Mass spectrometry identifies Taenia solium proteins in sera of patients with and without parenchymal neurocysticercosis

Abstract

Neurocysticercosis (NCC), a major cause of global acquired epilepsy, results from Taenia solium larval brain infection. T. solium adult worm releases large numbers of infective eggs into the environment contributing to high levels of exposure in endemic areas.

This study identifies T. solium proteins in the sera of individuals with and without NCC using mass spectrometry to examine exposure in endemic regions. Forty-seven patients (18–51 years), 24 parenchymal NCC (pNCC), 8 epilepsy of unknown etiology, 7 glioma, 8 brain tuberculoma and 7 healthy volunteers were studied. Trypsin digested sera were subject to liquid chromatography-tandem mass spectrometry and spectra of 375 to 1700 m/z matched against T. solium WormBase ParaSite database with Maxquant software to identify T. solium proteins.

Three hundred and nineteen T. solium proteins were identified in 87.5% of pNCC and 56.6% of non-NCC subjects. three hundred and four proteins were exclusive to pNCC sera, 7 to non-NCC sera, and 8 in both. Ten percent, exhibiting immune-modulatory properties, originated from the oncosphere and cyst vesicular fluid.

In conclusion, in endemic regions T. solium proteins are detected in sera of individuals with and without pNCC. The immunomodulatory nature of these proteins may influence susceptibility and course of infection.

Introduction:

The taeniasis/cysticercosis complex, caused by the parasite Taenia solium, constitutes a significant public health and economic burden in Low- and Middle-Income Countries across Africa, Asia, and Latin America.^1–2 The life cycle of T. solium involves humans and pigs, with the adult worm residing in the human small intestine (taeniasis) and producing thousands of eggs that are excreted in feces. Environmental contamination occurs through open defecation, with eggs surviving for extended periods.^3–5 In regions where open defecation is common, pigs are exposed to eggs while foraging, and humans become infected through ingestion of contaminated food or water or via person-to-person contact.⁴ Ingested eggs release oncospheres in the intestine, which develop into larvae in various tissues, including muscle and the brain (Neurocysticercosis: NCC).⁵

The prevalence of taeniasis in endemic populations varies widely, ranging from 0% to 17.25% ⁶, with NCC identified as a lead cause of acquired epilepsy in endemic area.⁷ Factors influencing cysticercosis prevalence include environmental egg contamination, host susceptibility, and immunological response to parasite antigens.^8–9 Acquired immunity may limit invasive infection, as evidenced by variations in exposure versus infection rates in different populations.¹⁰

T. solium proteins possess immune modulatory properties, potentially affecting infection outcomes.^11–13 These proteins may sensitise hosts to infection, thereby modulating infection rates in endemic regions. Some of these parasite proteins may be present at very low levels in the host and hence not detected by standard methods either in epidemiological studies or during diagnosis of the infection. Thus, while the T. solium antigen enzyme-linked immunosorbent assay (AgELISA) is commonly used for NCC diagnosis,^14–15 it may lack sensitivity for detecting low concentrations of parasite proteins in the serum resulting from exposure without infection.¹⁶

Recent advances in mass spectrometry allow high resolution of novel proteins from different sources including serum that have enabled better understanding of molecular mechanisms of pathogenesis.^17–18 Mass spectrometric proteomic characterization of excretory/secretory proteins from several helminths, including T. solium cyst fluid and oncosphere, has been carried out to understand their biology/pathogenesis, to identify associated diagnostic markers, new drug targets and potential vaccine targets.^19–23 However, these studies are based on examining the parasites themselves and not on identifying parasite molecules circulating in a patient’s bloodstream. This preliminary study aimed to identify T. solium proteins in the serum of infected (NCC) and non-infected (non-NCC) individuals residing in endemic regions using high-resolution tandem mass spectrometry. This would be towards understanding how endemicity of T. solium contributes to host immune responses and NCC pathogenesis.

Methods

Study population

Forty-seven patients with CNS disorders and seven healthy volunteers recruited to the study were residents of India. All study subjects were aged between 18 and 51 years and patients were recruited from the outpatient clinics of the Department of Neurological Sciences, Christian Medical College, Vellore. Informed consent was obtained from all subjects. The study protocol was approved by the Institutional Review Boards of Christian Medical College, Vellore, India and of the University of Oklahoma Health Sciences Center, USA.

The study groups included 24 parenchymal NCC (pNCC) patients who fulfilled the criteria for definitive diagnosis for NCC ²⁴ and 23 non-NCC epilepsy patients. Of the pNCC patients, 8 patients each had multiple granulomas (GMNCC), solitary cysticercus granuloma (SCG) and solitary calcified cysticercus (SCC). The 23 non-NCC epilepsy patients included 8 patients with brain tuberculoma ²⁵, 7 patients with glioma confirmed by histopathology ²⁶, 8 patients with epilepsy of unknown etiology (EUE) with no lesion on brain MRI. Seven healthy subjects who had no symptoms of any disease or co-morbid illness and were not on any medications were also included in the study. All patients except 4 with brain tuberculomas had seizures. The median duration of seizures in the 43 patients was 20 months (IQR 6 to 120 months), with at least one seizure in the last seven months. Demographic data of risk factors for T. solium infection were obtained from all subjects.

Serum of all subjects was assayed for cysticercus antibodies by enzyme-linked immunoelectrotransfer blot (EITB) and for cysticercus antigens by ELISA. ^{14, 27, 28} All non-NCC subjects included in the study were negative for cysticercus antibody and antigen serology.

Blood collection

Five milliliters peripheral blood was collected in BD Vacutainer RST Tubes from all participants by standard venipuncture and serum was separated and stored at −80°C until used.²⁹

Liquid chromatography tandem mass spectrometry of serum proteins

Serum (10 μl) was treated with 2% trichloroacetic acid and 100% isopropanol to precipitate proteins and remove high abundant proteins, albumin and immunoglobulins.³⁰ The pellets were re-suspended in 50μl 7M urea / 2M thiourea and protein concentration determined using a nanodrop (Thermo Scientific).

Twenty micrograms of precipitated serum proteins were reduced with 10 mM dithiothreitol at 37 °C for 30 min, alkylated with 30 mM iodoacetamide for 30 min at 25° C in the dark followed by trypsin digestion at 37.5°C for 16 hours at an enzyme/substrate ratio of 1:100 and the reaction stopped with 1% formic acid.³¹

The samples were cleaned using Ziptip C18 (Millipore) according to the manufacturer’s protocol and eluates lyophilised and re-suspended in 20μl 2% acetonitrile/1% formic acid.

Each sample (10μl) was subject to Liquid chromatography tandem mass spectrometry LC-MS/MS on an EASY-nLC 1200 system (Thermo Scientific) coupled to an Orbitrap Fusion Tribrid MS (Thermo Scientific). Peptides were first separated by HPLC on a trap Pep map TM 100 Nanoviper C18 column (3μm: 2 cm x 75μm; 100Å) and analytical EASY-Spray PepMap RSLC C18 column (3μm; 50cm x 75μm; 100Å) on a linear gradient of 0–98% acetonitrile in 0.1% formic acid at a flow rate of 250 nl/min over 120 minutes. Peptides were eluted between 25–60% acetonitrile.

The separated peptides were subject to MS/MS on the Thermo Orbitrap fusion instrument with a nano-ESI source to generate precursor ions of the peptides and those in the m/z 375–1700 range separated in the first MS at a mass resolution of 120000, positive polarity, Automatic Gain Control (AGC) target value set at 4 × 10⁵ and max injection time of 50ms.

The 20 most intense precursor ions with a 2–7 charge state detected in the first scan, were fragmented by higher energy collision induced dissociation of 30 ± 5% and subject to a second MS scan at a maximum injection time of 35 ms, AGC target set at 1 × 10⁴ and an isolation width of 1.2 m/z.

Protein identification

Proteins of the LC-MS/MS mass peaks between 375 and 1700 m/z were identified using MAXQUANT (version 1.4.1.2) set up to search the Taenia solium WormBase ParaSite database (2021). Decoy databases of modified reversed protein sequences were also considered in the search. Peptides were searched with a parent ion tolerance of 20ppm and a fragment ion mass tolerance of 7ppm. Trypsin was selected, allowing two missed cleavages; carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine as a variable modification.

T. solium proteins were identified by requiring a minimum of two protein unique peptide sequences and at a 5% false discovery rate. T. solium protein sequences identified were compared against all protein databases available in NCBI (National Center for Biotechnology Information) using pBLAST (protein-basic local alignment search tool) to confirm sequence identification to parasite database. The distribution of T. solium proteins between pNCC and non-NCC sera was depicted by an area-proportional Venn diagram created using BioVenn software.³² Gene ontology analysis of all identified T. solium proteins was carried out with BioMart software.³³

Statistical analysis

The characteristics between the pNCC and non-NCC study participants were analyzed for significant differences by the chi-square and Mann-Whitney U tests at p <0.05. Statistically significant differences between the LC-MS/MS mean label-free quantification (LFQ) peak intensities and number of all proteins identified in the pNCC and non-NCC groups were analyzed by the Mann Whitney U test and chi square test and between the pNCC subgroups (GMNCC, SCG and SCC) by the Kruskal Wallis test at p < 0.05.

Significant differences in the number of T. solium proteins detected by LC-MS/MS between Ag ELISA positive and negative sera of pNCC patients and significant differences in the number of T. solium proteins detected by LC-MS/MS between EITB cysticercus antibody positive and negative sera of pNCC patients was analyzed by the Mann-Whitney U test p <0.05.³⁴

Correlation between number of T. solium proteins detected by LC-MS/MS and Ag ELISA positive or EITB positive sera among pNCC patients was determined by Point-Biserial Correlation.

Results

Characteristics of study subjects

Among pNCC subjects (n=24), 62.5% and 37.5% were seropositive for cyst antibodies and antigen respectively (Table 1). Patients with pNCC were less well educated than those in the non-NCC group with a non-statistically significant trend (p = 0.06). (Table 1) Social situation and behaviors that might be associated with developing cysticercosis were more common in the pNCC group than in non-NCC group subjects, but the difference was only statistically significant for pork consumption (p = 0.009).

TABLE 1.

Socio behavioural characteristics of study subjects with pNCC and non-NCC brain disorders and healthy volunteers from India.

Variable	Category	pNCC (n = 24)	non-NCC (n = 30)	p value
Gender	Male : Female	19:05	17:13	0.081
Age (Years)	Median	29.5	29	0.61
	IQR	22–37.25	27–35.25
Socio-Behaviour History
Schooling	< Secondary school	6 (25%)	2 (7%)	0.06
Pork eating	Yes	14 (58%)	7 (23%)	0.009
Living near a house raising pigs	Yes	7 (29%)	4 (13%)	0.15
Owning Pigs	Yes	1 (4%)	0 (0%)	0.25
How often do you use the toilet to defecate?	Never	6 (25%)	3 (10%)	0.14
Serology
EITB	Positive (%)	15 (62.5%)	0 by design
AgELISA	Positive (%)	9 (37.5%)	0 by design

Abbrevations: AgELISA, antigen enzyme-linked immunosorbent assay; pNCC: EITB, enzyme-linked immunoelectrotransfer blot; IQR, interquartile range; non-NCC, other brain disorders (epilepsy of unknown etiology; brain tuberculoma; glioma) and healthy volunteers; pNCC, parenchymal neurocysticercosis.

IQR - Inter quartile range; EITB - Enzyme-linked immunoelectrotransfer blot; AgELISA - Antigen Enzyme-linked immunosorbent assay.

In non-NCC group three subjects who practiced open defecation also ate pork and one subject lived close to a pig rearing-house. None of the patients with EUE or healthy volunteers had factors that are associated with T. solium infection (pork consumption and living close to a pig rearing-house). A significantly high proportion of patients with tuberculomas (5/8; 62.5%) and smaller proportion of those with gliomas (2/7; 28.5%) had these factors (p = 0.01).

T. solium proteins in Sera of pNCC vs Non-NCC Groups

T. solium proteins were identified by LC-MS/MS in 38 of 54 (70.3%) study participants and in all groups including healthy volunteers. A total of 319 T. solium proteins were identified with 312 proteins in 21 of the 24 pNCC sera (87.5%) and 15 proteins in 17 of the 30 non-NCC sera (56.6%) (Table 2). Of the proteins identified in non-NCC sera, 8 eight were detected in both pNCC and non-NCC sera and 7 were found only in the non-NCC sera (Figure 1). Collectively, 95% (304) of the T. solium proteins were exclusive to the pNCC group (Figure 1 and Table S1).

TABLE 2.

Taenia solium proteins identified in sera of study subjects with pNCC and non-NCC brain disorders and healthy volunteers from India.

Feature	pNCC (n = 24)				Non-NCC (n = 30)
	GMNCC (n=8)	SCG (n=8)	SCC (n=8)	Total pNCC	EUE (n = 8)	Glioma (n = 7)	TB (n = 8)	HV (n = 7)	Total Non-NCC
Number of sera with T. solium proteins	7	7	7	21	5	4	6	2	17
Number of T. solium proteins detected Median (IQR)	254 24 (16–80)	197 35 (15–44)	149 22 (7–37)	312 24 (11–44)	7 1 (1–2)	5 1 (1–1.2)	6 1 (1–1)	4 2 (1.5–2.5)	15 1 (1–1)
p-value	p = 0.001 (pNCC vs Non-NCC)
	p = 0.69 (GMNCC vs SCG vs SCC)
Total T. solium protein LFQ peak intensity – Mean ± SD	2.197 ± 1.0				1.033 ± 0.5
Number of T. solium proteins detected	Number of sera
1	1	0	1	2	3	3	6	1	13
2	0	1	0	1	2	1	0	0	3
3–5	0	0	1	1	0	0	0	1	1
6–10	0	0	1	1	0	0	0	0	0
11–20	1	2	0	3	0	0	0	0	0
21–30	3	0	2	5	0	0	0	0	0
31–40	0	1	0	1	0	0	0	0	0
41–50	0	2	1	3	0	0	0	0	0
51–150	2	1	1	4	0	0	0	0	0

Abbreviations: GMNCC, multi-lesion NCC with granulomas; LFQ, label free quantification; non-NCC, other brain disorders (epilepsy of unknown etiology EUE; brain tuberculoma TB; glioma), healthy volunteers HV; pNCC, parenchymal Neurocysticercosis; SCC, solitary calcified cysticercus; SCG, solitary cysticercus granuloma.

Figure-1. Distribution of parasite proteins identified in sera of subjects from regions endemic for Taenia solium.

Venn diagram depicting vastly larger number of T. solium proteins detected in sera of patients with pNCC compared to those detected in the sera of non-NCC subjects. The overlap in the distribution of parasite proteins among the pNCC and non-NCC sera of study subjects living in regions endemic for T. solium is also shown.

Only one T. solium protein was detected in 2/24 pNCC sera (8.3%) but in a significantly higher number (13/30) of non-NCC sera (43.3%) (p<0.01). More than one T. solium protein was detected in 19 of the 24 pNCC sera (79.2%) with 13 sera (54.2%) showing more than 20 T. solium proteins. The highest number of T. solium proteins detected in a patient with pNCC was 147. The median number of T. solium proteins detected in pNCC sera was 27 (IQR, 11– 44) whereas it was 1 (IQR, 1–1) in the non-NCC sera (p = 0.001).

Among the different categories of NCC sera, patients with SCG had the highest median number of T. solium proteins followed by GMNCC with the lowest median being in the SCC group, but these differences were not statistically significant (p = 0.69).

In the non-NCC group, T. solium proteins were detected in the sera of all 7 (100%) subjects who ate pork as compared to 10 (43.4%) of 23 subjects who did not consume pork (p = 0.01). There was no correlation between the detection of T. solium proteins and the other socio-behavior features namely, living near a pig rearing house and open defecation. There was no significant difference in the number of T. solium proteins detected by LC-MS/MS between Ag ELISA positive and negative sera of pNCC patients nor in the number of T. solium proteins detected by LC-MS/MS between EITB positive and negative sera of pNCC patients. There was no correlation between the number of T. solium proteins detected by LC-MS/MS and EITB positivity in sera of pNCC patients. Similarly, no correlation was found between the number of T. solium proteins detected by LC-MS/MS and AgELISA positivity in sera of pNCC patients.

The mean LFQ peak intensities of the total T. solium proteins differed significantly between the pNCC and non-NCC groups (Table 2). The mean LFQ peak intensities of all proteins between pNCC subgroups (GMNCC, SCG and SCC) did not differ significantly and hence the groups were not considered separately in this study (Table S1).

Gene Ontology of T. solium Proteins Identified in Sera of an Endemic population.

Gene Ontology of the 319 T. solium proteins detected in the sera of the 38 participants showed that they are involved with a spectrum of cellular and metabolic processes, molecular functions and are from different cellular components of the parasite ( Tables S2 A–C). Forty per cent (124 of 312) of T. solium proteins detected in pNCC sera were annotated to biological processes of which the largest numbers relate to protein phosphorylation and cell adhesion. T. solium proteins involved in biological processes such as ubiquitin-dependent protein catabolism, G protein-coupled receptor signalling, ion transport and carbohydrate metabolism are important for parasite survival (Figure 2A). Four of 15 T. solium proteins identified in non-NCC sera were annotated to biological processes and involved in DNA replication, glutathione catabolism, response to pheromones, G protein-coupled receptor signalling and protein phosphorylation (Table S2A).

Figure 2: (A-C) Functional distribution of the 312 T. solium proteins identified in pNCC sera analysed by gene ontology.

Percentages are calculated based on 124 proteins annotated to biological functions (A), 182 proteins annotated to molecular functions (B) and 52 proteins annotated as cellular components (C).

Fifty-eight percent (182 out of 312) T. solium proteins identified in pNCC sera and 60% (9 out of 15) T. solium proteins in non-NCC sera were annotated to molecular functions, especially those involved in binding and catalytic activity. Most of the T. solium proteins identified in pNCC sera can be categorized to binding activities (protein binding, nucleic acis binding, calcium binding and metal ion binding), glycolysis (glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and fructose-bisphosphate aldolase) and as peptidases (threonine-type (proteasome subunits) and serine-type endopeptidase activity (trypsin-like protein) (Figure 2B).

T. solium proteins identified in non-NCC sera are also involved in binding (protein binding, metal ion binding) and enzymatic activity (peptidase activity, acyltransferase activity, transporter activity).

fifty-two of 312 (17%) T. solium proteins detected in pNCC sera were annotated to cellular components and related to membranes in over a third of the sera. Two of 15 proteins detected in non-NCC sera were cellular components of the parasite (Figure 2C).

Immune Modulatory Proteins

Ten percent (31 of 319) of T. solium proteins identified in all pNCC and non-NCC sera are involved with immune modulation. Twenty-six immune modulatory proteins from the oncosphere and larval cyst fluid and wall identified in pNCC sera span heat shock response, protein kinase activity, and cytoskeletal involvement and metabolic reactions (Table 3A). The 5 immune modulatory proteins identified in non-NCC sera are glutathione-specific gamma-glutamylcyclotransferase, intimal thickness related receptor, low-density lipoprotein receptor, E3 ubiquitin protein ligase TRIM56 and acyltransferase ChoActase/COT/CPT (Table 3B).

TABLE 3A.

Immune modulatory T. solium proteins identified only in pNCC sera.

S.No	Protein ID	Protein Name	Peptide counts	Number of sera pNCC (n=24)	LFQ Peak Intensity Mean± SD
1	TsM_000197500	Heat shock protein 70 family	2	2	1.44 ± 4.93
2	TsM_000336600	Heat Shock protein family	2	2	1.61 ± 5.48
3	TsM_000474500	Calpain	2	2	1.55 ± 5.28
4	TsM_000182700	Paramyosin	4	3	2.45 ± 6.66
5	TsM_000241200	Phosphatidylinositol 3-/4-kinase, catalytic domain	6	6	5.29 ± 9.43
6	TsM_000218700	Protein kinase domain	5	3	2.42 ± 6.56
7	TsM_000212400	Protein kinase domain	4	3	2.44 ± 6.66
8	TsM_000258700	Protein kinase domain	3	3	2.37 ± 6.57
9	TsM_000524400	6 phospho fructokinase	2	3	2.55 ± 6.95
10	TsM_000371100	Phosphatidylinositol 3-/4-kinase, catalytic domain	2	3	3.24 ± 8.86
11	TsM_000443200	Phosphatidylinositol 3-kinase Ras-binding (PI3K RBD)	2	2	1.96 ± 6.67
12	TsM_000523800	Proteasome component (PCI)	2	2	1.59 ± 5.4
13	TsM_000251200	cGMP-dependent protein kinase, interacting domain	2	2	1.9 ± 6.45
14	TsM_000222500	Protein kinase domain	2	2	1.66 ± 5.65
15	TsM_000179100	Protein kinase domain	2	2	2.05 ± 6.98
16	TsM_000274900	Protein kinase domain	2	2	1.67 ± 5.69
17	TsM_000512800	Protein kinase domain	2	2	1.63 ± 5.54
18	TsM_000234600	Protein kinase domain	2	2	1.54 ± 5.23
19	TsM_000324300	Protein kinase domain	2	2	1.47 ± 5
20	TsM_000523500	Filamin	3	4	4.11 ± 9.42
21	TsM_000495700	Filamin/ABP280 repeat	2;1	2	1.45 ± 4.91
22	TsM_000510200	Annexin	3	3	2.7 ± 7.33
23	TsM_000338800	Dynein heavy chain, domain-1	5	4	3.59 ± 8.21
24	TsM_000454600	Dynein heavy chain, domain-1	3	3	2.86 ± 8.08
25	TsM_000439500	Immunoglobulin subtype	3	2	1.79 ± 6.14
26	TsM_000257100	Immunoglobulin-like fold	8	7	6.43 ± 10.31

Abbreviations: LFQ, label free quantification; pNCC, parenchymal Neurocysticercosis.

TABLE 3B.

Immune modulatory T. solium proteins identified only in non-NCC sera.

S.No	Protein ID	Protein Name	Peptide counts	Number of sera Non-NCC (n=30)	LFQ Peak Intensity Mean± SD
1	TsM_000450300	Glutathione-specific gamma-glutamylcyclotransferase	2	1	0.8 ± 4.49
2	TsM_000326900	Intimal thickness related receptor, IRP	3	2	1.19 ± 4.64
3	TsM_000437000	Low-density lipoprotein (LDL) receptor class A repeat	2	1	0.77 ± 4.28
4	TsM_000772700	E3 ubiquitin protein ligase TRIM56	2	3	2.71 ± 8.41
5	TsM_000478700	Acyltransferase ChoActase/COT/CPT	2	1	0.63 ± 3.51

Abbreviations: LFQ, label free quantification; non-NCC, other brain disorders (epilepsy of unknown etiology EUE; brain tuberculoma TB; glioma), healthy volunteers HV.

Discussion

T. solium proteins in sera

Our findings provide insight into the T. solium proteome that may contribute to the pathophysiology of cysticercosis and NCC in endemic populations. A diverse array of over 300 T. solium proteins was detected, although at very low levels, in the sera of nearly 70.3% of Indian patients with and without NCC. While the presence of parasite proteins in the sera of patients with NCC is expected, their presence in over half the non-NCC participants is a striking and significant finding.

In Indian individuals with and without pNCC, the proteins detected were mostly from two life stages of T. solium, namely the oncosphere and larva. The high numbers of larval proteins in the peripheral blood of pNCC patients could originate from degenerating cysts in the brains of individuals with seizures via a breached blood-brain barrier.³⁵ Although the level of all T. solium proteins differed between pNCC and non-NCC sera the proteins of both groups share similar functional roles (G-protein couple receptor signalling, protein phosphorylation, protein and zion ion binding activity) that are important for survival of the cyst as well as parasite. The lack of correlation between the number of T. solium proteins identified by LC-MS/MS and EITB or AgELISA positivity may arise because antibodies detected by EITB are to T. solium lentil lectin specific glycoproteins and different from proteins detected by LC-MS/MS. Similarly, the T. solium protein antigens detected by the AgELISA are different from those proteins detected by LC-MS/MS.

Parasite proteins in sera of non-NCC subjects who are both AgELISA and EITB negative and whose brain images do not show T. solium cysts lesion could represent asymptomatic systemic infection or exposure but no infection to T. solium from living in an endemic region. A study from south Ecuador, also endemic for T. solium, found that 14% of the population were thought to be exposed to T. solium each year.¹⁰ The study used serological tests of antibody and antigen detection and Bayesian statistics to determine exposure. The notably higher exposure levels we report in an Indian population (56% in non-NCC) are likely due to the use of high-resolution LC-MS/MS that provides a sensitive platform for studying complex proteomes. The high sensitivity of LC-MS/MS in detection of proteins can also explain detection of T. solium proteins in 70.3% of all sera tested in this study while only 37.5% of the sera are positive for parasite proteins tested by AgELISA that has a lower sensitivity for T. solium protein detection.^36–38

In an incidental finding the serum mass spectra of 9 patients from Bangladesh who presented to our hospital with seizures and non-NCC CNS disorders (1 patient with glioma, 2 with brain tuberculoma and 6 patients with meningioma) did not show T. solium proteins. This finding from a country that is not endemic for T. solium lends support to our findings of T. solium proteins in the sera of an endemic population.

Roles of T. solium proteins detected in sera

Parasite proteins in the blood can provide information on disease pathogenesis and those that can be detected and measured may also be useful as diagnostic biomarkers.^39–41

Nearly 10% of T. solium proteins identified in the serum are immune modulatory proteins that could increase susceptibility to infection or generate an immune response that is protective against infection. An example of such proteins are the heat shock proteins induced after infection in response to cellular stresses (eg. oxidative stress) and enable the pathogen to adapt to a new environment. Among immune evasion strategies of the parasite, heat shock proteins are shown to have anti-inflammatory effects through inhibition of MAPKs and NF-κB signaling pathways leading to immune suppression.⁴² Both paramyosin, a major oncosphere protein that inhibits C1 complement and the classical complement activation pathway, and calpain, an immunomodulatory anti-inflammatory protein that can help evade immune clearance by the host, contribute to the establishment of latent and persistent cysticercus infection.^43–44 Additionally, T. solium paramyosin can activate host cellular immune responses that protect against infection.⁴⁵ The calcium-dependent phospholipid-binding annexins are known to induce apoptosis of host immune cells such as the eosinophils, an anti-inflammatory response that could favour parasite survival.^46–47

Protein kinases constitute 1.8% of the T. solium proteome.⁴⁸ Kinases play an indispensable role in cellular signalling in response to external and internal stimuli ⁴⁹ and are important in all stages of parasite development from embryogenesis as well as in immunity. They are also considered potential drug targets.⁴⁸

T. solium proteins such as the excretory/secretory proteins suppress host immune inflammation.⁴⁴ Parasite proteins present at low levels and over long periods of time in the serum of an endemic population due to chronic exposure or asymptomatic infections may induce suppressive host immune responses that allow infections to occur in the uninfected and persist in those infected.

Several T. solium proteins detected by LC-MS/MS in sera of pNCC patients in our study have been reported by others in their studies on the proteomes of active oncospheres, excretion/secretion products and larval cyst.^19–22 These include the oncosphere proteins proteinases (Ring-finger 11, Ubiquitin, and cysteine peptidase), stress-secretion proteins (Cyclophilin-type and Heat shock protein), signalling proteins (Annexin), and cytoskeleton and motility proteins (Paramyosin, Dynein heavy chain).¹⁹ Diaz-Masmela Y et al ⁵⁰ in a study on the proteomics of T. solium cysticerci vesicular fluid found annexin B1, which has phospholipid-binding abilities and anticoagulant/anti-inflammatory actions and cAMP-dependent protein kinase. Both annexin B1 and cAMP-dependent protein kinase are antigenic and have been examined as potential diagnostic targets for porcine cysticercosis.^{21, 50}

To our knowledge, this study is the first to report on the detection of T. solium proteome in the human sera of patients with and without NCC in an endemic region, using mass spectrometry. The findings have both clinical and epidemiological significance. The proteins may underlie host-parasite interactions and responses that impact on prevalence and pathogenesis of infection. The identification of parasite-specific proteins can significantly simplify the design for fast and inexpensive diagnosis. In asymptomatic individuals, it may indicate exposure and infection and they may benefit from a passive immunization. In individuals with travel history to an endemic area, presenting with seizures, detection of these proteins may be an adjunct in the diagnosis, if an enhancing or calcified lesion is detected on brain imaging. In addition, identifying potential vaccination targets (proteins) using immune informatics appears to be one of the leading ways to control parasitic disease and our findings could benefit this endeavour.⁵¹ Prior to extending the findings to understanding T. solium prevalence and infections and use in the clinic further study and validation of the findings in a larger number of individuals and from different regions of the country and from non-endemic countries are required.