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Comparative analysis of Brucella melitensis whole cell proteomes for diagnosis of human brucellosis
For correspondence: Dr Chiranjay Mukhopadhyay, Department of Microbiology, and Centre for Emerging and Tropical Diseases, Kasturba Medical College, Manipal 576 104, Karnataka, India e-mail: chiranjay.m@manipal.edu
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Received: ,
Accepted: ,
Abstract
Background & objectives
Brucellosis, an occupational zoonosis, poses notable difficulties in diagnosis because of the need for secure facilities and the risk of exposure when using culture-based methods. Conventional serological tests often suffer from cross-reactivity, highlighting the need for more specific diagnostic markers. The purpose of this study was to understand the differences in whole-cell protein expression of Brucella melitensis isolates from patients with acute and chronic brucellosis and to identify immunodominant proteins that could serve as potential diagnostic markers.
Methods
The study included 16 blood culture-confirmed patients with acute or chronic brucellosis from a cohort of 185 individuals. Clinical isolates of B. melitensis were subjected to two-dimensional gel electrophoresis (2-DE) and PDQuest software analysis to compare proteomic profiles. Differentially expressed proteins were identified using western blotting with infected human serum samples. Further identification of the protein spots was conducted using Matrix-Assisted Laser Desorption/Ionization - Time-of-flight Mass Spectrometry (MALDI-TOF MS).
Results
The proteomic analysis of B. melitensis identified an average of 249 protein spots. PDQuest software revealed 65 differentially expressed spots, with 17 of these spots successfully identified via MALDI-TOF MS. Western blot assays demonstrated that human sera containing anti-Brucella antibodies reacted with seven immunodominant protein spots. Among these, the outer membrane porin protein (omp25), aldehyde dehydrogenase, and universal stress protein showed potential for differentiating acute from chronic brucellosis.
Interpretation & conclusions
The findings indicate that omp25, aldehyde dehydrogenase, and universal stress protein are highly specific to B. melitensis infections and could serve as valuable targets for serodiagnosis.
Keywords
2-D gel electrophoresis
human brucellosis
MALDI-TOF
proteomics
western blot
Brucellosis is among the most common bacterial zoonoses globally, with >5 million new infections recorded each year 1. Despite its endemicity in various developing countries, including India, brucellosis continues to be under diagnosed and underreported1. Brucella melitensis is the most virulent and the primary cause of human brucellosis2. Diagnosing brucellosis has always been challenging because of its wide range of clinical manifestations, which often overlap with other infectious and non-infectious diseases. While bacterial culture and nucleic acid amplification tests (NAATs) can confirm the presence of the causative organism, they are frequently hazardous, time-consuming, and less sensitive3. Serological tests such as the standard agglutination test (SAT), Rose Bengal test (RBT), and enzyme-linked immunosorbent assay (ELISA) are commonly used for diagnosis3, 4. These tests detect antibodies against sonicated whole cell or smooth lipopolysaccharide (S-LPS) antigens of Brucella strains3, 4. Although S-LPS elicits a strong antibody response, its structural similarities to some Gram-negative bacteria, like Yersinia enterocolitica, Salmonella, Vibrio cholerae, Escherichia coli, Francisella tularensis, and others, can lead to cross-reactivity5, 6. Further, these antibodies can persist for over a year following an acute Brucella infection, making it difficult to identify between acute and chronic disease7, 8. Developing diagnostic assays based on specific protein antigens could help overcome these limitations.
Identifying immunodominant protein antigens is crucial for developing specific protein-based serological tests for brucellosis. Proteomic characterisation of whole-cell proteins provides valuable insights into the protein interactions, metabolic pathways, and phenotypic expression of the pathogen including Brucella9, 10. Recent studies have highlighted the variations in protein expression among virulent B. melitensis strain 16M and the attenuated vaccine strain Rev 1, leading to a clear understanding of the role of metabolic pathways involved in virulence attenuation11. Moreover, differences in bacterial protein expression and the host’s antibody response have been observed in acute versus chronic infections11. Few studies have focused on identifying immunodominant antigens and the associated antibody response in human B. melitensis infections12-14. In the present study, two-dimensional (2-D) gel electrophoresis was used to analyses variations in protein expression in B. melitensis isolates from patients with acute and chronic brucellosis. Immunodominant protein antigens were identified through Western blotting and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Materials & Methods
This study was undertaken by the department of Microbiology, Kasturba Medical College and Hospital, Manipal, Karnataka, India after obtaining the ethical clearance from the Institutional Ethics Committee.
Study population and setting
Adult patients (≥ 18 yr of age) with confirmed brucellosis by growth of Brucella spp. from clinical specimens like blood or body fluid were included in the study. Patients with other serious illnesses like HIV, undergoing chemotherapy, other immune system disorders, pregnant women and children were excluded. A total of 185 culture positive cases of brucellosis were diagnosed at Kasturba Medical College and Hospital, Manipal, Karnataka a tertiary care centre, between May 2017 and December 2020. Brucella spp. were isolated from all these 185 patients’ using BacT/ALERT (bioMerieux, France) for blood and body fluid specimens and conventional culture for other clinical specimens. We selected 16 patients diagnosed with acute and chronic brucellosis from this cohort of 185 cases. Informed consents were collected from each and every participants. Cases of acute and chronic brucellosis were identified based on the CDC guidelines4. Acute brucellosis is marked by a gradual onset of fever accompanied by one or more of the following symptoms: night sweats, joint pain, headaches, fatigue, loss of appetite, muscle pain, weight reduction, meningitis, arthritis or spondylitis, or involvement of specific organs such as endocarditis, orchitis or epididymitis, hepatomegaly, and splenomegaly lasting less than 8 wk. Chronic brucellosis is marked by persistent or recurrent symptoms like on and off fever, fatigue, joint pain and potential complications lasting longer than 52 wk4. Serum specimens were collected from each patient, and serological tests including the RBT and SAT were carried out. An antibody titre of ≥ 1:160 is considered significant titre for brucellosis in this study area. Paired sera samples were also collected to check for the 4-fold rise in the antibody titre. Serum specimens were stored at -80°C. Presumptively, Brucella isolates were identified by Gram staining, the oxidase test, and Christensen’s urease test. Further confirmation was performed using multiplex polymerase chain reaction (PCR) targeting the bcsp31 gene for the concurrent detection of genus Brucella (208 bp), B. abortus (498 bp), and B. melitensis (731 bp), followed by genus- and species-specific in-house real-time PCR15,16.
Whole cell-based protein extraction
Isolates were grown at 37°C in brain heart infusion (BHI) broth for 48–72 h. Bacterial cells were harvested and washed three times with phosphate-buffered saline (PBS). The protocol for protein extraction was modified from Rafie-Kolpin’smethod17. Cells were inactivated with 1%formaldehyde and lysed using a buffer containing urea (7M), thiourea (2M), CHAPS (4%), 50mM dithiothreitol (DTT), and 1% protease inhibitor cocktail (Sigma-Aldrich, Missouri, USA). The samples were sonicated (amplitude 60V, pulse 8 with 2-min intervals, for a total of half an hour). After centrifugation for 20 min at 10,000 rpm, proteins were precipitated by adding 10 per cent trichloroacetic acid (TCA) at 4°C. After overnight incubation, the precipitated proteins were washed twice in chilled acetone, centrifuged, and resuspended in rehydration buffer containing urea (7M), thiourea (2M), CHAPS (2%), DTT (0.2%), and carrier ampholyte (pH 3–10).
Protein concentration determination
Concentration of total extracted protein was determined by the Bradford assay, employing Bio-Rad protein stain with bovine serum albumin as the standard18.
First-dimension- Isoelectric focusing (IEF)
IEF was conducted using the 7 cm IPG strips (pH 4–7) and the PROTEAN i12 IEF system (Bio-Rad, California, USA) at 20°C for 8.5 h following the manufacturer’s protocol.
Second-dimension SDS-PAGE
Post focusing, the IPG strips were equilibrated in two buffers: equilibration buffer 1 (0.375 mM Tris-HCl, pH 8.8, 6M urea, 5 mM DTT, 20% glycerol, 2% SDS) for 15 minutes, followed by equilibration buffer 2 (0.375mM Tris-HCl, pH 8.8, 6M urea, 4% iodoacetamide (IAA), 20% glycerol, 2% SDS) for another 15 min. The strips were subjected to electrophoresis at 25°C using 12 per cent SDS-polyacrylamide gels, with a constant current: 10 mA/gel for 15 min, followed by 20 mA/gel for ⁓2 h). The staining of SDS-PAGE gels was performed using 1 per cent Coomassie Brilliant Blue R-25019.
Image analysis
Gel images were acquired from all 16 isolates, with each 2D electrophoresis (2-DE) experiment run in triplicate. The scanning of stained gels were done using a GS-900 calibrated densitometer (Bio-Rad, California, US), and the images were analysed using PDQuest Software, version 8.0 (Bio-Rad, California, US). An average gel was created by matching the protein spots. Spot quantification was based on spot intensity, calculated automatically by PDQuest. One-way ANOVA (P<0.05) was used to compare protein expression between the acute and chronic groups. Only spots reproducibly observed in at least two of the three gels for each isolate were considered differentially expressed20.
Western blotting
For the purpose of western blot, proteins were transferred to a nitrocellulose membrane utilising a semi-dry transfer unit (1mA/cm2 for 90 min). Blocking of the membranes was performed using 5 per cent skimmed milk for 90 min at 37°C and incubated with a 1:100 dilution of pooled sera from the 16 patients with brucellosis (diagnostic titre ≥1:160) as the primary antibody. After PBST wash, the membranes were incubated with horseradish peroxidase-conjugated polyclonal rabbit anti-human antibody (1:500) at 37°C for 1 h. The blots were developed using the chromogenic substrate, 3,3’-Diaminobenzidine (DAB)21.
Protein identification by MALDI-TOF MS and PMF
Excised protein spots were washed with deionised water, destained and in-gel trypsin digestion was performed as described previously21. Peptides were extracted, and a saturated solution of α-Cyano-4-hydroxycinnamic acid (CHCA) in a 1:1 ratio of deionised water to acetonitrile containing 0.1 per cent TFA was prepared and utilised as the matrix. The peptide extract and the matrix, was applied to the MALDI plate. The C18 ZipTip was employed to elute the dissolved peptides (Millipore, USA). The matrix and peptides were spotted onto a MALDI plate. The MS analysis were conducted utilising the MADLI-TOF instrument (Bruker Microflex LRF-20, Flex Control Workstation, Bremen, Germany), which is equipped with delayed extraction and a UV ionisation laser (N2, 337nm) featuring a pulse width of 3 nanosecond. The accelerating voltage was set at 20 Hz, while the grid voltage was adjusted to 18.3kV. For peptide mass fingerprinting, the instrument operated in reflector mode, averaging 500 laser shots per spectrum. The peptide calibration standards comprised a mixture of angiotensin I, angiotensin II, bombesin, substance P, ACTH clip 1-17, ACTH clip 18-39, and somatostatin 28, covering the m/z range of 500 - 5000. The spectra were analysed using the Flex analysis software (Bruker Microflex LRF-20, Flex Control Workstation, Bremen, Germany). The resulting spectrum was submitted for MASCOT search through Bio tools version 3.1. The parameters for the search included one missed cleavage, partial oxidation of methionine, a peptide mass tolerance of 100 ppm, and the chosen database was NCBI-PROT21. A molecular weight search (MOWSE) score >90 typically indicates a statistically significant (P<0.05) match between experimental mass spectrometry data and a protein sequence in a database.
Results
Clinical details of brucellosis patients
Total 16 blood culture-confirmed brucellosis patients at various stages of infection were included in the study (Table I). The common presenting symptoms were fever and back pain. The ages varied from 18 to 72 yr, with mean age of 42.7 (±15.7) yr. Patients were categorised as having acute (0–2 months) or chronic (>12 months) infection. All patients had direct contact with livestock, but none had consumed unpasteurised dairy products. Serum samples were positive for SAT, with titres ranging from 160 to 1280. B. melitensis was identified in all isolates using multiplex PCR. Of the 16 patients, 15 received appropriate therapy, with nine requiring a three-drug regimen (rifampicin, doxycycline, and gentamicin/streptomycin) due to complications. Six patients received a two-drug regimen (rifampicin and doxycycline) for uncomplicated cases. One patient with endocarditis died within 72 h before a diagnosis could be made.
| Characteristics | Number of patients | |
|---|---|---|
| Acute infection | Chronic infection | |
| Male, n (%) | 7 (87.5) | 6 (75) |
| Female, n (%) | 1 (12.5) | 2 (25) |
| Mean age (yr)±SD | 38 ±16 | 46 ±15 |
| Median duration of symptoms in days (IQR) | 15 (8-27) | 365 (365-639) |
| Fever, n (%) | 8 (100) | 6 (75) |
| Neck pain, n (%) | 1 (12.5) | 4 (50) |
| Back pain, n (%) | 3 (37.5) | 3 (37.5) |
| Headache, n (%) | 2 (25) | 1 (12.5) |
| Osteoarticular complications | 3 (37.5) | 5 (62.5) |
| Neurobrucellosis, n (%) | 1 (12.5) | 0 |
| Endocarditis, n (%) | 0 | 1 (12.5) |
| Died, n (%) | None | (12.5) |
Proteomic profile and analysis
The 2-DE gel images of clinical strains of B.melitensis isolated from acute and chronic brucellosis cases are shown in the figure. The proteome map, created using a 7 cm IPG strip (pH 4.0 – 7.0), revealed up to 249 protein spots. Differentially expressed proteins were selected based on a fold change ≥1.5 using PDQuest software. Supplementary figure shows a volcano plot with the negative log 10 t-test P-value against the log2 fold change, highlighting significant differences (P<0.001) between strains from acute and chronic infections. PDQuest analysis identified 65 differentially expressed proteins, with 17 spots identified by MALDI-TOF-MS (Table II). Due to low protein quantities, only 26 per cent were identified. Of the 65 proteins, 44 per cent (n=29) were overexpressed in acute isolates, while 56 per cent (n=36) were overexpressed in chronic isolates. Western blotting with human sera revealed nine immunodominant proteins, including omp25, aldehyde dehydrogenase, and universal stress protein, detected only in acute infections, suggesting potential diagnostic marker for distinguishing between acute and chronic brucellosis (Table III).
- 2-DE map of whole-cell soluble proteins of B. melitensis isolated from acute (A) and chronic (B) patients. The gel pictures were cropped to improve the clarity and conciseness of the presentation.
| Protein name | NCBI prot Accession number | Organism | pIa | Molecular weight (kDad) | Fold change | Matched peptides | MOWSE score | ||
|---|---|---|---|---|---|---|---|---|---|
| Expb | Theoc | Exptlb | Theoc | ||||||
| Porin family - omp25 | WP_006214086.1 | B.abortus | 4.9 | 4.87 | 24 | 24.4 | 3.8 in acute | 10 | 119 |
| Bacterioferrin | WP_006640801.1 | B. pinnipedialis | 5 | 4.81 | 15 | 16 | 3 in chronic | 7 | 98 |
| DNA starvation/stationary phase protection protein | ADZ67211.1 | B. melitensis | 5 | 4.8 | 17 | 17.6 | 1.6 in chronic | 8 | 97 |
| Peptidyl prolyl cis-trans isomerase | WP_065879767.1 | B.melitensis | 6 | 6.08 | 17 | 18 | 16.5 in chronic | 6 | 99 |
| Inorganic pyrophosphatase | WP_002965058.1 | Brucella spp. | 5 | 5.1 | 18 | 19.9 | 3.9 in chronic | 6 | 96 |
| Thiol-disulfide oxidoreductase | WP_075678875.1 | B. abortus | 5.1 | 5.2 | 17 | 18.22 | 2 in chronic | 7 | 95 |
| Porin family | WP_024767917.1 | B. melitensis | 5.3 | 5.18 | 17 | 18.78 | 1.6 in chronic | 7 | 95 |
| Universal stress protein | WP_017822078.1 | B. melitensis | 5.2 | 5.24 | 31 | 30.8 | 1.8 in acute | 13 | 101 |
| 3-mercapto-pyruvate sulfurtransferase | WP_023080644.1 | B. melitensis | 5.4 | 5.39 | 40 | 41.17 | 1.9 in chronic | 8 | 102 |
| ABC transporter – 1 | WP_006265950.1 | B. melitensis | 4.9 | 5.14 | 60 | 58 | - | 14 | 107 |
| ABC transporter – 2 | WP_008933731.1 | Brucella spp. | 5 | 5.29 | 50 | 47.994 | 4.9 in acute | 13 | 120 |
| Aldehyde dehydrogenase | WP_006191236.1 | Brucella spp. | 6 | 6.12 | 53 | 55 | 2 in acute | 11 | 104 |
| ABC transporter – 3 | WP_002966093.1 | 4.8 | 4.97 | 41 | 44.312 | 3.1 in acute | 15 | 107 | |
| Chaperonin | EEZ12907.1 | B. melitensis | 6 | 6.53 | 55 | 57.5 | 2.2 in acute | 60 | 96 |
| Glycerol-3-phosphate binding periplasmic protein | AAX75988.1 | B. abortus | 4.8 | 5 | 45 | 47.2 | 2 in chronic | 7 | 95 |
| Outer membrane protein (omp31) | AAB36693.1 | B. melitensis | 5 | 4.5 | 31 | 25.3 | 1.6 in acute | 9 | 103 |
| Enolase | AAL52032.1 | B. melitensis | 6 | 6.1 | 45 | 45.5 | 2 in chronic | 19 | 96 |
apI, isoelectric point; bExptl, experimental; cTheo, theoretical; dkDa, kilodalton;, upregulated; MOWSE, molecular weight search
| Protein name | NCBI prot Accession number | Acute sera | Chronic sera | Molecular weight (kDab) | pIa |
|---|---|---|---|---|---|
| Porin family – omp25 | WP_006214086.1 | + | - | 24.4 | 4.87 |
| DNA starvation protein (Dps) | ADZ67211.1 | + | + | 17.6 | 4.8 |
| Universal stress protein | WP_017822078.1 | + | - | 30.8 | 5.24 |
| 3-mercaptopyruvate sulfurtransferase | WP_023080644.1 | + | + | 31.17 | 5.39 |
| Bacterioferrin | WP_006640801.1 | + | + | 16 | 4.81 |
| Inorganic pyrophosphatase | WP_002965058.1 | + | + | 19.9 | 5.1 |
| Aldehyde dehydrogenase | WP_006191236.1 | + | _ | 55 | 6.12 |
| Chaperonin | EEZ12907.1 | + | + | 57 | 4.9 |
| 31kDa outer membrane protein (omp 31) | AAB36693.1 | + | + | 31 | 5 |
Discussion
The study identified porin protein (omp25), universal stress protein, and aldehyde dehydrogenase as immunogenic proteins, but only in strains isolated from patients with acute brucellosis. These antigens could be targeted for the development of specific serological diagnostic tests to differentiate between acute and chronic infections.
Clinical strains of B. melitensis isolated from acute and chronic brucellosis patients showed many similarities in terms of the number of protein spots and expression pattern, as expected, since the strains belonged to the same species and were cultured under similar conditions. However, differences in protein expression levels were observed for a few protein spots. These differences may reflect variations in the metabolic properties of the strains, contributing to their ability to cause either acute or chronic infection. Pathogens adapt their metabolism to compete against the resident flora and host immune system, regulating the expression of various virulence factors based on nutrient availability in specific environments22.
During infection, pathogens face harsh conditions, including nutrient scarcity due to the host’s immune response, which sequesters micronutrients like iron, zinc, and manganese. These elements act as co-factors for essential enzymes. The host immune system actively removes these nutrients from the pathogen’s environment, leading to ‘starvation’ and affecting cellular and metabolic processes within the pathogen. To overcome this, pathogens use specialised systems to acquire and store these essential nutrients. For example, Staphylococcus aureus produces haemolysins to acquire iron from red blood cells in low iron conditions created by lactoferrin. Similarly, many pathogens produce siderophores, which are small molecules that scavenge iron23.
In our study, a similar pattern of differential protein expression related to iron metabolism was observed in the two groups of B. melitensis strains. Bacterioferritin (Bfr), a protein involved in iron uptake and storage, was overexpressed in strains isolated from chronic infections, suggesting an enhanced capacity for iron assimilation. Bfr is crucial for storing and detoxifying intracellular iron, essential for pathogen survival within host cells. Host macrophages produce chelating agents that remove iron from the phagosome, where pathogens typically replicate. To survive in these conditions, pathogens produce proteins like Bfr to store iron and facilitate the synthesis of haeme-containing enzymes. This upregulation of Bfr in strains from chronic infections highlights the pathogen’s ability to store iron and support long-term survival24.
Another major finding was the difference in the expression of sugar-binding proteins (ABC transporters) among the acute and chronic infection groups. The increase in glucose-binding ABC transporters in strains from acute infections suggests that these strains rely on sugar transport systems to meet their higher energy demands during active infection. Glucose is a primary carbon and energy source for pathogens, and during acute infections, pathogens need a substantial supply of glucose to fuel their high metabolic activity. These transport systems help deliver glucose to the cytoplasm, supporting the pathogen’s ability to produce ATP and counteract the host’s immune response13.
Previous studies have primarily focused on immunogenic proteins of B. abortus or B. melitensis strains like 16M or Rev 1, using bovine or hyperimmune sera11. Fewer studies have employed human sera to identify immunogenic proteins from B. melitensis. A recent study used human sera from acute brucellosis patients to identify soluble immunodominant proteins of B. melitensis 16M13. In this study, human sera from patients with acute and chronic brucellosis were used to screen the whole-cell soluble protein antigens of B. melitensis, identifying seven immunoreactive protein spots. Our results suggest that combining highly immunogenic and specific proteins holds promise for improving serodiagnosis of brucellosis. Recent studies has reported Brucella express various immunogenic proteins which are required for survival in the animal host at the time of acute infection and shown the potential of immunoproteomics in understanding of the disease25.
Further investigation with larger sample size is needed to link protein expression patterns to clinical conditions/progression. This study highlights the potential of immunoproteomics for identifying virulence-related proteins and improving diagnostic tests for B. melitensis.
Acknowledgment
Authors acknowledge the support from the concerned clinical departments of Kasturba hospital for patient management.
Financial support & sponsorship
The study received financial support by Defence Research and Development Establishment (DRDE), Defence Research and Development Organisation (DRDO), Gwalior, India (grant no. DRDE/TC/05414/SUB-PROJECT/DRD-202/04/16) to carry out this work.
Conflicts of Interest
None
Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation
The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.
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