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.

Highly efficient base editing in bacteria using a Cas9-cytidine deaminase fusion.

The ability to precisely edit individual bases of bacterial genomes would accelerate the investigation of the function of genes. Here we utilized a nickase Cas9-cytidine deaminase fusion protein to direct the conversion of cytosine to thymine within prokaryotic cells, resulting in high mutagenesis frequencies in Escherichia coli and Brucella melitensis. Our study suggests that CRISPR/Cas9-guided base-editing is a viable alternative approach to generate mutant bacterial strains.

Expression of pseudorabies virus-encoded long noncoding RNAs in epithelial cells and neurons.

Long noncoding RNAs (lncRNAs) play important roles in regulating eukaryotic genome replication and gene expression in diverse biological systems. Here, we identified lncRNAs transcribed from pseudorabies virus (PRV)-infected PK-15 cells. Based on high-throughput sequencing data, we obtained 87,263,926 and 93,947,628 clean reads from mock-infected and PRV-infected PK-15 cells, respectively. Through a normalized analytic protocol, we identified three novel viral lncRNAs. According to an analysis of differential expression between the mock-infected and PRV-infected cells, 4151 host lncRNAs were significantly upregulated and 2327 host lncRNAs were significantly downregulated in the latter group. Viral lncRNAs and several host lncRNAs were verified by northern blotting and real-time PCR. The findings showed that the viral lncRNA LDI might regulate the expression of IE180, a potent transcriptional activator of viral genes. Furthermore, we characterized the expression of viral lncRNAs in a culture of infected primary chicken dorsal root ganglia (DRG). Collectively, the obtained data suggest that PRV generates lncRNAs in both epithelial cells and chick DRG neurons.

Proteomic Profiling of Sweat Exosome Suggests its Involvement in Skin Immunity.

Healthy human skin sustains an effective immune defense mechanism, formed by a complex physical and chemical epidermal barrier that coordinates with different cellular components of the skin immune system. However, the mechanism by which skin cells regulate local immune homeostasis in health and disease contexts is not well known. To investigate whether exosomes exist in sweat, sweat samples from healthy individuals were collected after aerobic exercise. Sweat exosome was isolated via differential ultracentrifugation, observed under transmission electron microscopy, measured by dynamic light scattering, and confirmed by immunoblot. Further, shotgun liquid chromatography (LC)-mass spectrometry (MS)/MS analysis was conducted to investigate the proteomic profiling of sweat exosome. Secreted exosome was detected in human sweat. A total of 1,062 proteins were identified in sweat exosome, including 997 different proteins compared with sweat proteomics and 896 unique proteins compared with urine, saliva, and plasma exosomes. Diverse antimicrobial peptides and immunological factors were found in sweat exosome, suggesting the involvement of exosome in skin immunity. This study provides direct evidence that secreted exosomes exist in human sweat. The proteomic profiling of sweat exosome provides insight into sweat features and the potential physiological significance of exosomes in immune homeostasis.

Comparison of pseudorabies virus China reference strain with emerging variants reveals independent virus evolution within specific geographic regions.

Pseudorabies virus (PRV) China reference strain Ea is genetically closely related to newly emerged variants; however, there is limited information about PRV Ea. Here, we compared PRV Ea with new variant strains by growth kinetics, genome sequencing, and protein expression analysis. Growth analysis showed that strain Ea forms smaller plaques than strain HNX. The full-length genome sequence of Ea revealed that it is clustered in the same subgroup as HNX. Ea and HNX strains exhibited similar extracellular virion protein polymorphisms, whereas strain Bartha expressed less VP26 and more GAPDH. In infected cells, strain Ea expressed high levels of IE180 protein, and Ea and HNX produced higher levels of UL21 protein than strain Bartha. These findings provide evidence that PRV China reference strain Ea is genetically closely related to the newly emerged variant strains, indicating that strain PRV China may have evolved independently leading to the emergence of a variant strain.

Characterization of ribonuclease III from Brucella.

Bacterial ribonuclease III (RNase III) is a highly conserved endonuclease, which plays pivotal roles in RNA maturation and decay pathways by cleaving double-stranded structure of RNAs. Here we cloned rncS gene from the genomic DNA of Brucella melitensis, and analyzed the cleavage properties of RNase III from Brucella. We identified Brucella-encoding small RNA (sRNA) by high-throughput sequencing and northern blot, and found that sRNA of Brucella and Homo miRNA precursor (pre-miRNA) can be bound and cleaved by B.melitensis ribonuclease III (Bm-RNase III). Cleavage activity of Bm-RNase III is bivalent metal cations- and alkaline buffer-dependent. We constructed several point mutations in Bm-RNase III, whose cleavage activity indicated that the 133th Glutamic acid residue was required for catalytic activity. Western blot revealed that Bm-RNase III was differently expressed in Brucella virulence strain 027 and vaccine strain M5-90. Collectively, our data suggest that Brucella RNase III can efficiently bind and cleave stem-loop structure of small RNA, and might participate in regulation of virulence in Brucella.

Immunogenic response to a recombinant pseudorabies virus carrying bp26 gene of Brucella melitensis in mice.

Brucellae are facultative intracellular bacterial pathogens of a zoonotic disease called brucellosis. Live attenuated vaccines are utilized for prophylaxis of brucellosis; however, they retain residual virulence to human and/or animals, as well as interfere with diagnosis. In this study, recombinant virus PRV ΔTK/ΔgE/bp26 was screened and purified. One-step growth curve assay showed that the titer of recombinant virus was comparable to the parent strain. Mice experiments showed the recombinant virus elicited high titer of humoral antibodies against Brucella detected by enzyme-linked immunosorbent assay and against PRV by serum neutralization test. The recombinant virus induced high level of Brucella-specific lymphocyte proliferation response and production of interferon gamma. Collectively, these data suggest that the bivalent virus was capable of inducing both humoral and cellular immunity, and had the potential to be a vaccine candidate to prevent Brucella and/or pseudorabies virus infections.