IRG and GBP Host Resistance Factors Target Aberrant, “Non-self” Vacuoles Characterized by the Missing of “Self” IRGM Proteins

Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with “non-self” PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on “self” organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of “self” IRGM proteins from these structures.     

The obligate intracellular pathogen Chlamydia trachomatis targets host lipid droplets

Lipid droplets (LDs) are ubiquitous but poorly understood neutral-lipid-rich eukaryotic organelles that may participate in functions as diverse as lipid homeostasis, membrane traffic, and signaling . We report that infection with the obligate intracellular pathogen Chlamydia trachomatis, the causative agent of trachoma and many sexually transmitted diseases , leads to the accumulation of neutral-lipid-rich structures with features of LDs at the cytoplasmic surface of the bacteria-containing vacuole. To identify bacterial factors that target these organelles, we screened a collection of yeast strains expressing GFP-tagged chlamydial ORFs and identified several proteins with tropism for eukaryotic LDs. We determined that three of these LD-associated (Lda) proteins are translocated into the mammalian host and associate with neutral-lipid-rich structures. Furthermore, the stability of one Lda protein is dependent on binding to LDs, and pharmacological inhibition of LD formation negatively impacted chlamydial replication. These results suggest that C. trachomatis targets LDs to enhance its survival and replication in infected cells. The co-option of mammalian LD function by a pathogenic bacterium represents a novel mechanism of eukaryotic organelle subversion and provides unique research opportunities to explore the function of these understudied organelles.     

EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.     

The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components

Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells’ antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.     

High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation

Accurate protein inventories are essential for understanding an organelle’s functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae. Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism.     

Chlamydia trachomatis targets mitochondrial dynamics to promote intracellular survival and proliferation

Chlamydia trachomatis is an obligate intracellular bacterium that scavenges host metabolic products for its replication. Mitochondria are the power plants of eukaryotic cells and provide most of the cellular ATP via oxidative phosphorylation. Several intracellular pathogens target mitochondria as part of their obligatory cellular reprogramming. This study was designed to analyse the mitochondrial morphological changes in response to C. trachomatis infection in HeLa cells. Mitochondrial elongation and fragmentation were found at the early stages and late stages of C. trachomatis infection, respectively. C. trachomatis infection‐induced mitochondrial elongation was associated with the increase of mitochondrial respiratory activity, ATP production, and intracellular growth of C. trachomatis. Silencing mitochondrial fusion mediator proteins abrogated the C. trachomatis infection‐induced elevation in the oxygen consumption rate and attenuated chlamydial proliferation. Mechanistically, C. trachomatis induced the elevation of intracellular cAMP at the early phase of infection, followed by the phosphorylation of fission‐inactive serine residue 637 (S637) of Drp1, resulting in mitochondrial elongation. Accordingly, treatment with adenylate cyclase inhibitor diminished mitochondrial elongation and bacterial growth in infected cells. Collectively, these results strongly indicate that C. trachomatis promotes its intracellular growth by targeting mitochondrial dynamics to regulate ATP synthesis via inhibition of the fission mediator Drp1.     

STIM1 Is a Novel Component of ER-Chlamydia trachomatis Inclusion Membrane Contact Sites

Productive developmental cycle of the obligate intracellular bacterial pathogen Chlamydia trachomatis depends on the interaction of the replicative vacuole, named the inclusion, with cellular organelles. We have recently reported the formation of ER-Inclusion membrane contact sites (MCSs), where the endoplasmic reticulum (ER) is in apposition to the inclusion membrane. These platforms contain the C. trachomatis inclusion membrane protein IncD, the mammalian ceramide transfer protein CERT and the ER resident proteins VAPA/B and were proposed to play a role in the non-vesicular trafficking of lipids to the inclusion. Here, we identify STIM1 as a novel component of ER-Inclusion MCSs. STIM1, an ER calcium (Ca²⁺) sensor that relocate to ER-Plasma Membrane (PM) MCSs upon Ca²⁺ store depletion, associated with C. trachomatis inclusion. STIM1, but not the general ER markers Rtn3C and Sec61ß, was enriched at the inclusion membrane. Ultra-structural studies demonstrated that STIM1 localized to ER-Inclusion MCSs. Time-course experiments showed that STIM1, CERT and VAPB co-localized throughout the developmental cycle. By contrast, Orai1, the PM Ca²⁺ channel that interacts with STIM1 at ER-PM MCSs, did not associate with C. trachomatis inclusion. Upon ER Ca²⁺ store depletion, a pool of STIM1 relocated to ER-PM MCSs, while the existing ER-Inclusion MCSs remained enriched in STIM1. Finally, we have identified the CAD domain, which mediates STIM1-Orai1 interaction, as the minimal domain required for STIM1 enrichment at ER-Inclusion MCSs. Altogether this study identifies STIM1 as a novel component of ER-C. trachomatis inclusion MCSs. We discuss the potential role(s) of STIM1 during the infection process.     

Chlamydia trachomatis Frequency in a Cohort of HPV-Infected Colombian Women

Background

Chlamydia trachomatis (C. trachomatis), an obligate intracellular bacterium, is the commonest infectious bacterial agent of sexual transmission throughout the world. It has been shown that the presence of this bacteria in the cervix represents a risk regarding HPV persistence and, thereafter, in developing cervical cancer (CC). Prevalence rates may vary from 2% to 17% in asymptomatic females, depending on the population being analysed. This study reports the identification of C. trachomatis in a cohort of 219 HPV-infected Colombian females.

Methods

C. trachomatis infection frequency was determined during each of the study’s follow-up visits; it was detected by amplifying the cryptic plasmid sequence by polymerase chain reaction (PCR) using two sets of primers: KL5/KL6 and KL1/KL2.Infection was defined as a positive PCR result using either set of primers at any time during the study. Cox proportional risk models were used for evaluating the association between the appearance of infection and a group of independent variables.

Results

Base line C. trachomatis infection frequency was 28% (n = 61). Most females infected by C. trachomatis were infected by multiple types of HPV (77.42%), greater prevalence occurring in females infected with HPV-16 (19.18%), followed by HPV-58 (17.81%). It was observed that females having had the most sexual partners (HR = 6.44: 1.59–26.05 95%CI) or infection with multiple types of HPV (HR = 2.85: 1.22–6.63 95%CI) had the greatest risk of developing C. trachomatis.

Conclusions

The study provides data regarding the epidemiology of C. trachomatis /HPV coinfection in different population groups of Colombian females and contributes towards understanding the natural history of C. trachomatis infection.     

The Relationship between Active Trachoma and Ocular Chlamydia trachomatis Infection before and after Mass Antibiotic Treatment

BackgroundTrachoma is a blinding disease, initiated in early childhood by repeated conjunctival infection with the obligate intracellular bacterium Chlamydia trachomatis. The population prevalence of the clinical signs of active trachoma; ‘‘follicular conjunctivitis” (TF) and/or ‘‘intense papillary inflammation” (TI), guide programmatic decisions regarding the initiation and cessation of mass drug administration (MDA). However, the persistence of TF following resolution of infection at both the individual and population level raises concerns over the suitability of this clinical sign as a marker for C. trachomatis infection.Conclusions/SignificancePrior to MDA, TF is a good indicator of the community prevalence of C. trachomatis infection. Following MDA, the prevalence of TF tends to overestimate the underlying infection prevalence. In order to prevent unnecessary additional rounds of MDA and to accurately ascertain when elimination goals have been reached, a cost-effective test for C. trachomatis that can be administered in low-resource settings remains desirable.     

Effector proteins of chlamydiae

This review summarizes the recently published data on the molecular mechanisms of Chlamydiae-host cell interaction, first of all, on chlamydial effector proteins. Such proteins, along with type III transport system proteins, which transfer many effector proteins into the host cytoplasm, are attractive targets for drug therapy of chlamydial infections. The majority of the data concerns two species, Chlamydia trachomatis and Chlamydophila pneumoniae. The C. trachomatis protein TARP, which is presynthesized in elementary bodies, plays an essential role in the initial stages of infection. The pathogen proteins that are involved in the next stage, which is the intracellular inclusion traffic to the centrosome, are C. trachomatis CT229 and C. pneumoniae Cpn0585, which interact with cell Rab GTPases. In C. trachomatis, IncA plays a key role in the fusion of chlamydial inclusions, CT847 modulates the life cycle of the host cell, and LDA3 is essential for the acquisition of nutrients. The protease CPAF and the inclusion membrane proteins IncG and CADD are involved in suppressing apoptosis of infected cells. The proteases CPAF and CT441 and the deubiquitinating protein ChlaDub1 help the pathogen to evade the immune response.     

Infection of epithelial cells with Chlamydia trachomatis inhibits TNF‐induced apoptosis at the level of receptor internalization while leaving non‐apoptotic TNF‐signalling intact

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of medical importance. C. trachomatis develops inside a membranous vacuole in the cytosol of epithelial cells but manipulates the host cell in numerous ways. One prominent effect of chlamydial infection is the inhibition of apoptosis in the host cell, but molecular aspects of this inhibition are unclear. Tumour necrosis factor (TNF) is a cytokine with important roles in immunity, which is produced by immune cells in chlamydial infection and which can have pro‐apoptotic and non‐apoptotic signalling activity. We here analysed the signalling through TNF in cells infected with C. trachomatis. The pro‐apoptotic signal of TNF involves the activation of caspase‐8 and is controlled by inhibitor of apoptosis proteins. We found that in C. trachomatis‐infected cells, TNF‐induced apoptosis was blocked upstream of caspase‐8 activation even when inhibitor of apoptosis proteins were inhibited or the inhibitor of caspase‐8 activation, cFLIP, was targeted by RNAi. However, when caspase‐8 was directly activated by experimental over‐expression of its upstream adapter Fas‐associated protein with death domain, C. trachomatis was unable to inhibit apoptosis. Non‐apoptotic TNF‐signalling, particularly the activation of NF‐κB, initiates at the plasma membrane, while the activation of caspase‐8 and pro‐apoptotic signalling occur subsequently to internalization of TNF receptor and the formation of a cytosolic signalling complex. In C. trachomatis‐infected cells, NF‐κB activation through TNF was unaffected, while the internalization of the TNF–TNF‐receptor complex was blocked, explaining the lack of caspase‐8 activation. These results identify a dichotomy of TNF signalling in C. trachomatis‐infected cells: Apoptosis is blocked at the internalization of the TNF receptor, but non‐apoptotic signalling through this receptor remains intact, permitting a response to this cytokine at sites of infection.     

Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector

Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide “proof of principle” that it is possible to “knock out” selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.     

Human Guanylate Binding Proteins Potentiate the Anti-Chlamydia Effects of Interferon-γ

Chlamydiae are obligate intracellular pathogens that are sensitive to pro-inflammatory cytokine interferon-γ. IFN-γ-inducible murine p47 GTPases have been demonstrated to function in resistance to chlamydia infection in vivo and in vitro. Because the human genome does not encode IFN-γ-inducible homologues of these proteins, the significance of the p47 GTPase findings to chlamydia pathogenesis in humans is unclear. Here we report a pair of IFN-γ-inducible proteins, the human guanylate binding proteins (hGBPs) 1 and 2 that potentiate the anti-chlamydial properties of IFN-γ. hGBP1 and 2 localize to the inclusion membrane, and their anti-chlamydial functions required the GTPase domain. Alone, hGBP1 or 2 have mild, but statistically significant and reproducible negative effects on the growth of Chlamydia trachomatis, whilst having potent anti-chlamydial activity in conjunction with treatment with a sub-inhibitory concentration of IFN-γ. Thus, hGBPs appear to potentiate the anti-chlamydial effects of IFN-γ. Indeed, depletion of hGBP1 and 2 in cells treated with IFN-γ led to an increase in inclusion size, indicative of better growth. Interestingly, chlamydia species/strains harboring the full-length version of the putative cytotoxin gene, which has been suggested to confer resistance to IFN-γ was not affected by hGBP overexpression. These findings identify the guanylate binding proteins as potentiators of IFN-γ inhibition of C. trachomatis growth, and may be the targets of the chlamydial cytotoxin.     

CT358蛋白在沙眼衣原体感染细胞中的定位及特性分析

确定沙眼衣原体CT358蛋白在衣原体感染细胞中的位置并初步鉴定其生物学功能．采用PCR方法从D型沙眼衣原体的基因组中扩增CT358基因,并克隆入pGEX和pDSRedC1表达载体中．将重组质粒pGEX-CT358转化到XL1-blue宿主菌,并诱导表达融合蛋白GST-CT358．纯化后的CT358融合蛋白免疫小鼠制备抗体,应用间接免疫荧光技术对CT358蛋白在衣原体感染细胞内的定位及表达模式进行分析．同时,pDSRedC1-CT358重组质粒瞬时转染HeLa细胞,观察CT358蛋白对衣原体感染的影响．实验结果证明CT358蛋白为沙眼衣原体包涵体膜蛋白．该蛋白质在衣原体感染12 h后就表达定位于包涵体膜上,直至持续到整个感染周期,转基因在胞浆表达的CT358融合蛋白不影响其后的衣原体感染．该研究为深入研究衣原体与宿主细胞间相互作用提供了新的线索,并可为衣原体性的治疗、预防提供新方向．     

Damage/Danger Associated Molecular Patterns (DAMPs) Modulate Chlamydia pecorum and C. trachomatis Serovar E Inclusion Development In Vitro

Persistence, more recently termed the chlamydial stress response, is a viable but non-infectious state constituting a divergence from the characteristic chlamydial biphasic developmental cycle. Damage/danger associated molecular patterns (DAMPs) are normal intracellular components or metabolites that, when released from cells, signal cellular damage/lysis. Purine metabolite DAMPs, including extracellular ATP and adenosine, inhibit chlamydial development in a species-specific manner. Viral co-infection has been shown to reversibly abrogate Chlamydia inclusion development, suggesting persistence/chlamydial stress. Because viral infection can cause host cell DAMP release, we hypothesized DAMPs may influence chlamydial development. Therefore, we examined the effect of extracellular ATP, adenosine, and cyclic AMP exposure, at 0 and 14 hours post infection, on C. pecorum and C. trachomatis serovar E development. In the absence of de novo host protein synthesis, exposure to DAMPs immediately post or at 14 hours post infection reduced inclusion size; however, the effect was less robust upon 14 hours post infection exposure. Additionally, upon exposure to DAMPs immediately post infection, bacteria per inclusion and subsequent infectivity were reduced in both Chlamydia species. These effects were reversible, and C. pecorum exhibited more pronounced recovery from DAMP exposure. Aberrant bodies, typical in virus-induced chlamydial persistence, were absent upon DAMP exposure. In the presence of de novo host protein synthesis, exposure to DAMPs immediately post infection reduced inclusion size, but only variably modulated chlamydial infectivity. Because chlamydial infection and other infections may increase local DAMP concentrations, DAMPs may influence Chlamydia infection in vivo, particularly in the context of poly-microbial infections.     

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues ^1,2. As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur ^3,4. Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell- culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo ⁵. Further limitations are given by the fact that central signaling cascades (e.g. IFN-γ mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans ^6,7. We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo ^8,9.For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue.     

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.     

Polyunsaturated fatty acids promote the rapid fusion of lipid droplets in Caenorhabditis elegans

Lipid droplets (LDs) are intracellular organelles that dynamically regulate lipids and energy homeostasis in the cell. LDs can grow through either local lipid synthesis or LD fusion. However, how lipids involving in LD fusion for LD growth is largely unknown. Here, we show that genetic mutation of acox-3 (acyl-CoA oxidase), maoc-1 (enoyl-CoA hydratase), dhs-28 (3-hydroxylacyl-CoA dehydrogenase), and daf-22 (3-ketoacyl-CoA thiolase), all involved in the peroxisomal β-oxidation pathway in Caenorhabditis elegans, led to rapid fusion of adjacent LDs to form giant LDs (gLDs). Mechanistically, we show that dysfunction of peroxisomal β-oxidation results in the accumulation of long-chain fatty acid-CoA and phosphocholine, which may activate the sterol-binding protein 1/sterol regulatory element–binding protein to promote gLD formation. Furthermore, we found that inactivation of either FAT-2 (delta-12 desaturase) or FAT-3 and FAT-1 (delta-15 desaturase and delta-6 desaturase, respectively) to block the biosynthesis of polyunsaturated fatty acids (PUFAs) with three or more double bonds (n≥3-PUFAs) fully repressed the formation of gLDs; in contrast, dietary supplementation of n≥3-PUFAs or phosphocholine bearing these PUFAs led to recovery of the formation of gLDs in peroxisomal β-oxidation–defective worms lacking PUFA biosynthesis. Thus, we conclude that n≥3-PUFAs, distinct from other well-known lipids and proteins, promote rapid LD fusion leading to LD growth.     

Specific chlamydial inclusion membrane proteins associate with active Src family kinases in microdomains that interact with the host microtubule network

Chlamydiae are Gram‐negative obligate intracellular bacteria that cause diseases with significant medical and economic impact. Chlamydia trachomatis replicates within a vacuole termed an inclusion, which is extensively modified by the insertion of a number of bacterial effector proteins known as inclusion membrane proteins (Incs). Once modified, the inclusion is trafficked in a dynein‐dependent manner to the microtubule‐organizing centre (MTOC), where it associates with host centrosomes. Here we describe a novel structure on the inclusion membrane comprised of both host and bacterial proteins. Members of the Src family of kinases are recruited to the chlamydial inclusion in an active form. These kinases display a distinct, localized punctate microdomain‐like staining pattern on the inclusion membrane that colocalizes with four chlamydial inclusion membrane proteins (Incs) and is enriched in cholesterol. Biochemical studies show that at least two of these Incs stably interact with one another. Furthermore, host centrosomes associate with these microdomain proteins in C. trachomatis‐infected cells and in uninfected cells exogenously expressing one of the chlamydial effectors. Together, the data suggest that a specific structure on the C. trachomatis inclusion membrane may be responsible for the known interactions of chlamydiae with the microtubule network and resultant effects on centrosome stability.