The Goat Cytotoxic T Lymphocyte-Associated Antigen-4 Gene: mRNA Expression and Association Analysis of Insertion/Deletion Variants with the Risk of Brucellosis.

The cytotoxic T lymphocyte-associated antigen-4 (CTLA4) gene, a member of the immunoglobulin superfamily, is crucial for maintaining immune homeostasis and preventing autoimmune diseases. Studies have shown that polymorphisms in the CTLA4 gene are linked to an increased risk of brucellosis in humans, but its association with brucellosis in goats remains unexplored. In this study, the tissue expression profile of CTLA4 in goats was investigated, and the correlation between InDel polymorphisms in the CTLA4 gene and susceptibility to brucellosis in goats was examined. The findings reveal the widespread expression of CTLA4 in goat tissues, particularly in the spleen and testes. The tested goat populations presented genotypes insertion/insertion (II), insertion/deletion (ID), and deletion/deletion (DD) at both the P1 and P2 loci, and an association analysis revealed significant differences in the distribution of genotypes and allele frequencies at the P1 and P2 loci of the CTLA4 gene between the Brucella goat case and the control groups (p < 0.05). Specifically, compared with the II genotype, the P1 and P2 loci were significantly associated with an elevated risk of brucellosis development in goats under both the codominant (ID/II) and dominant (ID + DD/II) models (P1, p = 0.042, p = 0.016; P2, p = 0.011, p = 0.014). Additionally, haplotype analysis indicated that haplotypes IP1DP2, DP1IP2, and DP1DP2 were significantly associated with an increased risk of brucellosis in goats compared to the reference haplotype IP1IP2 (p = 0.029, p = 0.012, p = 0.034). Importantly, the Lipopolysaccharide (LPS) stimulation of peripheral blood monocytes and/or macrophages from goats with the II, ID, and DD genotypes resulted in increased CTLA4 expression levels in the II genotype, leading to a robust LPS-induced inflammatory response. Through bioinformatic analysis, the observed effect of the InDel locus on Brucella pathogenesis risk in goats could be attributed to the differential binding of the transcription factors nuclear factor kappaB (NF-κB) and CCAAT/enhancer-binding protein α (C/EBPα). These findings offer potential insights for breeding strategies against brucellosis.

RNA-seq reveals the important role of transcriptional regulator DeoR1 in regulating Brucella abortus various pathways.

Brucella spp. is an intracellular bacterium that uses its transcriptional regulator DeoR1 to promote intracellular transport and survival, but the molecular mechanism remains unknown. To analyze the role of DeoR1 in the virulence of B. abortus and the genes regulated by DeoR1, we created a A19ΔdeoR1 mutant of B. abortus A19 (A19). Virulence assay was performed using a murine macrophage cell line (RAW264.7) and mice. We observed that A19ΔdeoR1 mutant is attenuated in RAW264.7 cells and mice. We performed RNA-seq whole transcriptome analysis of A19ΔdeoR1 and A19 from infected RAW264.7 cells. A total of 135 differentially expressed genes were identified, including 100 up-regulated and 35 down-regulated genes. These differentially expressed genes were involved in amino acid synthesis and metabolism, energy production and conversion, stress proteins, chaperonin, hypothetical proteins and protein of unknown function, cell wall/membrane/envelope, intracellular transporting and secretion, and transcriptional regulator. Interestingly, genes involved in the intracellular trafficking and secretion were significantly down-regulated in A19ΔdeoR1. Furthermore, selected RNA-seq results were experimentally confirmed by qRT-PCR. Overall, these results deciphered differential phenomena associated with virulence in A19ΔdeoR1 and A19 from infected RAW264.7 cells, which provided important information for understanding the detailed role of DeoR1 in Brucella pathogenesis. SIGNIFICANCE: Transcriptional regulators are predominant bacterial signal transduction factors. The pathogenicity of Brucella is due to its ability to regulate the expression of virulence related genes. Transcriptional regulators are designed to regulate gene expression and enact an appropriate adaptive physiological response. Here, a total of 135 differentially expressed genes were identified in transcriptional regulator deoR1 mutant.

Expression of cytokine and Apoptosis-Associated genes in mice bone Marrow-Derived Macrophages stimulated with Brucella recombinant type IV secretion effectors.

BACKGROUND: Brucellosis is an economically important infectious caused by most commonly by Brucella. Detection of infected animals at the early stage is important for controlling the disease. The diagnostic antigens, usually protein antigens, have attracted much interest. However, the accurate mechanism of immune response is still unknown. The secretory effectors (BPE005, BPE275, and BPE123) of the type IV secretion system (T4SS) were involved in the intracellular circulation process of Brucella and the immune responses of the host. METHODS: Genes encoding three B. abortus effector proteins (BPE005, BPE275, and BPE123) of T4SS were cloned and the recombinant proteins were expressed and purified. The purified recombinant proteins were named rBPE005, rBPE275 and rBPE123. Then, the expressions of Th1- and Th2-related cytokine genes were analyzed in mice bone marrow-derived macrophages (BMDMs) after stimulation with rBPE005, rBPE275, and rBPE123. Furthermore, four apoptosis-associated genes (Caspase-3, Caspase-8, Bax, and Bcl-2) were also detected to explore the damage of the proteins to the cells. RESULTS: Expressions of all Th1- and Th2-related cytokine genes were induced with three proteins, and different cytokine expression patterns induced by each protein depend on the stimulation time and dose of protein. However, expressions of apoptosis-related genes did not change. CONCLUSION: These results showed that the secreted antigens of Brucella induced an immune reaction via the production of Th1- and Th2-type cytokines in BMDMs without exerting any damage on the cells.

Beyond its preferential niche: Brucella abortus RNA down-modulates the IFN-γ-induced MHC-I expression in epithelial and endothelial cells.

Brucella abortus (Ba) is a pathogen that survives inside macrophages. Despite being its preferential niche, Ba infects other cells, as shown by the multiple signs and symptoms humans present. This pathogen can evade our immune system. Ba displays a mechanism of down-modulating MHC-I on monocytes/macrophages in the presence of IFN-γ (when Th1 response is triggered) without altering the total expression of MHC-I. The retained MHC-I proteins are located within the Golgi Apparatus (GA). The RNA of Ba is one of the PAMPs that trigger this phenomenon. However, we acknowledged whether this event could be triggered in other cells relevant during Ba infection. Here, we demonstrate that Ba RNA reduced the surface expression of MHC-I induced by IFN-γ in the human bronchial epithelium (Calu-6), the human alveolar epithelium (A-549) and the endothelial microvasculature (HMEC) cell lines. In Calu-6 and HMEC cells, Ba RNA induces the retention of MHC-I in the GA. This phenomenon was not observed in A-549 cells. We then evaluated the effect of Ba RNA on the secretion of IL-8, IL-6 and MCP-1, key cytokines in Ba infection. Contrary to our expectations, HMEC, Calu-6 and A-549 cells treated with Ba RNA had higher IL-8 and IL-6 levels compared to untreated cells. In addition, we showed that Ba RNA down-modulates the MHC-I surface expression induced by IFN-γ on human monocytes/macrophages via the pathway of the Epidermal Growth Factor Receptor (EGFR). So, cells were stimulated with an EGFR ligand-blocking antibody (Cetuximab) and Ba RNA. Neutralization of the EGFR to some extent reversed the down-modulation of MHC-I mediated by Ba RNA in HMEC and A-549 cells. In conclusion, this is the first study exploring a central immune evasion strategy, such as the downregulation of MHC-I surface expression, beyond monocytes and could shed light on how it persists effectively within the host, enduring unseen and escaping CD8+ T cell surveillance.

Deletion of the alr gene in Brucella suis S2 attenuates virulence by enhancing TLR4-NF-κB-NLRP3- mediated host inflammatory responses.

Brucella is an intracellular parasitic bacterium lacking typical virulence factors, and its pathogenicity primarily relies on replication within host cells. In this study, we observed a significant increase in spleen weight in mice immunized with a Brucella strain deleted of the gene for alanine racemase (Alr), the enzyme responsible for alanine racemization (Δalr). However, the bacterial load in the spleen markedly decreased in the mutant strain. Concurrently, the ratio of white pulp to red pulp in the spleen was increased, serum IgG levels were elevated, but no significant damage to other organs was observed. In addition, the inflammatory response was potentiated and the NF-κB-NLRP3 signaling pathway was activated in macrophages (RAW264.7 Cells and Bone Marrow-Derived Cells) infect ed with the Δalr mutant. Further investigation revealed that the Δalr mutant released substantial amounts of protein in a simulated intracellular environment which resulted in heightened inflammation and activation of the TLR4-NF-κB-NLRP3 pathway in macrophages. The consequent cytoplasmic exocytosis reduced intracellular Brucella survival. In summary, cytoplasmic exocytosis products resulting from infection with a Brucella strain deleted of the alr gene effectively activated the TLR4-NFκB-NLRP3 pathway, triggered a robust inflammatory response, and reduced bacterial survival within host cells. Moreover, the Δalr strain exhibits lower toxicity and stronger immunogenicity in mice.

Immunoinformatic-guided designing of multi-epitope vaccine construct against Brucella Suis 1300.

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.

Polymorphisms in vitamin D receptor genes and its relation with susceptibility to brucellosis: a case-control study.

OBJECTIVE: One of the systemic infections is Brucellosis which is caused by facultative intracellular bacteria of the genus Brucella. Vitamin D is a fat-soluble prohormone, that metabolizes enzymes and its intracellular receptor creates the active hormone and also mediate in responses of immune system. METHODS: Current research consists of 102 patients with brucellosis who were selected based on culture, PCR results serology, and clinical symptoms. The control group composed of 102 healthy people. The polymorphism of genes (Bsm I, Fok I, Taq I, Apa I) encoding Vitamin D receptor (VDR) were assessed by the PCR-RFLP method. RESULTS: The results showed that ff, tt, aa, and bb genotypes in Fok I, ApaI, TaqI, and BsmI were significant in case/control groups (P-value ≤ 0.0001). The genotype frequency AA in the control group is higher than that of the study group, while genotype frequency aa in the study group is more than the control. The odds ratio for brucellosis in individuals with ff genotype is 37 times higher than that of Ff genotype. Also, the odds ratio of brucellosis in individuals with genotype tt, aa, and bb was 12, 53, and 6 times higher than those of the Aa, Bb, and Tt genotypes. CONCLUSION: The genotypes aa and ff in the positions of the ApaI and FokI are of higher importance. The brucellosis risk in individuals accompanied aa genotype at Apa I is 53 times higher than that of the genotype AA, in other words, AA and BB, TT and FF genotypes are protective against the disease.

Differentially expressed long noncoding RNAs in RAW264.7 macrophages during Brucella infection and functional analysis on the bacterial intracellular replication.

Long noncoding RNAs (lncRNAs) are a group of functional RNA molecules without protein-coding potential and play vital roles in majority of biological processes. To date, the expression profiles of lncRNAs and their influence on Brucella replication in RAW264.7 cells are poorly understood. In this study, we performed high-throughput transcriptome analysis to investigate the differentially expressed lncRNAs associated with Brucella abortus S2308 infection. Of these, 8, 6, 130 and 94 cellular lncRNAs were differentially expressed at 4, 8, 24 and 48 h post-infection, respectively. Moreover, 1918 protein-coding genes are predicted as potential cis target genes of differentially expressed lncRNAs by searching protein-coding genes located at upstream and downstream of lncRNA loci on the chromosome DNA of Mus musculus. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that majority of lncRNA target genes were associated with B. abortus infection. Fourteen lncRNAs from transcriptome data were selected for qRT-PCR verification, confirming 13 were differentially expressed. Animal experiments revealed three were differentially expressed in vivo by qRT-PCR analysis. Furthermore, knockdown of LNC_000428 by CRISPR/dCas9 inhibition or Locked Nucleic Acids transfection downregulated Tnfrsf8 expression at mRNA level and increased Brucella intracellular replication. Thus, we provide a novel evidence that lncRNAs induced by Brucella-infection function on Brucella intracellular replication.

Allelic polymorphisms of the BoLA-DRB3 gene and resistance to brucellosis in Kazakh cattle.

The Authors investigated polymorphism of the bovine BoLA­DRB3 gene in connection with resistance or susceptibility to brucellosis of two Kazakh meat breeds, Auliekol and Kazakh Whiteheaded breeds, using PCR­RFLP. In Auliekol cattle (n = 158), 22 alleles were detected in the brucellosis group, and 24 alleles were shown in the healthy group. BoLA-DRB3 alleles *3, *4, *19, *21 were more common in healthy animals, while Brucella­positive cattle were more frequently carriers of alleles *7, *10, *18. In Kazakh Whiteheaded cattle (n = 146), 21 alleles were detected in infected and 23 alleles in healthy cattle. Alleles *3, *8, *21 significantly predominate in healthy cattle, while alleles *7, *11, *16 are typical for animals with brucellosis. This study identified BoLA-DRB3 alleles associated with genetic resistance (*3 and *21) and susceptibility (*7) to brucellosis; remarkably, resistance alleles are shared by two important meat breeds of Kazakhstan.

The regulon of Brucella abortus two-component system BvrR/BvrS reveals the coordination of metabolic pathways required for intracellular life.

Brucella abortus is a facultative intracellular pathogen causing a severe zoonotic disease worldwide. The two-component regulatory system (TCS) BvrR/BvrS of B. abortus is conserved in members of the Alphaproteobacteria class. It is related to the expression of genes required for host interaction and intracellular survival. Here we report that bvrR and bvrS are part of an operon composed of 16 genes encoding functions related to nitrogen metabolism, DNA repair and recombination, cell cycle arrest, and stress response. Synteny of this genomic region within close Alphaproteobacteria members suggests a conserved role in coordinating the expression of carbon and nitrogen metabolic pathways. In addition, we performed a ChIP-Seq analysis after exposure of bacteria to conditions that mimic the intracellular environment. Genes encoding enzymes at metabolic crossroads of the pentose phosphate shunt, gluconeogenesis, cell envelope homeostasis, nucleotide synthesis, cell division, and virulence are BvrR/BvrS direct targets. A 14 bp DNA BvrR binding motif was found and investigated in selected gene targets such as virB1, bvrR, pckA, omp25, and tamA. Understanding gene expression regulation is essential to elucidate how Brucella orchestrates a physiological response leading to a furtive pathogenic strategy.

Genome-wide transcription start site mapping in the facultative intracellular pathogen Brucella melitensis by Capping-seq.

Brucella melitensis (B. melitensis) is an important facultative intracellular bacterium that causes global zoonotic diseases. Continuous intracellular survival and replication are the main obstruction responsible for the accessibility of prevention and treatment of brucellosis. Bacteria respond to complex environment by regulating gene expression. Many regulatory factors function at loci where RNA polymerase initiates messenger RNA synthesis. However, limited gene annotation is a current obstacle for the research on expression regulation in bacteria. To improve annotation and explore potential functional sites, we proposed a novel genome-wide method called Capping-seq for transcription start site (TSS) mapping in B. melitensis. This technique combines capture of capped primary transcripts with Single Molecule Real-Time (SMRT) sequencing technology. We identified 2,369 TSSs at single nucleotide resolution by Capping-seq. TSSs analysis of Brucella transcripts showed a preference of purine on the TSS positions. Our results revealed that -35 and -10 elements of promoter contained consensus sequences of TTGNNN and TATNNN, respectively. The 5' ends analysis showed that 57% genes are associated with more than one TSS and 47% genes contain long leader regions, suggested potential complex regulation at the 5' ends of genes in B. melitensis. Moreover, we identified 52 leaderless genes that are mainly involved in the metabolic processes. Overall, Capping-seq technology provides a unique solution for TSS determination in prokaryotes. Our findings develop a systematic insight into the primary transcriptome characterization of B. melitensis. This study represents a critical basis for investigating gene regulation and pathogenesis of Brucella.

Evaluation of the genetic profiles of Brucella melitensis strain from Turkey using multilocus variable number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) techniques.

Brucellosis is caused by Brucella, and Brucella melitensis is highly prevalent in small ruminants in Turkey. Our aim was to genotype 50 B. melitensis strains isolated from sheep, goat, and cattle abortion samples from different farms in seven geographical regions of Turkey between 2009 and 2017. Forty-six different genotypes were detected in 50 isolates studied according to the MLVA-16. Thirty out of 50 isolate profiles matched profiles from the database exactly, and the remaining 20 were absent. Of these 30 isolates, 93.3% were identical to human isolates previously present in the database. All B. melitensis strains belonged to the eastern Mediterranean group. Genotype 43 was the most common isolate profile, and sequence typing (ST8) was dominant and detected in 39 strains. MLST analysis revealed a novel profile in 11 strains. On comparing the sequences of ST8 and the novel ST, a glucokinase gene variation was detected. In the MLST and MLVA analyses, no distinction was made between B. melitensis biovars. Moreover, there was no significant difference between the strains based on host, region, and year. Consequently, the discrimination power of MLVA was higher than that of MLST in this study. Contrastingly, MLST was useful in distinguishing strains according to geographic origins, as determined by performer studies. Profiles determined by MLVA were the same as those in humans. This raises concerns in regard to One Health and transition between hosts, as it is clear that protecting animal health is very important for human health.

Brucella melitensis biovar 1 isolation in a captive wildlife population in the United Arab Emirates. First isolation in the scimitar-horned Oryx (Oryx dammah).

In 2013, Brucella melitensis biovar 1 was recovered from the stomach contents of a scimitar-horned Oryx - SHO (Oryx dammah) aborted foetus, and from the articular fluid of a sand gazelle (Gazella marica) in a captive wildlife collection near Abu Dhabi, United Arab Emirates. Other evidence of exposure to the pathogen was collected through serological testing (Rose Bengal test) and B. melitensis-specific PCR of samples from captive wildlife kept in six different enclosures. A Multiple Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) using 15 markers showed that the two strains isolated in animals kept in enclosures, located 1300 m apart from each other, shared an identical genotype. The phylogenetic analysis of MLVA-15 profiles retrieved from the public database suggested that these strains belong to the African clade, clustering regionally in the UAE, Oman and Qatar. This is the first confirmed case of B. melitensis in a SHO, an African antelope extinct in the wild and warrants further investigation.

Assessment of Association between miR-146a Polymorphisms and Expression of miR-146a, TRAF-6, and IRAK-1 Genes in Patients with Brucellosis.

BACKGROUND: Brucellosis is a major zoonosis all over the world. MicroRNAs are significant gene expression regulators and could be involved during the infections and also genetic alterations in the miRNAs sequence can affect primary miRNAs and precursor miRNAs processing and thus alter miRNAs expression. Current research studied the impact of the miR-146a polymorphism on miR-146a, TRAF-6, and IRAK-1 genes expression in patients with brucellosis illness. METHODS AND RESULTS: In this research, 25 patients with brucellosis and 25 healthy participants with determined genotypes for miR-SNP rs2910164 and miR-SNP rs57095329 were recruited. IRAK-1, TRAF-6, and miR-146a expressions in peripheral blood mononuclear cells (PBMCs) were specified by quantitative real- time PCR (qRT-PCR). Moreover, interleukin-1ß (IL-1ß) and tumor necrosis factor- alpha (TNF-α) serum levels were assessed by a sandwich enzyme-linked immunosorbent assay (ELISA) technique. There was no significant difference in the expression level of miR-146a, IRAK-1, and TRAF-6, among the patients with brucellosis and control group. TRAF-6 PBMCs expression levels in the distinctive genotypes of rs2910164 were significantly observed in patients (P = 0.048). No significant distinctions were found in miR-146a, IRAK-1, and TRAF-6 expression levels and among the rs57095329 different genotypes in brucellosis patients and controls. Meanwhile, no significant relationship was found between the rs2910164 and rs57095329 genotypes and the serum level of cytokines mentioned between the two groups. We did not find any association between expression of TRAF-6, miR-146a, and IRAK-1 in PBMCs, and cytokines serum levels with two single nucleotide polymorphisms (SNPs) in miR-146a. CONCLUSIONS: To the best of writers' knowledge, this research is the first one evaluating the probable link between the miR-146a rs2910164 and rs57095329 variant with miRNAs, relevant cytokine levels, and target genes in brucellosis.

Expression of IL1ß Gene in the Placentas from Humans and Cows during Brucella Infection.

Brucellosis is an important contagious disease affecting most domestic and mature animals. Since the impact of IL-1ß in B. abortus invasion and survival remains elusive, the current study sought to elucidate the actual roles of these potent cytokines in the modulation of the initial immune response to Brucella infection. Therefore, this study aimed to detect Brucella abortus in the placenta of aborted women and cows and estimate the expression of the interleukin 1ß (IL1ß) gene associated with immune response mechanisms to Brucella abortus infection. The detection of Brucella abortus was performed by Rose Bengal Test (RBT) and Polymerase Chain Reaction based AlkB gene (AlkB-PCR) in the sera and placenta samples of aborted women and cows, respectively. The overall percentage of Brucella abortus infection was 13.1% and 5% as determined by RBT and AlkB-PCR in aborted women's sera and placentas, respectively. On the other hand, the overall percentage rates of Brucella abortus infection in the sera and placentas from aborted cows were 30% and 11% as estimated by RBT and AlkB-PCR, respectively. The results of RBT demonstrated that the association between Brucella abortus and abortion in cows was statistically significant. On the other hand, it was found that the association between Brucella abortus and abortion in women was not significant. Moreover, according to the results of AlkB-based PCR, the association between Brucella abortus and abortion was statistically significant in aborted cows, while it was not significant in aborted women. The sensitivity, specificity, and accuracy of RBT were calculated as 60.00, 53.85, and 54.55%, respectively. Moreover, positive and negative predictive values were reported as 14.33% and 91.28%, respectively. Regarding RBT for aborted cows, the sensitivity, specificity, and accuracy of the test were 81.82%, 57.78%, and 62.49%, respectively. The positive predictive value was reported as 32.08%, while the negative predictive value was reported as 92.88%. Quantitative PCR (qPCR) was carried out for the evaluation of Interleukin 1 Beta (IL1ß) gene expression. The qPCR result was presented as a fold change in gene expression. A significant increment of IL1ß gene expression was observed in aborted women (114.905±99.661) and cows (22.454 ±18.528), compared to non-aborted women (4.953±5.564) and cows (2.033±1.845). Statistical comparison of IL1ß gene expression between aborted women and cows illustrated a non-significant increment in IL1ß gene expression in aborted women (114.905±99.661), compared to aborted cows (22.454 ±18.528).

Association between risk of brucellosis and genetic variations in MicroRNA-146a.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most common types of DNA changes in the human genome that leading to phenotypic differences in humans. MicroRNAs (miRNAs) are usually affected by various bacterial infections, and they are involved in controlling the immune responses. MicroRNA-146a (miR-146a) plays an essential role in the development of infectious and inflammatory diseases. The aim of the present study was to investigate the association between risk of brucellosis and genetic variations in miR-146a. METHODS: This case-control study was conducted on 108 Brucellosis patients and 108 healthy controls. We genotyped two SNPs (rs2910164 and rs57095329) of the miR-146a using tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) and restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) methods. RESULTS: The rs2910164 SNP was significantly associated with brucellosis in co-dominant [OR = 4.27, 95% CI = (2.35-7.79, P = 0.001] and dominant [OR = 3.52, 95% CI = (1.97-6.30, P = 0.001] models. Co-dominant (P = 0.047) and recessive (P = 0.018) models were significant at position rs57095329 between the two groups of patient and healthy. The A C haplotype (rs2910164 and rs57095329) was associated with brucellosis in the assessed population [OR (95% CI) = 1.98 (1.22-3.20), P = 0.0059]. CONCLUSIONS: Consequently, our study demonstrated significant differences in genotype and haplotype frequencies of miR-146a variants between brucellosis patients and controls. Further studies on the larger sample sizes are required to verify the observed associations.

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence.

Brucella is a facultative extracellular-intracellular pathogen that belongs to the Alphaproteobacteria class. Precise sensing of environmental changes and a proper response mediated by a gene expression regulatory network are essential for this pathogen to survive. The plant-related Alphaproteobacteria Sinorhizobium meliloti and Agrobacterium tumefaciens also alternate from a free to a host-associated life, where a regulatory invasion switch is needed for this transition. This switch is composed of a two-component regulatory system (TCS) and a global inhibitor, ExoR. In B. abortus, the BvrR/BvrS TCS is essential for intracellular survival. However, the presence of a TCS inhibitor, such as ExoR, in Brucella is still unknown. In this work, we identified a genomic sequence similar to S. meliloti exoR in the B. abortus 2308W genome, constructed an exoR mutant strain, and performed its characterization through ex vivo and in vivo assays. Our findings indicate that ExoR is related to the BvrR phosphorylation state, and is related to the expression of known BvrR/BrvS gene targets, such as virB8, vjbR, and omp25 when grown in rich medium or starving conditions. Despite this, the exoR mutant strain showed no significant differences as compared to the wild-type strain, related to resistance to polymyxin B or human non-immune serum, intracellular replication, or infectivity in a mice model. ExoR in B. abortus is related to BvrR/BvrS as observed in other Rhizobiales; however, its function seems different from that observed for its orthologs described in A. tumefaciens and S. meliloti.

microRNAs in human brucellosis: A promising therapeutic approach and biomarker for diagnosis and treatment.

INTRODUCTION: Human brucellosis is a zoonotic bacterial disease with up to 500,000 new cases each year. The major evasion mechanisms from the host immune system by Brucella are restraint of complement pathway and Toll-like receptors signaling pathways, interference with efficient antigen presentation to CD4-positive T lymphocytes, selective subversion of autophagy pathways, inhibition of dendritic cell stimulation, inhibition of autophagolysosomal fusion, and macrophage apoptosis. Many molecular and cellular pathways contribute to brucellosis that microRNAs have a vital function in the immunopathogenesis of this disease. In this regard, these molecules apply for their roles by modulating various events like inflammatory reactions and immune defense. Recently, in the case of immunity to human brucellosis, it has been shown that microRNAs play an important role in immunity against these bacteria. METHODS AND RESULTS: In this study, we tried to review the immune defense and immunopathogenesis of Brucella infection and highlight the current knowledge of the microRNAs in infected cells by Brucella pathogens. The recent findings suggest that the regulation of microRNAs expression is impaired during brucellosis infection, which may contribute to disease progression or inhibition by modulating immune responses against this pathogen. CONCLUSIONS: The interplay between miRNAs and Brucella pathogens and the underlying process required comprehensive examination to unravel the novel therapeutic or diagnostic approaches.

Overexpression of ASMT likely enhances the resistance of transgenic sheep to brucellosis by influencing immune-related signaling pathways and gut microbiota.

Melatonin is a pleiotropic molecule with a variety of biological functions, which include its immunoregulatory action in mammals. Brucellosis is a worldwide endemic zoonotic disease caused by the Brucella, which not only causes huge economic losses for the livestock industry but also impacts human health. To target this problem, in current study, two marker-free transgenic sheep overexpressing melatonin synthetic enzyme ASMT (acetylserotonin O-methyltransferase) gene were generated and these melatonin enrich transgenic sheep were challenged by Brucella infection. The results showed that the serum melatonin concentration was significantly higher in transgenic sheep than that of wild type (726.92 ± 70.6074 vs 263.10 ± 34.60 pg/mL, P < .05). Brucella challenge test showed that two thirds (4/6) of the wild-type sheep had brucellosis, while none of the transgenic sheep were infected. Whole-blood RNA-seq results showed that differential expression genes (DEGs) were significantly enriched in natural killer cell-mediated cytotoxicity, phagosome, antigen processing, and presentation signaling pathways in overexpression sheep. The DEGs of toll-like receptors (TLRs) and NOD-like receptors (NLRs) families were verified by qPCR and it showed that TLR1, TLR2, TLR7, CD14, NAIP, and CXCL8 expression levels in overexpression sheep were significantly higher and NLRP1, NLRP3, and TNF expression levels were significantly lower than those of wild type. The rectal feces were subjected to 16S rDNA amplicon sequencing, and the microbial functional analysis showed that the transgenic sheep had significantly lower abundance of microbial genes related to infectious diseases compared to the wild type, indicating overexpression animals are likely more resistant to infectious diseases than wild type. Furthermore, exogenous melatonin treatment relieved brucellosis inflammation by upregulating anti-inflammatory cytokines IL-4 and downregulating pro-inflammatory IL-2, IL-6, and IFN-γ. Our preliminary results provide an informative reference for the study of the relationship between melatonin and brucellosis.

Small ubiquitin-related modifier 2 affects the intracellular survival of Brucella abortus 2308 by regulating activation of the NF-κB pathway.

Brucella is a genus of Gram-negative intracellular pathogens that cause animal and human diseases. Brucella survival and replication inside immune cells is critical for the establishment of chronic infections. Protein modifications by small ubiquitin-related modifier proteins and the NF-κB pathway are involved in many cellular activities, playing major roles in regulating protein function that is essential for pathogenic bacteria during infection. However, the relationship between them in the intracellular survival of Brucella is still largely unknown. We demonstrated that Brucella abortus 2308 infection can activate the expression of small ubiquitin-related modifier-2 proteins in a time-dependent manner. We found the production of Th1 cytokines (IFN-γ and TNF-α) and the transcription of NF-κB/p65 were promoted by overexpression and inhibited by interference of small ubiquitin-related modifier-2. In addition, we showed that small ubiquitin-related modifier-2 can inhibit intracellular survival of Brucella abortus 2308 by regulating activation of the NF-κB pathway. Taken together, this work shows that small ubiquitin-related modifier-2 modification of NF-κB2/p65 is essential for the survival of Brucella abortus 2308 inside macrophages. This work may help to unravel the pathogenic mechanisms of Brucella infections.