Central leptin signaling deficiency induced by leptin receptor antagonist leads to hypothalamic proteomic remodeling.

AIMS: Leptin irresponsiveness, which is often associated with obesity, can have significant impacts on the hypothalamic proteome of individuals, including those who are lean. While mounting evidence on leptin irresponsiveness has focused on obese individuals, understanding the early molecular and proteomic changes associated with deficient hypothalamic leptin signaling in lean individuals is essential for early intervention and prevention of metabolic disorders. Leptin receptor antagonists block the binding of leptin to its receptors, potentially reducing its effects and used in cases where excessive leptin activity might be harmful. MATERIALS AND METHODS: In this work, we blocked the central actions of leptin in lean male adult Wistar rat by chronically administering intracerebroventricularly the superactive leptin receptor antagonist (SLA) (D23L/L39A/D40A/F41A) and investigated its impact on the hypothalamic proteome using label-free sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) for quantitative proteomics. KEY FINDINGS: Our results show an accumulation of proteins involved in mRNA processing, mRNA stability, and translation in the hypothalamus of SLA-treated rats. Conversely, hypothalamic leptin signaling deficiency reduces the representation of proteins implicated in energy metabolism, neural circuitry, and neurotransmitter release. SIGNIFICANCE: The alterations in the adult rat hypothalamic proteome contribute to dysregulate appetite, metabolism, and energy balance, which are key factors in the development and progression of obesity and related metabolic disorders. Additionally, using bioinformatic analysis, we identified a series of transcription factors that are potentially involved in the upstream regulatory mechanisms responsible for the observed signature.

Subolesin knockdown in tick cells provides insights into vaccine protective mechanisms.

Ticks as obligate blood-feeding arthropod vectors of pathogenic viruses, bacteria, protozoa and helminths associated with prevalent tick-borne diseases (TBDs) worldwide. These arthropods constitute the second vector after mosquitoes that transmit pathogens to humans and the first vector in domestic animals. Vaccines constitute the safest and more effective approach to control tick infestations and TBDs, but research is needed to identify new antigens and improve vaccine formulations. The tick protein Subolesin (Sub) is a well-known vaccine protective antigen with a highly conserved sequence at both gene and protein levels in the Ixodidae and among arthropods and vertebrates. In this study, transcriptomics and proteomics analyses were conducted together with graph theory data analysis in wild type and Sub knockdown (KD) tick ISE6 cells in order to identify and characterize the functional implications of Sub in tick cells. The results support a key role for Sub in the regulation of gene expression in ticks and the relevance of this antigen in vaccine development against ticks and TBDs. Proteins with differential representation in response to Sub KD provide insights into vaccine protective mechanisms and candidate tick protective antigens.

Multi-omics analysis of zebrafish response to tick saliva reveals biological processes associated with alpha-Gal syndrome.

The alpha-Gal syndrome (AGS) is a tick-borne allergy. A multi-omics approach was used to determine the effect of tick saliva and mammalian meat consumption on zebrafish gut transcriptome and proteome. Bioinformatics analysis using R software was focused on significant biological and metabolic pathway changes associated with AGS. Ortholog mapping identified highly concordant human ortholog genes for the detection of disease-enriched pathways. Tick saliva treatment increased zebrafish mortality, incidence of hemorrhagic type allergic reactions and changes in behavior and feeding patterns. Transcriptomics analysis showed downregulation of biological and metabolic pathways correlated with anti-alpha-Gal IgE and allergic reactions to tick saliva affecting blood circulation, cardiac and vascular smooth muscle contraction, behavior and sensory perception. Disease enrichment analysis revealed downregulated orthologous genes associated with human disorders affecting nervous, musculoskeletal, and cardiovascular systems. Proteomics analysis revealed suppression of pathways associated with immune system production of reactive oxygen species and cardiac muscle contraction. Underrepresented proteins were mainly linked to nervous and metabolic human disorders. Multi-omics data revealed inhibition of pathways associated with adrenergic signaling in cardiomyocytes, and heart and muscle contraction. Results identify tick saliva-related biological pathways supporting multisystemic organ involvement and linking α-Gal sensitization with other illnesses for the identification of potential disease biomarkers.

Allergic reactions to tick saliva components in zebrafish model.

BACKGROUND: Alpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate. METHODS: Protein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA). RESULTS: Zebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles. CONCLUSIONS: Results support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate.

Low NETosis Induced in Anaplasma phagocytophilum-Infected Cells.

Anaplasma phagocytophilum are obligatory intracellular bacteria that preferentially replicate inside leukocytes by utilizing biological compounds and processes of these primary host defensive cells. In this study, bioinformatics analysis was conducted to further characterize A. phagocytophilum-host interactions using the neutrophil-like model of human Caucasian promyelocytic leukemia HL60 cells. We detected a hierarchy of molecules involved in A. phagocytophilum-HL60 interactions with overrepresentation in infected human cells of proteins involved in the reactive oxygen species (ROS) pathway and cell surface monocyte markers. As A. phagocytophilum phagocytosis by neutrophils is inhibited, the results suggested a possible explanation for our bioinformatics data: radical oxygen compounds could induce the killing of bacteria activating NETosis, a unique form of defense mechanism resulting in cell death that is characterized by the release of decondensed chromatin and granular contents to the extracellular space, forming neutrophil extracellular traps (NETs) to eliminate invading microorganisms. Thus, we confirmed the existence of a low NETosis induced in A. phagocytophilum-infected cells by immunofluorescence (IF) experiments. These results provide new insights into the complex mechanisms that govern immune response during A. phagocytophilum host interactions.

Correlates with Vaccine Protective Capacity and COVID-19 Disease Symptoms Identified by Serum Proteomics in Vaccinated Individuals.

In the last two years, the coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a scientific and social challenge worldwide. Vaccines have been the most effective intervention for reducing virus transmission and disease severity. However, genetic virus variants are still circulating among vaccinated individuals with different disease symptomatology. Understanding the protective- or disease-associated mechanisms in vaccinated individuals is relevant to advances in vaccine development and implementation. To address this objective, serum-protein profiles were characterized by quantitative proteomics and data-analysis algorithms in four cohorts of uninfected and SARS-CoV-2-infected vaccinated individuals with asymptomatic, non-severe, and severe disease symptomatology. The results show that immunoglobulins were the most overrepresented proteins in infected cohorts when compared to PCR-negative individuals. The immunoglobulin profile varied between different infected cohorts and correlated with protective- or disease-associated capacity. Overrepresented immunoglobulins in PCR-positive individuals correlated with protective response against SARS-CoV-2, other viruses, and thrombosis in asymptomatic cases. In non-severe cases, correlates of protection against SARS-CoV-2 and HBV together with risk of myasthenia gravis and allergy and autoantibodies were observed. Patients with severe symptoms presented risk for allergy, chronic idiopathic thrombocytopenic purpura, and autoantibodies. The analysis of underrepresented immunoglobulins in PCR-positive compared to PCR-negative individuals identified vaccine-induced protective epitopes in various coronavirus proteins, including the spike receptor-binding domain RBD. Non-immunoglobulin proteins were associated with COVID-19 symptoms and biological processes. These results evidence host-associated differences in response to vaccination and the possibility of improving vaccine efficacy against SARS-CoV-2.

Differentially Represented Proteins in Response to Infection with Mycobacterium tuberculosis Identified by Quantitative Serum Proteomics in Asian Elephants.

Tuberculosis is a major global concern. Tuberculosis in wildlife is a risk for zoonotic transmission and becoming one of the challenges for conservation globally. In elephants, the number of cases is likely rising. The aim of this study was to identify proteins related to tuberculosis infection in elephants, which could then be used for the development of diagnostic tools and/or vaccines. A serum proteomics approach was used to characterize differentially represented proteins in response to Mycobacterium tuberculosis in Asian elephants (Elaphas maximus). Blood samples were collected from eight elephants, four of which were antibody positive for tuberculosis and four were antibody negative. Proteomics analysis identified 26 significantly dysregulated proteins in response to tuberculosis. Of these, 10 (38%) were identified as immunoglobulin and 16 (62%) as non-immunoglobulin proteins. The results provided new information on the antibody response to mycobacterial infection and biomarkers associated with tuberculosis and protective response to mycobacteria in Asian elephants. Protective mechanisms included defense against infection (Alpha-1-B glycoprotein A1BG, Serpin family A member 1 SERPINA1, Transthyretin TTR), neuroprotection (TTR), and reduced risks of inflammation, infections, and cancer (SERPINA1, Keratin 10 KRT10). Using a translational biotechnology approach, the results provided information for the identification of candidate diagnostic, prognostic, and protective antigens for monitoring and control of tuberculosis in Asian elephants.

A Quantum Vaccinomics Approach Based on Protein-Protein Interactions.

Vaccines are the most effective preventive intervention to reduce the impact of infectious diseases worldwide. In particular, tick-borne diseases represent a growing burden for human and animal health worldwide and vaccines are the most effective and environmentally sound approach for the control of vector infestations and pathogen transmission. However, the development of effective vaccines for the control of tick-borne diseases with combined vector-derived and pathogen-derived antigens is one of the limitations for the development of effective vaccine formulations. Quantum biology arise from findings suggesting that living cells operate under non-trivial features of quantum mechanics, which has been proposed to be involved in DNA mutation biological process. Then, the electronic structure of the molecular interactions behind peptide immunogenicity led to quantum immunology and based on the definition of the photon as a quantum of light, the immune protective epitopes were proposed as the immunological quantum. Recently, a quantum vaccinomics approach was proposed based on the characterization of the immunological quantum to further advance the design of more effective and safe vaccines. In this chapter, we describe methods of the quantum vaccinomics approach based on proteins with key functions in cell interactome and regulome of vector-host-pathogen interactions for the identification by yeast two-hybrid screen and the characterization by in vitro protein-protein interactions and musical scores of protein interacting domains, and the characterization of conserved protective epitopes in protein interacting domains. These results can then be used for the design and production of chimeric protective antigens.

Akirin/Subolesin regulatory mechanisms at host/tick-pathogen interactions.

Ticks and tick-borne pathogens such as Anaplasma phagocytophilum affect human and animal health worldwide and thus the characterization of host/tick-pathogen interactions is important for the control of tick-borne diseases. The vertebrate regulatory proteins Akirins and its tick ortholog, Subolesin, are conserved throughout the metazoan and involved in the regulation of different biological processes such as immune response to pathogen infection. Akirin/Subolesin have a key role in host/tick-pathogen interactions and exert its regulatory function primarily through interacting proteins such as transcription factors, chromatin remodelers and RNA-associated proteins. Recent results have provided evidence of akirin/subolesin genetic interactions and the interaction of Akirin/Subolesin with histones, thus suggesting a role in direct chromatin remodeling. Finally, and still to be proven, some models suggest the possibility of direct Akirin/Subolesin protein interactions with DNA. Future research should advance the characterization of Akirin/Subolesin interactome and its functional role at the host/tick-pathogen interface. These results have implications for translational biotechnology and medicine for the development of new effective interventions for the control of ticks and tick-borne diseases.

Characterization by Quantitative Serum Proteomics of Immune-Related Prognostic Biomarkers for COVID-19 Symptomatology.

The COVID-19 pandemic caused by SARS-CoV-2 challenges the understanding of factors affecting disease progression and severity. The identification of prognostic biomarkers and physiological processes associated with disease symptoms is relevant for the development of new diagnostic and therapeutic interventions to contribute to the control of this pandemic. To address this challenge, in this study, we used a quantitative proteomics together with multiple data analysis algorithms to characterize serum protein profiles in five cohorts from healthy to SARS-CoV-2-infected recovered (hospital discharge), nonsevere (hospitalized), and severe [at the intensive care unit (ICU)] cases with increasing systemic inflammation in comparison with healthy individuals sampled prior to the COVID-19 pandemic. The results showed significantly dysregulated proteins and associated biological processes and disorders associated to COVID-19. These results corroborated previous findings in COVID-19 studies and highlighted how the representation of dysregulated serum proteins and associated BPs increases with COVID-19 disease symptomatology from asymptomatic to severe cases. The analysis was then focused on novel disease processes and biomarkers that were correlated with disease symptomatology. To contribute to translational medicine, results corroborated the predictive value of selected immune-related biomarkers for disease recovery [Selenoprotein P (SELENOP) and Serum paraoxonase/arylesterase 1 (PON1)], severity [Carboxypeptidase B2 (CBP2)], and symptomatology [Pregnancy zone protein (PZP)] using protein-specific ELISA tests. Our results contributed to the characterization of SARS-CoV-2-host molecular interactions with potential contributions to the monitoring and control of this pandemic by using immune-related biomarkers associated with disease symptomatology.

Function of cofactor Akirin2 in the regulation of gene expression in model human Caucasian neutrophil-like HL60 cells.

The Akirin family of transcription cofactors are involved throughout the metazoan in the regulation of different biological processes such as immunity, interdigital regression, muscle and neural development. Akirin do not have catalytic or DNA-binding capability and exert its regulatory function primarily through interacting proteins such as transcription factors, chromatin remodelers, and RNA-associated proteins. In this study, we focused on the human Akirin2 regulome and interactome in neutrophil-like model human Caucasian promyelocytic leukemia HL60 cells. Our hypothesis is that metazoan evolved to have Akirin2 functional complements and different Akirin2-mediated mechanisms for the regulation of gene expression. To address this hypothesis, experiments were conducted using transcriptomics, proteomics and systems biology approaches in akirin2 knockdown and wildtype HL60 cells to characterize Akirin2 gene/protein targets, functional complements and to provide evidence of different mechanisms that may be involved in Akirin2-mediated regulation of gene expression. The results revealed Akirin2 gene/protein targets in multiple biological processes with higher representation of immunity and identified immune response genes as candidate Akirin2 functional complements. In addition to linking chromatin remodelers with transcriptional activation, Akirin2 also interacts with histone H3.1 for regulation of gene expression.

Function of cofactor Akirin2 in the regulation of gene expression in model human Caucasian neutrophil-like HL60 cells.

The Akirin family of transcription cofactors are involved throughout the metazoan in the regulation of different biological processes (BPs) such as immunity, interdigital regression, muscle and neural development. Akirin do not have catalytic or DNA-binding capability and exert its regulatory function primarily through interacting proteins such as transcription factors, chromatin remodelers, and RNA-associated proteins. In the present study, we focused on the human Akirin2 regulome and interactome in neutrophil-like model human Caucasian promyelocytic leukemia HL60 cells. Our hypothesis is that metazoan evolved to have Akirin2 functional complements and different Akirin2-mediated mechanisms for the regulation of gene expression. To address this hypothesis, experiments were conducted using transcriptomics, proteomics and systems biology approaches in akirin2 knockdown and wildtype (WT) HL60 cells to characterize Akirin2 gene/protein targets, functional complements and to provide evidence of different mechanisms that may be involved in Akirin2-mediated regulation of gene expression. The results revealed Akirin2 gene/protein targets in multiple BPs with higher representation of immunity and identified immune response genes as candidate Akirin2 functional complements. In addition to linking chromatin remodelers with transcriptional activation, Akirin2 also interacts with histone H3.1 for regulation of gene expression.

Comparative analysis of Rhipicephalus tick salivary gland and cement elementome.

Rhipicephalus spp. (Acari: Ixodidae) ticks are obligate hematophagous arthropods, which constitute a model for the study of vector-host interactions. The chemical composition or elementome of salivary glands (SG) and cement provides information relevant for the study of protein-based complex multifunctional tissues with a key role in tick biology. In this study, we characterized the elementome of cement cones in Rhipicephalus sanguineus collected from naturally infested dogs and in SG and cement of R. bursa collected from experimentally infested rabbits at different feeding stages. The elementome was characterized using scanning electron microscopy (SEM) combined with energy dispersive X-ray spectroscopy (EDS). The results showed the identification of up to 14 chemical elements in the cement, and suggested tick/host-driven differences in the cement elementome between tick species and between SG and cement within the same species. By still unknown mechanisms, ticks may regulate cement elementome during feeding to affect various biological processes. Although these analyses are preliminary, the results suggested that N is a key component of the cement elementome with a likely origin in SG/salivary proteins (i.e., Glycine (C2H5NO2)-rich superfamily member proteins; GRPs) and other tick/host-derived components (i.e. NAPDH). Future research should be focused on tick elementome and its functional implications to better understand cement structure and function.

Tick Importin-α Is Implicated in the Interactome and Regulome of the Cofactor Subolesin.

Ticks and tick-borne diseases (TBDs) represent a burden for human and animal health worldwide. Currently, vaccines constitute the safest and most effective approach to control ticks and TBDs. Subolesin (SUB) has been identified as a vaccine antigen for the control of tick infestations and pathogen infection and transmission. The characterization of the molecular function of SUB and the identification of tick proteins interacting with SUB may provide the basis for the discovery of novel antigens and for the rational design of novel anti-tick vaccines. In the present study, we used the yeast two-hybrid system (Y2H) as an unbiased approach to identify tick SUB-interacting proteins in an Ixodes ricinus cDNA library, and studied the possible role of SUB as a chromatin remodeler through direct interaction with histones. The Y2H screening identified Importin-α as a potential SUB-interacting protein, which was confirmed in vitro in a protein pull-down assay. The sub gene expression levels in tick midgut and fat body were significantly higher in unfed than fed female ticks, however, the importin-α expression levels did not vary between unfed and fed ticks but tended to be higher in the ovary when compared to those in other organs. The effect of importin-α RNAi was characterized in I. ricinus under artificial feeding conditions. Both sub and importin-α gene knockdown was observed in all tick tissues and, while tick weight was significantly lower in sub RNAi-treated ticks than in controls, importin-α RNAi did not affect tick feeding or oviposition, suggesting that SUB is able to exert its function in the absence of Importin-α. Furthermore, SUB was shown to physically interact with histone 4, which was corroborated by protein pull-down and western blot analysis. These results confirm that by interacting with numerous tick proteins, SUB is a key cofactor of the tick interactome and regulome. Further studies are needed to elucidate the nature of the SUB-Importin-α interaction and the biological processes and functional implications that this interaction may have.

Coronavirus in cat flea: findings and questions regarding COVID-19.

The coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. Recent evidence raised the question about the possibility that cats may be a domestic host for SARS-CoV-2 with unknown implications in disease dissemination. Based on the fact that the domestic cat flea, Ctenocephalides felis, are abundant ectoparasites infesting humans, companion animals and wildlife and that coronavirus-like agents have been identified in the ectoparasite tick vector, Ixodes uriae of seabirds, herein we considered the presence of coronaviruses in general and SARS-CoV-2 in particular in C. felis. We identified coronavirus-derived and cell receptor angiotensin-converting enzyme RNA/proteins in C. felis. Although current evidence suggests that pets are probably dead-end-hosts with small risk of transmission to humans, our results suggested that cat flea may act as biological and/or mechanical vectors of SARS-CoV. Although preliminary, these results indicate a possibility of ectoparasites acting as reservoirs and vectors of SARS-CoV and related beta-coronavirus although with little disease risk due to systemic transmission route, low viremia, virus attenuation or other unknown factors. These results support the need to further study the role of animal SARS-CoV-2 hosts and their ectoparasite vectors in COVID-19 disease spread.

Tick and Host Derived Compounds Detected in the Cement Complex Substance.

Ticks are obligate hematophagous arthropods and vectors of pathogens affecting human and animal health worldwide. Cement is a complex protein polymerization substance secreted by ticks with antimicrobial properties and a possible role in host attachment, sealing the feeding lesion, facilitating feeding and pathogen transmission, and protection from host immune and inflammatory responses. The biochemical properties of tick cement during feeding have not been fully characterized. In this study, we characterized the proteome of Rhipicephalus microplus salivary glands (sialome) and cement (cementome) together with their physicochemical properties at different adult female parasitic stages. The results showed the combination of tick and host derived proteins and other biomolecules such as α-Gal in cement composition, which varied during the feeding process. We propose that these compounds may synergize in cement formation, solidification and maintenance to facilitate attachment, feeding, interference with host immune response and detachment. These results advanced our knowledge of the complex tick cement composition and suggested that tick and host derived compounds modulate cement properties throughout tick feeding.

Allergic Reactions and Immunity in Response to Tick Salivary Biogenic Substances and Red Meat Consumption in the Zebrafish Model.

Ticks are arthropod ectoparasite vectors of pathogens and the cause of allergic reactions affecting human health worldwide. In humans, tick bites can induce high levels of immunoglobulin E antibodies against the carbohydrate Galα1-3Galß1-(3)4GlcNAc-R (α-Gal) present in glycoproteins and glycolipids from tick saliva that mediate anaphylactic reactions known as the alpha-Gal syndrome (AGS) or red meat allergy. In this study, a new animal model was developed using zebrafish for the study of allergic reactions and the immune mechanisms in response to tick salivary biogenic substances and red meat consumption. The results showed allergic hemorrhagic anaphylactic-type reactions and abnormal behavior patterns likely in response to tick salivary toxic and anticoagulant biogenic compounds different from α-Gal. However, the results showed that only zebrafish previously exposed to tick saliva developed allergic reactions to red meat consumption with rapid desensitization and tolerance. These allergic reactions were associated with tissue-specific Toll-like receptor-mediated responses in types 1 and 2 T helper cells (TH1 and TH2) with a possible role for basophils in response to tick saliva. These results support previously proposed immune mechanisms triggering the AGS and provided evidence for new mechanisms also potentially involved in the AGS. These results support the use of the zebrafish animal model for the study of the AGS and other tick-borne allergies.

A Novel Combined Scientific and Artistic Approach for the Advanced Characterization of Interactomes: The Akirin/Subolesin Model.

The main objective of this study was to propose a novel methodology to approach challenges in molecular biology. Akirin/Subolesin (AKR/SUB) are vaccine protective antigens and are a model for the study of the interactome due to its conserved function in the regulation of different biological processes such as immunity and development throughout the metazoan. Herein, three visual artists and a music professor collaborated with scientists for the functional characterization of the AKR2 interactome in the regulation of the NF-κB pathway in human placenta cells. The results served as a methodological proof-of-concept to advance this research area. The results showed new perspectives on unexplored characteristics of AKR2 with functional implications. These results included protein dimerization, the physical interactions with different proteins simultaneously to regulate various biological processes defined by cell type-specific AKR-protein interactions, and how these interactions positively or negatively regulate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway in a biological context-dependent manner. These results suggested that AKR2-interacting proteins might constitute suitable secondary transcription factors for cell- and stimulus-specific regulation of NF-κB. Musical perspective supported AKR/SUB evolutionary conservation in different species and provided new mechanistic insights into the AKR2 interactome. The combined scientific and artistic perspectives resulted in a multidisciplinary approach, advancing our knowledge on AKR/SUB interactome, and provided new insights into the function of AKR2-protein interactions in the regulation of the NF-κB pathway. Additionally, herein we proposed an algorithm for quantum vaccinomics by focusing on the model proteins AKR/SUB.

The redox metabolic pathways function to limit Anaplasma phagocytophilum infection and multiplication while preserving fitness in tick vector cells.

Aerobic organisms evolved conserved mechanisms controlling the generation of reactive oxygen species (ROS) to maintain redox homeostasis signaling and modulate signal transduction, gene expression and cellular functional responses under physiological conditions. The production of ROS by mitochondria is essential in the oxidative stress associated with different pathologies and in response to pathogen infection. Anaplasma phagocytophilum is an intracellular pathogen transmitted by Ixodes scapularis ticks and causing human granulocytic anaplasmosis. Bacteria multiply in vertebrate neutrophils and infect first tick midgut cells and subsequently hemocytes and salivary glands from where transmission occurs. Previous results demonstrated that A. phagocytophilum does not induce the production of ROS as part of its survival strategy in human neutrophils. However, little is known about the role of ROS during pathogen infection in ticks. In this study, the role of tick oxidative stress during A. phagocytophilum infection was characterized through the function of different pathways involved in ROS production. The results showed that tick cells increase mitochondrial ROS production to limit A. phagocytophilum infection, while pathogen inhibits alternative ROS production pathways and apoptosis to preserve cell fitness and facilitate infection. The inhibition of NADPH oxidase-mediated ROS production by pathogen infection appears to occur in both neutrophils and tick cells, thus supporting that A. phagocytophilum uses common mechanisms for infection of ticks and vertebrate hosts. However, differences in ROS response to A. phagocytophilum infection between human and tick cells may reflect host-specific cell tropism that evolved during pathogen life cycle.

Anaplasma phagocytophilum modifies tick cell microRNA expression and upregulates isc-mir-79 to facilitate infection by targeting the Roundabout protein 2 pathway.

The microRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms including innate and adaptive immune pathways to control bacterial, parasite and viral infections, and pathogens could modify host miRNA profile to facilitate infection and multiplication. Therefore, understanding the function of host miRNAs in response to pathogen infection is relevant to characterize host-pathogen molecular interactions and to provide new targets for effective new interventions for the control infectious diseases. The objective of this study was to characterize the dynamics and functional significance of the miRNA response of the tick vector Ixodes scapularis in response to Anaplasma phagocytophilum infection, the causative agent of human and animal granulocytic anaplasmosis. To address this objective, the composition of tick miRNAs, functional annotation, and expression profiling was characterized using high throughout RNA sequencing in uninfected and A. phagocytophilum-infected I. scapularis ISE6 tick cells, a model for tick hemocytes involved in pathogen infection. The results provided new evidences on the role of tick miRNA during pathogen infection, and showed that A. phagocytophilum modifies I. scapularis tick cell miRNA profile and upregulates isc-mir-79 to facilitate infection by targeting the Roundabout protein 2 (Robo2) pathway. Furthermore, these results suggested new targets for interventions to control pathogen infection in ticks.