The molecular diversity of transcriptional factor TfoX is a determinant in natural transformation in Glaesserella parasuis

Natural transformation is a mechanism by which a particular bacterial species takes up foreign DNA and integrates it into its genome. The swine pathogen Glaesserella parasuis (G. parasuis) is a naturally transformable bacterium. The regulation of competence, however, is not fully understood. In this study, the natural transformability of 99 strains was investigated. Only 44% of the strains were transformable under laboratory conditions. Through a high-resolution melting curve and phylogenetic analysis, we found that genetic differences in the core regulator of natural transformation, the tfoX gene, leads to two distinct natural transformation phenotypes. In the absence of the tfoX gene, the highly transformable strain SC1401 lost its natural transformability. In addition, when the SC1401 tfoX gene was replaced by the tfoX of SH0165, which has no natural transformability, competence was also lost. These results suggest that TfoX is a core regulator of natural transformation in G. parasuis, and that differences in tfoX can be used as a molecular indicator of natural transformability. Transcriptomic and proteomic analyses of the SC1401 wildtype strain, and a tfoX gene deletion strain showed that differential gene expression and protein synthesis is mainly centered on pathways related to glucose metabolism. The results suggest that tfoX may mediate natural transformation by regulating the metabolism of carbon sources. Our study provides evidence that tfoX plays an important role in the natural transformation of G. parasuis

Fatigue characteristics of sand-cast AZ91D magnesium alloy

The fatigue characteristics of the AZ91D-T6 alloy samples taken from engine blocks have been investigated at 20 °C and elevated temperature (150 °C). The fatigue strength and cyclic stress amplitude of the alloy significantly decrease with the increase of the test temperature, although cyclic hardening occurs continuously until failure for both temperatures. With the increase of the temperature, the decreased fatigue life of the alloy tested at the same stress amplitude is mainly attributed to the decreased matrix strength and the increased hysteresis energies. Fatigue failure of the engine blocks made of AZ91D-T6 alloy is mainly controlled by casting defects. For the defect-free specimens, the crack initiation behavior is determined by the single-slip (20 °C) and by environment-assisted cyclic slip (150 °C) during fatigue, respectively. The low-cycle fatigue lives of the alloy can be predicted using the Coffin-Manson relation and Basquin laws, the three-parameter equation and the energy-based concepts, while the high-cycle fatigue lives of the alloy fitted well with the developed long crack life model and MSF life models. Keywords: Magnesium alloy, Engine blocks, Fatigue properties, Cyclic deformation behavior, Temperature, Fatigue life predictio

Sex differences in alarm response and predation risk in the fresh water snail Pomacea canaliculata

Many animals show alarm responses (ARs) to chemical cues released from predators or injured conspecifics. However, the prey often makes a trade-off between predation avoidance and reproduction, resulting in pronounced sex differences in AR and in sex-biased predation. This phenomenon has rarely been investigated in snails. The freshwater snail Pomacea canaliculata is reported to exhibit an AR to chemical cues released from predators or injured conspecifics. Here, we investigate the sex differences in AR in the snail to chemical cues released from its predator turtle Chinemys reevesii and injured conspecifics. By exposing adult females and males of equivalent size to turtles, we also evaluate the sex-biased predation in the snail. We find that females respond to chemical cues significantly more strongly than males. The predation experiment shows that more females survived than males after a week of predation. These results suggest that males may reduce their antipredator behaviour in order to increase the chance of mating, suggesting a trade-off between predation avoidance and reproduction

Metal Ion Periplasmic-Binding Protein YfeA of <i>Glaesserella parasuis</i> Induces the Secretion of Pro-Inflammatory Cytokines of Macrophages via MAPK and NF-κB Signaling through TLR2 and TLR4

The YfeA gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system Yfe, encodes the substrate-binding subunit of the iron, zinc, and manganese transport system in bacteria. As a potential vaccine candidate in Glaesserella parasuis, the functional mechanisms of YfeA in the infection process remain obscure. In this study, vaccination with YfeA effectively protected the C56BL6 mouse against the G. parasuis SC1401 challenge. Bioinformatics analysis suggests that YfeA is highly conserved in G. parasuis, and its metal-binding sites have been strictly conserved throughout evolution. Stimulation of RAW 264.7 macrophages with YfeA verified that toll-like receptors (TLR) 2 and 4 participated in the positive transcription and expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. The activation of TLR2 and TLR4 utilized the MyD88/MAL and TRIF/TRAM pairs to initiate TLRs signaling. Furthermore, YfeA was shown to stimulate nuclear translocation of NF-κB and activated diverse mitogen-activated protein (MAP) kinase signaling cascades, which are specific to the secretion of particular cytokine(s) in murine macrophages. Separate blocking TLR2, TLR4, MAPK, and RelA (p65) pathways significantly decreased YfeA-induced pro-inflammatory cytokine production. In addition, YfeA-stimulated RAW 264.7 produces the pro-inflammatory hallmark, reactive oxygen species (ROS). In conclusion, our findings indicate that YfeA is a novel pro-inflammatory mediator in G. parasuis and induces TLR2 and TLR4-dependent pro-inflammatory activity in RAW 264.7 macrophages through P38, JNK-MAPK, and NF-κB signaling pathways

A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformation

Haemophilus parasuis is a member of the family Pasteurellaceae and a major causative agent of Glässer’s disease. This bacterium is normally a benign swine commensal but may become a deadly pathogen upon penetration into multiple tissues, contributing to severe lesions in swine. We have established a successive natural transformation-based markerless mutation system in this species. However, the two-step mutation system requires screening of natural competent cells, and cannot delete genes which regulate natural competence per se. In this study, we successfully obtained streptomycin-resistant derivatives from H. parasuis wild type strain SC1401 by using ethyl methane sulfonate (EMS, CH3SO2OC2H5). Upon sequencing and site-directed mutations, we uncovered that the EMS-induced point mutation in rpsL at codon 43rd (AAA → AGA; K43R) or at 88th (AAA → AGA; K88R) confers a much higher streptomycin resistance than clinical isolates. We have applied the streptomycin resistance marker as a positive selection marker to perform homologous recombination through conjugation and successfully generated a double unmarked in-frame targeted mutant 1401D88△tfox△arcA. Combined with a natural transformation-based knockout system and this genetic technique, multiple deletion mutants or attenuated strains of H. parasuis can be easily constructed. Moreover, the mutant genetic marker rpsL and streptomycin resistant phenotypes can serve as an effective tool to select naturally competent strains, and to verify natural transformation quantitatively

Characterization, phylogenetic analysis, and pathogenicity of a novel genotype 2 porcine Enterovirus G

Enterovirus G belongs to the family Picornaviridae and are associated with a variety of animal diseases. We isolated and characterized a novel EV-G2 strain, CHN-SCMY2021, the first genotype 2 strain isolated in China. CHN-SCMY2021 is about 25 nm diameter with morphology typical of picornaviruses and its genome is 7341 nucleotides. Sequence alignment and phylogenetic analysis based on VP1 indicated that this isolate is a genotype 2 strain. The whole genome similarity between CHN-SCMY2021 and other EV-G genotype 2 strains is 78.3–86.4%, the greatest similarity is to EVG/Porcine/JPN/Iba26–506/2014/G2 (LC316792.1). Recombination analysis indicated that CHN-SCMY2021 resulted from recombination between 714,171/CaoLanh_VN (KT265894.2) and LP 54 (AF363455.1). Except for ST cells, CHN-SCMY2021 has a broad spectrum of cellular adaptations, which are susceptible to BHK-21, PK-15, IPEC-J2, LLC-PK and Vero cells. In piglets, CHN-SCMY2021 causes mild diarrhea and thinning of the intestinal wall. The virus was mainly distributed to intestinal tissue but was also found in heart, liver, spleen, lung, kidney, brain, and spinal cord. CHN-SCMY2021 is the first systematically characterized EV-G genotype 2 strain from China, our results enrich the information on the epidemiology, molecular evolution and pathogenicity associated with EV-G

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

Establishment of a Successive Markerless Mutation System in Haemophilus parasuis through Natural Transformation.

Haemophilus parasuis, belonging to the family Pasteurellaceae, is the causative agent of Glässer's disease leading to serious economic losses. In this study, a successive markerless mutation system for H. parasuis using two sequential steps of natural transformation was developed. By the first homologous recombination, the target genes were replaced by a cassette carrying kanamycin resistance gene and sacB (which confers sensitivity to sucrose) gene using kanamycin selection, followed by the second reconstruction to remove the selection cassette, with application of sucrose to further screen unmarked mutants. To improve DNA transformation frequency, several parameters have been analyzed further in this work. With this method, two unmarked deletions in one strain have been generated successfully. It is demonstrated that this system can be employed to construct multi-gene scarless deletions, which is of great help for developing live attenuated vaccines for H. parasuis

Polyamine Transport Protein PotD Protects Mice against Haemophilus parasuis and Elevates the Secretion of Pro-Inflammatory Cytokines of Macrophage via JNK–MAPK and NF–κB Signal Pathways through TLR4

The potD gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system. In this study, we found PotD in Haemophilus (Glaesserella) parasuis could actively stimulate both humoral immune and cellular immune responses and elevate lymphocyte proliferation, thus eliciting a Th1-type immune response in a murine immunity and infection model. Stimulation of Raw 264.7 macrophages with PotD validated that Toll-like receptor 4, rather than 2, participated in the positive transcription and expression of pro-inflammatory cytokines IL&ndash;1&beta;, IL&ndash;6, and TNF&ndash;&alpha; using qPCR and ELISA. Blocking signal-regulated JNK&ndash;MAPK and RelA(p65) pathways significantly decreased PotD-induced pro-inflammatory cytokine production. Overall, we conclude that vaccination of PotD could induce both humoral and cellular immune responses and provide immunoprotection against H. parasuis challenge. The data also suggest that Glaesserella PotD is a novel pro-inflammatory mediator and induces TLR4-dependent pro-inflammatory activity in Raw 264.7 macrophages through JNK&ndash;MAPK and RelA(p65) pathways