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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 -
Microorganisms Feb 2022sp. are the causative agents of brucellosis. One of the main characteristics of the genus concerns its very high genetic homogeneity. To date, classical bacteriology...
sp. are the causative agents of brucellosis. One of the main characteristics of the genus concerns its very high genetic homogeneity. To date, classical bacteriology typing is still considered as the gold standard assay for direct diagnosis of . Molecular approaches are routinely used for the identification of at the genus level. However, genotyping is more complex, and to date, no method exists to quickly assign a strain into species and biovar levels, and new approaches are required. Next generation sequencing (NGS) opened a new era into the diagnosis of bacterial diseases. In this study, we designed a high-resolution melting (HRM) method for the rapid screening of DNA and direct assignment into one of the 12 species of the genus. This method is based on 17 relevant single nucleotide polymorphisms (SNPs), identified and selected from a whole genome SNP (wgSNP) analysis based on 988 genomes (complete and drafts). These markers were tested against the collection of the European Reference Laboratory (EU-RL) for brucellosis (1440 DNAs extracted from strains). The results confirmed the reliability of the panel of 17 SNP markers, allowing the differentiation of each species of together with biovars 1, 2, and 3 of and vaccine strain Rev1 () within 3 h, which is a considerable gain of time for brucellosis diagnosis. Therefore, this genotyping tool provides a new and quick alternative for identification based on SNPs with the HRM-PCR assay.
PubMed: 35208791
DOI: 10.3390/microorganisms10020336 -
EcoHealth Dec 2020The northern European wild boar population has increased during the last decade. Highest wild boar numbers in Finland have been reported in the southeastern part near...
The northern European wild boar population has increased during the last decade. Highest wild boar numbers in Finland have been reported in the southeastern part near the Russian border. Wild boars may be infected with several human and animal pathogens. In this study, we investigated the presence of important foodborne pathogens in wild boars hunted in 2016 in Finland using serology, PCR and culturing. Seroprevalence of Salmonella (38%) and Yersinia (56%) infections was high in wild boars. Antibodies to hepatitis E virus, Toxoplasma gondii and Brucella were found in 18%, 9% and 9% of the wild boars, respectively. Trichinella antibodies were detected in 1% of the animals. We recorded no differences in the seroprevalence between males and females. However, Yersinia and T. gondii antibodies were detected significantly more often in adults than in young individuals. Listeria monocytogenes (48%) and stx-positive Escherichia coli (33%) determinants were frequently detected in the visceral organs (spleen and kidneys) by PCR. Yersinia pseudotuberculosis O:1 and L. monocytogenes 2a and 4b were identified by culturing from the PCR-positive samples. Brucella suis biovar 2 was isolated from visceral organs. No African swine fever, classical swine fever or Aujeszky's disease were detected in the wild boars. Our study shows that wild boars are important reservoirs of foodborne pathogens.
Topics: Animals; Female; Male; Seroepidemiologic Studies; Sus scrofa; Swine; Swine Diseases; Toxoplasma; Zoonoses
PubMed: 33326058
DOI: 10.1007/s10393-020-01509-5 -
Toxins Nov 2023is a notorious zoonotic disease caused by , which can lead to reproductive diseases in humans and animals, such as infertility and abortion. Lipopolysaccharides (LPS)...
is a notorious zoonotic disease caused by , which can lead to reproductive diseases in humans and animals, such as infertility and abortion. Lipopolysaccharides (LPS) are the main virulence factor of . LPS derived from are different and non-classical and are less toxic and less active than LPS isolated from However, the effects and possible mechanisms of LPS-caused pregnancy loss remain to be revealed. In the present study, we investigated the effects of S2 LPS on early pregnancy loss in mice. The results indicated that embryo implantation failure was induced by LPS treatment in a dose-dependent manner. The injection of LPS mainly resulted in fibrinolysis in the decidual area of the uterus on the 6th day post coition (dpc), infiltration of large granular cells among the decidual cells near the embryo on the 8th dpc, a large number of gaps in the decidual area, and cell necrosis around the embryo. In addition, the expression of Cyclin D3 mRNA in the uterus on the 7th and 8th dpc and IGFBP-1 mRNA and the progesterone receptor in the uterus on the 6th and 7th dpc were also inhibited. Moreover, the expression of decidualization marker Cyclin D3 and decidualization prolactin-associated protein (dPRP) in endometrial stromal cells were also inhibited by LPS treatment in vitro. In summary, LPS affect the process of endometrial decidualization in mice by affecting the structure of the decidua and the expression of decidual marker factors in endometrial stromal cells.
Topics: Pregnancy; Humans; Female; Mice; Animals; Decidua; Lipopolysaccharides; Brucella suis; Cyclin D3; Escherichia coli; Uterus; RNA, Messenger
PubMed: 37999525
DOI: 10.3390/toxins15110662 -
Frontiers in Cellular and Infection... 2021Neurobrucellosis is a chronic complication of human brucellosis that is caused by the presence of spp in the central nervous system (CNS) and the inflammation play a...
Neurobrucellosis is a chronic complication of human brucellosis that is caused by the presence of spp in the central nervous system (CNS) and the inflammation play a key role on the pathogenesis. Doxycycline (Dox) is a widely used antibiotic that induces apoptosis of bacteria-infected cells. However, the mechanisms of inhibition of microglial apoptosis and Dox induction of apoptosis are still poorly understood. In this study, we found that S2 strain ( S2) increased calreticulin (CALR) protein levels and inhbited HMC3 cell apoptosis. Hence, we constructed two HMC3 cell line variants, one with stable overexpression (HMC3-CALR) and one with low expression of CALR (HMC3-sh-CALR). CALR was found to decrease levels of p-JNK and p-p53 proteins, as well as suppress apoptosis in HMC3 cells. These findings suggest that CALR suppresses apoptosis by inhibiting the JNK/p53 signaling pathway. Next, we treated HMC3, HMC3-CALR and HMC3-sh-CALR cell lines with S2 or Dox. Our results demonstrate that S2 restrains the JNK/p53 signaling pathway to inhibit HMC3 cell apoptosis increasing CALR protein expression, while Dox plays the opposite role. Finally, we treated S2-infected HMC3 cells with Dox. Our results confirm that Dox induces JNK/p53-dependent apoptosis in S2-infected HMC3 cells through inhibition of CALR protein expression. Taken together, these results reveal that CALR and the JNK/p53 signaling pathway may serve as novel therapeutic targets for treatment of neurobrucellosis.
Topics: Apoptosis; Brucella suis; Brucellosis; Calreticulin; Doxycycline; Humans; MAP Kinase Signaling System; Microglia; Tumor Suppressor Protein p53
PubMed: 33996626
DOI: 10.3389/fcimb.2021.640847 -
Infection and Immunity Mar 2013Brucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence...
Brucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a nonadherent Escherichia coli strain and by phenotypic characterization of a B. suis ΔbtaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ΔbtaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockout btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, in Brucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process.
Topics: Adhesins, Bacterial; Animals; Antibodies, Bacterial; Bacterial Adhesion; Brucella suis; Brucellosis; Carrier Proteins; Cell Polarity; Escherichia coli; Gene Expression Regulation, Bacterial; Mice; Mice, Inbred BALB C; Multigene Family; Virulence
PubMed: 23319562
DOI: 10.1128/IAI.01241-12 -
Archives of Microbiology May 2021Gram-negative bacteria release nanovesicles, called outer membrane vesicles (OMVs), from their outer membrane. Proteomics has been used to determine their composition.... (Comparative Study)
Comparative Study
Gram-negative bacteria release nanovesicles, called outer membrane vesicles (OMVs), from their outer membrane. Proteomics has been used to determine their composition. OMVs contain proteins able to elicit an immune response, so they have been proposed as a model to develop acellular vaccines. In this study, OMVs of Brucella suis, B. ovis, B. canis, and B. neotomae were purified and analyzed by SDS-PAGE, transmission electron microscopy and liquid chromatography coupled to mass spectrometry to determine the pan-proteome of these vesicles. In addition, antigenic proteins were detected by western blot with anti-Brucella sera. The in silico analysis of the pan-proteome revealed many homologous proteins, such as Omp16, Omp25, Omp31, SodC, Omp2a, and BhuA. Proteins contained in the vesicles from different Brucella species were detected by anti-Brucella sera. The occurrence of previously described immunogenic proteins derived from OMVs supports the use of these vesicles as candidates to be evaluated as an acellular brucellosis vaccine.
Topics: Animals; Antigens, Bacterial; Bacterial Proteins; Brucella; Brucella canis; Brucella ovis; Brucella suis; Electrophoresis, Polyacrylamide Gel; Proteome; Proteomics
PubMed: 33432377
DOI: 10.1007/s00203-020-02170-w -
Pathogens (Basel, Switzerland) Apr 2023Invasive feral swine () are one of the most important wildlife species for disease surveillance in the United States, serving as a reservoir for various diseases of...
Invasive feral swine () are one of the most important wildlife species for disease surveillance in the United States, serving as a reservoir for various diseases of concern for the health of humans and domestic animals. , the causative agent of swine brucellosis, is one such pathogen carried and transmitted by feral swine. Serology assays are the preferred field diagnostic for infection, as whole blood can be readily collected and antibodies are highly stable. However, serological assays frequently have lower sensitivity and specificity, and few studies have validated serological assays for in feral swine. We conducted an experimental infection of Ossabaw Island Hogs (a breed re-domesticated from feral animals) as a disease-free proxy for feral swine to (1) improve understanding of bacterial dissemination and antibody response following infection and (2) evaluate potential changes in the performance of serological diagnostic assays over the course of infection. Animals were inoculated with and serially euthanized across a 16-week period, with samples collected at the time of euthanasia. The 8% card agglutination test performed best, whereas the fluorescence polarization assay demonstrated no capacity to differentiate true positive from true negative animals. From a disease surveillance perspective, using the 8% card agglutination test in parallel with either the buffered acidified plate antigen test or the / complement fixation test provided the best performance with the highest probability of a positive assay result. Application of these combinations of diagnostic assays for surveillance among feral swine would improve understanding of spillover risks at the national level.
PubMed: 37242308
DOI: 10.3390/pathogens12050638 -
BMC Veterinary Research Oct 2023Brucellosis is a common zoonotic disease caused by Brucella, which causes enormous economic losses and public burden to epidemic areas. Early and precise diagnosis and...
BACKGROUND
Brucellosis is a common zoonotic disease caused by Brucella, which causes enormous economic losses and public burden to epidemic areas. Early and precise diagnosis and timely culling of infected animals are crucial to prevent the infection and spread of Brucella. In recent years, RNA-guided CRISPR/Cas12a(Clustered Regularly Interspaced Short Palindromic Repeats and its associated protein 12a) nucleases have shown great promise in nucleic acid detection. This research aims to develop a CRISPR/CAST (CRISPR/Cas12a Test strip) package that can rapidly detect Brucella nucleic acid during on-site screening, especially on remote family pastures. The CRISPR/Cas12a system combined with recombinase polymerase amplification (RPA), and lateral flow read-out.
RESULTS
We selected the conserved gene bp26, which commonly used in Brucella infection detection and compared on Genbank with other Brucella species. The genomes of Brucella abortus 2308, Brucella suis S2, Brucella melitansis 16 M, and Brucella suis 1330, et al. were aligned, and the sequences were found to be consistent. Therefore, the experiments were only performed on B. melitensis. With the CRISPR/CAST package, the assay of Brucella nucleic acid can be completed within 30 min under isothermal temperature conditions, with a sensitivity of 10 copies/μl. Additionally, no antigen cross-reaction was observed against Yersinia enterocolitica O:9, Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensis. The serum samples of 398 sheep and 100 cattle were tested by the CRISPR/CAST package, of which 31 sheep and 8 cattle were Brucella DNA positive. The detection rate was consistent with the qPCR results and higher than that of the Rose Bengal Test (RBT, 19 sheep and 5 cattle were serum positive).
CONCLUSIONS
The CRISPR/CAST package can accurately detect Brucella DNA in infected livestock within 30 min and exhibits several advantages, including simplicity, speed, high sensitivity, and strong specificity with no window period. In addition, no expensive equipment, standard laboratory, or professional operators are needed for the package. It is an effective tool for screening in the field and obtaining early, rapid diagnoses of Brucella infection. The package is an efficient tool for preventing and controlling epidemics.
Topics: Animals; Cattle; Sheep; Livestock; CRISPR-Cas Systems; Brucellosis; Brucella abortus; DNA; Nucleic Acids; Cattle Diseases; Sheep Diseases
PubMed: 37833763
DOI: 10.1186/s12917-023-03767-1 -
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