The plant immune system: From discovery to deployment.

Plant diseases cause famines, drive human migration, and present challenges to agricultural sustainability as pathogen ranges shift under climate change. Plant breeders discovered Mendelian genetic loci conferring disease resistance to specific pathogen isolates over 100 years ago. Subsequent breeding for disease resistance underpins modern agriculture and, along with the emergence and focus on model plants for genetics and genomics research, has provided rich resources for molecular biological exploration over the last 50 years. These studies led to the identification of extracellular and intracellular receptors that convert recognition of extracellular microbe-encoded molecular patterns or intracellular pathogen-delivered virulence effectors into defense activation. These receptor systems, and downstream responses, define plant immune systems that have evolved since the migration of plants to land ∼500 million years ago. Our current understanding of plant immune systems provides the platform for development of rational resistance enhancement to control the many diseases that continue to plague crop production.

Pyroptosis modulation by bacterial effector proteins.

Pyroptosis is a proinflammatory form of programmed cell death featured with membrane pore formation that causes cellular swelling and allows the release of intracellular inflammatory mediators. This cell death process is elicited by the activation of the pore-forming proteins named gasdermins, and is intricately orchestrated by diverse regulatory factors in mammalian hosts to exert a prompt immune response against infections. However, growing evidence suggests that bacterial pathogens have evolved to regulate host pyroptosis for evading immune clearance and establishing progressive infection. In this review, we highlight current understandings of the functional role and regulatory network of pyroptosis in host antibacterial immunity. Thereafter, we further discuss the latest advances elucidating the mechanisms by which bacterial pathogens modulate pyroptosis through adopting their effector proteins to drive infections. A better understanding of regulatory mechanisms underlying pyroptosis at the interface of host-bacterial interactions will shed new light on the pathogenesis of infectious diseases and contribute to the development of promising therapeutic strategies against bacterial pathogens.

Mapping the global interactome of the ARF family reveals spatial organization in cellular signaling pathways.

The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ∼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.

DNA-binding and protein structure of nuclear factors likely acting in genetic information processing in the Paulinella chromatophore.

The chromatophores in Paulinella are evolutionary-early-stage photosynthetic organelles. Biological processes in chromatophores depend on a combination of chromatophore and nucleus-encoded proteins. Interestingly, besides proteins carrying chromatophore-targeting signals, a large arsenal of short chromatophore-targeted proteins (sCTPs; <90 amino acids) without recognizable targeting signals were found in chromatophores. This situation resembles endosymbionts in plants and insects that are manipulated by host-derived antimicrobial peptides. Previously, we identified an expanded family of sCTPs of unknown function, named here "DNA-binding (DB)-sCTPs". DB-sCTPs contain a ~45 amino acid motif that is conserved in some bacterial proteins with predicted functions in DNA processing. Here, we explored antimicrobial activity, DNA-binding capacity, and structures of three purified recombinant DB-sCTPs. All three proteins exhibited antimicrobial activity against bacteria involving membrane permeabilization, and bound to bacterial lipids in vitro. A combination of in vitro assays demonstrated binding of recombinant DB-sCTPs to chromatophore-derived genomic DNA sequences with an affinity in the low nM range. Additionally, we report the 1.2 Å crystal structure of one DB-sCTP. In silico docking studies suggest that helix α2 inserts into the DNA major grove and the exposed residues, that are highly variable between different DB-sCTPs, confer interaction with the DNA bases. Identification of photosystem II subunit CP43 as a potential interaction partner of one DB-sCTP, suggests DB-sCTPs to be involved in more complex regulatory mechanisms. We hypothesize that membrane binding of DB-sCTPs is related to their import into chromatophores. Once inside, they interact with the chromatophore genome potentially providing nuclear control over genetic information processing.

Legionella effectors SidC/SdcA ubiquitinate multiple small GTPases and SNARE proteins to promote phagosomal maturation.

Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.

Mimicry of Short Linear Motifs by Bacterial Pathogens: A Drugging Opportunity.

Bacterial pathogens have developed complex strategies to successfully survive and proliferate within their hosts. Throughout the infection cycle, direct interaction with host cells occurs. Many bacteria have been found to secrete proteins, such as effectors and toxins, directly into the host cell with the potential to interfere with cell regulatory processes, either enzymatically or through protein-protein interactions (PPIs). Short linear motifs (SLiMs) are abundant peptide modules in cell signaling proteins. Here, we cover the reported examples of eukaryotic-like SLiM mimicry being used by pathogenic bacteria to hijack host cell machinery and discuss how drugs targeting SLiM-regulated cell signaling networks are being evaluated for interference with bacterial infections. This emerging anti-infective opportunity may become an essential contributor to antibiotic replacement strategies.

Computational Prediction of Structure, Function, and Interaction of Myzus persicae (Green Peach Aphid) Salivary Effector Proteins.

Similar to plant pathogens, phloem-feeding insects such as aphids deliver effector proteins inside their hosts that act to promote host susceptibility and enable feeding and infestation. Despite exciting progress toward identifying and characterizing effector proteins from these insects, their functions remain largely unknown. The recent groundbreaking development in protein structure prediction algorithms, combined with the availability of proteomics and transcriptomic datasets for agriculturally important pests, provides new opportunities to explore the structural and functional diversity of effector repertoires. In this study, we sought to gain insight into the infection strategy used by the Myzus persicae (green peach aphid) by predicting and analyzing the structures of a set of 71 effector candidate proteins. We used two protein structure prediction methods, AlphaFold and OmegaFold, that produced mutually consistent results. We observed a wide continuous spectrum of structures among the effector candidates, from disordered proteins to globular enzymes. We made use of the structural information and state-of-the-art computational methods to predict M. persicae effector protein properties, including function and interaction with host plant proteins. Overall, our investigation provides novel insights into prediction of structure, function, and interaction of M. persicae effector proteins and will guide the necessary experimental characterization to address new hypotheses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Lack of evidence that Pseudomonas aeruginosa AmpDh3-PA0808 constitute a type VI secretion system effector-immunity pair.

Type VI secretion systems (T6SSs) are cell envelope-spanning protein complexes that Gram-negative bacteria use to inject a diverse arsenal of antibacterial toxins into competitor cells. Recently, Wang et al. reported that the H2-T6SS of Pseudomonas aeruginosa delivers the peptidoglycan recycling amidase, AmpDh3, into the periplasm of recipient cells where it is proposed to act as a peptidoglycan degrading toxin. They further reported that PA0808, the open reading frame downstream of AmpDh3, encodes an immunity protein that localizes to the periplasm where it binds to and inactivates intercellularly delivered AmpDh3, thus protecting against its toxic activity. Given that AmpDh3 has an established role in cell wall homeostasis and that no precedent exists for cytosolic enzymes moonlighting as T6SS effectors, we attempted to replicate these findings. We found that cells lacking PA0808 are not susceptible to bacterial killing by AmpDh3 and that PA0808 and AmpDh3 do not physically interact in vitro or in vivo. Additionally, we found no evidence that AmpDh3 is exported from cells, including by strains with a constitutively active H2-T6SS. Finally, subcellular fractionation experiments and a 1.97 Å crystal structure reveal that PA0808 does not contain a canonical signal peptide or localize to the correct cellular compartment to confer protection against a cell wall targeting toxin. Taken together, these results cast doubt on the assertion that AmpDh3-PA0808 constitutes an H2-T6SS effector-immunity pair.

Effector-triggered susceptibility by the rice blast fungus Magnaporthe oryzae.

Rice blast, the most destructive disease of cultivated rice world-wide, is caused by the filamentous fungus Magnaporthe oryzae. To cause disease in plants, M. oryzae secretes a diverse range of effector proteins to suppress plant defense responses, modulate cellular processes, and support pathogen growth. Some effectors can be secreted by appressoria even before host penetration, while others accumulate in the apoplast, or enter living plant cells where they target specific plant subcellular compartments. During plant infection, the blast fungus induces the formation of a specialized plant structure known as the biotrophic interfacial complex (BIC), which appears to be crucial for effector delivery into plant cells. Here, we review recent advances in the cell biology of M. oryzae-host interactions and show how new breakthroughs in disease control have stemmed from an increased understanding of effector proteins of M. oryzae are deployed and delivered into plant cells to enable pathogen invasion and host susceptibility.

Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor.

Plants use intracellular nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing immune receptors (NLRs) to detect pathogen-derived effector proteins. The Arabidopsis NLR pair RRS1-R/RPS4 confers disease resistance to different bacterial pathogens by perceiving the structurally distinct effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia solanacearum via an integrated WRKY domain in RRS1-R. How the WRKY domain of RRS1 (RRS1WRKY) perceives distinct classes of effector to initiate an immune response is unknown. Here, we report the crystal structure of the in planta processed C-terminal domain of AvrRps4 (AvrRps4C) in complex with RRS1WRKY Perception of AvrRps4C by RRS1WRKY is mediated by the ß2-ß3 segment of RRS1WRKY that binds an electronegative patch on the surface of AvrRps4C Structure-based mutations that disrupt AvrRps4C-RRS1WRKY interactions in vitro compromise RRS1/RPS4-dependent immune responses. We also show that AvrRps4C can associate with the WRKY domain of the related but distinct RRS1B/RPS4B NLR pair, and the DNA-binding domain of AtWRKY41, with similar binding affinities and how effector binding interferes with WRKY-W-box DNA interactions. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity.

Secreted Effector Proteins of Poplar Leaf Spot and Stem Canker Pathogen Sphaerulina musiva Manipulate Plant Immunity and Contribute to Virulence in Diverse Ways.

Fungal effectors play critical roles in manipulating plant immune responses and promoting colonization. Sphaerulina musiva is a heterothallic ascomycete fungus that causes Septoria leaf spot and stem canker disease in poplar (Populus spp.) plantations. This disease can result in premature defoliation, branch and stem breakage, increased mortality, and plantation failure. However, little is known about the interaction between S. musiva and poplar. Previous work predicted 142 candidate secreted effector proteins in S. musiva (SmCSEPs), 19 of which were selected for further functional characterization in this study. SmCSEP3 induced plant cell death in Nicotiana benthamiana, while 8 out of 19 tested SmCSEPs suppressed cell death. The signal peptides of these eight SmCSEPs exhibited secretory activity in a yeast signal sequence trap assay. Confocal microscopy revealed that four of these eight SmCSEPs target both the cytoplasm and the nucleus, whereas four predominantly localize to discrete punctate structures. Pathogen challenge assays in N. benthamiana demonstrated that the transient expression of six SmCSEPs promoted Fusarium proliferatum infection. The expression of these six SmCSEP genes were induced during infection. SmCSEP2, SmCSEP13, and SmCSEP25 suppressed chitin-triggered reactive oxygen species burst and callose deposition in N. benthamiana. The candidate secreted effector proteins of S. musiva target multiple compartments in the plant cell and modulate different pattern-triggered immunity pathways. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Long-term and rapid evolution in powdery mildew fungi.

The powdery mildew fungi (Erysiphaceae) are globally distributed plant pathogens with a range of more than 10,000 plant hosts. In this review, we discuss the long- and short-term evolution of these obligate biotrophic fungi and outline their diversity with respect to morphology, lifestyle, and host range. We highlight their remarkable ability to rapidly overcome plant immunity, evolve fungicide resistance, and broaden their host range, for example, through adaptation and hybridization. Recent advances in genomics and proteomics, particularly in cereal powdery mildews (genus Blumeria), provided first insights into mechanisms of genomic adaptation in these fungi. Transposable elements play key roles in shaping their genomes, where even close relatives exhibit diversified patterns of recent and ongoing transposon activity. These transposons are ubiquitously distributed in the powdery mildew genomes, resulting in a highly adaptive genome architecture lacking obvious regions of conserved gene space. Transposons can also be neofunctionalized to encode novel virulence factors, particularly candidate secreted effector proteins, which may undermine the plant immune system. In cereals like barley and wheat, some of these effectors are recognized by plant immune receptors encoded by resistance genes with numerous allelic variants. These effectors determine incompatibility ("avirulence") and evolve rapidly through sequence diversification and copy number variation. Altogether, powdery mildew fungi possess plastic genomes that enable their fast evolutionary adaptation towards overcoming plant immunity, host barriers, and chemical stress such as fungicides, foreshadowing future outbreaks, host range shifts and expansions as well as potential pandemics by these pathogens.

A Recipe for Success: Three Key Strategies Used by Aphids and Pseudomonas syringae to Colonize the Phyllosphere.

Aphids and Pseudomonas syringae are a permanent challenge for agriculture, causing severe losses to the crop industry worldwide. Despite the obvious phylogenetic distance between them, both have become predominant colonizers of the plant kingdom. In this study, we reviewed three key steps of spread and colonization that aphids and P. syringae have mastered to successfully colonize the phyllosphere. These steps involve (i) plant-to-plant movement for locating new nutritional sources, (ii) disruption and modification of the apoplast to facilitate nutrient acquisition, and (iii) suppression of host defenses through effector proteins. In addition, we will provide insights about the direct interaction between aphids and P. syringae and how this yet underrated phenomenon could bring new ecological implications for both organisms beyond their pathogenicity.

Detection of Secreted Effector Proteins from Phelipanche aegyptiaca During Invasion of Melon Roots.

Parasites can interact with their host plants through the induction and delivery of secreted effector proteins that facilitate plant colonization by decomposing plant cell walls and inhibiting plant immune response to weaken the defense ability of the host. Yet effectors mediating parasitic plant-host interactions are poorly understood. Phelipanche aegyptiaca is an obligate root parasite plant causing severe yield and economic losses in agricultural fields worldwide. Host resistance against P. aegyptiaca occurred during the attachment period of parasitism. Comparative transcriptomics was used to assess resistant and susceptible interactions simultaneously between P. aegyptiaca and two contrasting melon cultivars. In total, 2,740 secreted proteins from P. aegyptiaca were identified here. Combined with transcriptome profiling, 209 candidate secreted effector proteins (CSEPs) were predicted, with functional annotations such as cell wall degrading enzymes, protease inhibitors, transferases, kinases, and elicitor proteins. A heterogeneous expression system in Nicotiana benthamiana was used to investigate the functions of 20 putatively effector genes among the CSEPs. Cluster 15140.0 can suppress BAX-triggered programmed cell death in N. benthamiana. These findings showed that the prediction of P. aegyptiaca effector proteins based on transcriptomic analysis and multiple bioinformatics software is effective and more accurate, providing insights into understanding the essential molecular nature of effectors and laying the foundation of revealing the parasite mechanism of P. aegyptiaca, which is helpful in understanding parasite-host plant interaction.

Modulation of innate immune signaling by a Coxiella burnetii eukaryotic-like effector protein.

The Q fever agent Coxiella burnetii uses a defect in organelle trafficking/intracellular multiplication (Dot/Icm) type 4b secretion system (T4SS) to silence the host innate immune response during infection. By investigating C. burnetii effector proteins containing eukaryotic-like domains, here we identify NopA (nucleolar protein A), which displays four regulator of chromosome condensation (RCC) repeats, homologous to those found in the eukaryotic Ras-related nuclear protein (Ran) guanine nucleotide exchange factor (GEF) RCC1. Accordingly, NopA is found associated with the chromatin nuclear fraction of cells and uses the RCC-like domain to interact with Ran. Interestingly, NopA triggers an accumulation of Ran-GTP, which accumulates at nucleoli of transfected or infected cells, thus perturbing the nuclear import of transcription factors of the innate immune signaling pathway. Accordingly, qRT-PCR analysis on a panel of cytokines shows that cells exposed to the C. burnetii nopA::Tn or a Dot/Icm-defective dotA::Tn mutant strain present a functional innate immune response, as opposed to cells exposed to wild-type C. burnetii or the corresponding nopA complemented strain. Thus, NopA is an important regulator of the innate immune response allowing Coxiella to behave as a stealth pathogen.

A Functional Yeast-Based Screen Identifies the Host Microtubule Cytoskeleton as a Target of Numerous Chlamydia pneumoniae Proteins.

Bacterial pathogens have evolved intricate ways to manipulate the host to support infection. Here, we systematically assessed the importance of the microtubule cytoskeleton for infection by Chlamydiae, which are obligate intracellular bacteria that are of great importance for human health. The elimination of microtubules in human HEp-2 cells prior to C. pneumoniae infection profoundly attenuated the infection efficiency, demonstrating the need for microtubules for the early infection processes. To identify microtubule-modulating C. pneumoniae proteins, a screen in the model yeast Schizosaccharomyces pombe was performed. Unexpectedly, among 116 selected chlamydial proteins, more than 10%, namely, 13 proteins, massively altered the yeast interphase microtubule cytoskeleton. With two exceptions, these proteins were predicted to be inclusion membrane proteins. As proof of principle, we selected the conserved CPn0443 protein, which caused massive microtubule instability in yeast, for further analysis. CPn0443 bound and bundled microtubules in vitro and co-localized partially with microtubules in vivo in yeast and human cells. Furthermore, CPn0443-transfected U2OS cells had a significantly reduced infection rate by C. pneumoniae EBs. Thus, our yeast screen identified numerous proteins encoded using the highly reduced C. pneumoniae genome that modulated microtubule dynamics. Hijacking of the host microtubule cytoskeleton must be a vital part of chlamydial infection.

Characterization of Arbuscular Mycorrhizal Effector Proteins.

Plants are colonized by various fungi with both pathogenic and beneficial lifestyles. One type of colonization strategy is through the secretion of effector proteins that alter the plant's physiology to accommodate the fungus. The oldest plant symbionts, the arbuscular mycorrhizal fungi (AMF), may exploit effectors to their benefit. Genome analysis coupled with transcriptomic studies in different AMFs has intensified research on the effector function, evolution, and diversification of AMF. However, of the current 338 predicted effector proteins from the AM fungus Rhizophagus irregularis, only five have been characterized, of which merely two have been studied in detail to understand which plant proteins they associate with to affect the host physiology. Here, we review the most recent findings in AMF effector research and discuss the techniques used for the functional characterization of effector proteins, from their in silico prediction to their mode of action, with an emphasis on high-throughput approaches for the identification of plant targets of the effectors through which they manipulate their hosts.

Genomic Assessment of the Contribution of the Wolbachia Endosymbiont of Eurosta solidaginis to Gall Induction.

We explored the genome of the Wolbachia strain, wEsol, symbiotic with the plant-gall-inducing fly Eurosta solidaginis with the goal of determining if wEsol contributes to gall induction by its insect host. Gall induction by insects has been hypothesized to involve the secretion of the phytohormones cytokinin and auxin and/or proteinaceous effectors to stimulate cell division and growth in the host plant. We sequenced the metagenome of E. solidaginis and wEsol and assembled and annotated the genome of wEsol. The wEsol genome has an assembled length of 1.66 Mbp and contains 1878 protein-coding genes. The wEsol genome is replete with proteins encoded by mobile genetic elements and shows evidence of seven different prophages. We also detected evidence of multiple small insertions of wEsol genes into the genome of the host insect. Our characterization of the genome of wEsol indicates that it is compromised in the synthesis of dimethylallyl pyrophosphate (DMAPP) and S-adenosyl L-methionine (SAM), which are precursors required for the synthesis of cytokinins and methylthiolated cytokinins. wEsol is also incapable of synthesizing tryptophan, and its genome contains no enzymes in any of the known pathways for the synthesis of indole-3-acetic acid (IAA) from tryptophan. wEsol must steal DMAPP and L-methionine from its host and therefore is unlikely to provide cytokinin and auxin to its insect host for use in gall induction. Furthermore, in spite of its large repertoire of predicted Type IV secreted effector proteins, these effectors are more likely to contribute to the acquisition of nutrients and the manipulation of the host's cellular environment to contribute to growth and reproduction of wEsol than to aid E. solidaginis in manipulating its host plant. Combined with earlier work that shows that wEsol is absent from the salivary glands of E. solidaginis, our results suggest that wEsol does not contribute to gall induction by its host.

The Molecular Docking of MAX Fungal Effectors with Plant HMA Domain-Binding Proteins.

Fungal effector proteins are important in mediating disease infections in agriculturally important crops. These secreted small proteins are known to interact with their respective host receptor binding partners in the host, either inside the cells or in the apoplastic space, depending on the localisation of the effector proteins. Consequently, it is important to understand the interactions between fungal effector proteins and their target host receptor binding partners, particularly since this can be used for the selection of potential plant resistance or susceptibility-related proteins that can be applied to the breeding of new cultivars with disease resistance. In this study, molecular docking simulations were used to characterise protein-protein interactions between effector and plant receptors. Benchmarking was undertaken using available experimental structures of effector-host receptor complexes to optimise simulation parameters, which were then used to predict the structures and mediating interactions of effector proteins with host receptor binding partners that have not yet been characterised experimentally. Rigid docking was applied for both the so-called bound and unbound docking of MAX effectors with plant HMA domain protein partners. All bound complexes used for benchmarking were correctly predicted, with 84% being ranked as the top docking pose using the ZDOCK scoring function. In the case of unbound complexes, a minimum of 95% of known residues were predicted to be part of the interacting interface on the host receptor binding partner, and at least 87% of known residues were predicted to be part of the interacting interface on the effector protein. Hydrophobic interactions were found to dominate the formation of effector-plant protein complexes. An optimised set of docking parameters based on the use of ZDOCK and ZRANK scoring functions were established to enable the prediction of near-native docking poses involving different binding interfaces on plant HMA domain proteins. Whilst this study was limited by the availability of the experimentally determined complexed structures of effectors and host receptor binding partners, we demonstrated the potential of molecular docking simulations to predict the likely interactions between effectors and their respective host receptor binding partners. This computational approach may accelerate the process of the discovery of putative interacting plant partners of effector proteins and contribute to effector-assisted marker discovery, thereby supporting the breeding of disease-resistant crops.

The Chlamydia trachomatis Inclusion Membrane Protein CTL0390 Mediates Host Cell Exit via Lysis through STING Activation.

The obligate intracellular bacterium Chlamydia trachomatis is the causative agent of the most frequently reported bacterial sexually transmitted disease. Upon internalization into host cells, C. trachomatis remains within a membrane-bound compartment known as an inclusion, where it undergoes its developmental cycle. After completion of this cycle, bacteria exit the host cell. One mechanism of exit is lysis, whereby the inclusion and host cell rupture to release bacteria; however, the mechanism of lysis is not well characterized. A subset of C. trachomatis effectors, known as inclusion membrane proteins (Inc), are embedded within the inclusion membrane to facilitate host cell manipulation. The functions of many Inc proteins are unknown. We sought to characterize the Inc protein CTL0390. We determined that CTL0390 is expressed throughout the developmental cycle and that its C-terminal tail is exposed to the cytosol. To investigate the function of CTL0390, we generated a ctl0390 mutant complemented with ctl0390 on a plasmid. Loss of CTL0390 did not affect infectious progeny production but resulted in a reduction in lysis. Overexpression of CTL0390 induced premature lysis and host nuclear condensation, the latter of which could be reduced upon inhibition of the cGAS-STING DNA sensing pathway. Infection with the clt0390 mutant led to reduced Golgi translocation of STING, and chemical and genetic approaches to inactivate STING revealed that STING plays a role in lysis in a CTL0390-dependent manner. Together, these results reveal a role for CTL0390 in bacterial exit via lysis at late stages of the Chlamydia developmental cycle and through STING activation.