Pan-Genome Analysis of Vibrio cholerae and Vibrio metschnikovii Strains Isolated From Migratory Birds at Dali Nouer Lake in Chifeng, China.

Migratory birds are recently recognized as Vibrio disease vectors, but may be widespread transporters of Vibrio strains. We isolated Vibrio cholerae (V. cholerae) and Vibrio metschnikovii (V. metschnikovii) strains from migratory bird epidemic samples from 2017 to 2018 and isolated V. metschnikovii from migratory bird feces in 2019 from bird samples taken from the Inner Mongolia autonomous region of China. To investigate the evolution of these two Vibrio species, we sequenced the genomes of 40 V. cholerae strains and 34 V. metschnikovii strains isolated from the bird samples and compared these genomes with reference strain genomes. The pan-genome of all V. cholerae and V. metschnikovii genomes was large, with strains exhibiting considerable individual differences. A total of 2,130 and 1,352 core genes were identified in the V. cholerae and V. metschnikovii genomes, respectively, while dispensable genes accounted for 16,180 and 9,178 of all genes for the two strains, respectively. All V. cholerae strains isolated from the migratory birds that encoded T6SS and hlyA were non-O1/O139 serotypes without the ability to produce CTX. These strains also lacked the ability to produce the TCP fimbriae nor the extracellular matrix protein RbmA and could not metabolize trimetlylamine oxide (TMAO). Thus, these characteristics render them unlikely to be pandemic-inducing strains. However, a V. metschnikovii isolate encoding the complete T6SS system was isolated for the first time. These data provide new molecular insights into the diversity of V. cholerae and V. metschnikovii isolates recovered from migratory birds.

AMP-activated protein kinase negatively regulates heat treatment-induced lactate secretion in cultured boar sertoli cells.

Lactate secreted by Sertoli cells plays an important role in spermatogenesis. Heat stress changes AMP-activated protein kinase (AMPK) activity in many tissues and cells, and enhances lactate secretion in Sertoli cells. However, it is unclear whether heat stress affects lactate secretion by regulating the phosphorylation level of AMPK in boar immature Sertoli cells. In this study, immature boar Sertoli cells were treated at 43 °C for 30 min in an incubator. From 0 to 48 h post-heat stress, lactate secretion was enhanced and reached the maximum level (175% of the control) at 12 h. However, with increased recovery time, the phosphorylation level of AMPK decreased gradually, and reached the minimum level (58% of the control) at 12 h. Compared with heat treatment alone, pretreatment with the AMPK agonist AICAR (2 mmol/L, 2 h) reduced lactate secretion by 42.6%. Additionally, AICAR significantly decreased the lactate dehydrogenase (LDH) activity, and the mRNA and protein expression levels of GLUT3, LDHA, and MCT1. In addition, AMPK overexpression reduced lactate secretion by 22.5%, significantly decreased the LDH activity, and mRNA and protein expression levels of GLUT3, LDHA, and MCT1. These results showed that AMPK reduces heat-induced lactate secretion by decreasing the expression levels of GLUT3, LDHA and MCT1. The results also suggested that AMPK is a negative regulator of heat treatment-induced lactate secretion in cultured boar Sertoli cells.

ERK1/2 regulates heat stress-induced lactate production via enhancing the expression of HSP70 in immature boar Sertoli cells.

Lactate produced by Sertoli cells plays an important role in spermatogenesis, and heat stress induces lactate production in immature boar Sertoli cells. Extracellular signaling regulated kinase 1 and 2 (ERK1/2) participates in heat stress response. However, the effect of ERK1/2 on heat stress-induced lactate production is unclear. In the present study, Sertoli cells were isolated from immature boar testis and cultured at 32 °C. Heat stress was induced in a 43 °C incubator for 30 min. Proteins and RNAs were detected by western blotting and RT-PCR, respectively. Lactate production and lactate dehydrogenase (LDH) activity were detected using commercial kits. Heat stress promoted ERK1/2 phosphorylation, showing a reducing trend with increasing recovery time. In addition, heat stress increased heat shock protein 70 (HSP70), glucose transporter 3 (GLUT3), and lactate dehydrogenase A (LDHA) expressions, enhanced LDH activity and lactate production at 2-h post-heat stress. Pretreatment with U0126 (1 × 10-6 mol/L), a highly selective inhibitor of ERK1/2 phosphorylation, reduced HSP70, GLUT3, and LDHA expressions and decreased LDH activity and lactate production. Meanwhile, ERK2 siRNA1 reduced the mRNA level of ERK2 and weakened ERK1/2 phosphorylation. Additionally, ERK2 siRNA1 reduced HSP70, GLUT3, and LHDA expressions decreased LDH activity and lactate production. Furthermore, HSP70 siRNA3 downregulated GLUT3 and LDHA expressions and decreased LDH activity and lactate production. These results show that activated ERK1/2 increases heat stress-induced lactate production by enhancing HSP70 expression to promote the expressions of molecules related to lactate production (GLUT3 and LDHA). Our study reveals a new insight in reducing the negative effect of heat stress in boars.