Malaria is a significant public health burden with more than 3.2 billion people being at risk of infection, especially in the resource-poor settings (1). In 2020, 241 million malaria cases and 627,000 deaths were reported globally. In humans, malaria is exclusively caused by infection with a
Prompt and accurate diagnosis of malaria alongside reliable identification of
Traditional proteomic accesses can elucidate protein expression profiles, which may have promising applications to clinical cases, therapeutic responses, or investigation of the elucidating mechanisms of diseases such as cardiovascular diseases, autoimmune disorders, and various cancers (16, 17). Biomedical and clinical proteome research targeted at biomarker discovery is majorly founded on expression proteomics, which investigates the quantity of specific proteins in different conditions. Thus, proteomic studies are likely to be pivotal factors in accelerating the manifestation of new biomarkers.
Glycoprotein α1-antichymotrypsin (AACT) is a protein ascribed to the serine proteinase inhibitors superfamily, also known as serpins (18). AACT is involved in, inflammation, proteolysis, and the acute phase response. Although AACT is mainly synthesized in the liver, after which it is secreted into the blood, it is also synthesized in the brain, mainly by astrocytes (19, 20). In the brains of patients suffering from Alzheimer’s disease, AACT has been reported to bind amyloid-β peptides found in plaque cores and blood vessels and the protein is overexpressed in the brain of Alzheimer’s disease patients (21, 22). Its expression is controlled by proinflammatory cytokines including oncostatin M, interleukin (IL)-1, and complexes of IL-6, soluble IL-6 receptors, and transcriptional regulators such as activator protein 1 and nuclear factor 1-X (23). However, AACT’s potential as a biomarker for Alzheimer’s disease is controversial. These proteins inactivate proteinases with a serine residue in their active site (24). AACT is a major acute-phase reactant, and its concentration in plasma increases in response to trauma, surgery, and infection and its elevated level is widespread, but not universal (25, 26). The dysregulation of AACT expression and its glycosylation states are linked with tumor progression and recurrence and could be exploited as a biomarker for tracing tumors, including in diffuse large B-cell lymphoma, pancreatic cancer, liver cancer, ovarian cancer, and lung cancer (27). However, the expression changes in protein level, biological functions, and glycan modification of AACT is to be elusive.
With this purpose, in this study, we initially studied plasma proteins of patients suffering from vivax malaria alongside those of healthy specimen to deduce biomarkers for the discrimination between long-term and short-term latent malaria. We also sought to acquire a thorough elucidation of the pathophysiological mechanism of the infectious disease by utilizing confluences of depletion of abundant proteins, 2-dimensional gel electrophoresis (2-DE), gel-image analysis, and exact mass spectrometry. Though it failed to discriminate between short-term and long-term latent malaria, a plasma glycoprotein, AACT could be developed as a possible biomarker for
To identify new serologic biomarker candidates for malaria, we used plasma samples acquired from healthy donors and patients confirmed to be infected with vivax malaria. An ordinary 2-DE pattern of plasma from which 14 highly abundant proteins were removed as described previously is shown in Supplementary Fig. 1A. Although the primary goal of this study was to discover biomarkers to discriminate between short-term (S group) and long-term (L group) latent malaria, no differentially expressed proteins could be found in this study that would distinguish these two latent types of malaria. We leaved out those proteins that are previously known or involved in other diseases as well as those for which commercial antibodies are not available. Thus, AACT which was overexpressed in malaria plasma samples (2.5-fold in the L group compared with the control group, 2.6-fold in the S group compared with the control) was chosen as the sole candidate protein (Supplementary Fig. 1B). To verify AACT as a vivax malaria biomarker candidate and to assess its efficiency and specificity in diagnosing vivax malaria, we used 200 individual plasma samples from healthy donors (n = 100) and vivax malaria patients (n = 100); specifically, we evaluated differential AACT expression Western blot analysis, using a specific monoclonal antibody against AACT. As shown in Fig. 1, and Supplementary Fig. 2, the protein expression level of AACT in vivax malaria-patient samples was more than 2-fold higher than that in the control (means ± standard errors, Control: 1 ± 0.03 and vivax malaria patient: 2.83 ± 0.11, based on band intensities, Fig. 1D). These results indicates that AACT is highly expressed in the plasma of vivax malaria patients. Loading (30 μg) of each sample on the gel was normalized by staining with a Coomassie brilliant blue G250 (CBB G250) staining solution. Malaria is transmitted by female
Next, to elucidate the relative expression level of AACT in other mosquito-borne infectious diseases, we evaluated the comparative diagnostic specificity of AACT for malaria and other mosquito-borne infectious diseases including Tsutsugamushi disease and Dengue fever. We performed Western blot analyses using16 Dengue fever patient plasma samples (Fig. 4A) and 16 Tsutsugamushi disease patient plasma samples (Fig. 4B). As shown in Fig. 4, the expression pattern and level of AACT in Tsutsugamushi disease and Dengue fever samples differed from that of vivax and falciparum malaria plasma and was similar to that of healthy plasma.
Our results might confirm the malaria-specific up-expression of AACT in vivax and falciparum malaria. Malaria is presupposed primarily on the base of fever or a history of fever (4). There is no combination of symptoms or signs that reliably discriminates malaria from other causes of fever, and diagnosis relied only on clinical symptoms has very low specificity and results in bad treatment (9). To guide rational use of antimalaria medicines, the focus of malaria diagnoses should be to discriminate patients who truly suffer from malaria. The two practical tools used routinely for parasitological diagnosis of malaria are optical microscopy, the gold standard diagnosis for malaria, and immunochromatographic rapid diagnostic tests (RDTs). Optical microscopy of stained blood smears has important advantages including low direct cost, high sensitivity, differentiation of
Although this study demonstrated that AACT could serve as a novel biomarker for malaria caused by
In conclusion, we evaluated discriminatorily expressed proteins in the plasma of vivax malaria patients using traditional proteomic tools, which showed us to identify changes in AACT protein levels. Although the changes in AACT are likely not specific to vivax malaria patients, our study proposed the methodological achievements for a proteomic approach to examine plasma proteins in malaria patients. Additional in-depth investigation into the cellular and biochemical mechanism of AACT in malaria infection is warranted. These results have clinical implications in terms of the elucidation of
This study was performed under the regulation of the Institutional Review Board Committee of Konkuk University (No. 7001355-202007-BR-386). This research adhered to the tenets of the Declaration of Helsinki. The malaria patients’ and healthy donors’ plasma was obtained from the Global Resource Bank of Parasitic Protozoa Pathogens in Incheon National University. The vivax malaria patients’ plasma was obtained from 100 Korean patients and the falciparum malaria patients’ plasmas were from 8 Korean patients confirmed in the Inha University Hospital and Inha University Department of Tropical Medicine. The
Originally, we categorized the plasma samples into three groups: i) healthy donor (C group), ii) patients for whom the outbreak time coincides with the mosquito activity period (S group), and iii) patients for whom the outbreak time does not coincide with the mosquito activity period (L group). To remove 14 highly abundant proteins (albumin, immunoglobulin G (IgG), IgM, IgA, α1-antitrypsin, transferrin, haptoglobin, α2-macroglobulin, fibrinogen, complement C3, α1-acid glycoprotein (orosomucoid), HDL (apolipoproteins A-I and A-II), and LDL (mainly apolipoprotein B) from human plasma, the multiple affinity removal column system based on avian antibody-antigen interactions (SepproⓇ IgY14, Millipore Sigma, St. Louis, MO, USA) was routinely used according to the manufacturer’s recommended protocols. To search for a novel serologic indicator candidate for malaria, we carried out an integrated proteomic analysis using pooled plasma from healthy donors (C group) and vivax malaria patient groups (S and L groups) (39, 40). All procedures for the proteomic analysis, including 2-DE, image analysis, trypsin digestion, protein identification by LC-MS/MS, and data searches for protein identification were performed by Yonsei Proteome Research Center (Seoul, Korea) as previously described (39, 40).
Validation of some differentially expressed protein candidates was performed by Western blot analysis with the commercially available specific antibody. Total protein concentrations of plasma samples were estimated using a bicinchoninic acid-based protein assay system (Pierce, Rockford, IL, USA). Immunoreactive proteins on the membrane were detected using ECL Plus Western blotting detection reagents (GeneCure, Norcross, GA, USA). To evaluate band intensities, bands on the X-ray-films were imaged and analyzed using the ChemiDocTM XRS + System equipped with Image Lab SoftwareTM (Bio-Rad, Hercules, CA, USA).
Data were expressed as means ± standard errors and analyzed by a Student’s t-test. Statistical significance was accepted at P < 0.01. IBM SPSS Statistics ver. 27 (IBM, Somers, NY, USA) was used for all of the statistical analyses.
This study was supported by funding from the National Research Fund (NRF-2020R1F1A1070882 and NRF-2022R1F1A 1066481) in the Republic of Korea.
The authors have no conflicting interests.