Abstract

   One third of humans are infected lifelong with the brain-dwelling,
   protozoan parasite, Toxoplasma gondii. Approximately fifteen million of
   these have congenital toxoplasmosis. Although neurobehavioral disease
   is associated with seropositivity, causality is unproven. To better
   understand what this parasite does to human brains, we performed a
   comprehensive systems analysis of the infected brain: We identified
   susceptibility genes for congenital toxoplasmosis in our cohort of
   infected humans and found these genes are expressed in human brain.
   Transcriptomic and quantitative proteomic analyses of infected human,
   primary, neuronal stem and monocytic cells revealed effects on
   neurodevelopment and plasticity in neural, immune, and endocrine
   networks. These findings were supported by identification of protein
   and miRNA biomarkers in sera of ill children reflecting brain damage
   and T. gondii infection. These data were deconvoluted using three
   systems biology approaches: “Orbital-deconvolution” elucidated
   upstream, regulatory pathways interconnecting human susceptibility
   genes, biomarkers, proteomes, and transcriptomes.
   “Cluster-deconvolution” revealed visual protein-protein interaction
   clusters involved in processes affecting brain functions and circuitry,
   including lipid metabolism, leukocyte migration and olfaction. Finally,
   “disease-deconvolution” identified associations between the
   parasite-brain interactions and epilepsy, movement disorders,
   Alzheimer’s disease, and cancer. This “reconstruction-deconvolution”
   logic provides templates of progenitor cells’ potentiating effects, and
   components affecting human brain parasitism and diseases.

   Subject terms: Disease genetics, RNA sequencing, Predictive markers,
   Microbiology

Introduction

   The first half of the 20^th century achieved remarkable advances in
   control of some communicable diseases, with development of
   immunizations, antimicrobial therapies, and increasing ability to
   identify new pathogenic organisms^[87]1. The second half shifted to
   understanding chronic degenerative diseases as prevailing causes of
   death in older populations. One primary challenge for contemporary
   medicine is to control non-communicable diseases with complex
   gene-environment etiologies and progression^[88]2, [89]3, postulated to
   arise from complex interactive cascades of genetic and environmental
   factors. Historical efforts to find causes and cures for such
   illnesses, including brain diseases, have had a glaring omission of a
   significant environmental factor: Over 2 billion humans are infected
   with the neurotrophic parasite Toxoplasma gondii. Congenital and
   postnatal infections with T. gondii persist in all infected persons.
   The parasite interconverts between slow-growing, encysted bradyzoites
   and rapid-growing tachyzoites^[90]4. In mice, T. gondii creates a
   chronic intra-neuronal infection and an inflammatory process^[91]4.
   Mice with acute and chronic infection have alterations in
   neurotransmitters, memory, seizures, and neurobehaviors^[92]5, [93]6.
   Some epidemiologic-serologic studies show associations between
   seropositivity for T. gondii and human neurologic diseases, for
   example, Parkinson’s and Alzheimer’s diseases^[94]7, [95]8. Serologic
   studies of humans with diverse genetics are not optimal to detect
   strong associations or directionality. Epidemiologic associations also
   do not reveal parasite-modulated gene networks in human brain that
   could provide insights into how to cure and prevent resultant diseases.
   We need integrative approaches to examine relationships between brain
   parasitism and other brain diseases^[96]9, [97]10, to provide a
   foundation to identify key pathways and molecules for drug and vaccine
   design.

   To address these problems, we considered two central questions: (i) If
   chronic brain parasitism associates with other neurologic diseases,
   what are they? and (ii) Which macromolecular networks are modulated by
   the parasite in human brain that lead to neuropathology which could
   underpin and facilitate design of treatments? We hypothesized that a
   systems approach integrating multiple levels of host parasite
   interactions might resolve these questions. To gain insights into
   relationships between human T. gondii infections and brain disorders,
   we studied a unique cohort, the National Collaborative Chicago Based
   Congenital Toxoplasmosis Study (NCCCTS), and identified human
   susceptibility genes, as well as serologic biomarkers of active brain
   disease. The NCCCTS has diagnosed, treated and followed 246
   congenitally infected persons and their families continuously beginning
   in 1981^[98]11–[99]39. Next in our study, we obtained new
   transcriptomic and proteomic data from infections of primary, human,
   neuronal stem cells, and monocytic cells that infiltrate brain, to
   determine whether there are different phenotypic effects of Type I, II,
   or III T. gondii tachyzoites. We used these four data sets to construct
   an integrated molecular model of the infected human brain. In a second
   phase, the model was further analyzed using systems biology approaches
   to provide insights into the molecular mechanisms by which T. gondii
   infection may cause disease. The broader goal was to provide a robust
   database and informatic analysis for the scientific community to use
   for their research. This report described only a limited number of
   important observations. This effort to integrate multiple levels of
   intrinsic and extrinsic factors offers an original template to unravel
   pathogenesis of complex diseases in humans.

   Our studies presented here were performed to examine genetic and
   parasite effects we hypothesized would influence outcomes
   (Fig. [100]1a). We utilized a novel systems approach expanding from
   gene-environment paradigms to include a third component of development
   (Fig. [101]1a). In Fig. [102]1a, there is intersection/overlap between
   the circles representing components of the host-parasite interactions.
   These circles include host-parasite genetics, toxoplasmosis
   susceptibility genes, and serum biomarkers in children in the NCCCTS
   [T. gondii infection, red circle], human neuronal stem cell functional
   assays including transcriptomics and proteomics [Brain pathology
   mechanisms, blue circle], and disease pathogenesis/pathology
   susceptibility genes for other diseases [others, green circle]). This
   overlap/intersection in the Venn diagram indicates the circumstances in
   which we hypothesize that manifestations of other diseases will occur.
   To test this hypothesis, we isolated the infected brain as a system.
   This sequence of our work presented herein, and structure of our
   studies, and these results are shown in a flow chart (Fig. [103]1b),
   detailed outline (Fig. [104]1c), and schematic diagram of our model
   created based on this work (Fig. [105]1a and d). The overall plan was
   to gain access to the network interactome and biosignatures of T.
   gondii and the human brain, which first reconstructed the T. gondii
   brain infection. Our first steps of reconstructing T. gondii brain
   infection included discovery, integration and systems analysis of our
   original data. These systematic analyses of our novel human data sets
   used empiric studies of human T. gondii infections of persons with
   toxoplasmosis and their families, and infectomes of primary, human
   brain stem and monocytic cells (Fig. [106]1b,c). In our work, the human
   infectome is defined as the human host and parasite molecules, and
   pathways that are perturbed by the interaction of the human host and
   parasite T. gondii, as has been characterized by others for studies of
   other pathogens in earlier literature. An interactome is the whole set
   of molecular interactions such as protein-protein and genetic
   interactions. This can provide a global “Omic” view of molecular
   effects as in the resource APID interactome
   ([107]http://cicblade.dep.usal.es:8080/APID/init.action). To our
   knowledge, this study herein is the only single human cohort that is
   directly observed by a uniform group of examiners, longitudinally in a
   variety of ways, combined with human, primary, neuronal stem cell Omics
   Systems analysis for the T. gondii infectome, then interrogated for
   disease susceptibility genes/proteins. We obtained cellular data to
   test a few pathways relevant to pathogenesis. These first were
   considered individually and then together to determine whether we
   identified key biologic processes and biosignatures affected by T.
   gondii. Second, the integrated brain infectome became a global map that
   was deconvoluted to determine functional clusters and disease
   correlates (Fig. [108]1d). We termed this approach “Reconstruction and
   Deconvolution” (Fig. [109]1d). Specifically, Reconstruction was based
   on our unique cohort of infected persons, most of whom showed
   neuropathologic symptoms^[110]13, to identify novel human genetics of
   susceptibility to toxoplasmosis. Then, serologic biomarkers were
   studied for a limited number of infected humans to assay readout of an
   infected brain. We selected neural stem cells to uncover potential
   developmental mechanisms because of their multipotency central to
   neurodevelopment and neuroplasticity. Most brain diseases result from
   abnormal neurodevelopment (e.g., epilepsy) and neuroplasticity (e.g.,
   neurodegeneration). Hence, transcriptomic and proteomic infectomes of
   human neural stem cells were studied for parasite effects using primary
   cultures of cells from the hippocampus-temporal lobe. These datasets
   were integrated into a total brain infectome. They were unraveled, or
   “deconvoluted”, to identify functional and disease correlates. This was
   accomplished by analyzing upstream regulators in all our datasets.
   Thereby, we determined how different brain infectome components were
   interrelated. Cluster protein-protein interaction analysis revealed
   additional, functional correlates. Associations between the T. gondii
   brain infectome and other diseases that share these signature
   interactions were determined using our empirical, primary data.

Figure 1.

   Figure 1
   [111]Open in a new tab

   Methodology and analyses for understanding interaction of Toxoplasma
   gondii with human brain. (a) Gene-environment-pathology paradigm. The
   Venn diagram shows model of pathogenesis with confluence of permissive
   host and parasite genetics, and exposure. (b) Flow diagram of empirical
   genetic and biomarker data from NCCCTS, transcriptomics and proteomics.
   (c) Structure of the manuscript. This includes original empiric data,
   methods for analyses, and contributions of components to analyses in
   each figure. *Empiric but not from NCCCTS cohort; **Cell culture, IFA,
   microarray gene expression, mRNAseq, miRseq, quantitative proteomics,
   miR qPCR. d. Reconstruction and deconvolution analyses. Reconstruction
   is the discovery, integration and systems analysis of interrelatedness
   of four areas of primary, original data: genetics, transcriptomics and
   proteomics of infected cells and circulating serum biomarkers in ill
   persons. Deconvolution refers to the systems analysis that examines
   upstream regulatory genes, protein-protein cluster interactions and
   diseases with which biosignature pathways associate. These are the
   topics of the current work and are elaborated on throughout this
   manuscript. Image of family reproduced with their permission and also
   from “The Billion Brain Parasite”, Science Life (Easton, 2014).

Results

Reconstruction and deconvolution of Toxoplasma gondii brain infection

Reconstruction 1. Susceptibility genes expressed in human brain provide
insight into signature pathways of T. gondii in human brain

   Our unique NCCCTS cohort of persons with congenital toxoplasmosis and
   their families have been carefully characterized
   longitudinally^[112]13, [113]40–[114]44 (Fig. [115]1). Ongoing
   evaluations of this cohort contributes to the first phase of our
   present analyses as shown in the flow diagram in Fig. [116]1a.
   Figure [117]1b presents an outline of the work in this study including
   how the genetic and cohort analyses form a basis for the work.
   Figure [118]1c shows that these cohort and genetics analyses are
   integrated with other aspects of the current work in a model. Our
   NCCCTS cohort is the source of previously published
   analyses^[119]11–[120]39 which earlier provided a powerful tool to
   identify genes and pathways causing susceptibility to toxoplasmosis
   (Table [121]1^[122]11–[123]39, Figs [124]1 and [125]2). These
   susceptibility genes identified earlier are considered along with
   susceptibility genes newly identified herein. All these genes are part
   of our further analyses in this present manuscript. In our earlier
   work, the human susceptibility alleles of candidate genes identified
   for those in the NCCCTS were HLA Class I and II genes, ERAP 1, COL2A1,
   ABCA4, P2RX7, ALOX12, NALP1, IRAK4 (Table [126]1). Some of these
   gene/susceptibility associations were further confirmed using samples
   from the European Cohort study (EMSCOT)^[127]12, [128]14. One
   association, with NOD2^[129]39, was present in a Brazilian Cohort with
   eye disease, but not found in the NCCCTS. Characterization of
   mechanisms associated with ERAP1 were extended in studies of cross
   presentation of antigen^[130]45. Peptides interacting with MHC Class I
   genes of greater than usual octamer/nonamer lengths^[131]45 also were
   identified, suggesting that T. gondii subverts its host’s immune
   defenses with aberrant splicing of polypeptides^[132]46. These genetic
   data and their analysis are summarized in Table [133]1 and Fig. [134]2a
   and b. The newly identified genes or phenotypes identified herein are
   labeled “AM” or “OD” in Table [135]1. These susceptibility alleles
   indicated that the candidate genes were playing a role in
   susceptibility to toxoplasmosis and some have been studied for
   corresponding phenotypes to explain that susceptibility. The previously
   described genes are combined with newly identified genes for the
   analyses herein.

Table 1.

   Genes with Susceptibility/Resistance Alleles Defined with National
   Collaborative Toxoplasmosis Study and EMSCOT Cohorts.
   Gene SNP (Allele) P Value Reason Candidate Gene Replicate/Proof of
   Principle/Phenotype Reference/Supporting Data
   HLA Class II DQ3 <0.02 MD Hydrocephalous in children (DQ3), Fewer cysts
   in HLA transgenic mice (DQ1) [136]11
   DQ1 <0.0005
   COL2A1 rs6823 (G) <0.03 (brain) ED EMSCOT replicates, imprinted, brain
   and eye disease [137]12, [138]13
   rs2276455 (A) <0.03
   rs2276455 (G)* <0.0005
   rs1635544 (C) <0.03
   rs2070739 (T) <0.02
   rs2276454 (A) <0.007
   rs3803183 (T) <0.02
   rs3803183 (T)* <0.003
   ABCA4 rs952499 (C) <0.03 HC EMSCOT replicates, imprinted, localized in
   human brain [139]12, [140]13, HC
   rs952499 (T)* <0.005
   rs2297633 (G)* <0.0003
   rs1761375 (G)* <0.0001
   rs3112831 (C)* <0.02
   P2RX7 rs1621388(C1772T) <0.021 OI EMSCOT replicates for differing
   alleles; ATP mediated cell death, cytokine signaling, pro-inflammation
   [141]14, [142]15, OD
   rs1718119(T1068C) <0.015
   HLA Class I A <0.01 MD Genotype association and phenotypes humans and
   mice. PBMC from cohort. Peptides for HLA A2, A11, B7 confer protection
   [143]16–[144]22, OD
   B
   C
   ERAP1 rs149173(T/C) <0.0077 LfL Genotype association and phenotypes
   humans and mice [145]16, OD
   rs17481856(C/T) 0.0253
   IRAK4 rs1461567 <0.023 IOID Genotype; phenotype, cell death,
   inflammation [146]23
   rs4251513 <0.045
   NALP1 rs8081261 <0.002 MD TRNG Genotype (MD region; human); phenotype,
   cell death, inflammation; MD [147]24–[148]30
   rs11652907 <0.02
   rs9902174 <0.04
   ALOX12 rs6502997 <0.0003 MD TRNG Genotype and phenotype, cell death
   proinflammation [149]31
   rs312462 (C) <0.03
   rs6502998 (C) <0.03
   rs434473 <0.04
   TLR9 rs574386 (T1905C) <0.008 TLRs Brazil and Poland replicates;
   phenotype, ligand [150]32
   rs352140 (C) <0.0001 AM
   TIRAP rs8177374(S180L) <0.006 IOID Genotype; Phenotype TLR signaling
   and cytokines AM
   FOXQ1 rs920209 <0.02 HC Note NK cells mice [151]33, OD
   TREX1 rs 2242150(A/T) 0.02 SPD Related clinical & Type 1 IFN phenotype,
   LFL [152]34, [153]35, OD or AM
   NFκβ1 rs997476(C/A) <0.02 CtoPiEA Phenotype, nuclear localization,
   signaling pathway [154]36, OD
   TGFβ1 rs10417924 (G overtranscribed) 0.016 CtoPiEA, MD Phenotype
   transcriptomics, GRA1 [155]37, [156]38
   NOD2 (Brazil) rs3135499 (C/A)† <0.04 LfL Brazil Eye Disease, IL17, CD4+
   [157]39
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   Abbreviations: Reason for selection of gene to test sequentially as
   single candidate gene in NCCCTS (1981–2016): MD, Murine model data; ED,
   Eye disease in humans caused by mutation of this gene; HC,
   Hydrocephalous caused in humans by gene mutation and adult macular
   degeneration associated with allelic variants; OI, Implicated alleles
   for other infections; LfL, Logical to test from literature and other
   findings, for example MHC Class I presentation of antigen for ERAP1;
   IOID, Gene in other diseases in the literature; MD TRNG, Toxo 1 region,
   not gene initially in humans based on rat Toxo 1 region; TLRs, Testing
   TLR genes now replicated by other cohorts and proven to be important in
   mice; SPD, similar pattern of brain disease as AG brain disease due to
   DNA ligase mutations; CtoPIEA, Central to genetic pathway identified
   with original analysis led to TDT analysis herein. Note: LfL Brazil,
   Minas Gerais, did not replicate in US full cohort; no *, significant in
   NCCCTS not EMSCOT;* significant in EMSCOT cohort not NCCCTS. P values
   are nominal. Supporting Data [SD]: References (#) with narrative