Comprehensive analysis of transcriptome and metabolome analysis reveal new targets of Glaesserella parasuis glucose-specific enzyme IIBC (PtsG).

The ptsG (hpIIBCGlc) gene, belonging to the glucose-specific phosphotransferase system, encodes the bacterial glucose-specific enzyme IIBC. In this study, the effects of a deletion of the ptsG gene were investigated by metabolome and transcriptome analyses. At the transcriptional level, we identified 970 differentially expressed genes between ΔptsG and sc1401 (Padj<0.05) and 2072 co-expressed genes. Among these genes, those involved in methane metabolism, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, pyruvate metabolism, phosphotransferase system (PTS), biotin metabolism, Two-component system and Terpenoid backbone biosynthesis showed significant changes in the ΔptsG mutant strain. Metabolome analysis revealed that a total of 310 metabolites were identified, including 20 different metabolites (p < 0.05). Among them, 15 metabolites were upregulated and 5 were downregulated in ΔptsG mutant strain. Statistical analysis revealed there were 115 individual metabolites having correlation, of which 89 were positive and 26 negative. These metabolites include amino acids, phosphates, amines, esters, nucleotides, benzoic acid and adenosine, among which amino acids and phosphate metabolites dominate. However, not all of these changes were attributable to changes in mRNA levels and must also be caused by post-transcriptional regulatory processes. The knowledge gained from this lays the foundation for further study on the role of ptsG in the pathogenic process of Glaesserella parasuis (G.parasuis).

Comparative transcriptome analysis on candidate genes associated with fruiting body growth and development in Lyophyllum decastes.

Lyophyllum decastes is a mushroom that is highly regarded for its culinary and medicinal properties. Its delectable taste and texture make it a popular choice for consumption. To gain a deeper understanding of the molecular mechanisms involved in the development of the fruiting body of L. decastes, we used RNA sequencing to conduct a comparative transcriptome analysis. The analysis encompassed various developmental stages, including the vegetative mycelium, primordial initiation, young fruiting body, medium-size fruiting body, and mature fruiting body stages. A range of 40.1 to 60.6 million clean reads were obtained, and de novo assembly generated 15,451 unigenes with an average length of 1,462.68 bp. Functional annotation of transcriptomes matched 76.84% of the unigenes to known proteins available in at least one database. The gene expression analysis revealed a significant number of differentially expressed genes (DEGs) between each stage. These genes were annotated and subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Highly differentially expressed unigenes were also identified, including those that encode extracellular enzymes, transcription factors, and signaling pathways. The accuracy of the RNA-Seq and DEG analyses was validated using quantitative PCR. Enzyme activity analysis experiments demonstrated that the extracellular enzymes exhibited significant differences across different developmental stages. This study provides valuable insights into the molecular mechanisms that underlie the development of the fruiting body in L. decastes.

QseC Mediates Osmotic Stress Resistance and Biofilm Formation in Haemophilus parasuis.

Haemophilus parasuis is known as a commensal organism discovered in the upper respiratory tract of swine where the pathogenic bacteria survive in various adverse environmental stress. QseC, a histidine protein kinase of the two-component regulatory systems CheY/QseC, is involved in the environmental adaptation in bacteria. To investigate the role of QseC in coping with the adverse environment stresses and survive in the host, we constructed a qseC mutant of H. parasuis serovar 13 strain (ΔqseC), MY1902. In this study, we found that QseC was involved in stress tolerance of H. parasuis, by the ΔqseC exhibited a decreased resistance to osmotic pressure, oxidative stress, and heat shock. Moreover, the ΔqseC weakened the ability to take up iron and biofilm formation. We also found that the QseC participate in sensing the epinephrine in environment to regulate the density of H. parasuis.

OxyR of Haemophilus parasuis is a global transcriptional regulator important in oxidative stress resistance and growth.

Haemophilus parasuis is an opportunistic pathogen and the causative agent of Glässer's disease in swine. This disease has high morbidity and mortality rates in swine populations, and is responsible for major economic losses worldwide. Survival of H. parasuis within the host requires mechanisms for coping with oxidative stress conditions. In many bacteria, OxyR is known to mediate protection against oxidative stress; however, little is known about the role of OxyR in H. parasuis. In the current study, an oxyR mutant strain was constructed in H. parasuis strain SC1401 and designated H. parasuis SC1401∆oxyR. The oxyR mutant strain had a slower growth rate and impaired biofilm formation compared to the wild type strain. Complementation restored the growth-associated phenotypes to wild type levels. Oxidative stress susceptibility testing, using a range of concentrations of H2O2, indicated that H. parasuis SC1401∆oxyR was more sensitive to oxidative stress than the wild type strain. RNA sequencing transcriptome analysis comparing H. parasuis SC1401 with H. parasuis SC1401∆oxyR identified 466 differentially expressed genes. These genes were involved in a wide range of biological processes, including: oxidative stress, transcriptional regulation, and DNA replication, recombination, and repair. These findings provide a foundation for future research to examine the role of OxyR as a global transcriptional regulator and to better define its role in oxidative stress resistance in H. parasuis.

Effect of cheY deletion on growth and colonization in a Haemophilus parasuis serovar 13 clinical strain EP3.

CheY, a response regulator of controlling the bacterial chemotactic swimming, can be modulated by either phosphorylation or acetylation to generate clockwise rotation of the flagella. Here, we researched the biological characteristics of cheY deletion mutant in Haemophilus parasuis, and found that the growth rate of this mutant was significantly slower compared with serovar 13 wild strain EP3. Additionally, the cheY mutant didn't show obvious sensitivity to porcine sera. The results of biofilm formation assay showed that H. parasuis cheY mutant produced less biofilm mass compared with wild strain. The H. parasuis cheY mutant reduced autoagglutination obviously. These findings were vital for revealing the function of cheY in growth, biofilm formation and autoagglutination. Thus, cheY plays a crucial role in growth and colonization in vivo of H. parasuis.

Immunogenicity of the recombinant HxuCBA proteins encoded by hxuCBA gene cluster of Haemophilus parasuis in mice.

Haemophilus parasuis causes serious economic losses in pigs, which is the etiology of Glässer's disease. In this study we studied the immunogenicity of proteins encoded by the hxuCBA gene cluster in H. parasuis. Through bioinformatics analysis, HxuC, HxuB, and HxuA proteins were found that they might have strong antigenicity, with 31 putative cytotoxic T-lymphocyte (CTL) epitopes and multiple B-cell antigenic determinants. Western blotting assay indicated that all these antigens are highly immunogenic. The antibody levels and the levels of IL-2, IL-4, IFN-γ in the groups of HxuA, HxuB, HxuC, HxuCBA (include HxuC, HxuB and HxuA proteins), and M-3 were observed to significantly increase with time post vaccination. HxuC, HxuB, HxuCBA and H. parasuis M-3 vaccinated groups showed a strong immune response and protection against challenge with 6.5×10(9)cfu (5×LD50) of H. parasuis M-3 strain in a mouse model, but HxuA group showed only a low level protection. Additionally, the immune response induced by all of the proteins reduced histopathological lesions and their antisera could inhibit the growth of H. parasuis. We concluded that HxuC, HxuB and HxuCBA may have potential for use as a recombinant subunit vaccine against H. parasuis challenge.

The arcA gene contributes to the serum resistance and virulence of Haemophilus parasuis serovar 13 clinical strain EP3.

As a global transcriptional factor, ArcA regulates the expression of hundreds of genes involved in aerobic and anaerobic metabolism. Here we deleted arcA gene and investigated the biological characteristics of arcA deletion mutant (ΔarcA) in Haemophilus parasuis (H. parasuis) serovar 13 clinical strain EP3. Results indicated that deletion of arcA impaired growth of EP3 strain under anaerobic condition, and reduced virulence of EP3 strain in mice. Additionally, the ΔarcA strain showed greater sensitivity in porcine serum and produced less biofilm mass than the EP3 strain. Taken together, these findings suggested that the arcA gene may be involved in pathogenesis in Haemophilus parasuis.