Helicobacter pylori is a microaerophilic, gram-negative bacterial pathogen infecting greater than half of the worldwide population . The incidence of H. pylori ranges from 30 to 50% in developed international locations, while the incidence is far larger in growing international locations, starting from eighty five to 95% [2,three,4]. It induces an inflammatory response in the stomach by colonizing the gastric epithelium [5,6]. The pathogen persists for many years within the host and increased infection is associated with a quantity of diseases starting from mild to extreme infections similar to autoimmune atrophic gastritis, duodenal ulcer, and gastric cancer [7,eight,9,10]. Gastric ulcer develops in 10–20% of contaminated people and 1–3% could develop gastric most cancers . The risk of gastric cancer is 3–6-fold higher in H. pylori-infected individuals in comparison with non-infectious people [12,13].The prevalence of H. pylori in Pakistan may be very excessive (81%) when in comparability with different South Asian countries . This excessive prevalence is primarily due to factors similar to dietary habits and co-infection with other gastrointestinal pathogens . Numerous proteomic and genomic studies have recognized pathogenic variants responsible for inflicting a medical outcome within the host; nevertheless, genetic variety resulting in geographical variations of the pathogen is considered responsible for completely different phenotypes and illness onset [16,17]. It is nicely reported that genetic variety augments protein abundance, primarily via transcriptional and post-transcriptional signaling [18,19]. Proteomic investigation of the pathogen is a promising device to acquire protein profiles associated with a distinct clinical end result. Further proteome profiling of H. pylori is necessary to correlate epigenetic alterations with gene regulation and virulence mechanism. Similarly, protein signature amongst gastric-disease-causing H. pylori isolates validate the correlation between pathogen and illness sample [20,21,22].Investigation of transition from a light to a extreme type of the illness on the expression level is still elusive, and the molecular pathway just isn’t yet understood. This work investigated protein expression profiles of H. pylori isolated from gastritis, gastric ulcer, and gastric most cancers sufferers using LC-MS/MS analysis. Findings of the research could help to understand pathogenesis of H. pylori.
2. Materials and Methods
This research was carried out at the Department of Microbiology, Abdul Wali Khan University Mardan between 2019 and 2021. The research was permitted by the ethics committee of Hayatabad Medical Complex Peshawar (reference quantity:370/HEC/B&PSC/2020) to be used of human topics, the approval date is 28 November 2020. Data were collected through questionnaire and knowledgeable written consent was taken from the patients (n = 150) visiting the endoscopy section of the Gastrointestinal Department.
2.1. Identification of Helicobacter pylori
The schematic illustration of the methodology is given in Figure 1. For confirmation of Helicobacter pylori infection, contemporary biopsy samples from sufferers with gastric cancer (n = 35), gastric ulcer (n = 53), and gastritis (n = 62) have been collected. About 4 mm of tissue samples from the antrum of the stomach had been taken in a sterilized Petri plate and reduce into pieces using a sterile scalpel blade. Biopsy samples had been cultured on Columbia blood agar base (Oxoid, Hampshire, UK), supplemented with 5% sheep blood, utilizing DENT (Oxoid, Hampshire, UK) and CampyGen sachet (Thermo Fischer Scientific, Warrington, UK) and was morphologically and phenotypically recognized. Biochemical identification was carried out using oxidase, catalase, and urease checks . The findings have been confirmed by way of PCR concentrating on species-specific 16S rRNA genes utilizing primers (F-5′-GCGACCTGCTGGAACATTAC-3′), R (5′-CGTTAGCTGCATTACTGGAGA-3′). 2.2. Sample Preparation and Peptide Sequencing
PCR-positive H. pylori was sub-cultured in thioglycolate broth (Oxoid, Hampshire, UK) with CampyGen sachet for 72 h in an anaerobic jar. The broth was centrifuged at 4000× g for 20 min at four °C and a pellet was obtained. For label-free proteomics, proteins were extracted by resuspending the pellet in 800 μL of lysis buffer (Urea 7M, Thiourea 2M, 4% CHAPS and 1% DTT) and then 500 µL of protein resolution was desalinized utilizing Amicon Ultra-0.5 mL 3K-NMWL filter units (Merck Millipore Corporation, Darmstadt, Germany). The Bradford methodology was used to discover out the ultimate protein concentration. A measure of 500 μg of proteins were denatured with zero.1% RapiGest (RapiGest SF, Waters, Milford, CT, USA), then reduced with dithiothreitol (final focus 5 mM) and alkylated with iodoacetamide (final focus of 15 mM). For in-solution digestion, 60 µg of proteins from 10 pooled samples of every group (gastritis, gastric ulcer, and gastric cancer) was used. Trypsin digestion was carried out with sequencing Grade Modified Trypsin (Thermo Scientific™, Waltham, MA, USA) at a 1:a hundred (w/w) enzyme: protein ratio. After digestion, 1% (v/v) of trifluoroacetic acid was added to hydrolyze the RapiGest. The digested peptides were filtered using zero.2 µm spin filter (Corning, New York, NY, USA). Peptide sequencing was performed on QTOF G2 HDMS (Waters, Milford, CT, USA) at National Horizons Centre, Teesside UK. Chromatogram of the peptide fragments had been acquired by reverse-phase ultraperformance liquid chromatography (Acquity UPLC H-Class System, Waters, Milford, CT, USA). The separation was performed by way of an ACQUITY UPLC HSS T3 2.1 mm × 100 mm column, utilizing a binary gradient from 2 to 85% of acetonitrile with 1% formic acid (v/v) for sixty two, at a move rate of 250 μL/min. Data-independent scanning (MSE) experiments have been performed by switching between low and elevated collision energies (25–50 eV), and a scan time of 0.5 s was used for low- and high-energy scans from m/z 50 to 2000.
2.three. Data Analysis
The uncooked information from QTOF mass spectrometer were analyzed by Progenesis QI for proteomics (Waters, Milford, CT, USA). The reference protein sequences were downloaded from NCBI database to look MS/MS spectra towards the reference sequence. A false discovery rate of 4% was selected. Peptide tolerance and fragment tolerance were auto-selected. Proteins with 1 or extra peptides, three fragments per peptide or 7 fragments per protein identified were thought of quantifiable. Protein fold change was calculated using t-test, and cut-values of 1.5 at p-value , accessed on 22 April 2022) for significantly differentiated proteins have been downloaded and was uploaded on STRING version eleven.5 (-db.org, accessed on sixteen June 2022, Version quantity 11.5, Mardan, Pakistan) for protein–protein interplay networks and useful enrichment analysis. CELLO2GO net server (/cello2go/, accessed on sixteen June 2022) was used for the subcellular localization of differentiated proteins (Yu et al., 2014). The VaxiJen: Prediction of Protective Antigens and Subunit Vaccines (version 2.0) webserver was used for potent antigenicity prediction of differentially expressed proteins. InteractiVenn, a web-based software , was used for plotting Venn diagrams of overlapping proteins in three comparisons. Finally, protein fold change of overlapping proteins was plotted to match variation in the expression of proteins from one disease to a different and their possible interplay with one another. three. Results
The isolation of H. pylori from the biopsy samples of gastric sufferers was carried out via culturing. Results of cultured samples and subsequent microscopical examination along with biochemical and PCR amplification are depicted in Figure 2.Among the included topics (n = 150), forty two.7% of patients were more than 50 years of age, and 54% of patients had been females and 46% of sufferers have been males. Of the themes, 76.7% had no ulcer issues and a giant number of subjects, i.e., forty one.3%, have been affected by gastritis, followed by 35.3% of ulcer sufferers. Demographic elements are summarized in Table 1.Proteome investigation of H. pylori recognized a complete of twenty-two proteins (Supplementary Table S1) that comprised 6 significantly totally different proteins in cancer compared to gastritis with a fold change ranging from −2.52 to +4.81, whereas 9 proteins had been significantly regulated in cancer in comparison with ulcer, with a fold change starting from −2.sixty six to +10.19, while 8 proteins had been significantly regulated in ulcer compared to gastritis, starting from −6.16 to +4.18. The most importantly regulated protein was thioredoxin peroxidase, which was found to be downregulated with a worth of −6.sixteen from gastritis to ulcer, and −10.19 from ulcer to most cancers patients. Details of recognized proteins are tabulated in Table 2.Venn diagram reveals no overlapping protein amongst three groups; however, three different proteins had been found to be overlapping between the 2 teams. Compared to gastritis, both in ulcer and most cancers, hypA protein confirmed an upregulation which is involved in Nickle cation binding and performs a task in Pathogenesis.
This household protein, which is an unreviewed protein within the Uniprot database, was downregulated with illness severity, while HNH endonuclease family protein showed greater ranges of suppression in ulcer compared to cancer. Similarly, comparison of gastritis and cancer with ulcer indicated a suppression of thioredoxin peroxidase in the case of most cancers, and overexpression within the case of gastritis. A similar trend was observed in the case of DUF3972-domain-containing proteins, however there is a small diploma of lower in protein downregulation in comparison with thioredoxin peroxidase. Sec-independent protein translocase protein tatB confirmed a reverse trend compared to these of the opposite two proteins in the group. In the third comparability of overlapping proteins of cancer vs. ulcer and cancer vs. gastritis, nucleoside diphosphate kinase expression was lower in both gastritis and ulcer, indicating an increase in kinase exercise in extreme disease forms. An opposite development was noticed within the case of putative PZ21b protein and pseudaminic acid synthase protein, both of that are antigenic proteins. A Venn diagram of union and mobile localization of expressed proteins along with differential expression of overlapping proteins in each group is illustrated in Figure three.Protein–protein interaction by STRING database confirmed higher proportion of identities matched with H. pylori strain, however no interplay was discovered among these proteins (Figure 3). However, a few of these proteins are concerned in necessary molecular processes and pathways. For instance, hypA is concerned in nickel cation binding, tatB is a mobile part of the TAT protein transport complex, pseI is an element of O-Antigen nucleotide sugar biosynthesis, and ThiS family protein (C694_04105), which is a Molybdopterin-converting factor, as a small subunit, is involved within the sulfur relay system (Figure 4). Cellular localization of these proteins represented 14 (74%) as cytoplasmic, 2 (11%) internal membrane, 2 (10%) outer membrane, and 1 (5%) extracellular protein. Further exploring antigenicity of the proteins at threshold degree of 0.4, 7 out of 14 proteins were predicted as antigenic (Table 3).Investigating the position of expressed proteins revealed that they function cellular components corresponding to protein complexes, cytoplasm, cell membranes, and intracellular elements. Furthermore, these proteins are concerned in a wide selection of organic processes (transmembrane transport, macromolecular complex meeting, response to emphasize, pathogenesis, etc.), as properly as molecular functions such as oxidoreductase exercise, hydrolase activity, nuclease activity, etc. (Figure 5). 4. Discussion
In the current examine, the proteome profile of H. pylori, infecting the Pakistani population and causing a sequel of illnesses from the milder kind, i.e., gastritis, to the severe form, i.e., ulcers, and ultimately the deadly form, i.e., gastric cancer, was investigated. There are several epidemiological and genetic research on H. pylori reported from Pakistan; nonetheless, there’s a extreme lack of molecular data, similar to those addressed on this proteomic investigation. We recognized proteins liable for different illness phenotypes by way of differential protein expression utilizing nano LC-QTOF analysis.
For proteome profiling, 10 pooled samples of H. pylori every from gastritis, ulcer, and most cancers have been processed. Among the identified proteins, hypA, which is answerable for nickel cation binding, was upregulated in cancer and ulcer. Nickel is a virulence determinant as a outcome of it is a co-factor of urease, which is required for homing within the stomach by resisting acidity. A previous research indicated a 50-times-higher concentration of nickel in H. pylori compared to E. coli . Therefore, a provide of nickel is crucial for its survival within the stomach. In our research, the overexpression of hypA protein in most cancers and ulcer might improve nickel binding and urease exercise, and this has a role in illness development.Pseudaminic acid was upregulated in most cancers vs. ulcer, however downregulated in cancer vs. gastritis. It is reported that flagellar glycosylation is significant for flagellar perform [23,24], and the inactivation of genes answerable for post translational modifications, similar to glycosylation, may end result within the inactivation of flagella [25,26]. O-linked glycosylation attributable to synthesis of pseudaminic acid (Pse) is essential for useful flagella. In addition, Pse biosynthesis aids within the production of virulence elements corresponding to urease and lipopolysaccharide (LPS) [27,28]. It suppresses host immune response by masking the epitope of antigens of outer membrane proteins . Previous genetic research have reported elimination of practical flagella as a outcome of inactivation of the pseI gene of H. pylori, ultimately leading to reduced motility [30,31]. Increased expression of PseI in cancer vs. ulcer and decreased expression in cancer vs. gastritis might replicate its position within the preliminary and superior phases of disease onset brought on by H. pylori.Another enzyme expressed with the highest value of suppression is thioredoxin peroxidase. Protein–protein interplay analysis of thioredoxin peroxidase confirmed bacterioferritin co-migratory protein (bcp) in H. pylori strain as probably the most similar protein with an id rating of 98.7%. Bacterioferritin co-migratory protein (bcp) belongs to the peroxiredoxin household and is often generally identified as thiol peroxidase . The peroxidase enzyme utilizes lowered thioredoxin as an electron donor for its substrate catalytic reduction. Thioredoxins and thiol peroxidases play a task in resistance to bactericidal reactive oxygen species and reactive nitrogen species . However, bcp is proven to be concerned within the removing of fatty acid hydroperoxides generated by oxidative stress or by metabolism . Further mechanistic research to explore its function in an infection would help to know its inverse relationship with the severity of the disease.One of the protecting effector molecules is nucleoside diphosphate kinase (ndk) secreted by intracellular microorganisms during tissue colonization in the host. This effector molecule modulates signaling of host-derived small hazard molecules [35,36], establishing persistent infections in the host . Ndk-Purinergic signaling, by which ndk reduces extracellular purine signaling molecules such as extracellular ATP, is the primary target by which it modulates host immune response. Secretion of these effector molecules by H. pylori interact with oncogenic pathways and may induce carcinogenesis . It is of great importance because of a number of immune invasion methods during infection. In our research, upregulation of ndk in cancer-causing H. pylori may need a possible function in gastric adenocarcinoma.The twin-arginine translocation (Tat) pathway plays a exceptional role in the transport of folded proteins . This pathway transports folded proteins in a secretory (sec)-independent method  and is particularly concerned in the export of virulence proteins. Location and activity of hydrogenase and catalase is affected with impaired Tat machinery in H. pylori. In the current examine, the upregulation of tatB in cancer- and gastritis-causing H. pylori might need a possible function within the virulence mechanism of the pathogen.The proteins expressed in our research had been either immediately playing a task in virulence, similar to hypA, ndk, pseI, and bcp, by facilitating the pathogen in colonization throughout the acidic medium of the stomach, whereas tatB is concerned in the translocation of virulence proteins. However, the specific function of tatB and bcp in pathogenesis remains to be unknown. Further studies in regards to the specific position of these proteins in gastric pathologies would assist to know the molecular pathogenesis of H. pylori.
Differential proteome of the isolates revealed upregulation of hypA protein involved in bacterial colonization and tatB protein involved in protein translocation, whereas revealing downregulation of bacterioferritin co-migratory protein (bcp), that performs a job in resistance against reactive oxygen and nitrogen species. Functional characterization of the identified proteins would assist to explain the pathogenesis and virulence mechanism of H. pylori.