The T4bSS of Legionella features a two-step secretion pathway with an inner membrane intermediate for secretion of transmembrane effectors.

To promote intracellular survival and infection, Legionella spp. translocate hundreds of effector proteins into eukaryotic host cells using a type IV b protein secretion system (T4bSS). T4bSS are well known to translocate soluble as well as transmembrane domain-containing effector proteins (TMD-effectors) but the mechanisms of secretion are still poorly understood. Herein we investigated the secretion of hydrophobic TMD-effectors, of which about 80 were previously reported to be encoded by L. pneumophila. A proteomic analysis of fractionated membranes revealed that TMD-effectors are targeted to and inserted into the bacterial inner membranes of L. pneumophila independent of the presence of a functional T4bSS. While the T4bSS chaperones IcmS and IcmW were critical for secretion of all tested TMD-effectors, they did not influence inner membrane targeting of these proteins. As for soluble effector proteins, translocation of TMD-effectors into host cells depended on a C-terminal secretion signal and this signal needed to be presented towards the cytoplasmic side of the inner membrane. A different secretion behavior of TMD- and soluble effectors and the need for small periplasmic loops within TMD-effectors provided strong evidence that TMD-effectors are secreted in a two-step secretion process: Initially, an inner membrane intermediate is formed, that is extracted towards the cytoplasmic side, possibly by the help of the type IV coupling protein complex and subsequently secreted into eukaryotic host cells by the T4bSS core complex. Overall, our study highlights the amazing versatility of T4bSS to secrete soluble and TMD-effectors from different subcellular locations of the bacterial cell.

Musculoskeletal manifestations of syphilis in adults: secondary syphilis presenting with ankle inflammatory arthritis and bone involvement with calvarial and sternal lesions. What the rheumatologist needs to know.

Although the incidence of syphilis reached a historic low in 2000, the number of incident cases has since increased in men and women across the USA. In 2019, men who have sex with men (MSM) accounted for 57% of all primary and secondary (P&S) syphilis cases, and about half of MSM with P&S syphilis are living with human immunodeficiency virus (HIV) infection. Days after infection, Treponema pallidum disseminates and invades tissues distant from the site of inoculation. Once the spirochete disseminates, the host develops an inflammatory response; diagnosis requires a high level of suspicion since syphilis may affect the skin, musculoskeletal, cardiovascular, and central nervous systems. We report a 61-year-old man with virally suppressed HIV infection who presented with polyarthralgia, chest pain, and weight loss, diagnosed with secondary syphilis, manifesting with ankle inflammatory arthritis and bone involvement, of the calvarium and manubrium. Early and late syphilis in adults can manifest with articular and periarticular pathologies, including inflammatory arthritis, tenosynovitis, periostitis, and myositis. Higher clinical suspicion is needed for prompt diagnosis of syphilis in patients who are at risk and suspected of having an autoimmune disease. This report includes a review of the musculoskeletal manifestations of syphilis.

Evaluation of an automated phenotyping algorithm for rheumatoid arthritis.

To better understand the challenges of generally implementing and adapting computational phenotyping approaches, the performance of a Phenotype KnowledgeBase (PheKB) algorithm for rheumatoid arthritis (RA) was evaluated on a University of California, Los Angeles (UCLA) patient population, focusing on examining its performance on ambiguous cases. The algorithm was evaluated on a cohort of 4,766 patients, along with a chart review of 300 patients by rheumatologists against accepted diagnostic guidelines. The performance revealed low sensitivity towards specific subtypes of positive RA cases, which suggests revisions in features used for phenotyping. A close examination of select cases also indicated a significant portion of patients with missing data, drawing attention to the need to consider data integrity as an integral part of phenotyping pipelines, as well as issues around the usability of various codes for distinguishing cases. We use patterns in the PheKB algorithm's errors to further demonstrate important considerations when designing a phenotyping algorithm.

Proteins DotY and DotZ modulate the dynamics and localization of the type IVB coupling complex of Legionella pneumophila.

Legionella pneumophila is an opportunistic pathogen infecting alveolar macrophages and protozoa species. Legionella utilizes a Type IV Secretion System (T4SS) to translocate over 300 effector proteins into its host cell. In a recent study, we have isolated and solved the cryo-EM structure of the Type IV Coupling Complex (T4CC), a large cytoplasmic determinant associated with the inner membrane that recruits effector proteins for delivery to the T4SS for translocation. The T4CC is composed of a DotLMNYZ hetero-pentameric core from which the flexible IcmSW module flexibly protrudes. The DotY and DotZ proteins were newly reported members of this complex and their role remained elusive. In this study, we observed the effect of deleting DotY and DotZ on T4CC stability and localization. Furthermore, we found these two proteins are co-dependent, whereby the deletion of DotY resulted in DotZ absence from the coupling complex, and vice versa. Additional cryo-EM data analysis revealed the dynamic movement of the IcmSW module is modified by the DotY/Z proteins. We therefore determined the likely function of DotY and DotZ and revealed their importance on T4CC function.

Mechanism of effector capture and delivery by the type IV secretion system from Legionella pneumophila.

Legionella pneumophila is a bacterial pathogen that utilises a Type IV secretion (T4S) system to inject effector proteins into human macrophages. Essential to the recruitment and delivery of effectors to the T4S machinery is the membrane-embedded T4 coupling complex (T4CC). Here, we purify an intact T4CC from the Legionella membrane. It contains the DotL ATPase, the DotM and DotN proteins, the chaperone module IcmSW, and two previously uncharacterised proteins, DotY and DotZ. The atomic resolution structure reveals a DotLMNYZ hetero-pentameric core from which the flexible IcmSW module protrudes. Six of these hetero-pentameric complexes may assemble into a 1.6-MDa hexameric nanomachine, forming an inner membrane channel for effectors to pass through. Analysis of multiple cryo EM maps, further modelling and mutagenesis provide working models for the mechanism for binding and delivery of two essential classes of Legionella effectors, depending on IcmSW or DotM, respectively.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Apixaban as a Rare Cause of Leukocytoclastic Vasculitis.

Apixaban is a rare cause of leukocytoclastic vasculitis (LCV). To our knowledge, there is only one other reported case due to apixaban in the literature. We present a case of apixaban-induced leukocytoclastic vasculitis in a 95-year-old male. He had been started on apixaban 12 days prior to presentation and developed worsening palpable purpura of his lower extremities. Possible etiologies of this new rash were excluded, with biopsy showing extensive purpura with superficial perivascular neutrophilic infiltrate and leukocytoclasis. Apixaban was discontinued, and the patient was started on a slow prednisone taper with subsequent resolution of his rash.

Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes Induced by DotB Binding.

Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput cryoelectron tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane.IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced cryoelectron tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine.

A unique cytoplasmic ATPase complex defines the Legionella pneumophila type IV secretion channel.

Type IV secretion systems (T4SSs) are complex machines used by bacteria to deliver protein and DNA complexes into target host cells1-5. Conserved ATPases are essential for T4SS function, but how they coordinate their activities to promote substrate transfer remains poorly understood. Here, we show that the DotB ATPase associates with the Dot-Icm T4SS at the Legionella cell pole through interactions with the DotO ATPase. The structure of the Dot-Icm apparatus was solved in situ by cryo-electron tomography at 3.5 nm resolution and the cytoplasmic complex was solved at 3.0 nm resolution. These structures revealed a cell envelope-spanning channel that connects to the cytoplasmic complex. Further analysis revealed a hexameric assembly of DotO dimers associated with the inner membrane complex, and a DotB hexamer associated with the base of this cytoplasmic complex. The assembly of a DotB-DotO energy complex creates a cytoplasmic channel that directs the translocation of substrates through the T4SS. These data define distinct stages in Dot-Icm machine biogenesis, advance our understanding of channel activation, and identify an envelope-spanning T4SS channel.

Legionella DotM structure reveals a role in effector recruiting to the Type 4B secretion system.

Legionella pneumophila, a causative agent of pneumonia, utilizes the Type 4B secretion (T4BS) system to translocate over 300 effectors into the host cell during infection. T4BS systems are encoded by a large gene cluster termed dot/icm, three components of which, DotL, DotM, and DotN, form the "coupling complex", which serves as a platform for recruitment of effector proteins. One class of effectors includes proteins containing Glu-rich/E-block sequences at their C terminus. However, the protein or region of the coupling complex mediating recruitment of such effectors is unknown. Here we present the crystal structure of DotM. This all alpha-helical structure exhibits patches of positively charged residues. We show that these regions form binding sites for acidic Glu-rich peptides and that mutants targeting these patches are defective in vivo in the translocation of acidic Glu-rich motif-containing effectors. We conclude that DotM forms the interacting surface for recruitment of acidic Glu-rich motif-containing Legionella effectors.

Synaptojanin 2 is a druggable mediator of metastasis and the gene is overexpressed and amplified in breast cancer.

Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5'-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration.

Identification of two Legionella pneumophila effectors that manipulate host phospholipids biosynthesis.

The intracellular pathogen Legionella pneumophila translocates a large number of effector proteins into host cells via the Icm/Dot type-IVB secretion system. Some of these effectors were shown to cause lethal effect on yeast growth. Here we characterized one such effector (LecE) and identified yeast suppressors that reduced its lethal effect. The LecE lethal effect was found to be suppressed by the over expression of the yeast protein Dgk1 a diacylglycerol (DAG) kinase enzyme and by a deletion of the gene encoding for Pah1 a phosphatidic acid (PA) phosphatase that counteracts the activity of Dgk1. Genetic analysis using yeast deletion mutants, strains expressing relevant yeast genes and point mutations constructed in the Dgk1 and Pah1 conserved domains indicated that LecE functions similarly to the Nem1-Spo7 phosphatase complex that activates Pah1 in yeast. In addition, by using relevant yeast genetic backgrounds we examined several L. pneumophila effectors expected to be involved in phospholipids biosynthesis and identified an effector (LpdA) that contains a phospholipase-D (PLD) domain which caused lethal effect only in a dgk1 deletion mutant of yeast. Additionally, LpdA was found to enhance the lethal effect of LecE in yeast cells, a phenomenon which was found to be dependent on its PLD activity. Furthermore, to determine whether LecE and LpdA affect the levels or distribution of DAG and PA in-vivo in mammalian cells, we utilized fluorescent DAG and PA biosensors and validated the notion that LecE and LpdA affect the in-vivo levels and distribution of DAG and PA, respectively. Finally, we examined the intracellular localization of both LecE and LpdA in human macrophages during L. pneumophila infection and found that both effectors are localized to the bacterial phagosome. Our results suggest that L. pneumophila utilize at least two effectors to manipulate important steps in phospholipids biosynthesis.

Poor cerebral inflammatory response in eIF2B knock-in mice: implications for the aetiology of vanishing white matter disease.

BACKGROUND: Mutations in any of the five subunits of eukaryotic translation initiation factor 2B (eIF2B) can lead to an inherited chronic-progressive fatal brain disease of unknown aetiology termed leucoencephalopathy with vanishing white matter (VWM). VWM is one of the most prevalent childhood white matter disorders, which markedly deteriorates after inflammation or exposure to other stressors. eIF2B is a major housekeeping complex that governs the rate of global protein synthesis under normal and stress conditions. A previous study demonstrated that Eif2b5(R132H/R132H) mice suffer delayed white matter development and fail to recover from cuprizone-induced demyelination, although eIF2B enzymatic activity in the mutant brain is reduced by merely 20%. PRINCIPAL FINDINGS: Poor astrogliosis was observed in Eif2b5(R132H/R132H) mice brain in response to systemic stress induced by peripheral injections of lipopolysaccharide (LPS). Even with normal rates of protein synthesis under normal conditions, primary astrocytes and microglia isolated from mutant brains fail to adequately synthesise and secrete cytokines in response to LPS treatment despite proper induction of cytokine mRNAs. CONCLUSIONS: The mild reduction in eIF2B activity prevents the appropriate increase in translation rates upon exposure to the inflammatory stressor LPS. The data underscore the importance of fully-functional translation machinery for efficient cerebral inflammatory response upon insults. It highlights the magnitude of proficient translation rates in restoration of brain homeostasis via microglia-astrocyte crosstalk. This study is the first to suggest the involvement of microglia in the pathology of VWM disease. Importantly, it rationalises the deterioration of clinical symptoms upon exposure of VWM patients to physiological stressors and provides possible explanation for their high phenotypic variability.

Negative regulation of the endocytic adaptor disabled-2 (Dab2) in mitosis.

Mitotic cells undergo extensive changes in shape and size through the altered regulation and function of their membrane trafficking machinery. Disabled 2 (Dab2), a multidomain cargo-specific endocytic adaptor and a mediator of signal transduction, is a potential integrator of trafficking and signaling. Dab2 binds effectors of signaling and trafficking that localize to different intracellular compartments. Thus, differential localization is a putative regulatory mechanism of Dab2 function. Furthermore, Dab2 is phosphorylated in mitosis and is thus regulated in the cell cycle. However, a detailed description of the intracellular localization of Dab2 in the different phases of mitosis and an understanding of the functional consequences of its phosphorylation are lacking. Here, we show that Dab2 is progressively displaced from the membrane in mitosis. This phenomenon is paralleled by a loss of co-localization with clathrin. Both phenomena culminate in metaphase/anaphase and undergo partial recovery in cytokinesis. Treatment with 2-methoxyestradiol, which arrests cells at the spindle assembly checkpoint, induces the same effects observed in metaphase cells. Moreover, 2-methoxyestradiol also induced Dab2 phosphorylation and reduced Dab2/clathrin interactions, endocytic vesicle motility, clathrin exchange dynamics, and the internalization of a receptor endowed with an NPXY endocytic signal. Serine/threonine to alanine mutations, of residues localized to the central region of Dab2, attenuated its phosphorylation, reduced its membrane displacement, and maintained its endocytic abilities in mitosis. We propose that the negative regulation of Dab2 is part of an accommodation of the cell to the altered physicochemical conditions prevalent in mitosis, aimed at allowing endocytic activity throughout the cell cycle.

Monoubiquitinylation regulates endosomal localization of Lst2, a negative regulator of EGF receptor signaling.

Genetic screens performed in worms identified major regulators of the epidermal growth factor receptor (EGFR) pathway, including the ubiquitin ligase Cbl/SLI-1. Here we focus on the less-characterized Lst2 protein and confirm suppression of MAPK signals. Unexpectedly, human Lst2, a monoubiquitinylated phosphoprotein, does not localize to endosomes, despite an intrinsic phosphoinositol-binding FYVE domain. By constructing an ubiquitinylation-defective mutant and an ubiquitin fusion, we conclude that endosomal localization of Lst2, along with an ability to divert incoming EGFR molecules to degradation in lysosomes, is regulated by ubiquitinylation/deubiquitinylation cycles. Consistent with bifurcating roles, Lst2 physically binds Trim3/BERP, which interacts with Hrs and a complex that biases cargo recycling. These results establish an ubiquitin-based endosomal switch of receptor sorting, functionally equivalent to the mechanism inactivating Hrs via monoubiquitinylation.

Dab2 regulates clathrin assembly and cell spreading.

The recruitment of clathrin to the membrane and its assembly into coated pits results from its interaction with endocytic adaptors and other regulatory proteins in the context of a specific lipid microenvironment. Dab2 (disabled 2) is a mitotic phosphoprotein and a monomeric adaptor for clathrin-mediated endocytosis. In the present study, we employed GFP (green fluorescent protein) fusion constructs of different isoforms and mutants of rat Dab2 and characterized their effect on the size, distribution and dynamics of clathrin assemblies. Enhanced levels of expression of the p82 isoform of Dab2 in COS7 cells induced enlarged clathrin assemblies at the plasma membrane. p82-clathrin assemblies, which concentrate additional endocytic proteins, such as AP2 (adaptor protein 2) and epsin, are dynamic structures in which both p82 and clathrin exchange actively between the membrane-bound and cytosolic sub-populations. The ability of p82 to induce enlarged clathrin assemblies is dependent on the presence of a functional PTB domain (phosphotyrosine-binding domain), on binding to clathrin and phospholipids, and on a newly identified and evolutionarily conserved poly-lysine stretch which precedes the PTB domain. These same molecular features are required for Dab2 to enhance the spreading of COS7 cells on fibronectin. The ability of the p82 isoform of Dab2 to enhance cell spreading was confirmed in both HeLa cells and HBL cells (human breast epithelial cells). COS7 cells expressing GFP-p82 and plated on to fibronectin concentrate the beta1 integrin into clathrin-p82 assemblies. Furthermore, during cell spreading, p82-clathrin assemblies concentrate at the site of the initial cell-matrix contact and are absent from regions of intense membrane ruffling. We propose a role for Dab2 and clathrin in integrin-mediated cell spreading.

Protection of lipids from oxidation by epicatechin, trans-resveratrol, and gallic and caffeic acids in intestinal model systems.

Consumption of polyphenols is associated with health promotion through diet, although many are poorly absorbed in animals and humans alike. Lipid peroxides may reach the intestine and initiate deleterious oxidation. Here we measured inhibition of the oxidation of linoleic acid (LA) in authentic fluid from rat small intestine (RIF) by two dietary polyphenols, a flavonoid, epicatechin (EC), and a stilbene, resveratrol (RV), and by gallic (GA) and caffeic (CA) acids, and their partition coefficients. Both polyphenols inhibited 80%, and CA inhibited 65%, of the production of hexanal. GA was the weakest antioxidant in this assay. Interestingly, measuring peroxides production in RIF showed that only epicatechin inhibited the first stage of oxidation. The oxidizing agent, the antioxidant comound, the solution pH and lipophilicity are known to affect the total antioxidative activity. We suggest that the mechanism of this activity changes in accord with the environment: i.e., RV may act as a free radial scavenger, but here, in protecting lipids in intestinal fluid from oxidation, it acts as a hydrogen atom donor. Since the concentration of phenolics is much higher in the intestinal fluid than is ever achieved in plasma or other body tissues, it is suggested that their antioxidant activity could be exerted in the gastrointestinal tract (GIT), breaking the propagation of lipid peroxides oxidation and production of toxic compounds.