salmonella. In this study, we aimed at elucidating new aspects of TTSS function. The results obtained provide a comprehensive description of the dynamics of TTSS activity of EPEC and new insights into the mechanisms that control these changes. This knowledge sets the stage for further analysis of the system and may accelerate the development of new ways to treat EPEC and EHEC infections. Further, the newly described microscopy-based assay can be readily adapted to study the dynamics of TTSS activity in other pathogens.

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Salmonellaeffectors SseK1 and SseK3 target death domain proteins in the TNF and TRAIL signaling pathways

10.1101/359117 ◽

2018 ◽

Cited By ~ 1

Author(s):

Joshua P M Newson ◽

Nichollas E Scott ◽

Ivy Yeuk Wah Chung ◽

Tania Wong Fok Lung ◽

Cristina Giogha ◽

...

Keyword(s):

Death Receptor ◽

Distinct Type ◽

Effector Proteins ◽

Host Cells ◽

Death Domain ◽

Secretion Systems ◽

Host Proteins ◽

Tnf Superfamily ◽

Arginine Residues ◽

Death Receptor Signaling

AbstractStrains ofSalmonellautilise two distinct type three secretion systems to deliver effector proteins directly into host cells. TheSalmonellaeffectors SseK1 and SseK3 are arginine glycosyltransferases that modify mammalian death domain containing proteins with N-acetyl glucosamine (GlcNAc) when overexpressed ectopically or as recombinant protein fusions. Here, we combined Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry to identify host proteins GlcNAcylated by endogenous levels of SseK1 and SseK3 duringSalmonellainfection. We observed that SseK1 modified the mammalian signaling protein TRADD, but not FADD as previously reported. Overexpression of SseK1 greatly broadened substrate specificity, while ectopic co-expression of SseK1 and TRADD increased the range of modified arginine residues within the death domain of TRADD. In contrast, endogenous levels of SseK3 resulted in modification of the death domains of receptors of the mammalian TNF superfamily, TNFR1 and TRAILR, at residues Arg376and Arg293respectively. Structural studies on SseK3 showed that the enzyme displays a classic GT-A glycosyltransferase fold and binds UDP-GlcNAc in a narrow and deep cleft with the GlcNAc facing the surface. Together our data suggests that Salmonellae carryingsseK1andsseK3employ the glycosyltransferase effectors to antagonise different components of death receptor signaling.

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roles Hrp-dependent effector proteins and hrp gene regulation as determinants of virulence and host-specificity in Erwinia stewartii and E. herbicola pvs. gypsophilae and betae

10.32747/2005.7587216.bard ◽

2005 ◽

Author(s):

David L. Coplin ◽

Shulamit Manulis ◽

Isaac Barash

Keyword(s):

Gene Expression ◽

Host Specificity ◽

Pathogenic Bacteria ◽

Effector Proteins ◽

Host Cells ◽

Secretion Systems ◽

Gram Negative ◽

Regulatory Cascade ◽

Effector Genes ◽

Erwinia Stewartii

Gram-negative plant pathogenic bacteria employ specialized type-III secretion systems (TTSS) to deliver an arsenal of pathogenicity proteins directly into host cells. These secretion systems are encoded by hrp genes (for hypersensitive response and pathogenicity) and the effector proteins by so-called dsp or avr genes. The functions of effectors are to enable bacterial multiplication by damaging host cells and/or by blocking host defenses. We characterized essential hrp gene clusters in the Stewart's Wilt of maize pathogen, Pantoea stewartii subsp. stewartii (Pnss; formerly Erwinia stewartii) and the gall-forming bacterium, Pantoea agglomerans (formerly Erwinia herbicola) pvs. gypsophilae (Pag) and betae (Pab). We proposed that the virulence and host specificity of these pathogens is a function of a) the perception of specific host signals resulting in bacterial hrp gene expression and b) the action of specialized signal proteins (i.e. Hrp effectors) delivered into the plant cell. The specific objectives of the proposal were: 1) How is the expression of the hrp and effector genes regulated in response to host cell contact and the apoplastic environment? 2) What additional effector proteins are involved in pathogenicity? 3) Do the presently known Pantoea effector proteins enter host cells? 4) What host proteins interact with these effectors? We characterized the components of the hrp regulatory cascade (HrpXY ->7 HrpS ->7 HrpL ->7 hrp promoters), showed that they are conserved in both Pnss and Fag, and discovered that the regulation of the hrpS promoter (hrpSp) may be a key point in integrating apoplastic signals. We also analyzed the promoters recognized by HrpL and demonstrated the relationship between their composition and efficiency. Moreover, we showed that promoter strength can influence disease expression. In Pnss, we found that the HrpXY two-component signal system may sense the metabolic status of the bacterium and is required for full hrp gene expression in planta. In both species, acyl-homoserine lactone-mediated quorum sensing may also regulate epiphytic fitness and/or pathogenicity. A common Hrp effector protein, DspE/WtsE, is conserved and required for virulence of both species. When introduced into corn cells, Pnss WtsE protein caused water-soaked lesions. In other plants, it either caused cell death or acted as an Avr determinant. Using a yeast- two-hybrid system, WtsE was shown to interact with a number of maize signal transduction proteins that are likely to have roles in either programmed cell death or disease resistance. In Pag and Pab, we have characterized the effector proteins HsvG, HsvB and PthG. HsvG and HsvB are homologous proteins that determine host specificity of Pag and Pab on gypsophila and beet, respectively. Both possess a transcriptional activation domain that functions in yeast. PthG was found to act as an Avr determinant on multiple beet species, but was required for virulence on gypsophila. In addition, we demonstrated that PthG acts within the host cell. Additional effector genes have been characterized on the pathogenicity plasmid, pPATHₚₐg, in Pag. A screen for HrpL- regulated genes in Pnsspointed up 18 candidate effector proteins and four of these were required for full virulence. It is now well established that the virulence of Gram-negative plant pathogenic bacteria is governed by Hrp-dependent effector proteins. However; the mode of action of many effectors is still unresolved. This BARD supported research will significantly contribute to the understanding of how Hrp effectors operate in Pantoea spp. and how they control host specificity and affect symptom production. This may lead to novel approaches for genetically engineering plants resistant to a wide range of bacterial pathogens by inactivating the Hrp effectors with "plantabodies" or modifying their receptors, thereby blocking the induction of the susceptible response. Alternatively, innovative technologies could be used to interfere with the Hrp regulatory cascade by blocking a critical step or mimicking plant or quorum sensing signals.

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Using an Optimal Set of Features with a Machine Learning-Based Approach to Predict Effector Proteins forLegionella pneumophila

10.1101/383570 ◽

2018 ◽

Author(s):

Zhila Esna Ashari ◽

Kelly A. Brayton ◽

Shira L. Broschat

Keyword(s):

Machine Learning ◽

Legionella Pneumophila ◽

De Novo ◽

Effector Proteins ◽

Machine Learning Algorithms ◽

Host Cells ◽

Support Vector ◽

Secretion Systems ◽

Type Iv ◽

Optimal Set

AbstractType IV secretion systems exist in a number of bacterial pathogens and are used to secrete effector proteins directly into host cells in order to change their environment making the environment hospitable for the bacteria. In recent years, several machine learning algorithms have been developed to predict effector proteins, potentially facilitating experimental verification. However, inconsistencies exist between their results. Previously we analysed the disparate sets of predictive features used in these algorithms to determine an optimal set of 370 features for effector prediction. This work focuses on the best way to use these optimal features by designing three machine learning classifiers, comparing our results with those of others, and obtaining de novo results. We chose the pathogenLegionella pneumophilastrain Philadelphia-1, a cause of Legionnaires’ disease, because it has many validated effector proteins and others have developed machine learning prediction tools for it. While all of our models give good results indicating that our optimal features are quite robust, Model 1, which uses all 370 features with a support vector machine, has slightly better accuracy. Moreover, Model 1 predicted 760 effector proteins, more than any other study, 315 of which have been validated. Although the results of our three models agree well with those of other researchers, their models only predicted 126 and 311 candidate effectors.

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