J Cell Physiol. 2020 Sep 22 : 10.1002/jcp.30055.
doi: 10.1002/jcp.30055 [Epub ahead of print]


PMCID: PMC7536980
PMID: 32959892


Quantitative proteomics reveals a broad‐spectrum antiviral property of ivermectin, benefiting for COVID‐19 treatment

Na Li, 1 , 2 , 3 Lingfeng Zhao, 4 and Xianquan Zhan 1 , 2 , 3 , 5 , 6

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Abstract

Viruses such as human cytomegalovirus (HCMV), human papillomavirus (HPV), Epstein–Barr virus (EBV), human immunodeficiency virus (HIV), and coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2]) represent a great burden to human health worldwide. FDA‐approved anti‐parasite drug ivermectin is also an antibacterial, antiviral, and anticancer agent, which offers more potentiality to improve global public health, and it can effectively inhibit the replication of SARS‐CoV‐2 in vitro. This study sought to identify ivermectin‐related virus infection pathway alterations in human ovarian cancer cells. Stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomics was used to analyze human ovarian cancer cells TOV‐21G treated with and without ivermectin (20 μmol/L) for 24 h, which identified 4447 ivermectin‐related proteins in ovarian cancer cells. Pathway network analysis revealed four statistically significant antiviral pathways, including HCMV, HPV, EBV, and HIV1 infection pathways. Interestingly, compared with the reported 284 SARS‐CoV‐2/COVID‐19‐related genes from GencLip3, we identified 52 SARS‐CoV‐2/COVID‐19‐related protein alterations when treated with and without ivermectin. Protein–protein network (PPI) was constructed based on the interactions between 284 SARS‐CoV‐2/COVID‐19‐related genes and between 52 SARS‐CoV‐2/COVID‐19‐related proteins regulated by ivermectin. Molecular complex detection analysis of PPI network identified three hub modules, including cytokines and growth factor family, MAP kinase and G‐protein family, and HLA class proteins. Gene Ontology analysis revealed 10 statistically significant cellular components, 13 molecular functions, and 11 biological processes. These findings demonstrate the broad‐spectrum antiviral property of ivermectin benefiting for COVID‐19 treatment in the context of predictive, preventive, and personalized medicine in virus‐related diseases.

Keywords: ivermectin, quantitative proteomics, SARS‐CoV‐2/COVID‐19, stable isotope labeling by amino acids in cell culture, virus‐related pathways

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Abstract



  • 1.
    This study sought to identify ivermectin‐related virus infection pathway alterations in human cells.
  • 2.
    Quantitative proteomics revealed that ivermectin‐related proteins are involved in four statistically significant antiviral pathways, including human cytomegalovirus (HCMV), human papillomavirus (HPV), Epstein–Barr virus (EBV), human immunodeficiency virus 1 (HIV1), and COVID‐19 infection pathways.
  • 3.
    We identified 52 severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)/COVID‐19‐related protein alterations when treated with and without ivermectin, and these proteins were involved in cytokines and growth factor family, MAP kinase and G‐protein family, and HLA class proteins.
  • 4.
    These findings demonstrate the broad‐spectrum antiviral property of ivermectin benefiting for COVID‐19 treatment in the context of predictive, preventive, and personalized medicine in virus‐related diseases.







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1. INTRODUCTION

Ōmura discovered a unique and extraordinary microorganism that could produce ivermectin in 1973 (Burg et al., 1979). Ivermectin was subsequently commercialized because it showed great safety and effectivity in human health. The current status of ivermectin was continuing to surprise and excite scientists (Laing, Gillan, & Devaney, 2017). It was originally intended to be a broad‐spectrum antiparasitic agent, and treat onchocerciasis, strongyloidiasis, lymphatic filariasis, and scabies in veterinary and human medicine (Chabala et al., 1980). There was an outstanding advantage of ivermectin that no confirmed or increased drug resistance appears in parasites, even in those human populations who have been receiving ivermectin as a monotherapy for more than 30 years (van Wyk & Malan, 1988). In terms of mechanism, the primary target of ivermectin is glutamate‐gated chloride channels (Abdeltawab et al., 2020). However, it was increasingly believed that ivermectin was closely related to the immune defense mechanism and acted like immunomodulatory agents to help suppress the parasite's ability to evade the host's immune (Schaller et al., 2017). Today, the new use of ivermectin made it become a relatively unknown drug. Drug repurposing and repositioning has been shown to control a completely new range of diseases (Ashour, 2019). For example, orbital myiasis, trichinosis, malaria, leishmaniasis, African trypanosomiasis, asthma, epilepsy, neurological disease, antiviral (e.g., human immunodeficiency virus [HIV], dengue, encephalitis; Yang et al., 2020), antibacterial (tuberculosis and Buruli ulcer; Csóka et al., 2018), anticancer (breast cancer, leukemia, glioblastoma, cervical cancer, gastric cancer, ovarian cancer, colon cancer, melanoma, and lung cancer; Crump, 2017). Multifaceted ‘wonder’ ivermectin may become an even more exceptional drug in the future. An international patent ‘Use of ivermectin and derivatives thereof’ caught people's increasing attention to ivermectin those years. Ivermectin would be developed to use for metabolic‐related diseases (diabetes, hypercholesterolemia, insulin resistance, obesity, hypertriglyceridemia, and hyperglycemia), Famesoid X receptor‐mediated diseases (atherosclerosis, nonalcohol fatty liver disease, cholestasia, and gallstones), inflammation, and cancer (Crump, 2017).
Viruses such as HIV, human cytomegalovirus (HCMV), Epstein–Barr virus (EBV), human papillomavirus (HPV), and novel severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) represent a great burden for human health worldwide. For example, there were almost 37 million people infected with HIV‐1 in the whole world, and nearly 1 million patients died of human immunodeficiency virus infection and acquired immune deficiency syndrome (AIDS)‐related disease each year (Huynh & Gulick, 2020). HIV damaged the immune system and majorly killed CD4 cells to make patients vulnerable to various illnesses, including pneumonia, cytomegalovirus, cryptococcal meningitis, tuberculosis, cryptosporidiosis, oral thrush, toxoplasmosis, and cancer (Kaposi's sarcoma and lymphoma; (Nash & Robertson, 2019). HCMV is a β‐herpesvirus that closely has a prevalence of 55%–100% within the human population. HCMV is one of the most common infection in all live births (1%–2.5%) in the Western world (Buxmann, Hamprecht, Meyer‐Wittkopf, & Friese, 2017). HCMV intrauterine infection could lead to congenital abnormalities, including visual impairment, low birth weight, hearing loss, varying degrees of mental retardation, hepatosplenomegaly, and microcephaly (Zavattoni et al., 2014). HCMV acquired different mechanisms to evade the human immune response (Britt, 2008). For example, the HCMV prevented NK cell activity by virus UL16 and UL142 proteins. HCMV has acquired a viral homolog of IL‐10 to suppress anti‐cytomegalovirus immunity (Holder & Grant, 2019). HCMV also downregulated the expression of major histocompatibility complex to prevent the antigen processing and presentation by virus US11, US2, and US3 proteins (Britt, 2008). HCMV has also evolved proteins (UL36 and UL37) to prevent apoptosis of infected cells, which promoted HCMV dissemination within the host (Andoniou & Degli‐Esposti, 2006). EBV was a member of the herpesvirus family that could cause mononucleosis. Though lots of people were asymptomatic infections, but potential links between EBV and other lymphoproliferative diseases (nonmalignant, premalignant, and malignant diseases) were widely studied (Rezk, Zhao, & Weiss, 2018), such as Burkitt lymphoma, Hodgkin's lymphoma, hemophagocytic lymphohistiocytosis, gastric cancer, central nervous system lymphomas, acute cerebellar ataxia (Nussinovitch, Prais, Volovitz, Shapiro, & Amir, 2003), nasopharyngeal carcinoma, and hairy leukoplakia (Marques‐Piubelli et al., 2020). EBV can infect different kinds of cells, but viral tropism is preferred to B cells and epithelial cells. B cell membrane fusion was mediated by the three‐part glycoprotein complexes of gHgL gp42; although epithelial cell membrane fusion was mediated by the two‐part complexes of gHgL (Shannon‐Lowe, Rowe, 2014). About 90% of HPV were asymptomatic infections, but HPV infection would lead to either warts or precancerous lesions. The infected sites by HPV, especially the subtype HPV16 and HPV18, showed high risk of cancer, including cervix, vagina, vulva, mouth, penis, throat, and anus (Athanasiou et al., 2020). HPV was believed to cause cancers in nonintegrated episomes and integrating into DNA. Some of the HPV genes (E6 and E7), acted as oncogenes to promote malignant transformation (Hoppe‐Seyler, Bossler, Braun, Herrmann, & Hoppe‐Seyler, 2018). E6 protein bound to p53 protein and resulted in the inactivation of p53 (Almeida, Queiroz, Sousa, & Sousa, 2019). E7 acted as the transforming protein and competed between retinoblastoma protein (pRb) for binding to transcription factor E2F, which pushed the cell cycle forward (Almeida et al., 2019). SARS‐CoV‐2 lead to the outbreak of coronavirus disease 2019 (COVID‐19) and rapidly grew into a global pandemic. Scientists set out to develop a treatment for COVID‐19, but no anti‐SARS‐CoV‐2 drug or vaccine has been approved to solve the serious challenge (H. Li et al., 2020). In the whole world, more than 7 million people have infected SARS‐CoV‐2, including more than 400,000 deaths at the national level (Lai, Shih, Ko, Tang, & Hsueh, 2020). Further studies to develop the safest and most effective ways to combat viral infections were urgent. Ivermectin has been demonstrated to limit infection by a number of viruses with potential broad‐spectrum activity (Yang et al., 2020). For example, ivermectin has been reported anti‐HIV‐1 reliant on importin α/β nuclear import (Wagstaff, Sivakumaran, Heaton, Harrich, & Jans, 2012). Ivermectin could reduce MAPK pathway activation through the inhibition of PAK‐1 activity. The high content screening also identified ivermectin as a promising drug against EBV‐positive and EBV‐negative nasopharyngeal carcinoma cells (Gallardo, Mariamé, Gence, & Tilkin‐Mariamé, 2018). Herpes genitalis and infections, which are caused by HPV in males, might have an effective treatment choice for oral ivermectin, but it has not been officially approved until now (Buechner, 2002). More importantly, ivermectin was reported as an inhibitor of the SARS‐CoV‐2, which was able to produce an effect ~5000‐fold reduction in viral RNA with a single addition to cells infected with SARS‐CoV‐2 (Caly, Druce, Catton, Jans, & Wagstaff, 2020).
Viruses remain one of the least well‐understood pathogens. The lack of knowledge about mechanisms and host–parasite interactions limited success in developing vaccines. It is facing challenges on several fronts, including limitations in availability, high cost of production, high mutation probability, and high prevalence of resistance. Ivermectin, as an antiparasitic, anticancer, antibacterial, and antiviral agent, provided more potentiality to improve global public health. The present study used stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomics analysis to reveal ivermectin‐related proteomics profiling and molecular network alterations. We focused our attention on the virus‐related pathways, such as HCMV infection, HPV infection, EBV infection, and HIV1 infection. More interestingly, a large number of identified proteins were reported to be related to SARS‐CoV‐2/COVID‐19. These results indicated that ivermectin might be a broad‐spectrum antiviral drug. SILAC quantitative proteomics proved the molecular mechanisms of ivermectin in virus‐related pathways. Furthermore, protein–protein interaction (PPI)‐based hub modules for SARS‐CoV‐2‐related proteins discovered a key molecule in COVID‐19 disease in the context of predictive, preventive, and personalized medicine (PPPM) in COVID‐19.

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2. MATERIALS AND METHODS

2.1. SILAC‐treated cells

Human ovarian cell line (TOV‐21G; Keibai Academy of Science, Nanjing, China) was cultured with two different SILAC reagents (Thermo Fisher Scientific) (One was RPMI 1640 medium without l‐lysine [K] and l‐Arginine [R] supplemented with 100 mg/L l‐lysine‐2HCl and 100 mg/L l‐arginine‐HCl [“light” labeling reagent = L] and 10% fetal bovine serum [FBS; Gibco], and another was RPMI 1640 medium without l‐lysine [K] and l‐arginine [R] supplemented with 100 mg/L l‐lysine‐2HCl[13C6,15N2] and 100 mg/L l‐arginine‐HCl[13C6,15N4] [“heavy” labeling reagent = H; [13C6,15N2] means 8 mass units increased in residue K, [13C6,15N4] means 10 mass units increased in residue R] and 10% FBS), and maintained with 5% CO2 and 37°C and medium renewal every 2 days. A total of 10 passages were treated with SILAC reagents with 12C14N (light = L) and 13C15N (heavy = H)‐labeled amino acids to ensure complete incorporation of stable isotope into the cultured cells.

2.2. Ivermectin treatment of SILAC‐labeled cells

Our previous study found that when TOV‐21G cells were treated with ivermectin (0–60 μM) for 24 h, the IC50 was 22.54 μM for ivermectin, and also 20 μM ivermectin (it was less than IC50 22.54 μM) significantly suppressed cell proliferation and migration of TOV‐21G, and maintained TOV‐21G cells in good shape (N. Li & Zhan, 2020). Thus, TOV‐21G cells cultured in the H‐ and L‐stable isotope‐labeled media were treated with 20 μM ivermectin in dimethyl sulfoxide (DMSO) or with the same amount of the DMSO as control, for 24 h. Ivermectin‐treated TOV‐21G cells were centrifuged (800g), washed with PBS (×3), and then suspended (30 min, 4°C) in protein isolation buffer [7 M urea, 2 mM thiourea, 4% CHAPS (3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane), 100 mM dithiothreitol (DTT), and 2% ampholyte] with a vortex (×5). The extracted protein solution was centrifuged (13,000g, 20 min, 4°C). The supernatants were the extracted protein samples whose protein concentrations were examined with 2‐D quant kit.

2.3. SILAC‐labeling efficiency analysis

The extracted protein samples were ultrasonicated and centrifuged (14,000g, 25°C, 40 min). The H‐ and L‐stable isotope‐labeled proteins were equally mixed (1:1), separated with 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE; 20 μg/lane; constant current 14 mA, 90 min), and stained with Coomassie brilliant blue. SDS‐PAGE‐separated proteins were subjected to reduction, alkylation, digestion with trypsin, and identification with mass spectrometry (MS). The efficiency of SILAC‐isotope incorporation into proteins was estimated with Rappsilber's method (Rappsilber, Ishihama, & Mann, 2003). For this study, the SILAC labeling efficiency was up to 97%.

2.4. Protein digestion and LC‐fractionation

The extracted protein samples were treated with a final concentration of 100 mM DTT, boiled (water bath; 5 min), transferred to a 10 kD ultrafiltration centrifuge tube with 200 μl of 8 M urea in 0.1 M Tris–HCl, pH 8.5, and centrifuged (14,000g, 15 min; ×2). The protein samples in ultrafiltration centrifuge tube were treated (dark room, 30 min, room temperature) with 100 μl solution of 0.05 M iodoacetamide, 8 M urea, and 0.1 M Tris–HCl, pH 8.5), followed by centrifugation (14,000g, 15 min). The iodoacetamide‐treated protein sample was treated with 100 μl of 8 M urea in 0.1 M Tris–HCl, pH 8.5, and centrifuged (14,000g, 15 min; ×3), followed by treatment with 100 μl of 25 mM NH4HCO3 solution, and centrifugation (14,000g, 15 min; ×3). The treated protein samples were mixed (shaking with 600 rpm, 1 min) with 40 μl of 2 μg trypsin in 40 μl 100 mM NH4HCO3, shaked, stayed (37°C, 16–18 h), and transferred into a new collection tube for centrifugation (14,000g, 15 min), followed by mixing with 40 μl of 25 mM NH4HCO3, and centrifugation (14,000g, 15 min) to collect the filtrate as the tryptic peptide mixture. The peptide content was quantified (OD280). Liquid chromatography (LC) was used to fractionate the tryptic peptide mixture into 15 peptide fractions for reverse LC‐tandem mass spectrometry (LC‐MS/MS) analysis.

2.5. LC–MS/MS

Each peptide fraction was subjected to LC–MS/MS analysis for 60 min on an Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific) coupled with Q Exactive mass spectrometer (Thermo Fisher Scientific). The obtained MS/MS spectra data were used to identify and quantify proteins with MaxQuant software against the protein database. The intensities of the light and heavy isotopes were used to determine the protein differentially expressed levels between TOV‐21G cells treated with (heavy labeling = H) and without (light labeling = L) ivermectin.

2.6. Bioinformatics and statistical analysis

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed with clusterProfiler (https://bioconductor.org/packages/re...rProfiler.html; Yu, Wang, Han, & He, 2012) to find the signaling pathway based on the identified protein (p < .05, and adjusted p < .05). The reported SARS‐CoV‐2‐related genes were searched by GenCLiP3 (http://ci.smu.edu.cn/genclip3/; Wang et al., 2019). Cytoscape ClueGO (Bindea et al., 2009) was used to reveal the biological processes (BPs), molecular functions (MFs), and cellular components (CCs) enriched from identified proteins (two‐sided hypergeometric test, adjusted p < .05 corrected with Benjamini–Hochberg). The reported SARS‐CoV‐2‐related genes were analyzed by STRING 10.0 (http://string-db.org/cgi/input.pl; Szklarczyk et al., 2015) with the confidence of parameter (co‐expression score > 0.700) for PPI network construction. Then, the entire PPIs were analyzed with the molecular complex detection (MCODE; Bader & Hogue, 2003) using Cytoscape software (version 3.2.1; National Resource for Network Biology) to obtain hub modules (score > 6). The statistical significance was set as p < .05. A Benjamini–Hochberg was used to adjust p value for the probability of the association between the proteins in the pathway.


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3. RESULTS

3.1. SILAC quantitative proteomics analysis revealed a broad‐spectrum antiviral property of ivermectin

A total of 4447 ivermectin‐related proteins were identified in ovarian cancer cells treated with and without ivermectin with SILAC‐based quantitative proteomics (Table S1). Further KEGG pathway analysis revealed four virus‐related pathways (Table 1), including HCMV (Figure S1A), HPV (Figure S1b), EBV (Figure S1C), and HIV1 (Figure S1d) infection pathways, and eight bacteria‐related pathways (Table 1), including bacterial invasion of epithelial cells, vibrio cholerae infection, epithelial cell signaling in Helicobacter pylori infection, pathogenic Escherichia coli infection, shigellosis, salmonella infection, legionellosis, and yersinia infection (Figure S2). Those enriched pathways indicated that ivermectin was an antibacterial and antiviral agent, and provided clues to relevant mechanisms of ivermectin.
Table 1

Statistically significant bacteria‐ and virus‐related pathways identified with ivermectin‐related proteins by KEGG pathway enrichment analysis

Number Pathway ID Pathway name Ratio of matched versus total genes p value Adjusted p value q value Gene ID of ivermectin‐related proteins
1 hsa05100 Bacterial invasion of epithelial cells 44/2091 5.95E − 10 1.46E − 08 9.92E − 09 CLTB, CTNNB1, SEPTIN3, DNM1, FN1, CTNNA1, RAC1, CBL, ACTG1, CDC42, CRKL, RHOA, ARPC3, ARPC5, SEPTIN2, ARPC2, ARPC5L, ELMO2, SEPTIN11, SEPTIN9, SHC1, CD2AP, DNM2, PIK3R2, SRC, CLTC, ARPC1B, CRK, CTTN, PTK2, ACTB, PXN, ITGB1, MET, WASF1, SEPTIN8, WASF2, CAV1, CLTA, WASL, ARHGEF26, ITGA5, DOCK1, PIK3R1
2 hsa05110 Vibrio cholerae infection 28/2091 6.22E − 06 5.69E − 05 3.85E − 05 KDELR2, ATP6V0D1, ATP6V0A1, ATP6V1B2, ATP6V1C1, ATP6V1A, ATP6V1H, ACTG1, PRKCA, PRKACA, PRKACB, SLC12A2, ATP6V1E1, ARF1, ATP6V0C, TJP2, PLCG1, TJP1, ACTB, GNAS, SEC61G, ATP6V1G1, ATP6V1F, ERO1A, SEC61B, TCIRG1, SEC61A1, KDELR1
3 hsa05120 Epithelial cell signaling in Helicobacter pylori infection 33/2091 1.07E − 04 7.31E − 04 4.96E − 04 PAK1, F11R, ATP6V0D1, MAPK8, CHUK, IKBKG, ATP6V0A1, ATP6V1B2, RAC1, ATP6V1C1, ATP6V1A, ATP6V1H, ADAM10, LYN, CDC42, MAPK14, GIT1, ATP6V1E1, MAP2K4, RELA, ATP6V0C, CSK, SRC, PLCG1, TJP1, NFKB1, MET, PTPN11, EGFR, ATP6V1G1, CASP3, ATP6V1F, TCIRG1
4 hsa05130 Pathogenic Escherichia coli infection 90/2091 6.13E − 09 1.31E − 07 8.86E − 08 IRAK1, MYD88, MYH9, PAK1, ROCK1, TUBB1, TUBB2B, WIPF2, ARHGEF12, TMBIM6, BAIAP2, TUBB4A, CYFIP2, SEC24A, TUBA4A, MAPK8, CHUK, IKBKG, SLC9A3R1, TUBA1B, TUBB2A, MAPK1, RAC1, ABI1, ACTG1, LYN, CYFIP1, CDC42, FADD, BRK1, CYCS, SEC24C, MAPK14, PAK2, RHOA, ARPC3, ARPC5, ARHGEF2, TUBA1C, TUBB, NCL, RAB1A, ARPC2, ARPC5L, CASP8, MYO1B, RELA, ROCK2, ARF1, BAIAP2L1, TUBA1A, ABCF2, ARF6, SEC24B, TUBB3, MYH10, MYO1C, MYO1E, TJP1, ARPC1B, CTTN, SEC24D, TMED10, ACTB, EZR, NCKAP1, NFKB1, TUBAL3, TRAF2, ARHGEF1, RIPK1, TUBB8, ITGB1, MYH3, RPS3, CLDN1, PTPN11, WASF1, WASF2, CASP3, BAX, CASP7, CLDN11, WASL, TUBB6, MYO6, IL18, GNA13, TRADD, NCK1
5 hsa05131 Shigellosis 98/2091 2.10E‐07 2.68E − 06 1.82E − 06 BCL10, IRF3, MYD88, ROCK1, RPS6KB1, RPS6KB2, SEPTIN3, HKDC1, MAPK8, CHUK, PFN2, CBX3, IKBKG, MAPK1, RAC1, PRKCD, ACTG1, UBE2D3, CDC42, CRKL, PFN3, RBX1, TBK1, CYCS, GSK3B, MYL12A, AKT1, MAPK14, RHOA, RNF31, ARPC3, ARPC5, PPID, UBE2V2, ARHGEF2, CAST, SEPTIN2, ARPC2, ARPC5L, CUL1, ELMO2, FNBP1L, GLMN, PFN1, RELA, ROCK2, RPS6KA5, UBA52, VDAC1, ARF1, MTOR, SEPTIN11, SEPTIN9, ACTN1, PIK3R2, RPTOR, SKP1, SRC, TLN1, PLCG1, ACTN4, AKT1S1, ARPC1B, CRK, CTTN, PTK2, ACTB, NFKB1, PIK3C3, PXN, TRAF2, HK1, RIPK1, U2AF1L5, ITGB1, PLCB3, SQSTM1, DIAPH1, PLCD1, WASF1, CAPN1, EGFR, SEPTIN8, WASF2, RPS27A, BAX, WASL, ITPR3, CD44, ATG5, CAPN2, CAPNS1, IL18, ITGA5, DOCK1, TLN2, TRADD, PIK3R1
6 hsa05132 Salmonella infection 106/2091 7.10E − 14 2.84E − 12 1.92E − 12 ACBD3, CTNNB1, FBXO22, IRAK1, MAP2K1, MYD88, MYH9, MYL6, PAK1, DYNLL2, CYFIP2, MAPK8, CHUK, PFN2, TXN, IKBKG, MAPK1, RAC1, DYNC1I2, EXOC2, ABI1, ACTG1, CYFIP1, MAP2K2, CDC42, FADD, PFN3, BRK1, CYCS, MYL12A, AKT1, CSE1L, MAPK14, PKN1, RHOA, VPS39, ARPC3, ARPC5, KPNA1, KPNA4, RAB5B, ARPC2, ARPC5L, CASP8, DYNC1LI1, MAP2K4, PFN1, RELA, ROCK2, ARF1, FHOD1, KPNA3, AHNAK, ARF6, DNM2, FLNB, HSP90AB1, SKP1, VPS11, DYNC1H1, FLNA, MYH10, VPS18, ARPC1B, RAB9A, ACTB, EXOC7, MAP2K3, NCKAP1, NFKB1, PIK3C3, RAB5C, DYNLT1, TRAF2, DYNLRB2, RIPK1, MYH3, RPS3, DYNC1LI2, EXOC5, SNX9, KLC2, RHOB, RRAS, AHNAK2, RAB7A, CASP3, EXOC4, BAX, CASP7, FYCO1, RAB5A, S100A10, DYNLT3, WASL, GSDMD, MYO6, ARHGEF26, RALA, STX10, IL18, FLNC, VPS16, M6PR, VPS33A, TRADD
7 hsa05134 Legionellosis 26/2091 1.05E − 03 5.89E − 03 3.99E − 03 APAF1, HSPA1L, MYD88, EEF1A2, HSPA8, CYCS, HBS1L, BCL2L13, RAB1A, CASP8, RELA, SAR1B, ARF1, EEF1A1, EEF1G, HSPA1B, RAB1B, HSPD1, NFKB1, SEC22B, SAR1A, CASP3, CASP7, HSPA6, NFKB2, IL18
8 hsa05135 Yersinia infection 54/2091 4.64E − 06 4.58E − 05 3.10E − 05 IRAK1, IRF3, MAP2K1, MYD88, ROCK1, WIPF2, FN1, ARHGEF12, BAIAP2, RAC2, ARHGEF28, MAPK8, CHUK, IKBKG, MAPK1, RAC1, ACTG1, MAP2K2, CDC42, CRKL, TBK1, GSK3B, AKT1, MAPK14, PKN1, RHOA, ACTR3, MAP2K4, RELA, ROCK2, RPS6KA3, ARF6, PIK3R2, SRC, PLCG1, ACTR2, CRK, PTK2, ACTB, MAP2K3, NFKB1, PXN, TRAF2, ARHGEF1, ITGB1, PKN2, VAV2, WASF2, WASL, IL18, ITGA5, RPS6KA1, DOCK1, PIK3R1
9 hsa05163 Human cytomegalovirus infection 85/2091 5.57E − 05 4.15E − 04 2.81E − 04 CTNNB1, GNAO1, GNG2, HLA‐B, HLA‐C, IRF3, MAP2K1, PRKCB, ROCK1, RPS6KB1, RPS6KB2, PPP3R1, ARHGEF12, PPP3CA, PPP3CB, TSC2, RAC2, CHUK, IKBKG, HLA‐A, MAPK1, RAC1, GNB1, GNB2, CREB1, PRKCA, MAP2K2, STAT3, CCND1, CRKL, FADD, TBK1, CYCS, GSK3B, PRKACA, AKT1, MAPK14, RHOA, B2M, GNG12, PRKACB, GRB2, CALR, CASP8, RELA, ROCK2, GNG5, MTOR, CDKN2A, PIK3R2, SRC, GNAI2, ITGAV, CRK, GNAQ, PTK2, RHEB, GNAI1, NFKB1, CDK6, GNAS, PXN, TRAF2, ARHGEF1, RIPK1, NRAS, GNAI3, PLCB3, EGFR, BID, CASP3, BAX, GNA11, PDIA3, CALM3, ITPR3, RB1, TAP1, GNB4, GNA13, TAP2, TAPBP, TRADD, EIF4EBP1, PIK3R1
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