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Journal of Bacteriology May 2021The intracellular bacterial pathogen causes persistent infections in various mammalian species. To survive and replicate within macrophages, these bacteria must be able...
The intracellular bacterial pathogen causes persistent infections in various mammalian species. To survive and replicate within macrophages, these bacteria must be able to withstand oxidative stresses and express the type IV secretion system (T4SS) to evade host immune responses. The extracytoplasmic function (ECF) sigma factor system is a major signal transduction mechanism in bacteria that senses environmental cues and responds by regulating gene expression. In this study, we defined an ECF σ and its cognate anti-σ factor in Brucella melitensis M28 by conserved domain analysis and a protein interaction assay. BcrS directly activates an adjacent operon, , that encodes a methionine-rich peptide and a putative methionine sulfoxide reductase system, whereas AbcS is a negative regulator of and . The - and operons can be induced by hypochlorous acid and contribute to hypochlorous acid resistance . Next, RNA sequencing analysis and genome-wide recognition sequence search identified the regulons of BcrS and AbcS. Interestingly, we found that BcrS positively influences T4SS expression in an AbcS-dependent manner and that AbcS also affects T4SS expression independently of BcrS. Last, we demonstrate that is required for the maintenance of persistent infection, while is dispensable in a mouse infection model. Collectively, we conclude that BcrS and AbcS influence expression of multiple genes responsible for virulence traits. is a notorious intracellular pathogen that induces chronic infections in animals and humans. To survive and replicate within macrophages, these bacteria require a capacity to withstand oxidative stresses and to express the type IV secretion system (T4SS) to combat host immune responses. In this study, we characterized an extracytoplasmic function sigma/anti-sigma factor system that regulates resistance to reactive chlorine species and T4SS expression, thereby establishing a potential link between two crucial virulence traits of . Furthermore, the anti-sigma factor AbcS contributes to persistent infection of mice. Thus, this work provides novel insights into virulence regulation as well as a potential drug target for fighting infections.
Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Brucella melitensis; Gene Expression Regulation, Bacterial; Hypochlorous Acid; Models, Molecular; Protein Conformation; Sigma Factor; Type IV Secretion Systems
PubMed: 33820796
DOI: 10.1128/JB.00127-21 -
Scientific Reports Jun 2022Brucella is a typical facultative intracellular bacterium that can cause zoonotic infections. For Brucella, it is difficult to eliminate with current medical treatment....
Brucella is a typical facultative intracellular bacterium that can cause zoonotic infections. For Brucella, it is difficult to eliminate with current medical treatment. Therefore, a multi-epitope vaccine (MEV) should be designed to prevent Brucella infection. For this purpose, we applied the reverse vaccinology approach from Omp10, Omp25, Omp31 and BtpB. Finally, we obtained 13 cytotoxic T lymphocyte (CTL) epitopes, 17 helper T lymphocyte (HTL) epitopes, 9 linear B cell epitopes, and 2 conformational B cell epitopes for further study. To keep the protein folded normally, we linked AAY, GPGPG, and KK to CTL epitopes, HTL epitopes, and B cell epitopes, respectively. The N-terminal of the vaccine peptide is supplemented with appropriate adjuvants to enhance immunogenicity. To evaluate its immunogenicity, stability, safety, and feasibility, a final MEV containing 806 amino acids was constructed by linking linkers and adjuvants. In addition, molecular docking and molecular dynamics simulations were performed to verify the affinity and stability of the MEV-TLR4. Then, codon adaptation and in silico cloning studies were carried out to identify the possible codons for expressing the MEV. In animal experiments, the results demonstrated that the MEV had high immunogenicity. Collectively, this study provided a theoretical basis for the development of a Brucella vaccine.
Topics: Animals; Brucella melitensis; Computational Biology; Epitopes, B-Lymphocyte; Epitopes, T-Lymphocyte; Molecular Docking Simulation; Vaccines, Subunit
PubMed: 35710873
DOI: 10.1038/s41598-022-14427-z -
PLoS Neglected Tropical Diseases 2013The incidence of human brucellosis in Kyrgyzstan has been increasing in the last years and was identified as a priority disease needing most urgent control measures in...
The incidence of human brucellosis in Kyrgyzstan has been increasing in the last years and was identified as a priority disease needing most urgent control measures in the livestock population. The latest species identification of Brucella isolates in Kyrgyzstan was carried out in the 1960s and investigated the circulation of Brucella abortus, B. melitensis, B. ovis, and B. suis. However, supporting data and documentation of that experience are lacking. Therefore, typing of Brucella spp. and identification of the most important host species are necessary for the understanding of the main transmission routes and to adopt an effective brucellosis control policy in Kyrgyzstan. Overall, 17 B. melitensis strains from aborted fetuses of sheep and cattle isolated in the province of Naryn were studied. All strains were susceptible to trimethoprim-sulfamethoxazole, gentamicin, rifampin, ofloxacin, streptomycin, doxycycline, and ciprofloxacin. Multilocus variable number tandem repeat analysis showed low genetic diversity. Kyrgyz strains seem to be genetically associated with the Eastern Mediterranean group of the Brucella global phylogeny. We identified and confirmed transmission of B. melitensis to cattle and a close genetic relationship between B. melitensis strains isolated from sheep sharing the same pasture.
Topics: Animals; Anti-Bacterial Agents; Brucella melitensis; Brucellosis; Cattle; Cattle Diseases; Genetic Variation; Genotype; Kyrgyzstan; Microbial Sensitivity Tests; Minisatellite Repeats; Molecular Epidemiology; Molecular Typing; Sheep; Sheep Diseases
PubMed: 23469294
DOI: 10.1371/journal.pntd.0002047 -
Veterinary Medicine and Science Aug 2020In this study, Multiple Locus Variable Number Tandem Repeat Analysis (MLVA-16) was performed on 18 Brucella isolates identified bacteriologically and molecularly...
In this study, Multiple Locus Variable Number Tandem Repeat Analysis (MLVA-16) was performed on 18 Brucella isolates identified bacteriologically and molecularly (AMOS-PCR) as Brucella abortus (n = 6) and Brucella melitensis (n = 12). This was aimed to study the genetic association among some Egyptian Brucella genotypes isolated during the period from 2002 to 2013 along with the global genotypes database. MLVA-16 analysis for B. melitensis and B. abortus strains illustrates a total of 11, and 3 genotypes with 10 and 1 singleton genotypes, respectively. B. melitensis strains displayed greater markers diversity by VNTRs analysis of the 16 loci than B. abortus and this was attributed mainly to the diverging in panel 2B markers. B. melitensis genotype M4_Fayoum_Giza (3,5,3,13,1,1,3,3,8,21,8,7,5,9,5,3) was the only predominated genotype circulating between two different governorates. The most common B. abortus genotype, GT A3_Dakahlia (4,5,4,12,2,2,3,3,6,21,8,4,4,3,4,4), was present in three identical isolates. In phylogeny, Egyptian B. abortus bv1 genotypes were closely related to East Asian strain (for the first time), Western Mediterranean and Americas clonal lineages. B. melitensis local genotypes exhibit a genetic relatedness mostly to Western Mediterranean clonal lineage and one strain of Eastern Mediterranean clonal lineage. In conclusion, the geographic location is not the only factor stands behind the high genetic similarity of the Egyptian Brucella genotypes. These low variations may be a result of a stepwise mutational event of the most variable loci from a very limited number of ancestors especially during the transmission through non-preference hosts. The authors encourage the authorities in charge to establish pre-movement testing to reduce the risk of brucellosis spread.
Topics: Brucella abortus; Brucella melitensis; Egypt; Genetic Variation
PubMed: 32266769
DOI: 10.1002/vms3.260 -
BMC Complementary and Alternative... May 2018The current treatment for human brucellosis requires a combination of antibiotics for long periods of time, and the reported incidence and prevalence of the disease vary...
BACKGROUND
The current treatment for human brucellosis requires a combination of antibiotics for long periods of time, and the reported incidence and prevalence of the disease vary widely in nomadic livestock of Mongolia. The objective of the present study was to evaluate the in vivo antibacterial activity of the C. mongolica root extract against B. melitensis.
METHODS
In this study, we used of 6 groups of mice (n = 5). Five groups of BALB/c mice were inoculated intraperitoneally with the M16 strain of B. melintensis, as follows: (i) one group was used for pretreatment monitoring; (ii) the control group was administered 2% Tween 80 and was used as the non-treatment group; and the other three groups were treated with one oral gavage per day for 21 days with (iii) doxycycline (2 mg/day), (iv) doxycycline (1 mg/day) with root extract (20 mg/day), and (v) C. mongolica root extract (20 mg/day). The one group that was kept non-infected was used as a healthy control group.
RESULTS
This study demonstrated that daily treatment with doxycycline alone and in combination with C. mongolica root extract significantly reduced splenic infection at the end of treatment. However, the spleen index of both the doxycycline-treated and the combination-treated groups of mice decreased by approximately 50% compared to that of the healthy control mouse group. Treatment with the C. mongolica root extract resulted in a 1.47log reduction in splenic infection compared to the non-treatment group, and the spleen index of the C. mongolica-treated group of mice was the same as that of the normal mouse group. In all treatment groups, neutrophil phagocytic activity significantly decreased, and all treatment groups demonstrated splenic regeneration.
CONCLUSIONS
The present study showed that the C. mongolica root extract may be useful in the treatment of brucellosis patients, in combination with doxycycline or other antibiotics, to reduce the toxicity of high-dosage antibiotics, to prevent the development of antibiotic resistance and to prevent Brucella infection.
Topics: Animals; Brucella melitensis; Brucellosis; Female; Lamiaceae; Leukocyte Count; Mice; Mice, Inbred BALB C; Plant Extracts; Plant Roots; Spleen
PubMed: 29724202
DOI: 10.1186/s12906-018-2220-y -
Emerging Infectious Diseases Apr 2020We isolated Brucella melitensis biovar 3 from the spleen of an Asian badger (Meles leucurus) in Nilka County, northwestern China. Our investigation showed that this...
We isolated Brucella melitensis biovar 3 from the spleen of an Asian badger (Meles leucurus) in Nilka County, northwestern China. Our investigation showed that this isolate had a common multilocus variable-number tandem-repeat analysis 16 genotype, similar to bacterial isolates from local aborted sheep fetuses.
Topics: Animals; Brucella melitensis; Brucellosis; China; Genotype; Multilocus Sequence Typing; Mustelidae; Sheep
PubMed: 32187504
DOI: 10.3201/eid2604.190833 -
Infectious Diseases of Poverty Dec 2022Brucellosis, a zoonosis mainly transmitted by consumption of unpasteurized dairy products as well as direct contact with infected animals, is endemic in Kyrgyzstan....
BACKGROUND
Brucellosis, a zoonosis mainly transmitted by consumption of unpasteurized dairy products as well as direct contact with infected animals, is endemic in Kyrgyzstan. However, Brucella species in humans have not been investigated and the origin of the disease remains poorly known in wide parts of Сentral Asia. Thus, molecular characterization of the circulating strains is a critical first step in understanding Brucella diversity in the country.
METHODS
In this study, isolates were collected from patients with suspected brucellosis from different regions in Kyrgyzstan between 2019 and 2020. The detection and identification of Brucella was carried out by Bruce-ladder PCR. Next generation sequencing was used to sequence the 89 Brucella isolates, which were genotyped by cgSNP and cgMLST to identify epidemiological connection between Brucella isolates as well as placing them in the context of the global Brucella phylogeny.
RESULTS
The Brucella strains isolated from all regions of Kyrgyzstan were identified as B. melitensis. Based on cgSNP analysis, 18 sequence types were differentiated. The highest numbers of different sequence types were found in Batken (n = 8), Osh (n = 8) and Jalal-Abad (n = 6) oblasts. According to cgSNP and cgMLST analyses, different B. melitensis lineages circulate in Kyrgyzstan, all of them belonging to the Eastern Mediterranean group of the global Brucella phylogeny with the highest similarity to strains from Turkmenistan, Iran and Turkey.
CONCLUSION
In the present study, B. melitensis was identified as a causative agent of human brucellosis in Kyrgyzstan and different lineages could be identified. Since this study focused on isolates of human origin, the identity of Brucella species and lineages circulating among animal populations remains elusive. Implementing culture techniques and use of most recent molecular, bioinformatic and epidemiological tools are needed to set up a One Health approach to combat brucellosis in Kyrgyzstan. Further, other Сentral Asian countries need to take part in this effort as brucellosis is a transboundary disease in these regions.
Topics: Humans; Brucella melitensis; Kyrgyzstan; Asia; Computational Biology; Iran
PubMed: 36482466
DOI: 10.1186/s40249-022-01044-1 -
Infection and Immunity Sep 2014Brucella spp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed that Brucella melitensis is able to...
Brucella spp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed that Brucella melitensis is able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced following Brucella vaccination. In contrast to Bartonella, the type IV secretion system and flagellar expression are not critically required for the persistence of Brucella in blood. ImageStream analysis of blood cells showed that following a brief extracellular phase, Brucella is associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated that B. melitensis is able to invade erythrocytes in vivo. The bacteria do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open up new areas for investigation and should serve in the development of novel strategies for the treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the bacterial cells from the host's immune response and hamper antibiotic treatment and suggests possible Brucella transmission by bloodsucking insects in nature.
Topics: Animals; Bacterial Secretion Systems; Brucella Vaccine; Brucella melitensis; Brucellosis; Erythrocytes; Flagella; Mice; Mice, Inbred C57BL
PubMed: 25001604
DOI: 10.1128/IAI.01779-14 -
Scientific Reports Mar 2019Given brucellosis is a widespread zoonosis in the world, a safe and effective vaccine is urgently needed. Recent trend in vaccine design has shifted to epitope-based...
Given brucellosis is a widespread zoonosis in the world, a safe and effective vaccine is urgently needed. Recent trend in vaccine design has shifted to epitope-based vaccines that are safe and specific. In this study, peptide containing both T-cell and B-cell epitopes of OMP31 was synthesized and used to immunize the mice by nasal administration. The protective efficacy was evaluated. Mice immunized with the B epitope or TB epitope peptides of OMP31 had higher levels of IgG1 and IgG2a in the serum. While the BALB/c mice immunized with peptides containing T cell epitope or TB epitope of OMP31 showed high degree of IFN-γ-producing T cells in the lymphocytes from the respiratory draining lymph nodes and spleen. After intranasally challenged with 5 × 10 CFU of Brucella melitensis (strain 16 M), the bacterial loads in lung of the immunized mice were significantly lower than control group. These data demonstrate for the first time that peptides of OMP31 containing T epitope, B epitope or TB epitopes are of high immunogenicity and thus can protect host from Brucella melitensis infection in lung.
Topics: Administration, Intranasal; Animals; Antibodies, Bacterial; Bacterial Outer Membrane Proteins; Brucella melitensis; Brucellosis; Epitopes, B-Lymphocyte; Epitopes, T-Lymphocyte; Female; Immunity, Humoral; Immunity, Mucosal; Immunoglobulin G; Interferon-gamma; Mice; Mice, Inbred BALB C; Peptides; Spleen; T-Lymphocytes; Vaccination
PubMed: 30837598
DOI: 10.1038/s41598-019-40084-w -
Scientific Reports Jun 2018Brucellosis is a rarely encountered infection in Norway. The aim of this study was to explore all Brucella melitensis isolates collected in Norway from 1999 to 2016 in...
Brucellosis is a rarely encountered infection in Norway. The aim of this study was to explore all Brucella melitensis isolates collected in Norway from 1999 to 2016 in relation to origin of infection and antimicrobial resistance patterns. A total of 23 isolates were analysed by whole-genome sequencing and compared with selected sequences of B. melitensis available from NCBI. Additionally, SNP analysis in antibiotic resistance determining genes was performed. The majority belonged to the East Mediterranean clade (genotype II), while the remaining isolates belonged to the African clade (genotype III). These results indicate that human brucellosis in Norway is related to travels or migration from the Middle East, Asia or Africa, in accordance with results from Germany, Denmark and Sweden. Antibiotic susceptibility patterns were determined by broth microdilution method and/or gradient strip method. All isolates were susceptible for all tested antibiotics, except for rifampicin where phenotypical results indicated resistance or intermediate resistance in all isolates based on broth microdilution method, and in four isolates based on gradient strip testing. In contrast, screening of the rpoB gene did not reveal any mutations in the previously described rpoB "hot spot" regions related to rifampicin resistance, indicating overestimation of resistance based on phenotypical results.
Topics: Brucella melitensis; Brucellosis; Drug Resistance, Bacterial; Female; Humans; Male; Microbial Sensitivity Tests; Norway; Polymorphism, Single Nucleotide; Rifampin; Whole Genome Sequencing
PubMed: 29867163
DOI: 10.1038/s41598-018-26906-3