-
Vaccines Dec 2022Brucellosis is a zoonotic disease caused by Gram-negative bacteria. Most of the brucellosis vaccines in the application are whole-bacteria vaccines. Live-attenuated...
Brucellosis is a zoonotic disease caused by Gram-negative bacteria. Most of the brucellosis vaccines in the application are whole-bacteria vaccines. Live-attenuated vaccines are widely used for brucellosis prevention in sheep, goats, pigs, and cattle. Thus, there is also a need for an adjuvanted vaccine for human brucellosis, because the attenuated vaccines now utilized in animals cause human illness. Here, we developed a live-attenuated strain 2 vaccine (S2) adjuvanted with Ag85a (Ag85a-S2). We found that Ag85a-S2 activated cGAS-STING pathways both in intestinal mucosal cells in vivo and in the BMDM and U937 cell line in vitro. We demonstrated that the cGAS knockout significantly downregulated the abundance of interferon and other cytokines induced by Ag85a-S2. Moreover, Ag85a-S2 triggered a stronger cellular immune response compared to S2 alone. In sum, Ag85a-S2-mediated enhancement of immune responses was at least partially dependent on the cGAS-STING pathway. Our results provide a new candidate for preventing pathogens from livestock, which might reduce the dosage and potential toxicity compared to S2.
PubMed: 36560581
DOI: 10.3390/vaccines10122170 -
Frontiers in Cellular and Infection... 2022Brucellosis is a highly prevalent zoonotic disease caused by spp. S2 vaccination is an effective strategy to prevent animal brucellosis. However, S2 induces antibodies...
INTRODUCTION
Brucellosis is a highly prevalent zoonotic disease caused by spp. S2 vaccination is an effective strategy to prevent animal brucellosis. However, S2 induces antibodies against the smooth lipopolysaccharide,making it challenging to distinguish field infected from vaccinated livestock. Early and accurate diagnosis is essential for infection control and prevention. In this study, we aimed to develop a quick and accurate assay to distinguish the vaccine strain from closely related and .
METHODS
Whole-genome sequencing of was performed, and the sequence was compared with that of the genomes of and . One specific gene, , was selected as a marker to differentiate the S2vaccine strain from and . A loop-mediated isothermal amplification (LAMP) assay was developed, based on the gene, and then assessed for target specificity, lower limit of detection, and repeatability.
RESULTS
Our results revealed that there was no cross-reaction with other strains, and the LAMP assay displayed high sensitivity for detecting S2 with a minimum detection limit of 18.9×103 copies/µL DNA input, it is nearly 100 times higher than conventional PCR technology. Concordance between the LAMP assay and a conventional polymerase chain reaction method was assessed using 54 blood samples collected from sheep with suspected brucellosis. Total concordance between the two assays was 92.6%, without a significant difference (p > 0.05) in the test results.
CONCLUSION
This is the first report of a LAMP assay for the detection of the S2vaccine strain. Our approach can be helpful for the control and eradication of brucellosis, and its simplicity in requiring no specialized equipment or personnel makes it useful for implementation in resource-limited settings as well as for field use.
Topics: Animals; Sheep; Brucella Vaccine; Nucleic Acid Amplification Techniques; Brucellosis; Brucella suis; Brucella melitensis; Brucella abortus
PubMed: 36530431
DOI: 10.3389/fcimb.2022.1023243 -
Microbial Pathogenesis Jan 2023Brucellosis is a zoonotic disease caused by Gram-negative bacteria of the genus Brucella. These pathogens cause long-lasting infections, a process in which Brucella...
Brucellosis is a zoonotic disease caused by Gram-negative bacteria of the genus Brucella. These pathogens cause long-lasting infections, a process in which Brucella modifications in the lipopolysaccharide (LPS) and envelope lipids reduce pathogen-associated molecular pattern (PAMP) recognition, thus hampering innate immunity activation. In vivo models are essential to investigate bacterial virulence, mice being the most used model. However, ethical and practical considerations impede their use in high-throughput screening studies. Although lacking the complexity of the mammalian immune system, insects share key-aspects of innate immunity with mammals, and Galleria mellonella has been used increasingly as a model. G. mellonella larvae have been shown useful in virulence analyses, including Gram-negative pathogens like Klebsiella pneumoniae and Legionella pneumophila. To assess its potential to study Brucella virulence, we first evaluated larva survival upon infection with representative Brucella species (i.e.B. abortus 2308W, B. microti CCM4915 and B. suis biovar 2) and mutants in the VirB type-IV secretion system (T4SS) or in the LPS-O-polysaccharide (O-PS). As compared to K.pneumoniae, the Brucella spp. tested induced a delayed and less severe mortality profile consistent with an escape of innate immunity detection. Brucella replication within larvae was affected by the lack of O-PS, which is reminiscent of their attenuation in natural hosts. On the contrary, replication was not affected by T4SS dysfunction and the mutant induced only slightly less mortality (not statistically significant) than its parental strain. We also evaluated G. mellonella to efficiently recognise Brucella and their LPS by quantification of the pro-phenoloxidase system and melanisation activation, using Pseudomonas LPS as a positive control. Among the brucellae, only B. microti LPS triggered an early-melanisation response consistent with the slightly increased endotoxicity of this species in mice. Therefore, G. mellonella represents a tool to screen for potential Brucella factors modulating innate immunity, but its usefulness to investigate other mechanisms relevant in Brucella intracellular life is limited.
Topics: Animals; Mice; Moths; Brucella; Lipopolysaccharides; Larva; Host-Pathogen Interactions; Mammals
PubMed: 36496059
DOI: 10.1016/j.micpath.2022.105930 -
Immunologic Research Apr 2023Brucella suis mediates the transmission of brucellosis in humans and animals and a significant facultative zoonotic pathogen found in livestock. It has the capacity to...
Brucella suis mediates the transmission of brucellosis in humans and animals and a significant facultative zoonotic pathogen found in livestock. It has the capacity to survive and multiply in a phagocytic environment and to acquire resistance under hostile conditions thus becoming a threat globally. Antibiotic resistance is posing a substantial public health threat, hence there is an unmet and urgent clinical need for immune-based non-antibiotic methods to treat brucellosis. Hence, we aimed to explore the whole proteome of Brucella suis to predict antigenic proteins as a vaccine target and designed a novel chimeric vaccine (multi-epitope vaccine) through subtractive genomics-based reverse vaccinology approaches. The applied subsequent hierarchical shortlisting resulted in the identification of Multidrug efflux Resistance-nodulation-division (RND) transporter outer membrane subunit (gene BepC) that may act as a potential vaccine target. T-cell and B-cell epitopes have been predicted from target proteins using a number of immunoinformatic methods. Six MHC I, ten MHC II, and four B-cell epitopes were used to create a 324-amino-acid MEV construct, which was coupled with appropriate linkers and adjuvant. To boost the immunological response to the vaccine, the vaccine was combined with the TLR4 agonist HBHA protein. The MEV structure predicted was found to be highly antigenic, non-toxic, non-allergenic, flexible, stable, and soluble. To confirm the interactions with the receptors, a molecular docking simulation of the MEV was done using the human TLR4 (toll-like receptor 4) and HLAs. The stability and binding of the MEV-docked complexes with TLR4 were assessed using molecular dynamics (MD) simulation. Finally, MEV was reverse translated, its cDNA structure was evaluated, and then, in silico cloning into an E. coli expression host was conducted to promote maximum vaccine protein production with appropriate post-translational modifications. These comprehensive computer calculations backed up the efficacy of the suggested MEV in protecting against B. suis infections. However, more experimental validations are needed to adequately assess the vaccine candidate's potential. HIGHLIGHTS: • Subtractive genomic analysis and reverse vaccinology for the prioritization of novel vaccine target • Examination of chimeric vaccine in terms of allergenicity, antigenicity, MHC I, II binding efficacy, and structural-based studies • Molecular docking simulation method to rank based vaccine candidate and understand their binding modes.
Topics: Animals; Humans; Brucella suis; Brucellosis; Computational Biology; Epitopes, B-Lymphocyte; Epitopes, T-Lymphocyte; Escherichia coli; Molecular Docking Simulation; Toll-Like Receptor 4; Vaccines, Subunit; Drug Resistance, Bacterial; Proteome; Bacterial Proteins; Brucella Vaccine; Epitopes; Vaccine Development; Drug Design
PubMed: 36459272
DOI: 10.1007/s12026-022-09346-0 -
Infection and Immunity Jan 2023Bacteria use the twin arginine translocator (Tat) system to export folded proteins from the cytosol to the bacterial envelope or to the extracellular environment. As...
Bacteria use the twin arginine translocator (Tat) system to export folded proteins from the cytosol to the bacterial envelope or to the extracellular environment. As with most Gram-negative bacteria, the Tat system of the zoonotic pathogen Brucella spp. is encoded by a three-gene operon, Our attempts, using several different strategies, to create a Brucella suis strain 1330 mutant were all unsuccessful. This suggested that, for B. suis, Tat is essential, in contrast to a recent report for Brucella melitensis. This was supported by our findings that two molecules that inhibit the Pseudomonas aeruginosa Tat system also inhibit B. suis, B. melitensis, and Brucella abortus growth . In a bioinformatic screen of the B. suis 1330 proteome, we identified 28 proteins with putative Tat signal sequences. We used a heterologous reporter assay based on export of the Tat-dependent amidase AmiA by using the Tat signal sequences from the Brucella proteins to confirm that 20 of the 28 candidates can engage the Tat pathway.
Topics: Brucella suis; Bacterial Proteins; Twin-Arginine-Translocation System; Brucella melitensis; Protein Sorting Signals; Arginine
PubMed: 36448838
DOI: 10.1128/iai.00459-22 -
International Journal of Molecular... Nov 2022transfers effectors into host cells, manipulating cellular processes to its advantage; however, the mechanism by which effectors regulate cellular processes during...
transfers effectors into host cells, manipulating cellular processes to its advantage; however, the mechanism by which effectors regulate cellular processes during infection is poorly understood. A growing number of studies have shown that apoptosis and autophagy are critical mechanisms for target cells to cope with pathogens and maintain cellular homeostasis. BtpB is a type IV secretion system effector with a complex mechanism for manipulating host infection. Here, we show that the ectopic expression of BtpB promoted DNA fragmentation. In contrast, an isogenic mutant strain, Δ, inhibited apoptosis compared to the wild-type strain S2 in RAW264.7 cells. In addition, BtpB inhibited autophagy, as determined by LC3-II protein levels, the number of LC3 puncta, and p62 degradation. We also found that BtpB reduced autophagolysosome formation and blocked the complete autophagic flux. Moreover, our results revealed that the autophagy inhibitor, chloroquine, reduces 's intracellular survival. Overall, our data unveil new mechanisms of virulence implicating the effector BtpB in regulating host intracellular infection.
Topics: Mice; Animals; Brucella; Autophagy; Apoptosis; RAW 264.7 Cells; Type IV Secretion Systems
PubMed: 36430916
DOI: 10.3390/ijms232214439 -
Bioengineering (Basel, Switzerland) Nov 2022, one of the causative agents of brucellosis, is Gram-negative intracellular bacteria that may be found all over the globe and it is a significant facultative zoonotic...
, one of the causative agents of brucellosis, is Gram-negative intracellular bacteria that may be found all over the globe and it is a significant facultative zoonotic pathogen found in livestock. It may adapt to a phagocytic environment, reproduce, and develop resistance to harmful environments inside host cells, which is a crucial part of the Brucella life cycle making it a worldwide menace. The molecular underpinnings of Brucella pathogenicity have been substantially elucidated due to comprehensive methods such as proteomics. Therefore, we aim to explore the complete proteome to prioritize the novel proteins as drug targets via subtractive proteo-genomics analysis, an effort to conjecture the existence of distinct pathways in the development of brucellosis. Consequently, 38 unique metabolic pathways having 503 proteins were observed while among these 503 proteins, the non-homologs (n = 421), essential (n = 350), drug-like (n = 114), virulence (n = 45), resistance (n = 42), and unique to pathogen proteins were retrieved from . The applied subsequent hierarchical shortlisting resulted in a protein, i.e., isocitrate lyase, that may act as potential drug target, which was finalized after the extensive literature survey. The interacting partners for these shortlisted drug targets were identified through the STRING database. Moreover, structure-based studies were also performed on isocitrate lyase to further analyze its function. For that purpose, ~18,000 ZINC compounds were screened to identify new potent drug candidates against isocitrate lyase for brucellosis. It resulted in the shortlisting of six compounds, i.e., ZINC95543764, ZINC02688148, ZINC20115475, ZINC04232055, ZINC04231816, and ZINC04259566 that potentially inhibit isocitrate lyase. However, the ADMET profiling showed that all compounds fulfill ADMET properties except for ZINC20115475 showing positive Ames activity; whereas, ZINC02688148, ZINC04259566, ZINC04232055, and ZINC04231816 showed hepatoxicity while all compounds were observed to have no skin sensitization. In light of these parameters, we recommend ZINC95543764 compound for further experimental studies. According to the present research, which uses subtractive genomics, proteins that might serve as therapeutic targets and potential lead options for eradicating brucellosis have been narrowed down.
PubMed: 36354544
DOI: 10.3390/bioengineering9110633 -
Veterinary Sciences Oct 2022Brucellosis is one of the most important bacterial zoonotic diseases worldwide, characterized in domestic animals by long-term reproductive disorders. As known, wild...
Brucellosis is one of the most important bacterial zoonotic diseases worldwide, characterized in domestic animals by long-term reproductive disorders. As known, wild boars () are natural hosts for biovar 2, in which the infection passes in inapparent form, increasing the pathogen transmission risk to domestic pigs, other domestic animals and humans. So far, no studies regarding brucellosis in wild boars in Serbia have been published. During the hunting season 2020/2021, 480 sera of wild boars living in Serbia were collected and tested for the presence of anti- antibodies. For the serological survey, the Rose Bengal Test (RBT) and competitive enzyme-linked immunosorbent assay (c-ELISA) were used. Of the 480 sera, 45 sera tested positive, indicating the acquired seroprevalence in wild boars of 9.4%. The greatest numbers of seropositive animals were detected in the eastern parts of the country and in one of the central districts, i.e., Pomoravski, Branicevski, Borski and Juznobanatski. This study provides the first data regarding brucellosis in the wild boar population in Serbia, revealing the seroprevalence of , thus indicating that wild boars as natural hosts and/or vectors of likely present a risk for the infection of other animals.
PubMed: 36288188
DOI: 10.3390/vetsci9100575 -
Biomedicine & Pharmacotherapy =... Nov 2022Vaccines against Brucella abortus, B. melitensis and B. suis have been based on weakened or killed bacteria, however there is no recombinant vaccine for disease...
Vaccines against Brucella abortus, B. melitensis and B. suis have been based on weakened or killed bacteria, however there is no recombinant vaccine for disease prevention or therapy. This study attempted to predict IFN-γ epitopes, T cell cytotoxicity, and T lymphocytes in order to produce a multiepitope vaccine based on BtpA, Omp16, Omp28, virB10, Omp25, and Omp31 antigens against B. melitensis, B. abortus, and B. suis. AAY, GPGPG, and EAAAK peptides were used as epitope linkers, while the PADRE sequence was used as a Toll-like receptor 2 (TLR2) and TLR4 agonist. The final construct included 389 amino acids, and was a soluble protein with a molecular weight of 41.3 kDa, and nonallergenic and antigenic properties. Based on molecular docking studies, molecular dynamics simulations such as Gyration, RMSF, and RMSD, as well as tertiary structure validation methods, the modeled protein had a stable structure capable of interacting with TLR2/4. As a result, this novel vaccine may stimulate immune responses in B and T cells, and could prevent infection by B. suis, B. abortus, and B. melitensis.
Topics: Humans; Brucella melitensis; Toll-Like Receptor 2; Epitope Mapping; Epitopes, T-Lymphocyte; Brucellosis; Toll-Like Receptor 4; Molecular Docking Simulation; Antigens, Bacterial; Amino Acids
PubMed: 36115112
DOI: 10.1016/j.biopha.2022.113557 -
Microorganisms Aug 2022(1) Background: MALDI-TOF mass spectrometry (MS) is the gold standard for microbial fingerprinting, however, for phylogenetically closely related species, the resolution...
(1) Background: MALDI-TOF mass spectrometry (MS) is the gold standard for microbial fingerprinting, however, for phylogenetically closely related species, the resolution power drops down to the genus level. In this study, we analyzed MALDI-TOF spectra from 44 strains of , and to identify the optimal classification method within popular supervised and unsupervised machine learning (ML) algorithms. (2) Methods: A consensus feature selection strategy was applied to pinpoint from among the 500 MS features those that yielded the best ML model and that may play a role in species differentiation. Unsupervised -means and hierarchical agglomerative clustering were evaluated using the silhouette coefficient, while the supervised classifiers Random Forest, Support Vector Machine, Neural Network, and Multinomial Logistic Regression were explored in a fine-tuning manner using nested -fold cross validation (CV) with a feature reduction step between the two CV loops. (3) Results: Sixteen differentially expressed peaks were identified and used to feed ML classifiers. Unsupervised and optimized supervised models displayed excellent predictive performances with 100% accuracy. The suitability of the consensus feature selection strategy for learning system accuracy was shown. (4) Conclusion: A meaningful ML approach is here introduced, to enhance spp. classification using MALDI-TOF MS data.
PubMed: 36014076
DOI: 10.3390/microorganisms10081658