AhR ligands from LGG metabolites promote piglet intestinal ILC3 activation and IL-22 secretion to inhibit PEDV infection.

In maintaining organismal homeostasis, gut immunity plays a crucial role. The coordination between the microbiota and the immune system through bidirectional interactions regulates the impact of microorganisms on the host. Our research focused on understanding the relationships between substantial changes in jejunal intestinal flora and metabolites and intestinal immunity during porcine epidemic diarrhea virus (PEDV) infection in piglets. We discovered that Lactobacillus rhamnosus GG (LGG) could effectively prevent PEDV infection in piglets. Further investigation revealed that LGG metabolites interact with type 3 innate lymphoid cells (ILC3s) in the jejunum of piglets through the aryl hydrocarbon receptor (AhR). This interaction promotes the activation of ILC3s and the production of interleukin-22 (IL-22). Subsequently, IL-22 facilitates the proliferation of IPEC-J2 cells and activates the STAT3 signaling pathway, thereby preventing PEDV infection. Moreover, the AhR receptor influences various cell types within organoids, including intestinal stem cells (ISCs), Paneth cells, and enterocytes, to promote their growth and development, suggesting that AhR has a broad impact on intestinal health. In conclusion, our study demonstrated the ability of LGG to modulate intestinal immunity and effectively prevent PEDV infection in piglets. These findings highlight the potential application of LGG as a preventive measure against viral infections in livestock.IMPORTANCEWe observed high expression of the AhR receptor on pig and human ILC3s, although its expression was negligible in mouse ILC3s. ILC3s are closely related to the gut microbiota, particularly the secretion of IL-22 stimulated by microbial signals, which plays a crucial regulatory role in intestinal immunity. In our study, we found that metabolites produced by beneficial gut bacteria interact with ILC3s through AhR, thereby maintaining intestinal immune homeostasis in pigs. Moreover, LGG feeding can enhance the activation of ILC3s and promote IL-22 secretion in the intestines of piglets, ultimately preventing PEDV infection.

Lycium barbarum polysaccharide promotes the immunoprotective effects of a recombinant Lactobacillus plantarum vaccine expressing the Trichinella spiralis cathepsin F-like protease 1 gene.

Trichinellosis caused by Trichinella spiralis (T. spiralis) is a zoonotic disease that poses a substantial risk to human health. At present, vaccines used to prevent trichinellosis are effective, but the production of antibody levels and immunogenicity are low. Adjuvants can increase antibody levels and vaccine immunogenicity. As a result, it is critical to develop an effective adjuvant for the T. spiralis vaccine. Recent research has shown that traditional Chinese medicine polysaccharides with low-toxicity and biodegradability can act as adjuvants in vaccines. In this study, BALB/c mice were orally inoculated with a recombinant Lactobacillus plantarum (L. plantarum) vaccine expressing the T. spiralis cathepsin F-like protease 1 gene (rTs-CPF1), which was given three times at 10-day intervals. Lycium barbarum polysaccharide (LBP) was administered orally for 37 days. At 37 days after the first immunization, mice were infected with 350 T. spiralis muscle larvae (ML). Specific IgG and sIgA antibody levels against the T. spiralis CPF1 protein were increased in mice immunized with rTs-CPF1+LBP compared to those immunized with rTs-CPF1 alone. Furthermore, LBP increased IFN-γ and IL-4 expression levels, and the number of intestinal and intramuscular worms was significantly reduced in the rTs-CPF1+LBP group compared to that in the rTs-CPF1 group. In the rTs-CPF1+LBP group, the reduction rates of adult worms and muscle larvae were 47.31 % and 68.88 %, respectively. To summarize, LBP promotes the immunoprotective effects of the T. spiralis vaccine and may be considered as a novel adjuvant in parasitic vaccines.

VIM-encoding IncpSTY plasmids and chromosome-borne integrative and mobilizable elements (IMEs) and integrative and conjugative elements (ICEs) in Pseudomonas.

BACKGROUND: The carbapenem-resistance genes blaVIM are widely disseminated in Pseudomonas, and frequently harbored within class 1 integrons that reside within various mobile genetic elements (MGEs). However, there are few reports on detailed genetic dissection of blaVIM-carrying MGEs in Pseudomonas. METHODS: This study presented the complete sequences of five blaVIM-2/-4-carrying MGEs, including two plasmids, two chromosomal integrative and mobilizable elements (IMEs), and one chromosomal integrative and conjugative element (ICE) from five different Pseudomonas isolates. RESULTS: The two plasmids were assigned to a novel incompatibility (Inc) group IncpSTY, which included only seven available plasmids with determined complete sequences and could be further divided into three subgroups IncpSTY-1/2/3. A detailed sequence comparison was then applied to a collection of 15 MGEs belonging to four different groups: three representative IncpSTY plasmids, two Tn6916-related IMEs, two Tn6918-related IMEs, and eight Tn6417-related ICEs and ten of these 15 MGEs were first time identified. At least 22 genes involving resistance to seven different categories of antibiotics and heavy metals were identified within these 15 MGEs, and most of these resistance genes were located within the accessory modules integrated as exogenous DNA regions into these MGEs. Especially, eleven of these 15 MGEs carried the blaVIM genes, which were located within 11 different concise class 1 integrons. CONCLUSION: These blaVIM-carrying integrons were further integrated into the above plasmids, IMEs/ICEs with intercellular mobility. These MGEs could transfer between Pseudomonas isolates, which resulted in the accumulation and spread of blaVIM among Pseudomonas and thus was helpful for the bacteria to survival from the stress of antibiotics. Data presented here provided a deeper insight into the genetic diversification and evolution of VIM-encoding MGEs in Pseudomonas.

Comparative genomics of Tn6411 transposons carrying the blaIMP-1 gene in Pseudomonas aeruginosa.

We aimed to determine the molecular characteristics of carbapenem-resistant Pseudomonas aeruginosa strains 18081308 and 18083286, which were isolated from the urine and the sputum of two Chinese patients, respectively. Additionally, we conducted a comparative analysis between Tn6411 carrying blaIMP-1 in strain 18083286 and transposons from the same family available in GenBank. Bacterial genome sequencing was carried out on strains 18081308 and 18083286 to obtain their whole genome sequence. Average nucleotide identity (ANI) was used for their precise species identification. Serotyping and multilocus sequence typing were performed. Furthermore, the acquired drug resistance genes of these strains were identified. The carbapenem-resistant P. aeruginosa strains isolated in the present study were of sequence type ST865 and serotype O6. They all carried the same resistance genes (aacC2, tmrB, and blaIMP-1). Tn6411, a Tn7-like transposon carrying blaIMP-1, was found in strain 18083286 by single molecule real time (SMRT) sequencing. We also identified the presence of this transposon sequence in other chromosomes of P. aeruginosa and plasmids carried by Acinetobacter spp. in GenBank, indicating the necessity for heightening attention to the potential transferability of this transposon.

The E3 ligase ASB3 downregulates antiviral innate immunity by targeting MAVS for ubiquitin-proteasomal degradation.

E3 ubiquitin ligases are very important for regulating antiviral immunity during viral infection. Here, we discovered that Ankyrin repeat and SOCS box-containing protein 3 (ASB3), an E3 ligase, are upregulated in the presence of RNA viruses, particularly influenza A virus (IAV). Notably, overexpression of ASB3 inhibits type I IFN (IFN-I) responses induced by Sendai virus (SeV) and IAV, and ablation of ASB3 restores SeV and H9N2 infection-mediated transcription of IFN-ß and its downstream interferon-stimulated genes (ISGs). Interestingly, animals lacking ASB3 presented decreased susceptibility to H9N2 and H1N1 infections. Mechanistically, ASB3 interacts with MAVS and directly mediates K48-linked polyubiquitination and degradation of MAVS at K297, thereby inhibiting the phosphorylation of TBK1 and IRF3 and downregulating downstream antiviral signaling. These findings establish ASB3 as a critical negative regulator that controls the activation of antiviral signaling and describe a novel function of ASB3 that has not been previously reported.

A Wohlfahrtiimonas chitiniclastica with a novel type of blaVEB-1-carrying plasmid isolated from a zebra in China.

Background: Wohlfahrtiimonas chitiniclastica is an emerging fly-borne zoonotic pathogen, which causes infections in immunocompromised patients and some animals. Herein, we reported a W. chitiniclastica BM-Y from a dead zebra in China. Methods: The complete genome sequencing of BM-Y showed that this isolate carried one chromosome and one novel type of blaVEB-1-carrying plasmid. Detailed genetic dissection was applied to this plasmid to display the genetic environment of blaVEB-1. Results: Three novel insertion sequence (IS) elements, namely ISWoch1, ISWoch2, and ISWoch3, were found in this plasmid. aadB, aacA1, and gcuG were located downstream of blaVEB-1, composing a gene cassette array blaVEB-1-aadB-aacA1-gcuG bracketed by an intact ISWoch1 and a truncated one, which was named the blaVEB-1 region. The 5'-RACE experiments revealed that the transcription start site of the blaVEB-1 region was located in the intact ISWoch1 and this IS provided a strong promoter for the blaVEB-1 region. Conclusion: The spread of the blaVEB-1-carrying plasmid might enhance the ability of W. chitiniclastica to survive under drug selection pressure and aggravate the difficulty in treating infections caused by blaVEB-1-carrying W. chitiniclastica. To the best of our knowledge, this is the first report of the genetic characterization of a novel blaVEB-1-carrying plasmid with new ISs from W. chitiniclastica.

Vibrio parahaemolyticus from Migratory Birds in China Carries an Extra Copy of tRNA-Gly and Plasmid-Mediated Quinolone Resistance Gene qnrD.

Vibrio parahaemolyticus is a marine bacterium coming from estuarine environments, where the migratory birds can easily be colonized by V. parahaemolyticus. Migratory birds may be important reservoirs of V. parahaemolyticus by growth and re-entry into the environment. To further explore the spreading mechanism of V. parahaemolyticus among marine life, human beings, and migratory birds, we aimed to investigate the characteristics of the genetic diversity, antimicrobial resistance, virulence genes, and a potentially informative gene marker of V. parahaemolyticus isolated from migratory birds in China. This study recovered 124 (14.55%) V. parahaemolyticus isolates from 852 fecal and environmental (water) samples. All of the 124 strains were classified into 85 known sequence types (STs), of which ST-2738 was most frequently identified. Analysis of the population structure using whole-genome variation of the 124 isolates illustrated that they grouped into 27 different clonal groups (CGs) belonging to the previously defined geographical populations VppX and VppAsia. Even though these genomes have high diversity, an extra copy of tRNA-Gly was presented in all migratory bird-carried V. parahaemolyticus isolates, which could be used as a potentially informative marker of the V. parahaemolyticus strains derived from birds. Antibiotic sensitivity experiments revealed that 47 (37.10%) isolates were resistant to ampicillin. Five isolates harbored the plasmid-mediated quinolone resistance (PMQR) gene qnrD, which has not previously been identified in this species. The investigation of antibiotic resistance provides the basic knowledge to further evaluate the risk of enrichment and reintroduction of pathogenic V. parahaemolyticus strains in migratory birds. IMPORTANCE The presence of V. parahaemolyticus in migratory birds' fecal samples implies that the human pathogenic V. parahaemolyticus strains may also potentially infect birds and thus pose a risk for zoonotic infection and food safety associated with re-entry into the environment. Our study firstly highlights the extra copy of tRNA as a potentially informative marker for identifying the bird-carried V. parahaemolyticus strains. Also, we firstly identify the plasmid-mediated quinolone resistance (PMQR) gene qnrD in V. parahaemolyticus. To further evaluate the risk of enrichment and reintroduction of pathogenic strains carried by migratory birds, we suggest conducting estuarine environmental surveillance to monitor the antibiotic resistance and virulence factors of bird-carried V. parahaemolyticus isolates.

Characteristics of Carbapenem-resistant Klebsiella pneumoniae in sewage from a tertiary hospital in Jilin Province, China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection is a serious problem in hospitals worldwide. We monitored a tertiary hospital in Changchun, Jilin Province, China, and found that CRKP was the major species among the carbapenem-resistant isolates in sewage. Subsequently, we evaluated the drug susceptibility, resistance genes, virulence genes, outer pore membrane protein-related genes (OmpK35 & OmpK 36), multi-locus sequence typing and replicons, biofilm formation capabilities, and resistance to chlorine-containing disinfectants among KP isolates. Identification of drug sensitivity, multiple resistance profiles were observed including 77 (82.80%) multidrug resistant (MDR), 16 (17.20%) extensive drug resistant (XDR). Some antibiotic resistance genes were detected, the most prevalent carbapenemase gene was blaKPC, and 16 resistance genes were associated with other antibiotics. In addition, 3 (3.23%) CRKP isolates demonstrated loss of OmpK-35 and 2 (2.15%) demonstrated loss of OmpK-36. In the detection of multi-locus sequence typing (MLST), 11 ST11 isolates carried virulence genes. The most common replicon type was IncFII. Biofilm-forming capabilities were demonstrated by 68.8% of the isolates, all of which were resistant to chlorine-containing disinfectants. The results of the study showed that antibiotic-resistant isolates, especially CRKP, could resist disinfectants in hospital wastewater, and improper treatment of hospital wastewater may lead to the spread of drug-resistant bacteria and their genes. Thus, these bacteria must be eliminated before being discharged into the municipal sewage system.

Genetic Characterization of Four Groups of Chromosome-Borne Accessory Genetic Elements Carrying Drug Resistance Genes in Providencia.

Purpose: The aim of this study was to gain a deeper genomics and bioinformatics understanding of diversification of accessory genetic elements (AGEs) in Providencia. Methods: Herein, the complete genome sequences of five Providencia isolates from China were determined, and seven AGEs were identified from the chromosomes. Detailed genetic dissection and sequence comparison were applied to these seven AGEs, together with additional 10 chromosomal ones from GenBank (nine of them came from Providencia). Results: These 17 AGEs were divided into four groups: Tn6512 and its six derivatives, Tn6872 and its two derivatives, Tn6875 and its one derivative, and Tn7 and its four derivatives. These AGEs display high-level diversification in modular structures that had complex mosaic natures, and particularly different multidrug resistance (MDR) regions were presented in these AGEs. At least 52 drug resistance genes, involved in resistance to 15 different categories of antimicrobials and heavy metal, were found in 15 of these 17 AGEs. Conclusion: Integration of these AGEs into the Providencia chromosomes would contribute to the accumulation and distribution of drug resistance genes and enhance the ability of Providencia isolates to survive under drug selection pressure.

A Genomic and Bioinformatics View of the Classification and Evolution of Morganella Species and Their Chromosomal Accessory Genetic Elements Harboring Antimicrobial Resistance Genes.

In this study, draft-genome sequencing was conducted for 60 Chinese Morganella isolates, and furthermore, 12 of them were fully sequenced. Then, a total of 166 global sequenced Morganella isolates, including the above 60, were collected to perform average nucleotide identity-based genomic classification and core single nucleotide polymorphism-based phylogenomic analysis. A genome sequence-based species classification scheme for Morganella was established, and accordingly, the two conventional Morganella species were redefined as two complexes and further divided into four and two genospecies, respectively. At least 88 acquired antimicrobial resistance genes (ARGs) were disseminated in these 166 isolates and were prevalent mostly in the isolates from hospital settings. IS26/IS15DI, IS10 and IS1R, and Tn3-, Tn21-, and Tn7-subfamily unit transposons were frequently presented in these 166 isolates. Furthermore, a detailed sequence comparison was applied to 18 Morganella chromosomal accessory genetic elements (AGEs) from the fully sequenced 12 isolates, together with 5 prototype AGEs from GenBank. These 23 AGEs were divided into eight different groups belonging to composite/unit transposons, transposable prophages, integrative and mobilizable elements, and integrative and conjugative elements, and they harbored at least 52 ARGs involved in resistance to 15 categories of antimicrobials. Eleven of these 23 AGEs acquired large accessory modules, which exhibited complex mosaic structures and contained many antimicrobial resistance loci and associated ARGs. Integration of ARG-containing AGEs into Morganella chromosomes would contribute to the accumulation and dissemination of ARGs in Morganella and enhance the adaption and survival of Morganella under complex and diverse antimicrobial selection pressures. IMPORTANCE This study presents a comprehensive genomic epidemiology analysis on global sequenced Morganella isolates. First, a genome sequence-based species classification scheme for Morganella is established with a higher resolution and accuracy than those of the conventional scheme. Second, the prevalence of accessory genetic elements (AGEs) and associated antimicrobial resistance genes (ARGs) among Morganella isolates is disclosed based on genome sequences. Finally, a detailed sequence comparison of eight groups of 23 AGEs (including 19 Morganella chromosomal AGEs) reveals that Morganella chromosomes have evolved to acquire diverse AGEs harboring different profiles of ARGs and that some of these AGEs harbor large accessory modules that exhibit complex mosaic structures and contain a large number of ARGs. Data presented here provide a deeper understanding of the classification and evolution of Morganella species and also those of ARG-containing AGEs in Morganella at the genomic scale.

First Report of the Colistin Resistance Gene mcr-10.1 Carried by IncpA1763-KPC Plasmid pSL12517-mcr10.1 in Enterobacter cloacae in Sierra Leone.

Mobile colistin resistance (mcr) gene mcr-10.1 has been distributed widely since it was initially identified in 2020. The aim of this study was to report the first mcr-10.1 in Africa and the first mcr in Sierra Leone; furthermore, we presented diverse modular structures of mcr-10.1 loci. Here, the complete sequence of one mcr-10.1-carrying plasmid in one clinical Enterobacter cloacae isolate from Sierra Leone was determined. Detailed genetic dissection and comparison were applied to this plasmid, together with a homologous plasmid carrying mcr-10.1 from GenBank. Moreover, a genetic comparison of 19 mcr-10.1 loci was performed. In this study, mcr-10.1 was carried by an IncpA1763-KPC plasmid from one Enterobacter cloacae isolate. A total of 19 mcr-10.1 loci displayed diversification in modular structures through complex transposition and homologous recombination. A site-specific tyrosine recombinase XerC was located upstream of mcr-10.1, and at least one insertion sequence element was inserted adjacent to a conserved xerC-mcr-10.1-orf336-orf177 region. Integration of mcr-10.1 into a different gene context and carried by various Inc plasmids contributed to the wide distribution of mcr-10.1 and enhanced the ability of bacteria to survive under colistin selection pressure. IMPORTANCE Colistin is used as one of the last available choices of antibiotics for patients infected by carbapenem-resistant bacterial strains, but the unrestricted use of colistin aggravated the acquisition and dissemination of mobile colistin resistance (mcr) genes. So far, 10 mcr genes have been reported in four continents around the world. This study presented one mcr-10.1-carrying Enterobacter cloacae isolate from Sierra Leone. The mcr-10.1 gene was identified on an IncpA1763-KPC plasmid. According to the results of genetic comparison of 19 mcr-10.1 loci, the mcr-10.1 gene was found to be located in a conserved xerC-mcr-10.1-orf336-orf177 region, and at least one insertion sequence element was inserted adjacent to this region. To our knowledge, this is the first report of identifying the mcr-10.1 gene in Africa and the mcr gene in Sierra Leone.

A clinical Pseudomonas juntendi strain with bla IMP-1 carried by an integrative and conjugative element in China.

Objective: To precisely determine the species of a carbapenem-resistant Pseudomonas strain 1809276 isolated from the urine of a Chinese patient and analyze its integrative and conjugative element (ICE) 1276 formation mechanism. Methods: Single-molecule real-time (SMRT) sequencing was carried out on strain 18091276 to obtain the complete chromosome and plasmid (pCN1276) sequences, and average nucleotide identity (ANI) was used for precise species identification. The ICEs in GenBank with the same integrase structure as ICE 1276 were aligned. At the same time, the transfer ability of bla IMP-1 and the antibiotic sensitivity of Pseudomonas juntendi 18091276 were tested. Results: This bacterium was P. juntendi, and its drug resistance mechanism is the capture of the accA4' gene cassette by the Tn402-like type 1 integron (IntI1-bla IMP-1) to form In1886 before its capture by the ΔTn4662a-carrying ICE 1276. The acquisition of bla IMP-1 confers carbapenem resistance to P. juntendi 18091276. Conclusion: The formation of bla IMP-1-carrying ICE 1276, its further integration into the chromosomes, and transposition and recombination of other elements promote bacterial gene accumulation and transmission.

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.

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.

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.