Infection of Ixodes spp. tick cells with different Anaplasma phagocytophilum isolates induces the inhibition of apoptotic cell death

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The obligate intracellular pathogen, Anaplasma phagocytophilum, is the causative agent of life-threatening diseases in humans and animals. A. phagocytophilum is an emerging tick-borne pathogen in the United States, Europe, Africa and Asia, with increasing numbers of infected people and animals every year. It is increasingly recognized that intracellular pathogens modify host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. Recent reports have shown that amino acids are central to the host–pathogen metabolic interaction. In this study, a genome-wide search for components of amino acid metabolic pathways was performed in Ixodes scapularis, the main tick vector of A. phagocytophilum in the United States, for which the genome was recently published. The enzymes involved in the synthesis and degradation pathways of the twenty amino acids were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis amino acid metabolic pathway components in response to A. phagocytophilum infection of tick tissues and ISE6 tick cells. Our analysis was focused on the interplay between carbohydrate and amino acid metabolism during A. phagocytophilum infection in ISE6 cells. The results showed that tick cells increase the synthesis of phosphoenolpyruvate (PEP) from tyrosine to control A. phagocytophilum infection. Metabolic pathway analysis suggested that this is achieved by (i) increasing the transcript and protein levels of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), (ii) shunting tyrosine into the tricarboxylic acid (TCA) cycle to increase fumarate and oxaloacetate which will be converted into PEP by PEPCK-M, and (iii) blocking all the pathways that use PEP downstream gluconeogenesis (i.e., de novo serine synthesis pathway (SSP), glyceroneogenesis and gluconeogenesis). While sequestering host PEP may be critical for this bacterium because it cannot actively carry out glycolysis to produce PEP, excess of this metabolite may be toxic for A. phagocytophilum. The present work provides a more comprehensive view of the major amino acid metabolic pathways involved in the response to pathogen infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.

Section: Methodsmentioning

confidence: 99%

Ixodes scapularis Tick Cells Control Anaplasma phagocytophilum Infection by Increasing the Synthesis of Phosphoenolpyruvate from Tyrosine

Cabezas-Cruz

Espinosa

Obregón

et al. 2017

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“…The inhibitory effect of rabbit IgG antibodies against MSP4 and HSP70 recombinant proteins on A. phagocytophilum human NY18 (Asanovich et al, 1997) and sheep (Alberdi et al, 2015) isolates infection of HL60 human cells was conducted as described previously for tick cells (Villar et al, 2015b). The inhibitory effect of IgG antibodies purified from MSP4 and MSP4-HSP70 immunized and control sheep at 0 and 94 days post-infection (dpi) on A. phagocytophilum human NY18 isolate infection of HL60 human cells was conducted using the same experimental approach as for rabbit IgG.…”

Section: Methodsmentioning

confidence: 99%

“…Lambs were immunized three times on days 55, 30, and 10 before experimental infection with 1 ml doses of MSP4 (100 μg/dose), MSP4-HSP70 (100 μg of equal molar ratios of each protein/dose) or adjuvant/saline as control. The strain of A. phagocytophilum used for experimental infection originated from an infected lamb in Norway, which suffered TBF but was negative to other tick-borne pathogens (Alberdi et al, 2015; Stuen et al, 2015). The inoculum consisted of A. phagocytophilum infected heparinised blood that had been stored at −70°C with 10% dimethyl sulfoxide (DMSO).…”

Section: Methodsmentioning

confidence: 99%

Anaplasma phagocytophilum MSP4 and HSP70 Proteins Are Involved in Interactions with Host Cells during Pathogen Infection

Contreras

Alberdi

Mateos-Hernández

et al. 2017

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Anaplasma phagocytophilum transmembrane and surface proteins play a role during infection and multiplication in host neutrophils and tick vector cells. Recently, A. phagocytophilum Major surface protein 4 (MSP4) and Heat shock protein 70 (HSP70) were shown to be localized on the bacterial membrane, with a possible role during pathogen infection in ticks. In this study, we hypothesized that A. phagocytophilum MSP4 and HSP70 have similar functions in tick-pathogen and host-pathogen interactions. To address this hypothesis, herein we characterized the role of these bacterial proteins in interaction and infection of vertebrate host cells. The results showed that A. phagocytophilum MSP4 and HSP70 are involved in host-pathogen interactions, with a role for HSP70 during pathogen infection. The analysis of the potential protective capacity of MSP4 and MSP4-HSP70 antigens in immunized sheep showed that MSP4-HSP70 was only partially protective against pathogen infection. This limited protection may be associated with several factors, including the recognition of non-protective epitopes by IgG in immunized lambs. Nevertheless, these antigens may be combined with other candidate protective antigens for the development of vaccines for the control of human and animal granulocytic anaplasmosis. Focusing on the characterization of host protective immune mechanisms and protein-protein interactions at the host-pathogen interface may lead to the discovery and design of new effective protective antigens.

“…The I. scapularis embryo-derived tick cell line ISE6, provided by Ulrike Munderloh, University of Minnesota, USA, was cultured in L-15B300 medium as described previously (Kurtti et al, 1996 ; Munderloh et al, 1999 ; Villar et al, 2015a ). IRE/CTVM20 embryo-derived tick cells, provided by the Tick Cell Biobank, were maintained as described previously (Bell-Sakyi et al, 2007 ; Alberdi et al, 2015 ). Tick cells were first inoculated with A. phagocytophilum (human NY18 isolate; Asanovich et al, 1997 )-infected HL-60 cells and maintained according to Munderloh et al ( 1999 ).…”

Section: Methodsmentioning

confidence: 99%

Vaccinomics Approach to the Identification of Candidate Protective Antigens for the Control of Tick Vector Infestations and Anaplasma phagocytophilum Infection

Contreras

Alberdi

Mera

et al. 2017

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Anaplasma phagocytophilum is an emerging tick-borne pathogen causing human granulocytic anaplasmosis (HGA), tick-borne fever (TBF) in small ruminants, and other forms of anaplasmosis in different domestic and wild animals. The main vectors of this pathogen are Ixodes tick species, particularly I. scapularis in the United States and I. ricinus in Europe. One of the main limitations for the development of effective vaccines for the prevention and control of A. phagocytophilum infection and transmission is the identification of effective tick protective antigens. The objective of this study was to apply a vaccinomics approach to I. scapularis-A. phagocytophilum interactions for the identification and characterization of candidate tick protective antigens for the control of vector infestations and A. phagocytophilum infection. The vaccinomics pipeline included the use of quantitative transcriptomics and proteomics data from uninfected and A. phagocytophilum-infected I. scapularis ticks for the selection of candidate protective antigens based on the variation in tick mRNA and protein levels in response to infection, their putative biological function, and the effect of antibodies against these proteins on tick cell apoptosis and pathogen infection. The characterization of selected candidate tick protective antigens included the identification and characterization of I. ricinus homologs, functional characterization by different methodologies including RNA interference, immunofluorescence, gene expression profiling, and artificial tick feeding on rabbit antibodies against the recombinant antigens to select the candidates for vaccination trials. The vaccinomics pipeline developed in this study resulted in the identification of two candidate tick protective antigens that could be selected for future vaccination trials. The results showed that I. scapularis lipocalin (ISCW005600) and lectin pathway inhibitor (AAY66632) and I. ricinus homologs constitute candidate protective antigens for the control of vector infestations and A. phagocytophilum infection. Both antigens are involved in the tick evasion of host defense response and pathogen infection and transmission, but targeting different immune response pathways. The vaccinomics pipeline proposed here could be used to continue the identification and characterization of candidate tick protective antigens for the development of effective vaccines for the prevention and control of HGA, TBF, and other forms of anaplasmosis caused by A. phagocytophilum.