Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells

Background Using human brain microvascular endothelial cells (HBMECs) as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB) we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain). In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs) known as protease activated receptors (PARs) that might be implicated in calcium signaling by African trypanosomes. Methodology/Principal Findings Using RNA interference (RNAi) we found that in vitro PAR-2 gene (F2RL1) expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%–49%) and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Gαq with Pasteurella multocida toxin (PMT). PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain) and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. Conclusions/Significance Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Gαq-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.


Introduction
Human African trypanosomiasis (HAT), commonly called sleeping sickness, is a vector-borne disease for which death is inevitable if the patient is untreated [1,2,3]. HAT is caused by two subspecies of African trypanosomes, Trypanosoma brucei rhodesiense and T. b. gambiense causing East African and West African sleeping sickness, respectively. In classical late stage HAT (stage 2), the parasites invade the central nervous system (CNS) and the infected individuals suffer from progressive neurologic deterioration with concomitant psychiatric disorders, sleep disturbances, stupor, and coma. The role of the parasites in the pathogenesis of CNS lesions is not completely understood [4].
Using an in vitro model of the blood-brain barrier (BBB) consisting of human brain microvascular endothelial cells (HBMEC), we showed that human infective T. b. rhodesiense have a high potential for transendothelial migration, while animal infective T. b. brucei cross inefficiently [5]. We initially proposed that African trypanosome

HBMEC and trypanosomes
Primary HBMECs (# passage 13) were maintained as previously described [5,9,15]. The bloodstream form (BSF) T. b. rhodesiense used was originally obtained from the CSF from a Kenyan patient with sleeping sickness [5,9,10]. This parasite was formerly classified as T. b. gambiense IL1852, but has been reclassified as T. b. rhodesiense IL1852 based on the presence of the SRA gene [10]. The bloodstream form (BSF) trypanosomes were maintained in culture in HMI-9 [19]. For the microarray study, to inactivate brucipain activity, the parasites were pretreated for 30 min with 5 mM of the cathepsin-L inhibitor K11777 The parasites were then washed with medium to remove excess inhibitor prior to incubation with HBMEC monolayers [9,10].
Electrical cell-substrate impedance sensing for real-time transendothelial electrical resistance measurement and cell signaling The elevated transendothelial electrical resistance (TEER) and the lower paracellular permeability of the brain microvasculature is a characteristic feature that distinguishes it from non-brain endothelium. Measurement of TEER is a one of the most straightforward methods to access the barrier tightness using in vitro models [20]. Electrical Cell-Substrate Impedance Sensing (ECIS) gathers TEER data as an electrical method for assessing barrier function that detect changes in endothelial cell shape in real-time [21]. The Model 1600R ECIS system (Applied Biophysics) [22,23,24] was used to measure HBMEC TEER changes in real-time during exposure to African trypanosomes and their secreted products. HBMECs were grown on collagen-coated 8well single (8W1E) or multiple (8W10E + ) gold electrode ECIS arrays until confluent. While changes in relative TEER recorded by the single and multiple arrays were similar, absolute TEER values differ. Steady state TEER .10,000 ohms and .1,000 ohms were used for the 8W1E and 8W10E + arrays respectively (Applied Biophysics). The multiple electrode arrays in which HBMEC resistances are .1,000 ohms [4,5,25,26] record the activities of more cells over a larger region of the substrate. However, the 10fold lower capacitance of the 8W1E array leads to increased resistances (more than 10 times that of the multiple electrode arrays) and a higher signal to noise ratio (ECIS 1600R instruction manual). Changes in resistance of HBMEC monolayers were monitored every 80 sec in response to experimental variables. For the PMT experiments, HBMECs were simultaneously incubated with PMT (30 ng/ml) or pretreated with the toxin for 90 min then washed with fresh medium prior to incubation with parasites.

HBMEC gene silencing by RNAi and analysis by laser capture microdissection (LCM)
In single cell studies, when stimulated with PAR-2 agonists strong Ca 2+ signals are induced ( [15] in .60% of HBMEC (YV Kim, unpublished) suggesting a role for PAR-2 in parasite HBMEC traversal. We silenced the F2RL1 expression by co-transfecting a predesigned siRNA for F2RL1 and a GFP-expressing plasmid into

Author Summary
Human African trypanosomiasis, or sleeping sickness, occurs when single-cell trypanosome protozoan parasites spread from the blood to brain over the blood-brain barrier (BBB). This barrier is composed of brain microvascular endothelial cells (BMECs) especially designed to keep pathogens out. Safe drugs for treating sleeping sickness are lacking and alternative treatments are urgently required. Using our human BMEC BBB model, we previously found that a parasite protease, brucipain, induced calcium activation signals that allowed this barrier to open up to parasite crossing. Because human BMECs express protease-activated receptors (PARs) that trigger calcium signals in BMECs, we hypothesized a functional link between parasite brucipain and BMEC PARs. Utilizing RNA interference to block the production of one type of PAR called PAR-2, we hindered the ability of trypanosomes to both open up and cross human BMECs. Using geneprofiling methods to interrogate candidate BMEC pathways specifically triggered by brucipain, several pathways that potentially link brain inflammatory processes were identified, a finding congruent with the known role of PAR-2 as a mediator of inflammation. Overall, our data support a role for brucipain and BMEC PARs in trypanosome BBB transmigration, and as potential triggers for brain inflammation associated with the disease.
subconfluent HBMECs using Lipofectamine 2000 and standard protocols. A matched negative control siRNA (Ambion) was used as control. To determine the efficiency of gene silencing, HBMECs were grown on 35 mm LCM dishes (PALM) prior to RNAi silencing. Using laser capture microdissection (LCM), 15 individual GFP-HBMECs that expressed GFP in the F2RL1-silenced and control siRNA cultures were collected. The GFP-HBMECs were marked and catapulted using the following settings on the P.A.L.M. Microlaser (Bernied, Germany): energy-cut of 60; energy-lpc of 86; focus-cut of 75 and focus-lpc of 92 [27]. The RNA extracted using Ambion's RNAqueousH-Micro kit was then amplified using Ambion's MessageAmp TM II kit. qRT-PCR was done using predesigned F2RL1 primers (Invitrogen) and the data were normalized to ACTB transcripts.

Transcriptome microarray analysis
Samples from 2 independent experiments containing duplicate sets of infected (wild type or K11777 pretreated trypanosomes) and uninfected HBMECs were rapidly dissociated with trypsin/ EDTA, than washed. Total cellular RNA was isolated using the RNAeasy kit (QIAGEN) following the manufacturer's instructions. Purified RNA was treated with RNase-free DNase to remove contaminating genomic DNA. The integrity of RNA transcripts was verified by electrophoresis through denaturing agarose-formaldehyde gels followed by ethidium bromide staining [28].
cDNAs were radiolabeled with 32 P a-dCTP Isoblue (ICN) using SuperScript II Reverse Transcriptase (Invitrogen). Unbound label was separated using a Biospin P-30 spin column (Bio-Rad). Each cDNA probe was adjusted to 10 6 cpm/mL and hybridized to separate nylon MGC-1 microarrays [29] at 68uC overnight in Microhyb hybridization solution (Research Genetics). The MGC-1 microarray represents 9,600 different human gene features including those encoding cytokines and other immunological regulatory proteins such as chemokines, growth factors, and cellular receptors [29]. Membranes were washed three times in 26SSC (16SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% SDS for 30 min at 68uC and twice in 0.16 SSC-0.5% SDS for 30 min at 68uC. Membranes were exposed overnight and scanned on a Molecular Dynamics STORM phosphoimager set to 50micron resolution. mRNA expression levels were analyzed by scanning densitometry using ArrayPro imaging software. Differential patterns of gene expression were assessed by preparing RNA from both uninfected control HBMEC or HBMEC that were coincubated with wild-type (WT) T. b. rhodesiense IL1852 or T. b. rhodesiense K11777 inhibited for brucipain activity for 3 and 6 hours. These cDNAs were hybridized in parallel to pairs of identical microarrays.
Raw microarray data were subjected to Z normalization and tested for significant changes as previously described [30]. Genes were determined to be differentially expressed after calculating the Z ratio, which indicates the fold-difference between experimental groups, and false discovery rate (fdr), which controls for the expected proportion of falsely rejected hypotheses. Individual genes with p value #0.05, absolute value of Z ratio.1.5 and fdr,0.3 were considered significantly changed. Hierarchical cluster method with complete algorithm and K-mean cluster were employed to identify clustering within groups. Array data for each experimental condition were hierarchically clustered with the DIANE 6.0 software in JMP 6.0 environment and R programs. Pilot studies showed no signal detected when trypanosome cDNA was hybridized to the microarrays.
Probability scores for each network or functional Parametric analysis of gene set enrichment (PAGE ) [31] gene set analysis was performed by using the PAGE algorithm and PERL/R code with MYSQL database. The archive of gene sets used with this algorithm for this analysis is from the Molecular Signatures Database (MSigDB) (http://www.broad.mit.edu/gsea/msigdb/ genesets.jsp?collection=CP) [32]. Significance of functions and pathways was calculated using the z-test between each function group genes and the genes in the whole sample. The p-value was calculated by comparing the number of user-specified genes of interest participating in a given function or pathway relative to the total number of occurrences of these genes in all functional/ pathway annotations stored in the knowledge base. The Enrichment score, which is called pathway z-scores, were calculated by the difference of the mean z-ratio of the selected function groups with the mean of z-ratio of the whole sample genes to represent the pathway change altitude.
Array data from infected samples are presented as relative changes to uninfected controls in mRNA expression following normalization of gene signals to total signal and levels of housekeeping gene mRNA to ensure analysis of equivalent amounts of RNA. This approach facilitated the direct comparison of mRNA levels between control and the parasite-treated HBMEC.

Results/Discussion
The role of PAR-2 in trypanosome transmigration across HBMECs After silencing of F2RL1 by RNAi, based on qRT-PCR targeting F2RL1 transcripts normalized to ACTB (b-actin transcripts), we found that PAR-2 expression was reduced by over 95% (Fig 1A) in LCM-isolated HBMECs. To verify whether the ability of T. b. rhodesiense to cross HBMECs requires host cell PAR-2 signaling, we incubated T. b. rhodesiense IL1852 for 16h with HBMEC monolayers silenced for F2RL1 expression (F2RL1 siRNA), transfected with a matched scrambled siRNA control (control siRNA), or with untreated HBMECs (no Rx control) ( Fig 1B) and examined parasite transmigration. There was no statistical difference in parasite transmigration between the 2 control samples: in medium alone and or medium with control siRNA (p = 0.077; 2-tail Student t-test). Considering a 95% reduction in F2RL1 expression in the ,60% of the HBMEC that express PAR-2, significant differences were observed between F2RL1-silenced HBMECs compared to both control conditions: 39% inhibition (p = 0.040) versus untreated HBMECs (no Rx    The Ga-specific toxin from Pasteurella multocida blocks trypanosome-induced changes in TEER in HBMEC PARs, including PAR-2, are GPCRs known to mediate their cellular effects through the activation of Ga q/11 , Ga 12/13 and Ga i bc signaling pathways [33,34,35,36]. The protein toxin from Pasteurella multocida (PMT) has been shown to target Ga q [37,38,39], Ga 12/13 [40] and Ga i [41] heterotrimeric G proteins in eukaryotic cells. PMT potentiates Ga q protein-mediated GPCR responses to ligands by primarily activating phospholipase C (i.e. PLC-b1, 3,4) [37,38,39,42]. Accordingly, this leads to calcium mobilization and activation of PKCs, as well as activation of mitogenic pathways, including MAPK (ERK1/2, p38) activation [39,42,43]. PMT enters cells via receptor-mediated endocytosis and acts intracellularly to activate Ga q [37,38,39,44]. This is subsequently followed by uncoupling of Ga q signaling when cellular Ga q -mediated responses then become refractory to further stimulation [45,46]. This also occurs when HBMECs are incubated with PMT. As shown in Fig. 3A, when incubated together with HBMECs and T. b. rhodesiense, PMT (30 ng/ml) initially does not inhibit the parasite-induced drop in TEER by ECIS. However, TEER increases above control levels with parasites after 2 hours, consistent with uncoupling of Ga q signaling by PMT. When HBMECs are pretreated for 3h with PMT to allow for Ga q uncoupling prior to trypanosome addition, the toxin clearly inhibited the ability of the parasites to compromise the HBMEC monolayers ( Fig 3B). Since PAR-2 is a GPCR that can act via Gaq signaling, taken together, the data strongly suggest a role for PAR-2 and host Gaq-mediated calcium signaling in parasite interactions with the human BBB. While not yet tested would it be interesting to see the effect of PMT treatment on PAR-2 RNAi treated cells; i.e. are there additive effects of PMT treatment in these cells or is the PMT effect occluded by PAR-2 knockdown.
Gene expression pathways in HBMEC in response to African trypanosomes lacking brucipain activity PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles [47,48,49,50] and our data implicate a role for brucipain and PAR-2 in African trypanosome/HBMEC interactions. Therefore, we used transcription-profiling methods to interrogate candidate HBMEC pathways with particular attention paid to pathways that are specifically triggered by brucipain and that potentially link cellular processes to physiologic (i.e. CNS passage across the BBB) and pathophysiologic (neuroinflammation) processes associated with   CNS HAT [4]. T. b. rhodesiense inhibited for brucipain activity via pretreatment with K11777 (a cell-permeable class-specific irreversible inhibitor of brucipain) are defective in crossing HBMECs [5,9,10,16,17]. We interrogated candidate HBMEC pathways whose expression was modulated by exposure to T. b. rhodesiense pretreated with the brucipain inhibitor K11777. Because the halflife of brucipain is not known, the experimental time frame was kept to 6 hours, the approximate doubling time of the parasite. This was done to minimize the potential problems in data interpretation because of the contribution of decreasing drug within the parasites that were doubling. Gene set enrichment analysis of HBMEC based on the known canonical pathways showed that WT and modified African trypanosomes differentially altered the expression of genes represented in 99 pathways relative to the uninfected controls (Fig 4; Table 1). While the 99 pathways clustered into 15 groups according to their gene expression profiles, 28 pathways clustered into 4 unique cluster groups that were specifically expressed by HBMECs only after exposure to the K11777-pretreated trypanosomes (Table 1). A functional overview of the MSigDB gene sets was then done to categorize a small number of selected ''gene families'' whose members shared a common feature such as homology or biochemical activity, although not necessarily having common origins. Analysis of the gene functions within the HBMEC pathways showed 47 genes differentially expressed by HBMECs in response to trypanosome infection ( Table 2). From this gene subset, 30 genes were expressed exclusively by HBMEC in response to brucipain-inhibited parasites (Tables 2 and 3). Of these, 30 genes functionally i) 3 encoded cytokines, ii) 16 encoded transcription factors, iii) 8 encoded kinases, iv) 3 encoded for translocated genes, and v) 4 encoded for oncogenes. Interestingly, HBMEC genes encoding for cell surface markers and tumor suppressors in response to wild-type trypanosomes, were not expressed by brucipain-inhibited parasites ( Table 2).
Analysis of the HBMEC pathways specifically altered by the brucipain-inhibited parasites included those found only within down-regulated Clusters-1 and Cluster-15, or up-regulated Cluster-8 and Cluster-11. Cluster-1 consisted of 6 different pathways that had negative Z-score values (and were therefore positively effected by brucipain) by 3-hours, and that returned to control levels by 6h ( Figure 4, Table 1). While roles for the metabolic/degradation pathways in Cluster-1 in HAT are not clear, the data suggest brucipain alters the CARM1 and Leptin pathways. Leptin is a protein hormone typically produced by adipose tissue that is known to regulate appetite via binding to the leptin receptor (LEPR) in the hypothalamus after crossing the BBB [51,52]. It has been suggested that compromised leptin transport into the CNS resulting in low leptin levels in the hippocampus could lead to cognitive deficits [53]. There is evidence that the secretion of photoperiodic hormones such as melatonin is inversely regulated by leptin [54]. It is also tempting to speculate a role for leptin in HAT considering that disturbance in the circadian rhythm of the melatonin-generating systems in the pathogenesis of African sleeping sickness has been demonstrated [55].
Brucipain may also alter processes linked to arginine methylation. CARM1 is a protein arginine N-methlytransferase that plays a role in protein arginine methylation, a process that is implicated in signal transduction, nascent pre-RNA metabolism and transcriptional activation [56]. These data suggest that CARM1, as a promoter-specific regulator of NF-kB-dependent gene expression [57], could play a role in the inflammatory responses associated with CNS HAT.
In contrast to the Cluster-1 pathways, 3 hours exposure to the brucipain-inhibited trypanosomes upregulated 9 pathways represented in Cluster-8 ( Figure 1, Table 1). Inhibiting brucipain activity appeared to activate the eukaryotic initiation factor-2 (EIF2)-pathway. EIF2 binding to GTP and Met-tRNA would in turn initiate translation by transferring the Met-tRNA to the 40S ribosomal subunit. If brucipain plays a role in downregulating this pathway, the event could shut-down cellular protein synthesis and could have consequences to overall cell viability [58].
The response to activation of PAR-2 is the elevation of intracellular Ca 2+ via the PLC/IP 3 pathway [59,60], which leads to downstream increases in intracellular Ca 2+ , activation of PKC,   [47,48,49]. Because of the above characteristics associated with PAR-2 activation, it is remarkable that the most dramatic changes that happened between 3 and 6 hours in the pathways were associated with Cluster-11 and Cluster-15 (Table 1). Cluster-11 contained 4 pathways with strong positive Z-scores in response to the brucipain-inhibited trypanosomes. Of these 4 pathways, 3 were involved either with cell signaling (BREAST_CANCER_-ESTROGEN_SIGNALING) or inflammatory responses (CLAS-SIC_PATHWAY, IL7_PATHWAY). In contrast to Cluster 11, all pathways in Cluster 15 displayed negative Z-scores, indicating downregulation. In some respects, Cluster 15 is the most interesting as it contained pathways that conceivably play a role in parasite transmigration of the BBB as an early event, and in the subsequent later inflammatory responses associated with HAT. The involvement of the ST_GAQ_PATHWAY (Fig. 4, Table 1) is interesting given that calcium signaling may play an important role in trypanosome / BBB associated events. Furthermore, the changes in the ST_GA13-SA_TRKA_RECEPTOR, ST_DIC-TOYOSTELIUM_DISCOIDEUM_CAMP_CHEMOTAXIS, and CELL_2_CELL-pathways also parallel roles for PI3K, MAPK and cell cytoskeleton. A role for brucipain as an inducer of inflammatory responses [4] is also predicted (i.e., EPO_NFKB_PATHWAY, RNA_PATHWAY).

Conclusions
We studied parasite proteases in the interaction of T. b. rhodesiense with HBMECs. Overall, our RNAi, PMT and DNAmicroarray data support an important role for brucipain, HBMEC PAR-2 (and possibly other PARs) and G q signaling for trypanosome transmigration across the BBB. Owing to PAR-2's role in neuroinflammation, these data also suggest a role for this GPCR in CNS HAT. In murine models of HAT the neuroinflammatory response [1,65] is likely a balance between pro-inflammatory cytokines such as interferon-c (IFN-c), interleukin-1(IL-1) and tumour necrosis factor-a (TNF-a), and counter-inflammatory cytokines such as IL-10 [66]. A role for cytokines in determining entry of trypanosomes into the CNS was provided by a seminal study in knockout mice in which the gene for IFN-c had been disrupted [67]. Following systemic infection, it was found that trypanosomes accumulated in the perivascular regions, 'trapped' between the endothelial and the parenchymal basement membranes. While these findings suggested that lymphocyte-derived IFN-c is required for trypanosome traversal across cerebral blood vessels [67], precisely how IFN-c facilitates BBB traversal by the parasites has yet to be determined. Interestingly, minocycline, a tetracycline antibiotic, was also found to impede the penetration of leukocytes and trypanosomes into the brain parenchyma [68]. It is tempting to speculate a role for PAR-2 in these processes. It has been shown that the inflammatory response in mouse colonic tissue mediated by PAR-2 activation (using PAR-2 agonists) that leads to i) increased tissue permeability, ii) increased IFN-c and TNF-a expression, and iv) decreased IL-10 expression, are abolished in IFN-c deficient B6 mice [69]. More recently, minocycline has been shown to block the PAR-2-mediated TNFa-induced production of IL-8 proinflammatory response in epidermal keratinocytes (Ishikawa) [70].
Our previous work strongly suggested that T. b. rhodesiense crosses the human BBB by generating Ca 2+ activation signals in HBMECs through the activity of parasite cysteine proteases [5,9]. Using T. b. brucei silenced for (RNAi) it was later found that parasite cathepsin L (brucipain) could be the parasite factor initiating transmigration and increased vascular permeability [11]. While a singular role has not been established for this process of parasite transmigration in vivo, GPCR PAR2 is one of the molecular targets for brucipain as an activator of Gq-mediated calcium-signaling involving the downstream effectors PLC and PKC [5,9]. The in vivo consequence of these events, similar to our in vitro findings, is predicted to be increased permeability of the BBB to parasite transmigration, an event precursory to CNS Figure 5. Proposed model for African trypanosome-induced BBB dysfunction. We hypothesize that parasite proteases trigger GPCRs (i.e. PARs?) via Gaq activation, which leads to PLC-mediated Ca 2+ release from intracellular stores. The increase in intracellular calcium leads to calmodulin (CaM) activation of myosin light chain kinase (MLCK), ultimately leading to cytoskeletal changes and barrier dysfunction. Ca 2+ -independent activation of the cytoskeleton mediated by Ras-superfamily GTPases (i.e. RhoA) is also possible via p63RhoGEF. Parasite and/or host-derived proteases may also contribute by degrading or altering adherens junction (AJ) and tight junction (TJ) proteins. doi:10.1371/journal.pntd.0000479.g005 Role of PAR in African Trypanosome BBB Interaction www.plosntds.org disease [4,11]. The critical role of brucipain raises the possibility for this protein as an attractive drug or vaccine target.
Based on our published and findings reported here [4,5,9,10], we hypothesize that African trypanosome-mediated BBB dysfunction is linked to the interactions of parasite and/or parasite protease activity with BMEC GPCRs (i.e. PARs) (Fig. 5). Activation of downstream effectors then enable African trypanosome transmigration through the BBB after cytoskeletal rearrangements that induce cell retraction and loosening of junctional complexes. We predict that trypanosomes/parasite/proteases trigger Gaq activation of PLC-b, in turn generating inositol-1,4,5-triphosphate (IP 3 ) and diacylglycerol (DAG) from phosphatidylinositol-4,5-bisphosphate (PIP 2 ). Binding of IP 3 to its receptor on the endoplasmic reticulum releases Ca 2+ from intracellular stores. The increase in intracellular calcium leads to calmodulin (CaM) activation of myosin light chain (MLC) kinase (MLCK) and/or other effectors ultimately leading to cytoskeletal changes and barrier dysfunction. Ca 2+ -independent activation of the cytoskeleton by Ras-superfamily GTPases (i.e RhoA) traditionally by Ga 12/13 GPCRs, is also possible via Gaq activation of p63RhoGEF [42,71].
Although not yet determined, parasite and/or host-derived proteases may also contribute by degrading or altering adherens junction (AJ) and tight junction (TJ) proteins.
Clearly, a study on the contribution of the products of gene expression identified with the biochemical pathways needs to be investigated as well as testing using established animal models of HAT [4]. An understanding of these responses at a molecular level will help identify candidates for the early diagnosis, treatment, and prevention of CNS invasion with HAT.