Wheat Leaf Rust Fungus Effector Protein Pt1641 Is Avirulent to TcLr1.

Wheat leaf rust fungus is an obligate parasitic fungus that can absorb nutrients from its host plant through haustoria and secrete effector proteins into host cells. The effector proteins are crucial factors for pathogenesis as well as targets for host disease resistance protein recognition. Exploring the role of effector proteins in the pathogenic process of Puccinia triticina Eriks. (Pt) is of great significance for unraveling its pathogenic mechanisms. We previously found that a cysteine-rich effector protein, Pt1641, is highly expressed during the interaction between wheat and Pt, but its specific role in pathogenesis remains unclear. Therefore, this study employed techniques such as heterologous expression, qRT-PCR analysis, and host-induced gene silencing (HIGS) to investigate the role of Pt1641 in the pathogenic process of Pt. The results indicate that Pt1641 is an effector protein with a secretory function and can inhibit BAX-induced programmed cell death in Nicotiana benthamiana. qRT-PCR analyses showed that expression levels of Pt1641 were different during the interaction between the high-virulence strain THTT and low-virulence strains FGD and Thatcher, respectively. The highest expression level in the low-virulence strain FGD was four times that of the high-virulence strain THTT. The overexpression of Pt1641 in wheat near-isogenic line TcLr1 induced callose deposition and H2O2 production on TcLr1. After silencing Pt1641 in the Pt low-virulence strain FGD on wheat near-isogenic line TcLr1, the pathogenic phenotype of Pt physiological race FGD on TcLr1 changed from ";" to "3", indicating that Pt1641 plays a non-toxic function in the pathogenicity of FGD to TcLr1. This study helps to reveal the pathogenic mechanism of wheat leaf rust and provides important guidance for the mining and application of Pt avirulent genes.

The multifunction Coxiella effector Vice stimulates macropinocytosis and interferes with the ESCRT machinery.

Intracellular bacterial pathogens divert multiple cellular pathways to establish their niche and persist inside their host. Coxiella burnetii, the causative agent of Q fever, secretes bacterial effector proteins via its Type 4 secretion system to generate a Coxiella-containing vacuole (CCV). Manipulation of lipid and protein trafficking by these effectors is essential for bacterial replication and virulence. Here, we have characterized the lipid composition of CCVs and found that the effector Vice interacts with phosphoinositides and membranes enriched in phosphatidylserine and lysobisphosphatidic acid. Remarkably, eukaryotic cells ectopically expressing Vice present compartments that resemble early CCVs in both morphology and composition. We found that the biogenesis of these compartments relies on the double function of Vice. The effector protein initially localizes at the plasma membrane of eukaryotic cells where it triggers the internalization of large vacuoles by macropinocytosis. Then, Vice stabilizes these compartments by perturbing the ESCRT machinery. Collectively, our results reveal that Vice is an essential C. burnetii effector protein capable of hijacking two major cellular pathways to shape the bacterial replicative niche.

A phytoplasma effector destabilizes chloroplastic glutamine synthetase inducing chlorotic leaves that attract leafhopper vectors.

Leaf yellowing is a well-known phenotype that attracts phloem-feeding insects. However, it remains unclear how insect-vectored plant pathogens induce host leaf yellowing to facilitate their own transmission by insect vectors. Here, we report that an effector protein secreted by rice orange leaf phytoplasma (ROLP) inhibits chlorophyll biosynthesis and induces leaf yellowing to attract leafhopper vectors, thereby presumably promoting pathogen transmission. This effector, designated secreted ROLP protein 1 (SRP1), first secreted into rice phloem by ROLP, was subsequently translocated to chloroplasts by interacting with the chloroplastic glutamine synthetase (GS2). The direct interaction between SRP1 and GS2 disrupts the decamer formation of the GS2 holoenzyme, attenuating its enzymatic activity, thereby suppressing the synthesis of chlorophyll precursors glutamate and glutamine. Transgenic expression of SRP1 in rice plants decreased GS2 activity and chlorophyll precursor accumulation, finally inducing leaf yellowing. This process is correlated with the previous evidence that the knockout of GS2 expression in rice plants causes a similar yellow chlorosis phenotype. Consistently, these yellowing leaves attracted higher numbers of leafhopper vectors, caused the vectors to probe more frequently, and presumably facilitate more efficient phytoplasma transmission. Together, these results uncover the mechanism used by phytoplasmas to manipulate the leaf color of infected plants for the purpose of enhancing attractiveness to insect vectors.

Bacterial ubiquitin ligases hijack the host deubiquitinase OTUB1 to inhibit MTORC1 signaling and promote autophagy.

Many bacterial pathogens have evolved effective strategies to interfere with the ubiquitination network to evade clearance by the innate immune system. Here, we report that OTUB1, one of the most abundant deubiquitinases (DUBs) in mammalian cells, is subjected to both canonical and noncanonical ubiquitination during Legionella pneumophila infection. The effectors SidC and SdcA catalyze OTUB1 ubiquitination at multiple lysine residues, resulting in its association with a Legionella-containing vacuole. Lysine ubiquitination by SidC and SdcA promotes interactions between OTUB1 and DEPTOR, an inhibitor of the MTORC1 pathway, thus suppressing MTORC1 signaling. The inhibition of MTORC1 leads to suppression of host protein synthesis and promotion of host macroautophagy/autophagy during L. pneumophila infection. In addition, members of the SidE family effectors (SidEs) induce phosphoribosyl (PR)-linked ubiquitination of OTUB1 at Ser16 and Ser18 and block its DUB activity. The levels of the lysine and serine ubiquitination of OTUB1 are further regulated by effectors that function to antagonize the activities of SidC, SdcA and SidEs, including Lem27, DupA, DupB, SidJ and SdjA. Our study reveals an effectors-mediated complicated mechanism in regulating the activity of a host DUB.Abbreviations: BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; DUB: deubiquitinase; Dot/Icm: defective for organelle trafficking/intracellular multiplication; DEPTOR: DEP domain containing MTOR interacting protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L. pneumophila: Legionella pneumophila; LCV: Legionella-containing vacuole; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTORC1: mechanistic target of rapamycin kinase complex 1; OTUB1: OTU deubiquitinase, ubiquitin aldehyde binding 1; PR-Ub: phosphoribosyl (PR)-linked ubiquitin; PTM: posttranslational modification; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SidEs: SidE family effectors; Ub: ubiquitin.

Characterization of Microbotryum lychnidis-dioicae Secreted Effector Proteins, Their Potential Host Targets, and Localization in a Heterologous Host Plant.

Microbotryum lychnidis-dioicae is an obligate fungal species colonizing the plant host, Silene latifolia. The fungus synthesizes and secretes effector proteins into the plant host during infection to manipulate the host for completion of the fungal lifecycle. The goal of this study was to continue functional characterization of such M. lychnidis-dioicae effectors. Here, we identified three putative effectors and their putative host-plant target proteins. MVLG_02245 is highly upregulated in M. lychnidis-dioicae during infection; yeast two-hybrid analysis suggests it targets a tubulin α-1 chain protein ortholog in the host, Silene latifolia. A potential plant protein interacting with MVLG_06175 was identified as CASP-like protein 2C1 (CASPL2C1), which facilitates the polymerization of the Casparian strip at the endodermal cells. Proteins interacting with MVLG_05122 were identified as CSN5a or 5b, involved in protein turnover. Fluorescently labelled MVLG_06175 and MVLG_05122 were expressed in the heterologous plant, Arabidopsis thaliana. MVLG_06175 formed clustered granules at the tips of trichomes on leaves and in root caps, while MVLG_05122 formed a band structure at the base of leaf trichomes. Plants expressing MVLG_05122 alone were more resistant to infection with Fusarium oxysporum. These results indicate that the fungus might affect the formation of the Casparian strip in the roots and the development of trichomes during infection as well as alter plant innate immunity.

Functional Divergence of the Paralog Salmonella Effector Proteins SopD and SopD2 and Their Contributions to Infection.

Salmonella enterica is a leading cause of bacterial food-borne illness in humans and is responsible for millions of cases annually. A critical strategy for the survival of this pathogen is the translocation of bacterial virulence factors termed effectors into host cells, which primarily function via protein-protein interactions with host proteins. The Salmonella genome encodes several paralogous effectors believed to have arisen from duplication events throughout the course of evolution. These paralogs can share structural similarities and enzymatic activities but have also demonstrated divergence in host cell targets or interaction partners and contributions to the intracellular lifecycle of Salmonella. The paralog effectors SopD and SopD2 share 63% amino acid sequence similarity and extensive structural homology yet have demonstrated divergence in secretion kinetics, intracellular localization, host targets, and roles in infection. SopD and SopD2 target host Rab GTPases, which represent critical regulators of intracellular trafficking that mediate diverse cellular functions. While SopD and SopD2 both manipulate Rab function, these paralogs display differences in Rab specificity, and the effectors have also evolved multiple mechanisms of action for GTPase manipulation. Here, we highlight this intriguing pair of paralog effectors in the context of host-pathogen interactions and discuss how this research has presented valuable insights into effector evolution.

Brucella-driven host N-glycome remodeling controls infection.

Many powerful methods have been employed to elucidate the global transcriptomic, proteomic, or metabolic responses to pathogen-infected host cells. However, the host glycome responses to bacterial infection remain largely unexplored, and hence, our understanding of the molecular mechanisms by which bacterial pathogens manipulate the host glycome to favor infection remains incomplete. Here, we address this gap by performing a systematic analysis of the host glycome during infection by the bacterial pathogen Brucella spp. that cause brucellosis. We discover, surprisingly, that a Brucella effector protein (EP) Rhg1 induces global reprogramming of the host cell N-glycome by interacting with components of the oligosaccharide transferase complex that controls N-linked protein glycosylation, and Rhg1 regulates Brucella replication and tissue colonization in a mouse model of brucellosis, demonstrating that Brucella exploits the EP Rhg1 to reprogram the host N-glycome and promote bacterial intracellular parasitism, thereby providing a paradigm for bacterial control of host cell infection.

The Helicobacter pylori cag pathogenicity island as a determinant of gastric cancer risk.

Helicobacter pylori strains can be broadly classified into two groups based on whether they contain or lack a chromosomal region known as the cag pathogenicity island (cag PAI). Colonization of the human stomach with cag PAI-positive strains is associated with an increased risk of gastric cancer and peptic ulcer disease, compared to colonization with cag PAI-negative strains. The cag PAI encodes a secreted effector protein (CagA) and components of a type IV secretion system (Cag T4SS) that delivers CagA and non-protein substrates into host cells. Animal model experiments indicate that CagA and the Cag T4SS stimulate a gastric mucosal inflammatory response and contribute to the development of gastric cancer. In this review, we discuss recent studies defining structural and functional features of CagA and the Cag T4SS and mechanisms by which H. pylori strains containing the cag PAI promote the development of gastric cancer and peptic ulcer disease.

Brucella targets the host ubiquitin-specific protease, Usp8, through the effector protein, TcpB, for facilitating infection of macrophages.

Brucella species are Gram-negative intracellular bacterial pathogens that cause the worldwide zoonotic disease brucellosis. Brucella can infect many mammals, including humans and domestic and wild animals. Brucella manipulates various host cellular processes to invade and multiply in professional and non-professional phagocytic cells. However, the host targets and their modulation by Brucella to facilitate the infection process remain obscure. Here, we report that the host ubiquitin-specific protease, USP8, negatively regulates the invasion of Brucella into macrophages through the plasma membrane receptor, CXCR4. Upon silencing or chemical inhibition of USP8, the membrane localization of the CXCR4 receptor was enriched, which augmented the invasion of Brucella into macrophages. Activation of USP8 through chemical inhibition of 14-3-3 protein affected the invasion of Brucella into macrophages. Brucella suppressed the expression of Usp8 at its early stage of infection in the infected macrophages. Furthermore, we found that only live Brucella could negatively regulate the expression of Usp8, suggesting the role of secreted effector protein of Brucella in modulating the gene expression. Subsequent studies revealed that the Brucella effector protein, TIR-domain containing protein from Brucella, TcpB, plays a significant role in downregulating the expression of Usp8 by targeting the cyclic-AMP response element-binding protein pathway. Treatment of mice with USP8 inhibitor resulted in enhanced survival of B. melitensis, whereas mice treated with CXCR4 or 14-3-3 antagonists showed a diminished bacterial load. Our experimental data demonstrate a novel role of Usp8 in the host defense against microbial intrusion. The present study provides insights into the microbial subversion of host defenses, and this information may ultimately help to develop novel therapeutic interventions for infectious diseases.

Improving the Comprehension of Pathogenicity and Phylogeny in 'Candidatus Phytoplasma meliae' through Genome Characterization.

'Candidatus Phytoplasma meliae' is a pathogen associated with chinaberry yellowing disease, which has become a major phytosanitary problem for chinaberry forestry production in Argentina. Despite its economic impact, no genome information of this phytoplasma has been published, which has hindered its characterization at the genomic level. In this study, we used a metagenomics approach to analyze the draft genome of the 'Ca. P. meliae' strain ChTYXIII. The draft assembly consisted of twenty-one contigs with a total length of 751.949 bp, and annotation revealed 669 CDSs, 34 tRNAs, and 1 set of rRNA operons. The metabolic pathways analysis showed that ChTYXIII contains the complete core genes for glycolysis and a functional Sec system for protein translocation. Our phylogenomic analysis based on 133 single-copy genes and genome-to-genome metrics supports the classification as unique 'Ca. P. species' within the MPV clade. We also identified 31 putative effectors, including a homolog to SAP11 and others that have only been described in this pathogen. Our ortholog analysis revealed 37 PMU core genes in the genome of 'Ca. P. meliae' ChTYXIII, leading to the identification of 2 intact PMUs. Our work provides important genomic information for 'Ca. P. meliae' and others phytoplasmas for the 16SrXIII (MPV) group.

Small secreted effector protein from Fusarium sacchari suppresses host immune response by inhibiting ScPi21-induced cell death.

Fusarium sacchari is one of the primary pathogens causing pokkah boeng disease, which impairs the yield and quality of sugarcane around the world. Understanding the molecular mechanisms of the F. sacchari effectors that regulate plant immunity is of great importance for the development of novel strategies for the persistent control of pokkah boeng disease. In a previous study, Fs00367 was identified to inhibit BAX-induced cell death. In this study, Fs00367nsp (without signal peptide) was found to suppress BAX-induced cell death, reactive oxygen species bursts and callose accumulation. The amino acid region 113-142 of Fs00367nsp is the functional region. Gene mutagenesis indicated that Fs00367 is important for the full virulence of F. sacchari. A yeast two-hybrid assay revealed an interaction between Fs00367nsp and sugarcane ScPi21 in yeast that was further confirmed using bimolecular fluorescence complementation, pull-down assay and co-immunoprecipitation. ScPi21 can induce plant immunity, but this effect could be blunted by Fs00367nsp. These results suggest that Fs00367 is a core pathogenicity factor that suppresses plant immunity through inhibiting ScPi21-induced cell death. The findings of this study provide new insights into the molecular mechanisms of effectors in regulating plant immunity.

Mitochondrial-targeting effector RsIA_CtaG/Cox11 in Rhizoctonia solani AG-1 IA has two functions: plant immunity suppression and cell death induction mediated by a rice cytochrome c oxidase subunit.

Rhizoctonia solani AG-1 IA causes a necrotrophic rice disease and is a serious threat to rice production. To date, only a few effectors have been characterized in AG-1 IA. We previously identified RsIA_CtaG/Cox11 and showed that infiltration of the recombinant protein into rice leaves caused disease-like symptoms. In the present study, we further characterized the functionality of RsIA_CtaG/Cox11. RsIA_CtaG/Cox11 is an alternative transcript of cytochrome c oxidase copper chaperone Cox11 that starts from the second AUG codon, but contains a functional secretion signal peptide. RNA interference with RsIA_CtaG/Cox11 reduced the pathogenicity of AG-1 IA towards rice and Nicotiana benthamiana without affecting its fitness or mycelial morphology. Transient expression of the RsIA_CtaG/Cox11-GFP fusion protein demonstrated the localization of RsIA_CtaG/Cox11 to mitochondria. Agro-infiltration of RsIA_CtaG/Cox11 into N. benthamiana leaves inhibited cell death by BAX and INF1. In contrast to rice, agro-infiltration of RsIA_CtaG/Cox11 did not induce cell death in N. benthamiana. However, cell death was observed when it was coinfiltrated with Os_CoxVIIa, which encodes a subunit of cytochrome c oxidase. Os_CoxVIIa appeared to interact with RsIA_CtaG/Cox11. The cell death triggered by coexpression of RsIA_CtaG/Cox11 and Os_CoxVIIa is independent of the leucine-rich repeat receptor kinases BAK1/SOBIR1 and enhanced the susceptibility of N. benthamiana to AG-1 IA. Two of the three evolutionarily conserved cysteine residues at positions 25 and 126 of RsIA_CtaG/Cox11 were essential for its immunosuppressive activity, but not for cell death induction. This report suggests that RsIA_CtaG/Cox11 appears to have a dual role in immunosuppression and cell death induction during pathogenesis.

The fungal pathogen Ustilago maydis targets the maize corepressor RELK2 to modulate host transcription for tumorigenesis.

Ustilago maydis is a biotrophic fungus that causes tumor formation on all aerial parts of maize. U. maydis secretes effector proteins during penetration and colonization to successfully overcome the plant immune response and reprogram host physiology to promote infection. In this study, we functionally characterized the U. maydis effector protein Topless (TPL) interacting protein 6 (Tip6). We found that Tip6 interacts with the N-terminus of RELK2 through its two Ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs. We show that the EAR motifs are essential for the virulence function of Tip6 and critical for altering the nuclear distribution pattern of RELK2. We propose that Tip6 mimics the recruitment of RELK2 by plant repressor proteins, thus disrupting host transcriptional regulation. We show that a large group of AP2/ERF B1 subfamily transcription factors are misregulated in the presence of Tip6. Our study suggests a regulatory mechanism where the U. maydis effector Tip6 utilizes repressive domains to recruit the corepressor RELK2 to disrupt the transcriptional networks of the host plant.

Bimodular architecture of bacterial effector SAP05 that drives ubiquitin-independent targeted protein degradation.

In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously identified that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05-Rpn10vWA complex at 2.17 Å resolution and of the SAP05-SPL5ZnF complex at 2.20 Å resolution. Structural analyses revealed that SAP05 displays a remarkable bimodular architecture with two distinct nonoverlapping surfaces, a "loop surface" with three protruding loops that form electrostatic interactions with ZnF, and a "sheet surface" featuring two ß-sheets, loops, and α-helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical ubiquitin-proteasome proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of TPD technologies.

Identification of common fungal extracellular membrane (CFEM) proteins in Fusarium sacchari that inhibit plant immunity and contribute to virulence.

IMPORTANCE: Common fungal extracellular membrane (CFEM) domain-containing protein has long been considered an essential effector, playing a crucial role in the interaction of pathogens and plant. Strategies aimed at understanding the pathogenicity mechanism of F. sacchari are eagerly anticipated to ultimately end the spread of pokkah boeng disease. Twenty FsCFEM proteins in the genome of F. sacchari have been identified, and four FsCFEM effector proteins have been found to suppress BCL2-associated X protein-triggered programmed cell death in N. benthamiana. These four effector proteins have the ability to enter plant cells and inhibit plant immunity. Furthermore, the expression of these four FsCFEM effector proteins significantly increases during the infection stage, with the three of them playing an essential role in achieving full virulence. These study findings provide a direction toward further exploration of the immune response in sugarcane. By applying these discoveries, we can potentially control the spread of disease through techniques such as host-induced gene silencing.

Biodegradable Hydrogen-Bonded Organic Framework for Cytosolic Protein Delivery.

Hydrogen-bonded organic frameworks (HOFs) are a novel class of porous nanomaterials that show great potential for intracellular delivery of protein therapeutics. However, the inherent challenges in interfacing protein with HOFs, and the need for spatiotemporally controlling the release of protein within cells, have constrained their therapeutic potential. In this study, we report novel biodegradable hydrogen-bonded organic frameworks, termed DS-HOFs, specially designed for the cytosolic delivery of protein therapeutics in cancer cells. The synthesis of DS-HOFs involves the self-assembly of 4-[tris(4-carbamimidoylphenyl) methyl] benzenecarboximidamide (TAM) and 4,4'-dithiobisbenzoic acid (DTBA), governed by intermolecular hydrogen-bonding interactions. DS-HOFs exhibit high efficiency in encapsulating a diverse range of protein cargos, underpinned by the hydrogen-bonding interactions between the protein residue and DS-HOF subcomponents. Notably, DS-HOFs are selectively degraded in cancer cells triggered by the distinct intracellular reductive microenvironments, enabling an enhanced and selective release of protein inside cancer cells. Additionally, we demonstrate that the efficient delivery of bacterial effector protein DUF5 using DS-HOFs depletes the mutant RAS in cancer cells to prohibit tumor cell growth both in vitro and in vivo. The design of biodegradable HOFs for cytosolic protein delivery provides a powerful and promising strategy to expand the therapeutic potential of proteins for cancer therapy.

The interaction of two Puccinia striiformis f. sp. tritici effectors modulates high-temperature seedling-plant resistance in wheat.

Wheat cultivar Xiaoyan 6 (XY6) has high-temperature seedling-plant (HTSP) resistance to Puccinia striiformis f. sp. tritici (Pst). However, the molecular mechanism of Pst effectors involved in HTSP resistance remains unclear. In this study, we determined the interaction between two Pst effectors, PstCEP1 and PSTG_11208, through yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and pull-down assays. Transient overexpression of PSTG_11208 enhanced HTSP resistance in different temperature treatments. The interaction between PstCEP1 and PSTG_11208 inhibited the resistance enhancement by PSTG_11208. Furthermore, the wheat apoplastic thaumatin-like protein 1 (TaTLP1) appeared to recognize Pst invasion by interacting with PSTG_11208 and initiate the downstream defence response by the pathogenesis-related protein TaPR1. Silencing of TaTLP1 and TaPR1 separately or simultaneously reduced HTSP resistance to Pst in XY6. Moreover, we found that PstCEP1 targeted wheat ferredoxin 1 (TaFd1), a homologous protein of rice OsFd1. Silencing of TaFd1 affected the stability of photosynthesis in wheat plants, resulting in chlorosis on the leaves and reducing HTSP resistance. Our findings revealed the synergistic mechanism of effector proteins in the process of pathogen infection.

Three small partner proteins facilitate the type VII-dependent secretion of an antibacterial nuclease.

IMPORTANCE: Staphylococcus aureus is an opportunistic human pathogen associated with severe infections and antimicrobial resistance. S. aureus strains utilize a type VII secretion system to secrete toxins targeting competitor bacteria, likely facilitating colonization. EsaD is a nuclease toxin secreted by the type VII secretion system in many strains of S. aureus as well as other related bacterial species. Here, we identify three small proteins of previously unknown function as export factors, required for efficient secretion of EsaD. We show that these proteins bind to the transport domain of EsaD, forming a complex with a striking cane-like conformation.

Ubiquitination of Sec22b by a novel Legionella pneumophila ubiquitin E3 ligase.

Legionella pneumophila is a facultative intracellular pathogen that causes legionellosis. The key to its virulence is the delivery of hundreds of effector proteins into host cells via the defective in organelle trafficking/intracellular multiplication type IV secretion system. These effectors modulate numerous host signaling pathways to create a niche called the Legionella-containing vacuole (LCV) permissive for its intracellular replication. Previous investigation revealed that exploitation of the host ubiquitin system is among the most important strategies used by L. pneumophila to coopt host processes for its benefit. Here, we show that the effector Legionella ubiquitin ligase gene 15 (Lug15) (Lpg2327), which has no detectable homology with any enzyme involved in ubiquitin signaling, is an E3 ligase. In L. pneumophila-infected cells, Lug15 is localized on the LCV and impacts its association with polyubiquitinated proteins. We also demonstrate that Sec22b is ubiquitinated and recruited to the LCV by Lug15. Thus, our results establish Lug15 as a novel E3 ligase that functions to recruit a SNARE protein to remodel the L. pneumophila phagosome.IMPORTANCEProtein ubiquitination is one of the most important post-translational modifications that plays critical roles in the regulation of a wide range of eukaryotic signaling pathways. Many successful intracellular bacterial pathogens can hijack host ubiquitination machinery through the action of effector proteins that are injected into host cells by secretion systems. Legionella pneumophila is the etiological agent of legionellosis that is able to survive and replicate in various host cells. The defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IV secretion system of L. pneumophila injects over 330 effectors into infected cells to create an optimal environment permissive for its intracellular proliferation. To date, at least 26 Dot/Icm substrates have been shown to manipulate ubiquitin signaling via diverse mechanisms. Among these, 14 are E3 ligases that either cooperate with host E1 and E2 enzymes or adopt E1/E2-independent catalytic mechanisms. In the present study, we demonstrate that the L. pneumophila effector Legionella ubiquitin ligase gene 15 (Lug15) is a novel ubiquitin E3 ligase. Lug15 is involved in the remodeling of LCV with polyubiquitinated species. Moreover, Lug15 catalyzes the ubiquitination of host SNARE protein Sec22b and mediates its recruitment to the LCV. Ubiquitination of Sec22b by Lug15 promotes its noncanonical pairing with plasma membrane-derived syntaxins (e.g., Stx3). Our study further reveals the complexity of strategies utilized by L. pneumophila to interfere with host functions by hijacking host ubiquitin signaling.

Anaplasma phagocytophilum Ats-1 enhances exosome secretion through Syntenin-1.

Anaplasma phagocytophilum is an intracellular obligate parasite that causes granulocytic anaplasmosis. Effector Ats-1 is an important virulence factor of A. phagocytophilum. Multiomics screening and validation has been used to determine that Ats-1 regulates host cell apoptosis and energy metabolism through the respiratory chain mPTP axis. In this study, a total of 19 potential binding proteins of Ats-1 in host cells were preliminarily screened using a yeast two-hybrid assay, and the interaction between syntenin-1 (SDCBP) and Ats-1 was identified through immunoprecipitation. Bioinformatics analysis showed that SDCBP interacted with SDC1, SDC2, and SDC4 and participated in the host exosome secretion pathway. Further studies confirmed that Ats-1 induced the expression of SDC1, SDC2, and SDC4 in HEK293T cells through SDCBP and increased the exosome secretion of these cells. This indicated that SDCBP played an important role in Ats-1 regulating the exosome secretion of the host cells. These findings expand our understanding of the intracellular regulatory mechanism of A. phagocytophilum, which may enhance its own infection and proliferation by regulating host exosome pathways.