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Many intracellular pathogens structurally disrupt the Golgi apparatus as an evolutionarily conserved promicrobial strategy. Yet, the host factors and signaling processes involved are often poorly understood, particularly for Anaplasma phagocytophilum , the agent of human granulocytic anaplasmosis. We found that A. phagocytophilum elevated cellular levels of the bioactive sphingolipid, ceramide-1-phosphate (C1P), to promote Golgi fragmentation that enables bacterial proliferation, conversion from its non-infectious to infectious form, and productive infection. A. phagocytophilum poorly infected mice deficient in ceramide kinase, the Golgi-localized enzyme responsible for C1P biosynthesis. C1P regulated Golgi morphology via activation of a PKCα/Cdc42/JNK signaling axis that culminates in phosphorylation of Golgi structural proteins, GRASP55 and GRASP65. siRNA-mediated depletion of Cdc42 blocked A. phagocytophilum from altering Golgi morphology, which impaired anterograde trafficking of trans -Golgi vesicles into and maturation of the pathogen-occupied vacuole. Cells overexpressing phosphorylation-resistant versions of GRASP55 and GRASP65 presented with suppressed C1P- and A. phagocytophilum -induced Golgi fragmentation and poorly supported infection by the bacterium. By studying A. phagocytophilum , we identify C1P as a regulator of Golgi structure and a host factor that is relevant to disease progression associated with Golgi fragmentation.IMPORTANCECeramide-1-phosphate (C1P), a bioactive sphingolipid that regulates diverse processes vital to mammalian physiology, is linked to disease states such as cancer, inflammation, and wound healing. By studying the obligate intracellular bacterium Anaplasma phagocytophilum , we discovered that C1P is a major regulator of Golgi morphology. A. phagocytophilum elevated C1P levels to induce signaling events that promote Golgi fragmentation and increase vesicular traffic into the pathogen-occupied vacuole that the bacterium parasitizes. As several intracellular microbial pathogens destabilize the Golgi to drive their infection cycles and changes in Golgi morphology is also linked to cancer and neurodegenerative disorder progression, this study identifies C1P as a potential broad-spectrum therapeutic target for infectious and non-infectious diseases., Competing Interests: The authors declare no conflict of interest.

Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis. It poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine that functionally inhibits ASM. Whether inhibition or genetic deletion of ASM is bacteriostatic or bactericidal for A. phagocytophilum and desipramine's ability to lower pathogen burden requires a competent immune system were unknown. Anaplasma phagocytophilum-infected severe combined immunodeficiency disorder (SCID) mice were administered desipramine or PBS, followed by the transfer of blood to naïve wild-type mice. Next, infected wild-type mice were given desipramine or PBS followed by transfer of blood to naïve SCID mice. Finally, wild-type or ASM-deficient mice were infected and blood transferred to naïve SCID mice. The percentage of infected neutrophils was significantly reduced in all desipramine-treated or ASM-deficient mice and in all recipients of blood from these mice. Infection was markedly lower in ASM-deficient and desipramine-treated wild-type mice versus desipramine-treated SCID mice. Yet, infection was never ablated. Thus, ASM activity contributes to optimal A. phagocytophilum infection in vivo, pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner and A. phagocytophilum is capable of co-opting ASM-independent lipid sources., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society for Nutrition.)

The authors wish to make the following corrections to this paper [...].

Intracellular bacteria that live in host cell-derived vacuoles are significant causes of human disease. Parasitism of low-density lipoprotein (LDL) cholesterol is essential for many vacuole-adapted bacteria. Acid sphingomyelinase (ASM) influences LDL cholesterol egress from the lysosome. Using functional inhibitors of ASM (FIASMAs), we show that ASM activity is key for infection cycles of vacuole-adapted bacteria that target cholesterol trafficking- Anaplasma phagocytophilum , Coxiella burnetii , Chlamydia trachomatis , and Chlamydia pneumoniae. Vacuole maturation, replication, and infectious progeny generation by A. phagocytophilum , which exclusively hijacks LDL cholesterol, are halted and C. burnetii , for which lysosomal cholesterol accumulation is bactericidal, is killed by FIASMAs. Infection cycles of Chlamydiae, which hijack LDL cholesterol and other lipid sources, are suppressed but less so than A. phagocytophilum or C. burnetii A. phagocytophilum fails to productively infect ASM -/- or FIASMA-treated mice. These findings establish the importance of ASM for infection by intracellular bacteria and identify FIASMAs as potential host-directed therapies for diseases caused by pathogens that manipulate LDL cholesterol., (© 2019 Cockburn et al.)

Human granulocytic anaplasmosis (HGA) is a debilitating, non-specific febrile illness caused by the granulocytotropic obligate intracellular bacterium called Anaplasma phagocytophilum . Surveillance studies indicate a higher prevalence of HGA in male versus female patients. Whether this discrepancy correlates with differential susceptibility of males and females to A. phagocytophilum infection is unknown. Laboratory mice have long been used to study granulocytic anaplasmosis. Yet, sex as a biological variable (SABV) in this model has not been evaluated. In this paper, groups of male and female C57Bl/6 mice that had been infected with A. phagocytophilum were assessed for the bacterial DNA load in the peripheral blood, the percentage of neutrophils harboring bacterial inclusions called morulae, and splenomegaly. Infected male mice exhibited as much as a 1.85-fold increase in the number of infected neutrophils, which is up to a 1.88-fold increase in the A. phagocytophilum DNA load, and a significant increase in spleen size when compared to infected female mice. The propensity of male mice to develop a higher level of A. phagocytophilum infection is relevant for studies utilizing the mouse model. This stresses the importance of including SABV and aligns with the observed higher incidence of infection in male versus female patients.

Background: Allergy and allergic asthma are significant health burdens in developed countries and are increasing in prevalence. Dendritic cells (DCs) initiate immune responses to common aeroallergens, and ADAM10 has been demonstrated to be important for the development of adaptive responses. This study's objective was to understand the role of ADAM10 on DCs in the development of allergic and anaphylactic responses., Methods: In this study, we used mouse models of allergic airway inflammation (house dust mice and Alternaria alternata) and OVA-induced models of active anaphylaxis to determine the DC-specific function of ADAM10 and Notch signaling. To examine T H 1 and T H 17 immunity infection with Anaplasma phagocytophilum and Citrobacter rodentium respectively, were used., Results: Mice, which have ADAM10 deleted from DCs, have dramatic reductions in IgE production and do not develop significant T H 2 immune responses. Further, ADAM10 DC -/- mice are resistant to IgE-mediated anaphylaxis. This response is selective for T H 2 immunity as T H 1 and T H 17 immunity is largely unaffected. Notch1, a known ADAM10 substrate, when knocked out of DCs (Notch1 DC -/- ) demonstrated a similar reduction in anaphylaxis and IgE. Without ADAM10 and Notch1 signaling, DCs were unable to make cytokines that stimulate T H 2 cells and cytokines. Anaphylaxis and allergic lung inflammation were restored in ADAM10 DC -/- with the overexpression of the Notch1-intracellular domain, confirming the role of Notch signaling., Conclusions: Targeting ADAM10 and Notch1 on DCs represent a novel strategy for modulating T H 2 immune responses and IgE production., (© 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Anaplasma phagocytophilum is an obligate intracellular bacterial pathogen of humans and animals. The A. phagocytophium -occupied vacuole (ApV) is a critical host-pathogen interface. Here, we report that the intermediate filaments, keratin and vimentin, assemble on the ApV early and remain associated with the ApV throughout infection. Microtubules localize to the ApV to a lesser extent. Vimentin, keratin-8, and keratin-18 but not tubulin expression is upregulated in A. phagocytophilum infected cells. SUMO-2/3 but not SUMO-1 colocalizes with vimentin filaments that surround ApVs. PolySUMOylation of vimentin by SUMO-2/3 but not SUMO-1 decreases vimentin solubility. Consistent with this, more vimentin exists in an insoluble state in A. phagocytophilum infected cells than in uninfected cells. Knocking down the SUMO-conjugating enzyme, Ubc9, abrogates vimentin assembly at the ApV but has no effect on the bacterial load. Bacterial protein synthesis is dispensable for maintaining vimentin and SUMO-2/3 at the ApV. Withaferin A, which inhibits soluble vimentin, reduces vimentin recruitment to the ApV, optimal ApV formation, and the bacterial load when administered prior to infection but is ineffective once vimentin has assembled on the ApV. Thus, A. phagocytophilum modulates cytoskeletal component expression and co-opts polySUMOylated vimentin to aid construction of its vacuolar niche and promote optimal survival.

The genus Anaplasma consists of tick-transmitted obligate intracellular bacteria that invade white or red blood cells to cause debilitating and potentially fatal infections. A. phagocytophilum, a human and veterinary pathogen, infects neutrophils to cause granulocytic anaplasmosis. A. marginale invades bovine erythrocytes. Evidence suggests that both species may also infect endothelial cells in vivo. In mammalian and arthropod host cells, A. phagocytophilum and A. marginale reside in host cell derived pathogen-occupied vacuoles (POVs). While it was recently demonstrated that the A. phagocytophilum-occupied vacuole (ApV) intercepts membrane traffic from the trans-Golgi network, it is unclear if it or the A. marginale-occupied vacuole (AmV) interacts with other secretory organelles. Here, we demonstrate that the ApV and AmV extensively interact with the host endoplasmic reticulum (ER) in endothelial, myeloid, and/or tick cells. ER lumen markers, calreticulin, and protein disulfide isomerase, and the ER membrane marker, derlin-1, were pronouncedly recruited to the peripheries of both POVs. ApV association with the ER initiated early and continued throughout the infection cycle. Both the ApV and AmV interacted with the rough ER and smooth ER. However, only derlin-1-positive rough ER derived vesicles were delivered into the ApV lumen where they localized with intravacuolar bacteria. Transmission electron microscopy identified multiple ER-POV membrane contact sites on the cytosolic faces of both species' vacuoles that corresponded to areas on the vacuoles' lumenal faces where intravacuolar Anaplasma organisms closely associated. A. phagocytophilum is known to hijack Rab10, a GTPase that regulates ER dynamics and morphology. Yet, ApV-ER interactions were unhindered in cells in which Rab10 had been knocked down, demonstrating that the GTPase is dispensable for the bacterium to parasitize the ER. These data establish the ApV and AmV as pathogen-host interfaces that directly engage the ER in vertebrate and invertebrate host cells and evidence the conservation of ER parasitism between two Anaplasma species.

Anaplasma phagocytophilum is an emerging human pathogen and obligate intracellular bacterium. It inhabits a host cell-derived vacuole and cycles between replicative reticulate cell (RC) and infectious dense-cored (DC) morphotypes. Host-pathogen interactions that are critical for RC-to-DC conversion are undefined. We previously reported that A. phagocytophilum recruits green fluorescent protein (GFP)-tagged Rab10, a GTPase that directs exocytic traffic from the sphingolipid-rich trans-Golgi network (TGN) to its vacuole in a guanine nucleotide-independent manner. Here, we demonstrate that endogenous Rab10-positive TGN vesicles are not only routed to but also delivered into the A. phagocytophilum-occupied vacuole (ApV). Consistent with this finding, A. phagocytophilum incorporates sphingolipids while intracellular and retains them when naturally released from host cells. TGN vesicle delivery into the ApV is Rab10 dependent, up-regulates expression of the DC-specific marker, APH1235, and is critical for the production of infectious progeny. The A. phagocytophilum surface protein, uridine monophosphate kinase, was identified as a guanine nucleotide-independent, Rab10-specific ligand. These data delineate why Rab10 is important for the A. phagocytophilum infection cycle and expand the understanding of the benefits that exploiting host cell membrane traffic affords intracellular bacterial pathogens., (© 2015 John Wiley & Sons Ltd.)

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

Sodium valproate (VPA) is clinically employed as an anticonvulsant and, to a lesser extent, as a mood stabilizer. While the incidence of toxicity associated with the clinical use of valproate is low, serious hepatotoxicity makes up a significant percentage of these rare adverse effects, with fatalities occurring mainly in children receiving polypharmacy. Previous studies have highlighted the different pharmacological effects of acute valproate exposure, a combination of which are likely to underpin its observed broad-spectrum anticonvulsant efficacy. However, limited studies have been undertaken to investigate the subacute effects of this compound and how genomic effects may underlie the observed hepatotoxic effects. Investigation into the mild hepatoxicity observed in rats exposed to high doses of VPA may provide important information on the human situation. Male Sprague-Dawley rats were dosed with 500 mg/kg/day sodium valproate: after necropsy, mRNA was subjected to suppression PCR subtractive hybridization, identifying 8 up-regulated and 14 down-regulated mRNA species. The down-regulation of several mRNA species coding for enzymes involved in cellular energetics (e.g., succinate dehydrogenase, aldolase B) was of particular interest, as mitochondrial dysfunction is a key feature of valproate hepatotoxicity. In vitro studies were then undertaken to examine the dose and time dependence of these changes and also their effect on the overall energy levels within the cell. We demonstrate that, both in vivo and in vitro, valproate exposure in rats results in a significant decrease in pathways involved in cellular energy homeostasis. These changes may provide insight into the rare human hepatoxicity associated with this compound.

Background: Belonging to the Actinobacteria phylum, members of the Rhodococcus genus thrive in soil, water, and even intracellularly. While most species are non-pathogenic, several cause respiratory disease in animals and, more rarely, in humans. Over 100 phages that infect Rhodococcus species have been isolated but despite their importance for Rhodococcus ecology and biotechnology applications, little is known regarding the molecular genetic interactions between phage and host during infection. To address this need, we report RNA-Seq analysis of a novel Rhodococcus erythopolis phage, WC1, analyzing both the phage and host transcriptome at various stages throughout the infection process. Results: By five minutes post-infection WC1 showed upregulation of a CAS-4 family exonuclease, putative immunity repressor, an anti-restriction protein, while the host showed strong upregulation of DNA replication, SOS repair, and ribosomal protein genes. By 30 min post-infection, WC1 DNA synthesis genes were strongly upregulated while the host showed increased expression of transcriptional and translational machinery and downregulation of genes involved in carbon, energy, and lipid metabolism pathways. By 60 min WC1 strongly upregulated structural genes while the host showed a dramatic disruption of metal ion homeostasis. There was significant expression of both host and phage non-coding genes at all time points. While host gene expression declined over the course of infection, our results indicate that phage may exert more selective control, preserving the host's regulatory mechanisms to create an environment conducive for virion production. Conclusions: The Rhodococcus genus is well recognized for its ability to synthesize valuable compounds, particularly steroids, as well as its capacity to degrade a wide range of harmful environmental pollutants. A detailed understanding of these phage-host interactions and gene expression is not only essential for understanding the ecology of this important genus, but will also facilitate development of phage-mediated strategies for bioremediation as well as biocontrol in industrial processes and biomedical applications. Given the current lack of detailed global gene expression studies on any Rhodococcus species, our study addresses a pressing need to identify tools and genes, such as F6 and rpf, that can enhance the capacity of Rhodococcus species for bioremediation, biosynthesis and pathogen control. [ABSTRACT FROM AUTHOR]

Background: The blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the US. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied compared to other arthropod vectors. Ixodes scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility. Methods: Using BLAST, an in silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. Ixodes scapularis nymphs were injected with small interfering RNAs to knock down rab27 and then fed on naïve and A. phagocytophilum-infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition. Results: Our BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis. Conclusions: Rab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Endosomal Sorting Complex Required for Transport (ESCRT) machinery consists of multiple protein complexes that coordinate vesicle budding away from the host cytosol. ESCRTs function in many fundamental cellular processes including the biogenesis of multivesicular bodies and exosomes, membrane repair and restoration, and cell abscission during cytokinesis. Work over the past 2 decades has shown that a diverse cohort of viruses critically rely upon host ESCRT machinery for virus replication and envelopment. More recent studies reported that intracellular bacteria and the intracellular parasite Toxoplasma gondii benefit from, antagonize, or exploit host ESCRT machinery to preserve their intracellular niche, gain resources, or egress from infected cells. Here, we review how intracellular pathogens interact with the ESCRT machinery of their hosts, highlighting the variety of strategies they use to bind ESCRT complexes using short linear amino acid motifs like those used by ESCRTs to sequentially assemble on target membranes. Future work exposing new mechanisms of this molecular mimicry will yield novel insight of how pathogens exploit host ESCRT machinery and how ESCRTs facilitate key cellular processes. [ABSTRACT FROM AUTHOR]

Acid sphingomyelinase (ASM) promotes atherogenesis and acute cardiovascular events. We previously demonstrated ASM inhibitor desipramine attenuated oxidized-LDL-induced macrophage apoptosis in vitro. Here, we aim to determine whether ASM-mediated apoptosis in plaque improves stability in vivo. In this study, rabbits with abdominal aorta balloon injury and a 12-week high-cholesterol diet (HCD) were used to simulate an atherosclerotic plaque model. Atherosclerotic rabbits received oral administration of saline (Control group), atorvastatin (Ator group), or desipramine (DES group). ASM activity and ceramide level were measured by ultra-performance liquid chromatography (UPLC). Plaque morphology was assessed by histochemistry and immunohistochemistry. Apoptosis was evaluated by SPECT/CT imaging of 99mTc-duramycin uptake and TUNEL. We found that increasing ASM activity and ceramide level in atherosclerotic rabbits was abated by additional atorvastatin and desipramine treatment. Meanwhile, the DES and Ator groups were similar in plaque stability, with smaller plaque size, areas of macrophages, higher smooth muscle cell content, and decreased apoptosis and matrix metalloproteinase (MMP) activities relative to the Control group. 99mTc-duramycin uptake of rabbit aorta was significantly higher in Control than in the Normal group, while it was reduced by desipramine and atorvastatin administration. Moreover, the uptake of 99mTc-duramycin positively correlated with apoptotic cell number, macrophage infiltration, and plaque instability. The present study demonstrated that desipramine exerted plaque-stabilizing effects partially by suppressing apoptosis and MMP activity in a rabbit model. And 99mTc-duramycin SPECT/CT imaging allowed noninvasively monitoring of atherosclerotic disease and evaluation of anti-atherosclerotic therapy. [ABSTRACT FROM AUTHOR]

Highly immunogenic exotoxins are used as carrier proteins because they efficiently improve the immunogenicity of polysaccharides. However, their efficiency with protein antigens remains unclear. In the current study, the candidate antigen PA0833 from Pseudomonas aeruginosa was fused to the α-hemolysin mutant HlaH35A from Staphylococcus aureus to form a HlaH35A-PA0833 fusion protein (HPF). Immunization with HPF resulted in increased PA0833-specific antibody titers, higher protective efficacy, and decreased bacterial burden and pro-inflammatory cytokine secretion compared with PA0833 immunization alone. Using fluorescently labeled antigens to track antigen uptake and delivery, we found that HlaH35A fusion significantly improved antigen uptake in injected muscles and antigen delivery to draining lymph nodes. Both in vivo and in vitro studies demonstrated that the increased antigen uptake after immunization with HPF was mainly due to monocyte- and macrophage-dependent macropinocytosis, which was probably the result of HPF binding to ADAM10, the Hla host receptor. Furthermore, a transcriptome analysis showed that several immune signaling pathways were activated by HPF, shedding light on the mechanism whereby HlaH35A fusion improves immunogenicity. Finally, the improvement in immunogenicity by HlaH35A fusion was also confirmed with two other antigens, GlnH from Klebsiella pneumoniae and the model antigen OVA, indicating that HlaH35A could serve as a universal carrier protein to improve the immunogenicity of protein antigens. Author summary: Pore-forming toxins, a kind of exotoxins utilized by many pathogens as immune escaping weapons that targeting the immune cells and disturbing the immune system, are conventionally deemed as perfect antigens for vaccine development against infectious diseases. In this study, we reported that fusion of HlaH35A, a typical pore-forming toxin toxoid, to candidate protein antigens from different species resulted in improved immunogenicity and protective efficacy. The improvement was mainly due to the increased antigen uptake and activating of immune-associated signaling pathways, probably by targeting ADAM10, the receptor of Hla on host immune cells. The importance of this work was to demonstrate the possible mechanisms of that pore-forming toxin function as atypical carrier protein to improve the immunogenicity of other proteins and confirm the potential of non-conservative but highly immunogenic exotoxins derived from hyper-virulent clinical strains for application in rational antigen design and vaccines development. [ABSTRACT FROM AUTHOR]

Ixodes scapularis ticks feed on humans and other vertebrate hosts and transmit several pathogens of public health concern. Tick saliva is a complex mixture of bioactive proteins, lipids and immunomodulators, such as I. scapularis sphingomyelinase (IsSMase)‐like protein, an ortholog of dermonecrotoxin SMase D found in the venom of Loxosceles spp. of spiders. IsSMase modulates the host immune response towards Th2, which suppresses Th1‐mediated cytokines to facilitate pathogen transmission. Arboviruses utilize exosomes for their transmission from tick to the vertebrate host, and exosomes derived from tick saliva/salivary glands suppress C‐X‐C motif chemokine ligand 12 and interleukin‐8 immune response(s) in human skin to delay wound healing and repair processes. IsSMase affects also viral replication and exosome biogenesis, thereby inhibiting tick‐to‐vertebrate host transmission of pathogenic exosomes. In this review, we elaborate on exosomes and their biogenesis as potential candidates for developing novel control measure(s) to combat tick‐borne diseases. Such targets could help with the development of an efficient anti‐tick vaccine for preventing the transmission of tick‐borne pathogens. [ABSTRACT FROM AUTHOR]

Background: Porcine reproductive and respiratory syndrome (PRRS) is a threat to pig production worldwide. Our objective was to understand mechanisms of persistence of PRRS virus (PRRSV) in tonsil. Transcriptome data from tonsil samples collected at 42 days post infection (dpi) were generated by RNA-seq and NanoString on 51 pigs that were selected to contrast the two PRRSV isolates used, NVSL and KS06, high and low tonsil viral level at 42 dpi, and the favorable and unfavorable genotypes at a genetic marker (WUR) for the putative PRRSV resistance gene GBP5. Results: The number of differentially expressed genes (DEGs) differed markedly between models with and without accounting for cell-type enrichments (CE) in the samples that were predicted from the RNA-seq data. This indicates that differences in cell composition in tissues that consist of multiple cell types, such as tonsil, can have a large impact on observed differences in gene expression. Based on both the NanoString and the RNA-seq data, KS06-infected pigs showed greater activation, or less inhibition, of immune response in tonsils at 42 dpi than NVSL-infected pigs, with and without accounting for CE. This suggests that the NVSL virus may be better than the KS06 virus at evading host immune response and persists in tonsils by weakening, or preventing, host immune responses. Pigs with high viral levels showed larger CE of immune cells than low viral level pigs, potentially to trigger stronger immune responses. Presence of high tonsil virus was associated with a stronger immune response, especially innate immune response through interferon signaling, but these differences were not significant when accounting for CE. Genotype at WUR was associated with different effects on immune response in tonsils of pigs during the persistence stage, depending on viral isolate and tonsil viral level. Conclusions: Results of this study provide insights into the effects of PRRSV isolate, tonsil viral level, and WUR genotype on host immune response and into potential mechanisms of PRRSV persistence in tonsils that could be targeted to improve strategies to reduce viral rebreaks. Finally, to understand transcriptome responses in tissues that consist of multiple cell types, it is important to consider differences in cell composition. [ABSTRACT FROM AUTHOR]

There has been extensive progress in understanding the cellular and molecular mechanisms of inflammation and immune regulation in allergic diseases of the skin and lungs during the last few years. Asthma and atopic dermatitis (AD) are typical diseases of type 2 immune responses. interleukin (IL)‐25, IL‐33, and thymic stromal lymphopoietin are essential cytokines of epithelial cells that are activated by allergens, pollutants, viruses, bacteria, and toxins that derive type 2 responses. Th2 cells and innate lymphoid cells (ILC) produce and secrete type 2 cytokines such as IL‐4, IL‐5, IL‐9, and IL‐13. IL‐4 and IL‐13 activate B cells to class‐switch to IgE and also play a role in T‐cell and eosinophil migration to allergic inflammatory tissues. IL‐13 contributes to maturation, activation, nitric oxide production and differentiation of epithelia, production of mucus as well as smooth muscle contraction, and extracellular matrix generation. IL‐4 and IL‐13 open tight junction barrier and cause barrier leakiness in the skin and lungs. IL‐5 acts on activation, recruitment, and survival of eosinophils. IL‐9 contributes to general allergic phenotype by enhancing all of the aspects, such as IgE and eosinophilia. Type 2 ILC contribute to inflammation in AD and asthma by enhancing the activity of Th2 cells, eosinophils, and their cytokines. Currently, five biologics are licensed to suppress type 2 inflammation via IgE, IL‐5 and its receptor, and IL‐4 receptor alpha. Some patients with severe atopic disease have little evidence of type 2 hyperactivity and do not respond to biologics which target this pathway. Studies in responder and nonresponder patients demonstrate the complexity of these diseases. In addition, primary immune deficiency diseases related to T‐cell maturation, regulatory T‐cell development, and T‐cell signaling, such as Omenn syndrome, severe combined immune deficiencies, immunodysregulation, polyendocrinopathy, enteropathy, X‐linked syndrome, and DOCK8, STAT3, and CARD11 deficiencies, help in our understanding of the importance and redundancy of various type 2 immune components. The present review aims to highlight recent advances in type 2 immunity and discuss the cellular sources, targets, and roles of type 2 mechanisms in asthma and AD. [ABSTRACT FROM AUTHOR]

Fragmentation of the Golgi apparatus is observed during a number of physiological processes including mitosis and apoptosis, but also occurs in pathological states such as neurodegenerative diseases and some infectious diseases. Here we show that highly virulent strains of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, induce selective fragmentation of the trans-Golgi network (TGN) soon after infection of host cells by secretion of the effector protein Rickettsial Ankyrin Repeat Protein 2 (RARP2). Remarkably, this fragmentation is pronounced for the trans-Golgi network but the cis-Golgi remains largely intact and appropriately localized. Thus R. rickettsii targets specifically the TGN and not the entire Golgi apparatus. Dispersal of the TGN is mediated by the secreted effector protein RARP2, a recently identified type IV secreted effector that is a member of the clan CD cysteine proteases. Site-directed mutagenesis of a predicted cysteine protease active site in RARP2 prevents TGN disruption. General protein transport to the cell surface is severely impacted in cells infected with virulent strains of R. rickettsii. These findings suggest a novel manipulation of cellular organization by an obligate intracellular bacterium to determine interactions with the host cell. Author summary: Intracellular pathogens have evolved multiple mechanisms to modify the host cell to promote pathogen evasion of host defense mechanisms and enhance pathogen replication. Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, secretes a protease into the host cytosol to cause a fragmentation of the trans-Golgi network (TGN) with minimal effects on the cis-Golgi. Dispersal of the TGN disrupts trafficking of host cellular proteins to the plasma membrane. This includes inhibition of Major Histocompatibility Complex class I molecules to the cell surface thus providing R. rickettsii with a potential means of evading host immune surveillance. [ABSTRACT FROM AUTHOR]

Anaplasma phagocytophilum, an obligate intracellular bacterium that propagates within host granulocytes, is considered to modify the host intracellular environment for pathogenesis. However, the mechanism(s) underlying such host modifications remain unclear. Here, we aimed to investigate the relation between A. phagocytophilum and endoplasmic reticulum (ER) stress in THP‐1 cells. A. phagocytophilum activated the three ER stress sensors: inositol‐requiring enzyme‐1 (IRE1), protein kinase RNA‐like endoplasmic reticulum kinase (PERK), and activating transcription factor‐6 (ATF6). IRE1 activation occurred immediately after host cell invasion by A. phagocytophilum; however, the activated IRE1‐induced splicing of X‐box‐binding protein 1 was not promoted during A. phagocytophilum infection. This suppression was sustained even after the doxycycline‐mediated elimination of intracellular A. phagocytophilum. IRE1 knockdown accelerated A. phagocytophilum‐induced apoptosis and decreased intracellular A. phagocytophilum. These data suggest that A. phagocytophilum utilizes IRE1 activation to promote its own intracellular proliferation. Moreover, PERK and ATF6 partially mediated A. phagocytophilum‐induced apoptosis by promoting the expression of CCAAT/enhancer‐binding protein homologous protein, which induces the transcription of several proapoptotic genes. Thus, A. phagocytophilum possibly manipulates the host ER stress signals to facilitate intracellular proliferation and infection of surrounding cells before/after host cell apoptosis. [ABSTRACT FROM AUTHOR]

Atherosclerosis is a chronic autoimmune inflammatory disease that can cause coronary artery disease, stroke, peripheral artery disease, depending on which arteries are affected. At the beginning of atherosclerosis plasma lipoproteins accumulate in the sub-endothelial space. In response, monocytes migrate from the circulation through the endothelium into the intima where they differentiate into macrophages. These early events trigger a complex immune response that eventually involves many cellular subtypes of both innate and adaptive immunity. The Notch signaling pathway is an evolutionary conserved cell signaling system that mediates cell-to-cell communication. Recent studies have revealed that Notch modulate atherosclerosis by controlling macrophages polarization into M1 or M2 subtypes. Furthermore, it is known that Notch signaling controls differentiation and activity of T-helper and cytotoxic T-cells in inflammatory diseases. In this review, we will discuss the role of Notch in modulating immunity in the context of atherosclerosis and whether targeting Notch may represent a therapeutic strategy. [ABSTRACT FROM AUTHOR]

A single epitope of Leishmania analog of the receptors for activated C kinase (LACK) from Leishmania major, the polypeptide LACK156–173, is recognized by Vβ4+/Vα8+ T cells, and activate these cells that drives the subsequent T helper (Th)2 response. This study was undertaken to investigate the therapeutic potential of the LACK156–173 epitope in murine autoimmune arthritis models. To explore the influence of the LACK156–173 epitope on murine collagen antibody-induced arthritis, as well as its immunological mechanism, we vaccinated or treated mice with a LACK156–173 epitope expression plasmid or polypeptide. The effect of LACK156–173 epitope was then evaluated by clinical scores, histopathology, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Using flow cytometry, we measured the subsets and maturity of CD11c+ dendritic cells (DCs), as well as T cell polarization, in co-culture experiments. We also measured cytokine gene expression and production. The murine macrophage-like cell line RAW264.7 was used to identify the receptor for the epitope. Vaccination or treatment of the mice with the LACK156–173 epitope expression plasmid or polypeptide ameliorated the severity of arthritis. qRT-PCR analysis revealed that the LACK156–173 epitope improved the balance of effector T cells in synovial tissue compared to that in untreated arthritis controls. Toll-like receptor (TLR) 4 expression was diminished by LACK156–173. The epitope also influenced T cell polarization by regulating the differentiation, maturation, and functions of CD11c+ DCs and upregulating Jagged1 ligand expression. Blocking the mannose receptor (MR) significantly attenuated LACK156–173 epitope-induced macrophage activation. Our data indicate that vaccination or treatment with a single microorganism epitope, LACK156–173, is a highly efficient therapy for murine autoimmune arthritis. The therapeutic effects are mediated by the regulation of the differentiation, maturation, and functions of DCs via MR, resulting in the upregulation of Jagged1 expression and Th2 cell polarization. Our results demonstrate the therapeutic potential of the LACK156–173 epitope in rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitous transmembrane protein that functions as a “molecular scissor” to cleave the extracellular regions from its transmembrane target proteins. ADAM10 is well characterized as the ligand-dependent activator of Notch proteins, which control cell fate decisions. Indeed, conditional knockouts of ADAM10 in mice reveal impaired B-, T-, and myeloid cell development and/or function. ADAM10 cleaves many other leukocyte-expressed substrates. On B-cells, ADAM10 cleavage of the low-affinity IgE receptor CD23 promotes allergy and asthma, cleavage of ICOS ligand impairs antibody responses, and cleavage of the BAFF–APRIL receptor transmembrane activator and CAML interactor, and BAFF receptor, reduce B-cell survival. On microglia, increased ADAM10 cleavage of a rare variant of the scavenger receptor triggering receptor expressed on myeloid cells 2 may increase susceptibility to Alzheimer’s disease. We and others recently showed that ADAM10 interacts with one of six different regulatory tetraspanin membrane proteins, which we termed the TspanC8 subgroup, comprising Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33. The TspanC8s are required for ADAM10 exit from the endoplasmic reticulum, and emerging evidence suggests that they dictate ADAM10 subcellular localization and substrate specificity. Therefore, we propose that ADAM10 should not be regarded as a single scissor, but as six different scissors with distinct substrate specificities, depending on the associated TspanC8. In this review, we collate recent transcriptomic data to present the TspanC8 repertoires of leukocytes, and we discuss the potential role of the six TspanC8/ADAM10 scissors in leukocyte development and function. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a ‘cage’ around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection. [ABSTRACT FROM AUTHOR]

Human granulocytic anaplasmosis, caused by the tick-transmitted Anaplasma phagocytophilum, is not controlled by innate immunity, and induces a proinflammatory disease state with innate immune cell activation. In A. phagocytophilum murine infection models, hepatic injury occurs with production of IFNγ thought to be derived from NK, NKT cells, and CD8 T lymphocytes. Specific A. phagocytophilum ligands that drive inflammation and disease are not known, but suggest a clinical and pathophysiologic basis strikingly like macrophage activation syndrome (MAS) and hemophagocytic syndrome (HPS). We studied in vivo responses of NK, NKT, and CD8 T lymphocytes from infected animals for correlates of lymphocyte-mediated cytotoxicity and examined in vitro interactions with A. phagocytophilum-loaded antigen-presenting cells (APCs). Murine splenocytes were examined and found deficient in cytotoxicity as determined by CD107a expression in vitro for specific CTL effector subsets as determined by flow cytometry. Moreover, A. phagocytophilum-loaded APCs did not lead to IFNγ production among CTLs in vitro. These findings support the concept of impaired cytotoxicity with A. phagocytophilum presentation by APCs that express MHC class I and that interact with innate and adaptive immune cells with or after infection. The findings strengthen the concept of an enhanced proinflammatory phenotype, such as MAS and HPS disease states as the basis of disease and severity with A. phagocytophilum infection, and perhaps by other obligate intracellular bacteria. [ABSTRACT FROM AUTHOR]

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1β and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum. [ABSTRACT FROM AUTHOR]

Phages promise to be important and unprecedented bioresources for use as tools. Rapid identification of new phages is pivotal for phage resource collection. However, current methods for their characterization are usually laborious and complex. Conserved gene sharing by the same phage group can be exploited to expedite this process. In this study, mycobacteriophages were used as a model phage group to test this approach. First, we obtained the core genome of mycobacteriophages, which was divided into six groups based on homology. Second, we found that one kind of gene belonging to group A (TM4 gene 23-like) was conserved among all mycobacteriophages, and the primers targeting such genes were designed to interrogate the diversity of mycobacteriophages. The obtained data indicate that the conserved genes present in groups of closely-related phages could be used to simplify and streamline new phage identification. [ABSTRACT FROM AUTHOR]

Tick-borne encephalitis virus (TBEV) is a zoonotic pathogen which may cause tick-borne encephalitis (TBE) in humans and animals. More than 10,000 cases of TBE are reported annually in Europe and Asia. However, the knowledge on TBE in animals is limited. Co-infection with Anaplasma phagocytophilum and louping ill virus (LIV), a close relative to TBEV, in sheep has been found to cause more severe disease than single LIV or A. phagocytophilum infection. The aim of this study was to investigate TBEV infection and co-infection of TBEV and A. phagocytophilum in lambs. A total of 30 lambs, aged five to six months, were used. The experiment was divided into two. In part one, pre- and post-infection of TBEV and A. phagocytophilum was investigated (group 1 to 4), while in part two, co-infection of TBEV and A. phagocytophilum was investigated (group 5 and 6). Blood samples were drawn, and rectal temperature was measured daily. Lambs inoculated with TBEV displayed no clinical symptoms, but had a short or non-detectable viremia by reverse transcription real-time PCR. All lambs inoculated with TBEV developed neutralizing TBEV antibodies. Our study is in accordance with previous studies, and indicates that TBEV rarely causes symptomatic disease in ruminants. All lambs inoculated with A. phagocytophilum developed fever and clinical symptoms of tick-borne fever, and A. phagocytophilum was present in the blood samples of all infected lambs, shown by qPCR. Significantly higher mean TBEV titer was detected in the group co-infected with TBEV and A. phagocytophilum, compared to the groups pre- or post-infected with A. phagocytophilum. These results indicate that co-infection with TBEV and A. phagocytophilum in sheep stimulates an increased TBEV antibody response. [ABSTRACT FROM AUTHOR]