Pal Affects the Proliferation in Macrophages and Virulence of Brucella, and as Mucosal Adjuvants, Provides an Effective Protection to Mice Against Salmonella Enteritidis.

The purpose of this study was to elucidate the roles of peptidoglycan-associated lipoprotein (Pal protein) in the proliferation of Brucella in macrophage and bacterial virulence, and to evaluate the immune effect of Pal protein to Salmonella enteritidis. Murine macrophage-like cell line Raw264.7 was stimulated by recombinant Pal protein, and the expression of TNF-α and IFN-γ were up-regulated, but not it of IL-1ß and IL-6. The macrophages infection and in vitro simulated stress assays showed that deletion of pal gene reduced the proliferation of Brucella in macrophages, the survival in acidic, oxidative and polymyxin B-contained environment. The mice infection assay showed that mice challenged with the pal mutant strain were found to have more severe splenomegaly, but less bacterial load. After oral immunization of mice, Pal protein induced a higher titer of mucosal and humoral antibody (IgA and IgG) against heat-killed Salmonella enteritidis, and a stronger Th1 cellular immune response. The challengte experiments showed Pal protein elevated the survival rate and reduced the bacterial load of spleens in immunized mice. In conclusion, our results revealed the important roles of pal gene in Brucella virulence, and Pal protein was a potentially valuable adjuvant against mucosal pathogens, such as Salmonella enteritidis.

H9N2 swine influenza virus infection-induced damage is mediated by TRPM2 channels in mouse pulmonary microvascular endothelial cells.

Oxidative stress is implicated in the pathogenesis of influenza virus infection. Increasing evidences show that transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable non-selective cation channel, plays an important role in the pathomechanism of reactive oxygen species (ROS)-coupled diseases. The present study investigated the role of TRPM2 in pulmonary microvascular endothelial cells (PMVECs) during H9N2 influenza virus infection. We knocked down TRPM2 in PMVECs using TRPM2 shRNA lentiviral particles. Subsequently, we utilized enzyme-linked immunosorbent assay and flow cytometry to compare ROS levels, DNA damage, mitochondrial integrity, apoptosis, and inflammatory factors between control and TRPM2-knockdown PMVECs following H9N2 influenza virus infection. Inhibition of TRPM2 channels reduced H9N2 virus-induced intracellular ROS production, decreased DNA damage, and inhibited H9N2-induced cellular apoptosis. This study shows that the inhibition of TRPM2 channels may protect PMVECs from the damage caused by H9N2 virus infection. Our results highlight the importance of TRPM2 in modulating ROS production, apoptosis, mitochondrial dysfunction, cytokine expression, and DNA damage in H9N2 virus-infected PMVECs, and suggest that TRPM2 may be a potential antiviral target.

First isolation and characterization of Brucella suis from yak.

Brucella spp., facultative intracellular pathogens that can persistently colonize animal host cells and cause zoonosis, affect public health and safety. A Brucella strain was isolated from yak in Qinghai Province. To detect whether this isolate could cause an outbreak of brucellosis and to reveal its genetic characteristics, several typing and whole-genome sequencing methods were applied to identify its species and genetic characteristics. Phylogenetic analysis based on MLVA and whole-genome sequencing revealed the genetic characteristics of the isolated strain. The results showed that the isolated strain is a B. suis biovar 1 smooth strain, and this isolate was named B. suis QH05. The results of comparative genomics and MLVA showed that B. suis QH05 is not a vaccine strain. Comparison with other B. suis strains isolated from humans and animals indicated that B. suis QH05 may be linked to specific animal and human sources. In conclusion, B. suis QH05 does not belong to the Brucella epidemic species in China, and as the first isolation of B. suis from yak, this strain expands the host range of B. suis.

Transcriptional analysis reveals the relativity of acid tolerance and antimicrobial peptide resistance of Salmonella.

The objective of this study was to comprehensively identify the target genes induced by acid stimulation in Salmonella, and to clarify the relativity of acid tolerance and antimicrobial peptide resistance. A clinical S. Typhimurium strain, S6, was selected and performed a transcriptome analysis under the acid tolerance response. In total, we found 1461 genes to be differentially expressed, including 721 up-regulated and 740 down-regulated genes. Functional annotation revealed differentially expressed genes to be associated with regulation, metabolism, transport, virulence, and motility. Interestingly, KEGG pathway analysis demonstrated that the induced genes by acid were enriched in cationic antimicrobial peptide resistance, sulfur relay system, ABC transporters, and two-component system pathway. Therein, PhoQ belonging to the two-component system PhoP-PhoQ that promotes virulence by detecting the macrophage phagosome and controls the transcript levels of many genes associated with the resistance to AMPs; MarA, a multiple antibiotic resistance factor; SapA, one of the encoding gene of sapABCDF operon that confers resistance to small cationic peptides of Salmonella; YejB, one of the encoding gene of yejABEF operon that confers resistance to antimicrobial peptides and contributes to the virulence of Salmonella, were all induced by acid stimulation, and could potentially explain that there is a correlation between acid tolerance and AMPs resistance, and finally affects the virulence of intracellular pathogenic bacteria.

Relation of the pdxB-usg-truA-dedA Operon and the truA Gene to the Intracellular Survival of Salmonella enterica Serovar Typhimurium.

Salmonella is the genus of Gram-negative, facultative intracellular pathogens that have the ability to infect large numbers of animal or human hosts. The S. enterica usg gene is associated with intracellular survival based on ortholog screening and identification. In this study, the λ-Red recombination system was used to construct gene deletion strains and to investigate whether the identified operon was related to intracellular survival. The pdxB-usg-truA-dedA operon enhanced the intracellular survival of S. enterica by resisting the oxidative environment and the usg and truA gene expression was induced by H2O2. Moreover, the genes in this operon (except for dedA) contributed to virulence in mice. These findings indicate that the pdxB-usg-truA-dedA operon functions in resistance to oxidative environments during intracellular survival and is required for in vivo S. enterica virulence. This study provides insight toward a better understand of the characteristics of intracellular pathogens and explores the gene modules involved in their intracellular survival.

Genome-wide sequence transposon insertion sites and analyze the essential genes of Brucella melitensis.

A transposon mutant library of B. melitensis NI including 32,640 transposon mutants was established. By sequencing the transposon insertion sites, 10,832 mutants were successfully defined for their insertion sites. Analysis of the mutants with defined transposon insertion sites (DTIS) indicated that the insertions were well spread through the two genomes. In addition, 948 genes with no detectable transposon insertions were taken as the candidate for identification of essential genes. In comparison with the Bacterial Database of Essential Genes and by using comparative genomics analysis, 183 potential essential genes of B. melitensis NI cultured in vitro were found and they were conserved in the common bacteria. This work was focused on screening of the essential genes of B. melitensis NI, which may provide a foundation for identification of the novel drug targets against brucellosis. Besides, the sequence-defined transposon library should serve as a resource for screening of different function genes of Brucella.

Analysis of pan-genome to identify the core genes and essential genes of Brucella spp.

Brucella spp. are facultative intracellular pathogens, that cause a contagious zoonotic disease, that can result in such outcomes as abortion or sterility in susceptible animal hosts and grave, debilitating illness in humans. For deciphering the survival mechanism of Brucella spp. in vivo, 42 Brucella complete genomes from NCBI were analyzed for the pan-genome and core genome by identification of their composition and function of Brucella genomes. The results showed that the total 132,143 protein-coding genes in these genomes were divided into 5369 clusters. Among these, 1710 clusters were associated with the core genome, 1182 clusters with strain-specific genes and 2477 clusters with dispensable genomes. COG analysis indicated that 44 % of the core genes were devoted to metabolism, which were mainly responsible for energy production and conversion (COG category C), and amino acid transport and metabolism (COG category E). Meanwhile, approximately 35 % of the core genes were in positive selection. In addition, 1252 potential essential genes were predicted in the core genome by comparison with a prokaryote database of essential genes. The results suggested that the core genes in Brucella genomes are relatively conservation, and the energy and amino acid metabolism play a more important role in the process of growth and reproduction in Brucella spp. This study might help us to better understand the mechanisms of Brucella persistent infection and provide some clues for further exploring the gene modules of the intracellular survival in Brucella spp.

Development of an improved competitive ELISA based on a monoclonal antibody against lipopolysaccharide for the detection of bovine brucellosis.

BACKGROUND: Brucellosis is the most common bacterial zoonosis, and serological tests are routinely used in brucellosis control and eradication programs. In order to improve the accuracy of serological diagnostic method used in bovine brucellosis detection, this study developed an improved competitive ELISA with higher specificity and good sensitivity. RESULTS: This study prepared 12 monoclonal antibodies against smooth Brucella lipopolysaccharide. One monoclonal antibody 3 F9, presented C epitope specificity, was used to develop a competitive ELISA for the serological detection of bovine brucellosis. The competitive ELISA, a commercial competitive ELISA kit, the rose-bengal plate agglutination test, and a microplate agglutination test were all used in the detection of 6 hyperimmune antisera against other commonly cross-reacted bacterial pathogens and 110 clinical bovine serum samples. The results of the test comparisons indicated that the competitive ELISA had higher specificity than the commercial competitive ELISA kit and RBT, and comparable sensitivity with the commercial ELISA kit. CONCLUSIONS: This study provided a valuable detection tool with high specificity and good sensitivity, which prevent the wrong-culling of bovines in the eradication campaigns of bovine brucellosis.

The effects of RegM on stress responses in Brucella melitensis.

Brucella melitensis is a facultative intracellular pathogen. The regM gene encodes a sensory transduction protein kinase in B. melitensis 16M, and genes orthologous to regM have been found to exist in many bacterial species. However, little is known about the regulation function of this gene in Brucella. In order to characterize this gene, we constructed a marked deletion mutant of regM as well as its complemented strain. The mutant was less able to withstand acid and hyperosmotic conditions than wild-type strain but shown no significant difference with wild-type strain when challenged by elevated temperature and hypotonic conditions. In addition, inactivation of regM did not affect virulence in B. melitensis 16M in macrophage and mice infection models.

Complete genome sequence of Brucella melitensis biovar 3 strain NI, isolated from an aborted bovine fetus.

From an aborted bovine fetus in China, a bacterial strain named NI was isolated and identified as Brucella melitensis by a PCR assay. Strain NI was further characterized as B. melitensis biovar 3 using biochemical assays. Here we report the complete genome sequence of strain NI.

Genome-Wide Analysis of the Response of Brucella melitensis NI to Polymyxin B.

BACKGROUND: The ability of pathogenic bacteria to survive Antimicrobial Peptides (AMPs) in various host niches may contribute to their virulence. Polymyxin B is a cationic AMP, and polymyxin drugs are considered to be the "last line of defense" in the clinical treatment of bacterial infections. OBJECTIVE: The objectives of this study were to comprehensively study the response of Brucella melitensis strain NI to polymyxin B treatment and to identify the target genes in Brucella induced by polymyxin B stimulation. METHODS: Following treatment with polymyxin B, differentially expressed genes in Brucella were detected using RNA-seq and validated using qRT-PCR. RESULTS: In total, 874 differentially expressed genes were identified, including 560 up-regulated and 314 down-regulated genes. Functional annotation and KEGG pathway analysis revealed that many of these genes are involved in metabolism, two-component systems, transcriptional regulation, transport/ membrane proteins, and virulence factors. Expression of genes involved in T4SS and flagellar biosynthesis and assembly, which are important virulence factors in Brucella, were up-regulated by polymyxin B treatment. DISCUSSION: Additionally, genes encoding the ABC transporters YejABEF and the cold-shock protein CspA were also up-regulated. These genes confer resistance to AMPs and contribute to the virulence of Brucella. The NIΔsufC, NIΔsufD, NIΔompW, NIΔexbB, NIΔtetR, and NIΔcspA mutants were also more sensitive than B. melitensis NI to polymyxin B. CONCLUSION: The results of this study provide important insights into the comprehensive response of Brucella in response to polymyxin B stimulation.

Whole-Genome Resequencing to Study Brucellosis Susceptibility in Sheep.

Brucellosis is a zoonotic disease and a major public health problem. However, the genetic mechanism of brucellosis in sheep remains unclear. In this study, serum samples were collected from 6,358 sheep from the F2 population (Dorper sheep ♂ × Hu sheep ♀), and antibody levels were continuously measured at 14 days and 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 months after administration of brucellosis vaccine. Finally, 19 brucellosis-resistant group (BRG) sheep and 22 brucellosis-susceptible group sheep (BSG) were screened for whole-genome sequencing. Using the fixation index, Fisher's exact test, and chi-square test, a total of 205 candidate SNP sites were identified. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis suggested that 138 candidate genes were significantly enriched in adherens junction (CTNNA3, PARD3, and PTPRM), cell adhesion molecules (NLGN1, CNTNAP2, NCAM1, and PTPRM), salivary secretion (LOC101102109, PRKG1, and ADCY2), and hippo signaling pathway (CTNNA3, YAP1, and PARD3). These findings provide valuable molecular markers for brucellosis resistance breeding in sheep and novel insights into the genetic mechanism of brucellosis resistance.

Cell membrane components of Brucella melitensis play important roles in the resistance of low-level rifampicin.

Brucella spp. are facultative intracellular pathogens that can persistently colonize host cells and cause the zoonosis- brucellosis. The WHO recommended a treatment for brucellosis that involves a combination of doxycycline, rifampicin, or streptomycin. The aim of this study was to screen rifampicin-resistance related genes by transcriptomic analysis and gene recombination method at low rifampicin concentrations and to predict the major rifampicin- resistance pathways in Brucella spp. The results showed that the MIC value of rifampicin for B. melitensis bv.3 Ether was 0.5 µg / mL. Meanwhile, B. melitensis had an adaptive response to the resistance of low rifampicin in the early stages of growth, while the SNPs changed in the rpoB gene in the late stages of growth when incubated at 37°C with shaking. The transcriptome results of rifampicin induction showed that the functions of significant differentially expressed genes were focused on metabolic process, catalytic activity and membrane and membrane part. The VirB operon, ß-resistance genes, ABC transporters, quorum-sensing genes, DNA repair- and replication -related genes were associated with rifampicin resistance when no variations of the in rpoB were detected. Among the VirB operons, VirB7-11 may play a central role in rifampicin resistance. This study provided new insights for screening rifampicin resistance-related genes and also provided basic data for the prevention and control of rifampicin-resistant Brucella isolates.

Identification of Small-Molecule Inhibitors of Brucella Diaminopimelate Decarboxylase by Using a High-Throughput Screening Assay.

Brucellosis, caused by intracellular gram-negative pathogens of the genus Brucella, continues to be one of the most pandemic zoonotic diseases in most countries. At present, the therapeutic treatment of brucellosis relies on a combination of multiple antibiotics that involves a long course of treatment, easy relapse, and high side effects from the use of certain antibiotics (such as streptomycin). Thus, the need to identify novel drugs or targets to control this disease is urgent. Diaminopimelate decarboxylase (DAPDC), a key enzyme involved in the bacterial diaminopimelate (DAP) biosynthetic pathway, was suggested to be a promising anti-Brucella target in our previous study. In this work, the biological activity of Brucella melitensis DAPDC was characterized, and a library of 1,591 compounds was screened for inhibitors of DAPDC. The results of a high-throughput screening (HTS) assay showed that 24 compounds inhibited DAPDC activity. In a further in vitro bacterial inhibition experiment, five compounds exhibited anti-Brucella activity (SID3, SID4, SID14, SID15, and SID20). These results suggested that the identified compounds can be used as potent molecules against brucellosis and that the application ranges of these approved drugs can be expanded in the future.

Cytotoxicity in macrophages infected with rough Brucella mutants is type IV secretion system dependent.

Smooth Brucella spp. inhibit macrophage apoptosis, whereas rough Brucella mutants induce macrophage oncotic and necrotic cell death. However, the mechanisms and genes responsible for Brucella cytotoxicity have not been identified. In the current study, a random mutagenesis approach was used to create a mutant bank consisting of 11,354 mutants by mariner transposon mutagenesis using Brucella melitensis rough mutant 16M delta manBA as the parental strain. Subsequent screening identified 56 mutants (0.49% of the mutant bank) that failed to cause macrophage cell death (release of 10% or less of the lactate dehydrogenase). The absence of cytotoxicity during infection with these mutants was independent of demonstrable defects in in vitro bacterial growth or uptake and survival in macrophages. Interrupted genes in 51 mutants were identified by DNA sequence analysis, and the mutations included interruptions in virB encoding the type IV secretion system (T4SS) (n = 36) and in vjbR encoding a LuxR-like regulatory element previously shown to be required for virB expression (n = 3), as well as additional mutations (n = 12), one of which also has predicted roles in virB expression. These results suggest that the T4SS is associated with Brucella cytotoxicity in macrophages. To verify this, deletion mutants were constructed in B. melitensis 16M by removing genes encoding phosphomannomutase/phosphomannoisomerase (delta manBA) and the T4SS (delta virB). As predicted, deletion of virB from 16M delta manBA and 16M resulted in a complete loss of cytotoxicity in rough strains, as well as the low level cytotoxicity observed with smooth strains at extreme multiplicities of infection (>1,000). Taken together, these results demonstrate that Brucella cytotoxicity in macrophages is T4SS dependent.

Screening and Identification of ssDNA Aptamers against HN Protein for Detection of Bovine Parainfluenza Virus Type 3 Antibodies in Serum.

BACKGROUND: Bovine Parainfluenza Virus type 3 (BPIV3) is a major but often overlooked pathogen that causes respiratory disease in cattle, especially during transportation and in feedlot situations. There is a demand for the rapid detection and serological diagnosis of BPIV3 to monitor the presence of the virus and its antibodies in cattle, which is critical in designing suitable interventions and control. METHODS: In the present study, ssDNA aptamers with high affinity and specificity against the HN protein of BPIV3 were selected using microplates as the matrix. RESULTS: After eleven rounds selection, thirty-four different DNA sequences were obtained in total, wherein w-32, w-33, and w-34 were repeated seven, eleven, and nine times, and with Kd values of 56.57 ± 2.7 nM, 24.64 ± 2.84 nM, and 31.3 ± 3.32 nM, respectively. Two-dimensional structural analysis showed that the three aptamers had several loop structures that were probably more energetically favorable for target binding. Of the three candidates, aptamer w-33 showed the best affinity in an indirect enzyme-linked aptamer assay (ELAA). The percent inhibition cutoff value of the ELAA, assessed using twenty negative sera, was 31%. CONCLUSION: In a comparative study with commercial ELISA kits, the positive detection rate of the ELAA was slightly higher than that of the commercial ELISA kits, and the coincidence rate of ELAA and ELISA was 88%. Further optimization of the ELAA method with more serums is needed.

Transcriptional analysis reveals the critical role of RNA polymerase-binding transcription factor, DksA, in regulating multi-drug resistance of Escherichia coli.

The objective of this study was to comprehensively identify the target genes regulated by the RNA polymerase-binding transcription factor DksA in Escherichia coli, and to clarify the role of DksA in multi-drug resistance. A clinical E. coli strain, E8, was selected to construct the dksA gene deletion mutant by using the Red recombination system. The minimum inhibitory concentrations of 12 antibiotics in the E8ΔdksA (mutant) were markedly lower than those in the wild-type strain, E8. Genes expressed differentially in the wild-type and dksA mutant were detected using RNA-Seq, and were validated by performing quantitative real-time polymerase chain reaction. In total, 168 differentially expressed genes were identified in E8ΔdksA, including 81 upregulated and 87 downregulated genes. Many of the genes identified are involved in metabolism, two-component systems, transcriptional regulators and transport/membrane proteins. Interestingly, genes encoding the transcriptional regulator, MarR, which is known to repress the multiple drug resistance operon, marRAB; MdfA, a transport protein that exhibits multi-drug efflux activities; and oligopeptide transport system proteins OppA and OppD were among those differentially expressed, and could potentially contribute to the increased drug susceptibility of E8ΔdksA. In conclusion, DksA plays an important role in the multi-drug resistance of this E. coli strain, and directly or indirectly regulates the expression of several genes related to antibiotic resistance.

Ortholog-based screening and identification of genes related to intracellular survival.

Bioinformatics and comparative genomics analysis methods were used to predict unknown pathogen genes based on homology with identified or functionally clustered genes. In this study, the genes of common pathogens were analyzed to screen and identify genes associated with intracellular survival through sequence similarity, phylogenetic tree analysis and the λ-Red recombination system test method. The total 38,952 protein-coding genes of common pathogens were divided into 19,775 clusters. As demonstrated through a COG analysis, information storage and processing genes might play an important role intracellular survival. Only 19 clusters were present in facultative intracellular pathogens, and not all were present in extracellular pathogens. Construction of a phylogenetic tree selected 18 of these 19 clusters. Comparisons with the DEG database and previous research revealed that seven other clusters are considered essential gene clusters and that seven other clusters are associated with intracellular survival. Moreover, this study confirmed that clusters screened by orthologs with similar function could be replaced with an approved uvrY gene and its orthologs, and the results revealed that the usg gene is associated with intracellular survival. The study improves the current understanding of intracellular pathogens characteristics and allows further exploration of the intracellular survival-related gene modules in these pathogens.

BASI74, a Virulence-Related sRNA in Brucella abortus.

Brucella spp. are intracellular pathogens that infect a wide variety of mammals including humans, posing threats to the livestock industry and human health in developing countries. A number of genes associated with the intracellular trafficking and multiplication have so far been identified in Brucella spp. However, the sophisticated post-transcriptional regulation and coordination of gene expression that enable Brucella spp. to adapt to changes in environment and to evade host cell defenses are not fully understood. Bacteria small RNAs (sRNAs) play a significant role in post-transcriptional regulation, which has already been confirmed in a number of bacteria but the role of sRNAs in Brucella remains elusive. In this study, we identified several different sRNAs in Brucella spp., and found that over-expression of a sRNA, tentatively termed BASI74, led to alternation in virulence of Brucella in macrophage infection model. The expression level of BASI74 increased while Brucella abortus 2308 was grown in acidic media. In addition, BASI74 affected the growth ratio of the Brucella cells in minimal media and iron limiting medium. Using a two-plasmid reporter system, we identified four genes as the target of BASI74. One target gene, BABI1154, was predicted to encode a cytosine-N4-specific DNA methyltransferase, which protects cellular DNA from the restriction endonuclease in Brucella. These results show that BASI74 plays an important role in Brucella survival in macrophage infection model, speculatively by its connection with stress response or impact on restriction-modification system. Our study promotes the understanding of Brucella sRNAs, as well as the mechanism by which sRNAs use to influence Brucella physiology and pathogenesis.

Complete Genome Sequences of Four Brucella Strains Isolated from China.

Brucella spp. are facultative intracellular pathogens that cause a contagious zoonotic disease. Twelve different species are currently identified. This study presents the complete genome sequences of four Brucella strains. These complete genomes were annotated and the contents compared to those of other strains isolated from China.