Quantitative proteomic analysis of the tizoxanide effect in vero cells

Nitazoxanide (NTZ) is effective against helminths and numerous microorganisms, including bacteria and viruses. In vivo, NTZ is metabolized into Tizoxanide (TIZ), which is the active circulating metabolite. With the emergence of SARS-Cov-2 as a Pandemic agent, NTZ became one of the molecules already approved for human use to engage clinical trials, due to results in vitro showing that NTZ was highly effective against the SARS-Cov-2, agent of COVID-19. There are currently several ongoing clinical trials mainly in the USA and Brazil involving NTZ due not only to the in vitro results, but also for its long-known safety. Here, we study the response of Vero cells to TIZ treatment and unveil possible mechanisms for its antimicrobial effect, using a label-free proteomic approach (LC/MS/MS) analysis to compare the proteomic profile between untreated- and TIZ-treated cells. Fifteen differentially expressed proteins were observed related to various biological processes, including translation, intracellular trafficking, RNA processing and modification, and signal transduction. The broad antimicrobial range of TIZ points towards its overall effect in lowering cell metabolism and RNA processing and modification. The decreased levels of FASN, HNRNPH and HNRNPK with the treatment appear to be important for antiviral activity.

www.nature.com/scientificreports/ The broad-spectrum nature of NTZ/TIZ also suggests that its ability to inhibit different viruses could be a consequence of its action on the host-regulated processes rather than its direct effect on the virus processes themselves. It seems plausible then, that the effect of these molecules could not only be a result of the direct action of the drug upon the virus or its proteins but might also stem from its influence on the way that the cell responds to the virus.
In December of 2019, China reported the outbreak of a new disease, caused by a new Coronavirus that was later named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The disease was named COVID-19. On March 11, the World Health Organization declared COVID-19 a pandemic. Since then and to this date, SARS-CoV-2 has over 3.7 million confirmed cases and has killed over 260 thousand people. Although there are speculations about a vaccine on a fast track, most specialists are not very optimistic about a quick vaccine solution for COVID- 19. In vitro studies with nitazoxanide and tizoxanide were performed with canine coronavirus strain S-378, with a murine strain of coronavirus (MHV-A59) and a bovine coronavirus strain (BCoV-L9) with promising antiviral activity 24,25 . NTZ and TIZ had also its antiviral activity tested in vitro against the Middle East Respiratory Syndrome CoV (MERS-CoV) showing also effectivity 26 . Furthermore, Nitazoxanide was tested in China against SARS-CoV-2 (COVID-19 agent) using Vero E-6 cells and showed high antiviral activity 27 .
As the clinical trial results utilizing Nitazoxanide against SARS-Cov-2 infections start to build up, the more we know about the mode of action of this molecule, the better we can approach and rationalize about its use not only for COVId-19 but also for the other viruses being studied with this molecule.
In this work, we aimed to examine the response of Vero cells to TIZ treatment and unveil possible mechanisms for its antimicrobial effect. To this end, a label-free proteomic approach using liquid chromatography-tandem mass spectrometry (LC/MS/MS) was used as strategy. We performed a comparative proteomic analysis between untreated-or TIZ-treated Vero cells, highlighting differential abundance of proteins involved in cell processes. TIZ-treated Vero cells caused a decrease in several protein abundance levels related to different biological processes. It is our understanding that the way TIZ influences a broad range of cells and pathogens can bring new insights in the development of molecules with broad anti-pathogen action.

Results
cell viability assay and teM. In order to evaluate the toxicity of TIZ in Vero cells, neutral red dye-uptake method was performed. The cytotoxicity assay showed no interference of the DMSO at the concentrations used in the samples in Vero cells and the TIZ influenced cell viability in a dose-dependent manner as shown in Fig. 2. The calculated CC 50% of TIZ was 1.77 μg/mL. A non-toxic concentration of TIZ, 0.5 μg/mL, was selected as a concentration for downstream experiments. TEM of ultrathin sections of Vero cells were also performed in an attempt to observe morphological changes or other phenomena such as inclusion bodies, alterations in cell membrane, or increases in the number of cytoplasmic vesicles the TIZ treatment could cause in comparison to untreated cells. However, no significant disturbances were observed (Fig. 3).  www.nature.com/scientificreports/ 1D electrophoresis. To visualize the changes in protein profile of Vero cell in response to TIZ treatment, total protein of confluent Vero cells untreated and TIZ-treated were extracted, dosed and loaded for SDS-PAGE. Fluorescent and Coomassie blue staining showed that the proteins were successfully extracted (Fig. 4). As indicated in Fig. 4, it is possible to observe several bands in the SDS-PAGE gel becoming highly intense or almost disappearing with TIZ treatment compared to the untreated control. These differences in band intensity slightly changed the protein profile of Vero cells mainly above 28 kDa and were similar in all replicates.
Protein identification and functional protein identification and functional categorization. In order to concentrate the sample and quantify the differences in overall protein abundance levels in untreatedand TIZ-treated Vero cells, FASP digestion using a 30 kDa cut-off filter was performed. A total of 1,303 proteins were identified (Supplementary Table 1). Gene Ontology analysis identified 133 functional subcategories of proteins (www.agbas e.mssta te.edu), and three primary categories: cellular component (28), molecular function (39), and biological process (66) (Fig. 5). The greatest differences were observed in the biological process subcategory: ribonucleoprotein complex assembly, nucleocytoplasmic transport, tRNa metabolic, circulatory system, cofactor metabolic and cellular amino acid metabolic process. They presented more than a 100% increase in the number of proteins in treated cells compared to the untreated cells.  www.nature.com/scientificreports/ For cellular component, TIZ-treated cells had an increase in the number of proteins associated with cilium, nuclear chromosome and cytoplasmic membrane-bounded vesicle while ribosome proteins were decreased. In the molecular function category, more proteins were associated with methyltransferase activity, ubiquitin-like protein binding and GTPase activity and fewer proteins associated with structural constituent ribosome, transferase activity, transferring glycosyl groups, lipid binding and ATPase activity in TIZ-treated cells. teins in both samples, using the parameters described in the Methods section. According to the statistical analysis, FASP method was able to identify 15 differentially expressed proteins at the p < 0.05 level, 12 of them with higher abundance in mock-treated samples and 3 in TIZ-treated samples, involved in different biological process such as translation, ribosomal structure and biogenesis, intracellular trafficking, secretion and vesicular transport, signal transduction, chromatin structure and dynamics, RNA processing and modification and cytoskeleton (Table 1).

Discussion
NTZ is a safe clinical approved antiparasitic agent indicated for the treatment of a variety of gastrointestinal infections and, along with its active form, TIZ, has been shown to inhibit a broad range of viruses and microorganisms. With the ongoing COVID-19 pandemic, clinical studies with Nitazoxanide and its efficacy against SARS-CoV-2 infections (COVID-19) are currently underway. In this context, more information on how the host cell responds to TIZ would be interesting to unravel other intracellular mechanisms responsible for its antiviral and antimicrobial activity. In 2015, MS-based proteomic approaches were used as a new and promising tool to help understanding cellular changes in metabolism due to a treatment exposure 28 . In this study, a label free MS-based proteomic approach was used to identify proteins whose abundance is altered by TIZ treatment and to show possible cell processes disturbed or enhanced by it. A filter-aid sample preparation (FASP) method was adopted for protein digestion strategy. Cell viability assay was performed to assess the Vero cells response to TIZ treatment and to help us set our treatment protocol while TEM was performed for visualization of cell morphology and 1D electrophoresis for visualizations of proteome profile. It has been reported that the group of proteins with altered abundance in proteomic studies of virus-infected cells or cells exposed to some level of stress, such as drug action, is small. Besides, the changes can be subtle and, sometimes, with fold change less than 2 29 . Even using different approaches as isotopic labelling or Differential Gel Electrophoresis (DIGE), the number of detected proteins with altered abundance does not increase dramatically 30,31 . When we considered a p value < 0.05, as demonstrated in Table 1, the FASP label-free LC/MS/ MS methodology was able to identify 15 cellular proteins with different abundance levels when treated with TIZ (3 had increased levels and 12 had decreased levels). This indicates a subtle effect of TIZ on the cell.
Among the decreased proteins in metabolism, we highlight fatty acid synthase (FASN), a protein responsible to synthesize palmitate from acetyl-CoA and malonyl-CoA in a reaction that requires NADPH. Palmitate, the core of fatty acid production, can be further metabolized into a variety of long chain fatty acids that will be used in lipid production for membrane biosynthesis and lipid droplet formation 32 . Importantly, many viruses induce and require fatty acid synthesis at some stage of their replication cycle and the interference in this pathway could affect the virus production. The use of critical inhibitors of enzymes for fatty acid synthesis such as acetyl-CoA www.nature.com/scientificreports/ carboxylase and fatty acid synthase led to a significant decrease in the production of infectious cytomegalovirus, influenza A, hepatitis C and dengue viruses 33 . In the case of dengue virus, non-structural protein NS3 seems to be responsible for FASN recruitment, and lipidomics of mosquito infected cells showed that some sphingolipids and phospholipids were upregulated by dengue infection 34 . Therefore, FASN inhibition caused by TIZ treatment could explain the wide antiviral action of this substance. However, the specific mechanism by which inhibition of fatty acid biosynthesis interferes with the replication of these viruses has not yet been determined. Studies suggest that possible changes in membrane composition required for viral replication, assembly or budding, decreases in envelope phospholipid synthesis or delays in the modification of fatty acids from proteins would be among the possible targets caused by inhibition of FASN in infected cells 33,35 . Ras-related protein Rap-1a, a signal transduction-related protein, was detected in lower levels with the TIZ treatment. Signal transduction processes are, in many aspects, protein-driven events. Changes in protein level, activity, localization or interactions allow cells to react to a specific event and also to vary the sensitivity, duration and dynamics of the response 36 . Ras-related protein Rap-1a have been described to interact with several different pathways, namely MAPK, ERK, GRK2, (JAK)/STAT, and NF-kB [37][38][39] . In addition, they have been implicated in other biological events, including Intracellular trafficking, secretion and vesicular transport, Transcription, replication, recombination and repair, and defense mechanisms. Thus, more information is needed about these complex interactions at both the cellular and whole organism levels for a better understanding of the TIZ antimicrobial activity.
The abundance levels of two proteins related to RNA processing and modification were also altered with the TIZ treatment. Newly synthesized RNA molecules undergo different modifications before their translation in the cytoplasm. These processing steps include specific modifications of RNA nucleotides at the ends of the primary transcript or at internal positions, and removal of internal extra RNA sequences 40 . Levels of spliceosome RNA helicase DDX39B and heterogeneous nuclear ribonucleoprotein H (HNRNPH) were decreased with the treatment. These numbers are in accordance to the overall number of ribosome proteins, decreased with the treatment.
HNRNPs are a large Family of RNA-binding proteins involved in alternative splicing, mRNA stabilization, and transcriptional and translational regulation [reviewed in Ref. 41 ], and they have been reported to interact with several virus proteins and RNA. Studies demonstrate that HNRNPH is upregulated with dengue infection in cell culture in addition to interacting with dengue non-structural protein 1, helping the virus to propagate in the cell 42 . HNRNPH also seems to be necessary to bind to specific retroviruses proteins to control the RNA splicing needed to HIV-1 and Rous sarcoma virus life cycle 43 . Therefore, decrease in the amount of HNRNPH account for part of the broad-spectrum antiviral activity of TIZ.
In addition, three proteins with increased levels with the treatment stood out: dynactin subunit 2, eukaryotic translation initiation factor 6 (EIF6) and cytoskeleton associated protein 4 (CKAP4).
Dynactin subunit 2, one of the increased proteins in intracellular trafficking, secretion and vesicular transport, is part of the dynactin complex, acting as cofactor for the dynein, a minus-ended-directed microtubule associated motor responsible for retrograde transport in eukaryotic cells. The dynactin-dynein motor complex has been implicated in several important subcellular functions involving intracellular organelle transport 44 , including the transport of many viruses from cytosol to their site of replication 45 . Due to the great diversity of viruses and their replication mechanisms and requirements, each antiviral activity must be evaluated individually. On the other hand, Harrinson et al. 46 demonstrated that full maturation of phagosomes of the murine monocyte/ macrophage line depends on dynein-dynactin association to acquire its antimicrobial properties required for pathogen elimination.
EIF6 is a protein from the translation initiation group that does not function as an initiation factor and is known to be important in ribosome biogenesis by regulating cellular levels of free 60S subunit 47  CKAP4 also known as cytoskeleton-linking membrane protein 63 (CLIMP-63) or p63, is a stable and abundant type II transmembrane protein 51 predominantly located in the RER and present in higher eukaryotes 52 . It has multiple functions including maintaining ER structure, ribosome anchoring in the RER, RER anchoring to the cytoskeleton via microtubule interaction, besides acting as a receptor for different ligands. In addition, Li et al. 53 demonstrated that expression of CKAP4 discouraged cell cycle progression and reduced the proliferation ability of hepatocellular carcinoma cells.
The current results in this work confirm the safe clinical use of TIZ. It presented low toxicity and subtle changes in protein profile were observed. These changes do not seem to be responsible for triggering a specific protein for antimicrobial effect in general, but to act on the cell as a whole. Nevertheless, the decreased levels of FASN, HNRNPH and HNRNPK with the treatment appear to be in part responsible for the antiviral success against several viruses. The biosynthetic pathway of fatty acids has already been suggested as targets for development of therapeutics that inhibit the replication of DENV and other enveloped viruses 58 . However, most of the studies are focused on virus-cell interaction and there is still a lot to understand about how cellular metabolism changes influence on host response to viral infection at the organism level, from the production of hormones to immune responses.
In this work, we report for the first time a differential proteomic analysis between Vero cell cultures mockand TIZ-treated by label-free LC/MS/MS analysis. This proteomics study provides valuable data for a better Scientific RepoRtS | (2020) 10:14733 | https://doi.org/10.1038/s41598-020-71634-2 www.nature.com/scientificreports/ understanding of the roles played by host cell proteins during TIZ treatment. The broad antimicrobial range of TIZ points towards its overall effect in cell metabolism and RNA processing and modification. Nevertheless, further functional analysis is necessary. The list of proteins is likely to be further extended by improving the proteomic analysis, i.e. by increasing the number of replicates in the analysis, providing some level of protein fractionation, or performing a longer peptide separation coupled to a more sensitive and faster scanning mass spectrometer. Our data provides evidence that the knowledge of the functional expression of proteins may be of value for therapeutic purposes. www.nature.com/scientificreports/ concentrated onto a 2 cm × 100 µm i.d. Pepmap C18 (5 µm particle size) (Thermo Scientific), and then eluted onto and separated using a self-packed PicoFrit (New Objective, Woburn, MA, USA) 75 µm id × 25 cm Magic C18 column (3 µm particle size) with a 60 min linear gradient from 2% mobile phase B to 40% mobile phase B (A = 2% acetonitrile in water, 0.1% formic acid; B = acetonitrile, 0.1% formic acid). An electrospray voltage of 2.8 kV was applied to the PicoFrit column to ionise peptides in the nanoelectrospray ion source of the Obitrap Elite with a heated capillary temperature of 275 °C. The data acquisition of MS (scan range of m/z 400-2000) and MS/MS (scan range of m/z 140-2000) were collected utilizing the Orbitrap analyser. A top 5 method with higher-energy collisional activation (HCD) for product ion generation in the HCD cell was used with normalized collision energy setting of 27 V to induce precursor ion fragmentation (+ 1 charge states were excluded).

Methods
Protein identification and analysis. Raw LC/MS/MS datafiles were processed using MaxQuant software 57 and database searched using the integrated Andromeda 58 search engine against the non-redundant database of the National Center of Biotechnology Information (NCBI) containing entries from Chlorocebus sabaeus as well as the two protein standards bovine serum albumin and rabbit glycogen phosphorylase (62,148 entries). Trypsin was defined as the digesting enzyme, along with a maximum of two missed tryptic sites, whereas fixed carbamidomethyl Cys modification and variable oxidized Met modification were permitted. A false discovery rate (FDR) of 0.05 was utilized. Otherwise, default MaxQuant and Andromeda parameters were used for processing and searching. For statistical analysis, the MaxQuant ProteinGroups report was imported into Perseus (www.perse us-frame work.org), and protein label free quantification (LFQ) intensities based on extracted ion chromatograms were used to compare expression differences between sample groups. Data preprocessing prior to analysis of variance (ANOVA) included the following steps: Log(2) transformation of protein intensities and replacing missing data with a value approximating the lower limit of detection. Only proteins that were observed in three out of three replicates for at least one treatment group were retained and subjected to ANOVA. Statistically significant proteins were retained at the p < 0.1 significance level.
The identified and validated proteins from protein digestion were classified according to gene ontology (GO) terms in cell component, biological process and molecular function by AgBase software (www.agbas e.mssta te.edu). The uncharacterized proteins were classified according to GO terms and protein function from Homo sapiens proteins searched at Universal Protein Resource (Uniprot) catalog (www.unipr ot.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database (www.genom e.jp). To reduce the large number of GO terms, the biological process was divided into 15 terms: Cell cycle control and cell division, chromatin structure and dynamics, cytoskeleton, defense mechanisms, energy production and conversion, Inorganic ion transport and metabolism, intracellular trafficking, secretion, and vesicular transport, membrane organization, metabolism (including carbohydrate, amino acids and lipids transport and metabolism), post-translational modification, protein turnover, and chaperones, RNA processing and modification, secondary metabolites biosynthesis, transport and catabolism, signal transduction, transcription, replication, recombination and repair, translation, ribosomal structure and biogenesis, and other function.

Data availability
All data generated or analyzed during this study are included in this published article and the Supplementary  Table and deposited