Tandem Mass Tag-Based Quantitative Proteomics and Virulence Phenotype of Hemolymph-Treated Bacillus thuringiensis kurstaki Cells Reveal New Insights on Bacterial Pathogenesis in Insects.

The spore-forming bacterium Bacillus thuringiensis (Bt) of the Bacillus cereus group uses toxin-opened breaches at the insect midgut epithelium to infest the hemolymph, where it can rapidly propagate despite antimicrobial host defenses and induce host death by acute septicemia. The response of Bt to host hemolymph and the latter's role in bacterial pathogenesis is an area that needs clarification. Here, we report a proteomic analysis of the Bt kurstaki strain HD73 (Btk) hemolymph stimulon showing significant changes in 60 (34 up- and 26 downregulated) differentially accumulated proteins (DAPs). Gene ontology (GO) enrichment analysis revealed that DAPs were mainly related to glutamate metabolism, transketolase activity, and ATP-dependent transmembrane transport. KEGG analysis disclosed that DAPs were highly enriched in the biosynthesis of bacterial secondary metabolites, ansamycins. Interestingly, about 30% of all DAPs were in silico predicted as putative virulence factors. Further characterization of hemolymph effects on Btk showed enhanced autoaggregation in liquid cultures and biofilm formation in microtiter polystyrene plates. Hemolymph-exposed Btk cells were less immunogenic in mice, suggesting epitope masking of selected surface proteins. Bioassays with intrahemocoelically infected Bombyx mori larvae showed that hemolymph preexposure significantly increased Btk toxicity and reproduction within the insect (spore count per cadaver) at low inoculum doses, possibly due to 'virulence priming'. Collectively, our findings suggest that the Btk hemolymph stimulon could be partially responsible for bacterial survival and propagation within the hemolymph of infected insects, contributing to its remarkable success as an entomopathogen. All mass spectrometry data are available via ProteomeXchange with identifier PXD021830. IMPORTANCE After ingestion by a susceptible insect and damaging its midgut epithelium, the bacterium Bacillus thuringiensis (Bt) reaches the insect blood (hemolymph), where it propagates despite the host's antimicrobial defenses and induces insect death by acute septicemia. Although the hemolymph stage of the Bt toxic pathway is determinant for the infested insects' fate, the response of Bt to hemolymph and the latter's role in bacterial pathogenesis has been poorly explored. In this study, we identified the bacterial proteins differentially expressed by Bt after hemolymph exposure. We found that about 30% of hemolymph-regulated Bt proteins were potential virulence factors, including manganese superoxide dismutase, a described inhibitor of hemocyte respiratory burst. Additionally, contact with hemolymph enhanced Bt virulence phenotypes, such as cell aggregation and biofilm formation, altered bacterial immunogenicity, and increased Bt toxicity to intrahemocoelically injected insects.

Characterization of two newly emerged torque teno sus virus isolates from a large-scale pig farm in China, in 2018.

Torque teno sus virus (TTSuV) infection is common in China's pig herd. Although of uncertain pathogenicity, TTSuVs have been reported as a worsening factor of other porcine diseases, including porcine circovirus associated disease (PCVAD), porcine respiratory diseases complex (PRDC) or porcine dermatitis and nephropathy syndrome (PDNS). To better understand the genetic diversity in TTSuVs, the complete genomes of two newly emerged isolates, referred to as HeN1-A9 and HeN1-A11, collected from pig samples at a large-scale pig farm in China, were analyzed. Phylogenetic relationships of TTSuV sequences separated TTSuV1 and TTSuVk2a groups and divided TTSuV1 into two major subtypes, including TTSuV1a and TTSuV1b; HeN1-A9 and HeN1-A11 strains classified into the TTSuV1a subtype. Recombination analysis demonstrated HeN1-A9 and HeN1-A11 were generated via recombination in the overlapping ORF1/ORF3 region of TTSuV1a genome, which we report for the first time. Furthermore, we found that HeN1-A9 could be replicated in cultured MARC-145 cells for 18 passages. Our findings may be useful for elucidating the characteristics and epidemic status of TTSuVs in China.

Investigation of the Selectivity of L-Type Voltage-Gated Calcium Channels 1.3 for Pyrimidine-2,4,6-Triones Derivatives Based on Molecular Dynamics Simulation.

Human Cav1.3 (hCav1.3) is of great interest as a potential target for Parkinson's disease. However, common medications like dihydropyridines (DHPs), a kind of classic calcium channel blocker, have poor selectivity to hCav1.3 in clinical treatment, mainly due to being implicated in cardiovascular side-effects mediated by human Cav1.2 (hCav1.2). Recently, pyrimidine-2,4,6-triones (PYTs) have received extensive attention as prominent selective inhibitors to hCav1.3. In this study, we describe the selectivity mechanism of PYTs for hCav1.2 and hCav1.3 based on molecular dynamic simulation methods. Our results reveal that the van der Waals (vdW) interaction was the most important force affecting selectivity. Moreover, the hydrophobic interaction was more conducive to the combination. The highly hydrophobic amino acid residues on hCav1.3, such as V162 (IR1), L303 (IR2), M481 (IR3), and F484 (IR3), provided the greatest contributions in the binding free energy. On the other hand, the substituents of a halogen-substituted aromatic ring, cycloalkyl and norbornyl on PYTs, which are pertinent to the steric hindrance of the compounds, played core roles in the selectivity and affinity for hCav1.3, whereas strong polar substituents needed to be avoided. The findings could provide valuable information for designing more effective and safe medicines for Parkinson's disease.

Hypermutations in porcine respiratory and reproductive syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS), which is caused by the PRRS virus (PRRSV), has resulted in large economic losses for the swine industry. The virus has shown remarkable genetic diversity since its discovery. In our study, we investigated mutation types in the evolution of PRRSV for both in vivo and in vitro passaging of the virus. Sequence alignment analysis demonstrated that the most common hypermutations expressed were A→G/U→C and G→A/C→U. The data provide a new theoretical basis for PRRSV evolution.

Le syndrome reproducteur et respiratoire porcin (SRRP), qui est causé par le virus SRRP (VSRRP), a entrainé de grosses pertes économiques à l'industrie porcine. Le virus a démontré une remarquable diversité génétique depuis sa mise en évidence. Dans notre étude, nous avons examiné les types de mutation dans l'évolution du VSRRP lors du passage in vivo et in vitro du virus. L'analyse d'alignement des séquences a démontré que les hypermutations les plus fréquemment exprimées étaient A→G/U→C et G→A/C→U. Ces données fournissent une nouvelle base théorique pour l'évolution du VSRRP.(Traduit par Docteur Serge Messier).

Characterization of newly emerged NADC30-like strains of porcine reproductive and respiratory syndrome virus in China.

Different strains of porcine reproductive and respiratory syndrome virus (PRRSV) have emerged and circulated in different regions of mainland China since 1996, particularly after 2006. In 2012, NADC30-like PRRSV was first isolated in Henan Province. By 2016, it had spread to most provinces in China. In the present study, the whole genomes (excluding the poly(A) tails) of 13 newly emerged NADC30-like PRRSV strains were sequenced and analyzed. Furthermore, the pathogenicity of SD53-1603, one of the 13 PRRSV strains, was assessed. Phylogenetic analysis showed that these 13 newly emerged NADC30-like PRRSV strains, together with some reference strains, formed a new subgroup (subgroup 5), characterized by a predicted 131-amino-acid deletion in the nonstructural protein (NSP) 2. However, low levels of whole-genome similarity and a wide variety of recombination patterns complicated the classification of the NADC30-like PRRSV isolates. Interestingly, almost all of the recombination breakpoints found in these 13 PRRSV isolates and other NADC30-like PRRSV isolates occurred in genes encoding NSPs and/or minor structural proteins. In addition, piglets infected with the newly emerged NADC30-like strain SD53-1603 displayed clear clinical respiratory symptoms and underwent typical pathological changes. The findings may be useful for elucidating the characteristics and epidemic status of NADC30-like PRRSV in China.