Jianchao Wei

Detection and new genetic environment of the pleuromutilin–lincosamide–streptogramin A resistance gene lsa(E) in methicillin-resistant Staphylococcus aureus of swine origin

OBJECTIVESnTo investigate the genetic basis of pleuromutilin resistance in porcine methicillin-resistant Staphylococcus aureus (MRSA) and to map the genetic environment of the identified plasmid-borne resistance gene.nnnMETHODSnSeventy porcine MRSA isolates, which exhibited high MICs of tiamulin, valnemulin and retapamulin, were investigated for pleuromutilin resistance genes and mutations. They were characterized by staphylococcal cassette chromosome mec (SCCmec) typing, spa typing and multilocus sequence typing (MLST). Plasmid DNA was extracted from the lsa(E)-positive strains and transferred to S. aureus RN4220 for selection of resistance plasmids. The plasmid-borne lsa(E) gene region was sequenced and 10 overlapping PCR assays for the analysis of the genetic environment of lsa(E) were developed.nnnRESULTSnAll 70 MRSA isolates were ST9 (MLST)-t899 (spa)-IVa (SCCmec). Sixteen isolates carried the lsa(E) gene; all others were negative for known pleuromutilin resistance mechanisms. An lsa(E)-carrying plasmid of ∼41 kb was detected in a single isolate. Sequence analysis revealed that the lsa(E) gene was located in a multiresistance gene cluster, which showed partial homology to clusters identified in MRSA, methicillin-susceptible S. aureus (MSSA) and Enterococcus faecalis. PCR analysis of the remaining isolates revealed a partly deleted multiresistance gene cluster in 6/15 isolates and solely the lsa(E) gene without the known flanking regions in 9/15 isolates.nnnCONCLUSIONSnWe identified the pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E) in porcine MRSA isolates. The multiresistance gene cluster in which lsa(E) was located differed from the previously described ones found in human MRSA/MSSA or in E. faecalis. The location of lsa(E) on a multiresistance plasmid facilitates its persistence and dissemination.

Nonstructural Protein 1 of Influenza A Virus Interacts with Human Guanylate-Binding Protein 1 to Antagonize Antiviral Activity

Human guanylate-binding protein 1 (hGBP1) is an interferon-inducible protein involved in the host immune response against viral infection. In response to infection by influenza A virus (IAV), hGBP1 transcript and protein were significantly upregulated. Overexpression of hGBP1 inhibited IAV replication in a dose-dependent manner in vitro. The lysine residue at position 51 (K51) of hGBP1 was essential for inhibition of IAV replication. Mutation of K51 resulted in an hGBP1 that was unable to inhibit IAV replication. The viral nonstructural protein 1 (NS1) was found to interact directly with hGBP1. K51 of hGBP1 and a region between residues 123 and 144 in NS1 were demonstrated to be essential for the interaction between NS1 and hGBP1. Binding of NS1 to hGBP1 resulted in a significant reduction in both GTPase activity and the anti-IAV activity of hGBP1. These findings indicated that hGBP1 contributed to the host immune response against IAV replication and that hGBP1-mediated antiviral activity was antagonized by NS1 via binding to hGBP1.

Stabilization of p53 in influenza A virus-infected cells is associated with compromised MDM2-mediated ubiquitination of p53.

Backgound: p53 is accumulated and activated in response to influenza virus infection. Results: p53 accumulation results from protein stabilization, which is associated with compromised Mdm2-mediated p53 ubiquitination. Viral nucleoprotein binds to p53 and impairs Mdm2-mediated p53 ubiquitination. Conclusion: p53 stabilization results from compromised Mdm2-mediated p53 ubiquitination. Significance: First time to demonstrate the mechanism of p53 stabilization in influenza virus-infected cells. Influenza A virus (IAV) induces apoptosis of infected cells. In response to IAV infection, p53, a tumor suppressor involved in regulating apoptosis and host antiviral defense, accumulates and becomes activated. This study was undertaken to examine the mechanism of p53 accumulation in IAV-infected cells. Here we show that p53 accumulation in IAV-infected cells results from protein stabilization, which was associated with compromised Mdm2-mediated ubiquitination of p53. In IAV-infected cells, p53 was stabilized and its half-life was remarkably extended. The ladders of polyubiquitinated p53 were not detectable in the presence of the proteasome inhibitor MG132 and were less sensitive to proteasome-mediated degradation. IAV infection did not affect the abundance of Mdm2, a major ubiquitin E3 ligase responsible for regulating p53 ubiquitination and degradation, but weakened the interaction between p53 and Mdm2. Viral nucleoprotein (NP) was able to increase the transcriptional activity and stability of p53. Furthermore, NP was found to associate with p53 and to impair the p53-Mdm2 interaction and Mdm2-mediated p53 ubiquitination, demonstrating its role in inhibiting Mdm2-mediated p53 ubiquitination and degradation.

Characterization of a novel small plasmid carrying the florfenicol resistance gene floR in Haemophilus parasuis

Sir, The Gram-negative bacterium Haemophilus parasuis is the causative agent of Glässer’s disease, which is characterized by various combinations of meningoencephalitis, polyserositis, polyarthritis and bacterial pneumonia, resulting in major economic losses to the swine industry worldwide. Florfenicol is used exclusively in veterinary medicine and was approved for the treatment of porcine respiratory diseases in China in 1999. So far, five florfenicol resistance genes (floR, fexA, fexB, cfr and optrA) have been reported in bacteria of animal origin. In Gram-negative bacteria, the floR gene is the most important contributor to florfenicol resistance and has been described in various species. To date, studies on the antimicrobial susceptibility of H. parasuis have been carried out in several countries (China, Denmark, the UK, Spain, the Czech Republic and Australia), which reported that all of the clinical isolates were susceptible to florfenicol. Here, we report the emergence of florfenicol-resistant H. parasuis isolates in China, which is attributable to a novel small plasmid bearing the floR gene. A total of 62 H. parasuis isolates were collected from pigs with respiratory diseases in Shanghai and Jiangsu province, China, from March 2013 to May 2014. Conventional biochemical testing, diagnostic PCR analysis and 16S rDNA sequencing were used to identify the isolates. Since there is no standard method for susceptibility testing of H. parasuis, broth microdilution was performed according to the recommendations of the CLSI (VET01-A4, 2013) for Actinobacillus pleuropneumoniae. A. pleuropneumoniae ATCC 27090 served as the quality control strain. Of the 62 H. parasuis isolates, 3 (ASB6W, ASB17W and A4) showed high florfenicol MIC values of 8 mg/L, while the remaining 59 strains exhibited low MIC values of florfenicol, ranging from 0.25 to 1 mg/L. The isolates ASB6W and ASB17W were obtained from a farm in Jiangsu province and the A4 isolate was from a farm in Shanghai. The three isolates were tentatively considered as florfenicol resistant according to the distribution of MIC values of the 62 H. parasuis isolates and the CLSI breakpoint of florfenicol for A. pleuropneumoniae. The presence of the floR gene in the three strains was confirmed by PCR with the primers floRF (5′-GCGATATTCATTACTTTGGC-3′) and floRR (5′-TAGGATGAAGG TGAGGAATG-3′) and subsequent sequencing of the amplicon. Plasmid DNA was extracted using the Qiagen Plasmid Mini Kit (Qiagen, Hilden, Germany) and profiling analysis revealed that each of the three florfenicol-resistant H. parasuis isolates harboured only a single plasmid of 6 kb. The aforementioned PCR fragment of the floR gene was labelled using the DIG High Prime DNA Labelling and Detection Kit (Roche Diagnostics, Mannheim, Germany). Southern blot analysis performed with the floR-specific probe showed that the floR gene was located on the 6 kb plasmids in the three isolates (data not shown). Subsequently, the three plasmids from the three isolates were completely sequenced by primer walking, starting with the floR-specific primers described above. Sequence comparisons revealed that the three plasmids were identical. This plasmid, designated as pHPSF1, was transformed into the plasmid-free H. parasuis isolate D20 by electrotransformation. The transformant exhibited elevated MICs of florfenicol (0.5–8 mg/L) and chloramphenicol (0.5–8 mg/L) when compared with those of the recipient strain. These results suggested that the plasmid pHPSF1 carried the floR gene and was responsible for florfenicol resistance in the three H. parasuis isolates. The plasmid pHPSF1 was 6328 bp (GenBank accession number KR262062) and consisted of five ORFs encoding the florfenicol resistance protein FloR, which consists of 404 amino acids, the 109 amino acid putative transcriptional regulator LysR, a potential Rep protein of 337 amino acids involved in plasmid replication and two Mob proteins associated with plasmid mobilization (MobC of 97 amino acids and MobA/L of 450 amino acids). The FloR protein of plasmid pHPSF1 had a typical size of 404 amino acids and 12 putative transmembrane domains, which is same as other functionally active members of the FloR family. It showed amino acid identity of 88.4% (to the FloR variant of Stenotrophomonas maltophilia, accession number AIU94575) to 97.5% (to the FloR of Escherichia coli, accession number CEL26452) with the FloR proteins in the GenBank database. Database searches identified three plasmids that showed high sequence homology with pHPSF1, including the 5486 bp plasmid p11745 carrying the tet(B) tetracycline resistance gene (GenBank accession number DQ176855) of A. pleuropneumoniae isolated from a pig in Spain, the 10 874 bp floR-carrying plasmid

Transcriptional analysis of immune-related gene expression in p53-deficient mice with increased susceptibility to influenza A virus infection

Backgroundp53 is a tumor suppressor that contributes to the host immune response against viral infections in addition to its well-established protective role against cancer development. In response to influenza A virus (IAV) infection, p53 is activated and plays an essential role in inhibiting IAV replication. As a transcription factor, p53 regulates the expression of a range of downstream responsive genes either directly or indirectly in response to viral infection. We compared the expression profiles of immune-related genes between IAV-infected wild-type p53 (p53WT) and p53-deficient (p53KO) mice to gain an insight into the basis of p53-mediated antiviral response.Methodsp53KO and p53WT mice were infected with influenza A/Puerto Rico/8/1934 (PR8) strain. Clinical symptoms and body weight changes were monitored daily. Lung specimens of IAV-infected mice were collected for analysis of virus titers and gene expression profiles. The difference in immune-related gene expression levels between IAV-infected p53KO and p53WT mice was comparatively determined using microarray analysis and confirmed by quantitative real-time reverse transcription polymerase chain reaction.Resultsp53KO mice showed an increased susceptibility to IAV infection compared to p53WT mice. Microarray analysis of gene expression profiles in the lungs of IAV-infected mice indicated that the increased susceptibility was associated with significantly changed expression levels in a range of immune-related genes in IAV-infected p53KO mice. A significantly attenuated expression of Ifng (encoding interferon (IFN)-gamma), Irf7 (encoding IFN regulator factor 7), and antiviral genes, such as Mx2 and Eif2ak2 (encoding PKR), were observed in IAV-infected p53KO mice, suggesting an impaired IFN-mediated immune response against IAV infection in the absence of p53. In addition, dysregulated expression levels of proinflammatory cytokines and chemokines, such as Ccl2 (encoding MCP-1), Cxcl9, Cxcl10 (encoding IP-10), and Tnf, were detected in IAV-infected p53KO mice during early IAV infection, reflecting an aberrant inflammatory response.ConclusionLack of p53 resulted in the impaired expression of genes involved in IFN signaling and the dysregulated expression of cytokine and chemokine genes in IAV-infected mice, suggesting an essential role of p53 in the regulation of antiviral and inflammatory responses during IAV infection.

Characterization of nonstructural protein 3 of a neurovirulent Japanese encephalitis virus strain isolated from a pig

BackgroundJapanese encephalitis virus (JEV), as a re-emerging virus that causes 10,000-15,000 human deaths from encephalitis in the world each year, has had a significant impact on public health. Pigs are the natural reservoirs of JEV and play an important role in the amplification, dispersal and epidemiology of JEV. The nonstructural protein 3 (NS3) of JEV possesses enzymatic activities of serine protease, helicase and nucleoside 5-triphosphatase, and plays important roles in viral replication and pathogenesis.ResultsWe characterized the NS3 protein of a neurovirulent strain of JEV (SH-JEV01) isolated from a field-infected pig. The NS3 gene of the JEV SH-JEV01 strain is 1857 bp in length and encodes protein of approximately 72 kDa with 99% amino acid sequence identity to that of the representative immunotype strain JaGAr 01. The NS3 protein was detectable 12 h post-infection in a mouse neuroblastoma cell line, Neuro-2a, and was distributed in the cytoplasm of cells infected with the SH-JEV01 strain of JEV. In the brain of mice infected with the SH-JEV01 strain of JEV, NS3 was detected in the cytoplasm of neuronal cells, including pyramidal neurons of the cerebrum, granule cells, small cells and Purkinje cells of the cerebellum.ConclusionsThe NS3 protein of a neurovirulent strain of JEV isolated from a pig was characterized. It is an approximately 72 kDa protein and distributed in the cytoplasm of infected cells. The Purkinje cell of the cerebellum is one of the target cells of JEV infection. Our data should provide some basic information for the study of the role of NS3 in the pathogenesis of JEV and the immune response.

Glycyrrhetinic acid extracted from Glycyrrhiza uralensis Fisch. induces the expression of Toll-like receptor 4 in Ana-1 murine macrophages

Glycyrrhetinic acid (GA) is an active component of licorice root that has long been used as a herbal medicine for the treatment of peptic ulcer, hepatitis, and pulmonary and skin diseases in Asia and Europe. In this study, we analyzed the effect of GA extracted from Glycyrrhiza uralensis Fisch. on the expression of Toll-like receptors (TLRs) that play key roles in regulating the innate immune response against invading pathogens. Stimulation of Ana-1 murine macrophages with GA induced a significant dose-dependent expression of TLR-4, and its mRNA expression that increased from 3-h post-treatment was approximately fivefold over the level in the mock-treated cells. No endotoxin contamination contributed to the GA-induced TLR-4 expression, because polymyxin B treatment did not alter the upregulated expression of TLR-4 in GA-treated cells. Several molecules, such as myeloid differentiation factor 88, interferon-β, and interleukin-6, which are involved in the TLR-4 downstream signaling pathway, were upregulated significantly in response to GA stimulation. Our findings demonstrate that GA is able to induce the expression of TLR-4 and activate its downstream signaling pathway.

Identification of human guanylate-binding protein 1 gene (hGBP1) as a direct transcriptional target gene of p53

Human guanylate-binding protein 1 (hGBP1) plays an important role in antitumor and antiviral immune responses. Here, we show that tumor suppressor p53 positively regulated hGBP1 transcription via binding to the p53 response element (p53RE) present in the hGBP1 promoter region. p53 activation by 5-fluorouracil significantly increased hGBP1 expression in wild-type p53 cells, but not in p53-null cells. Knockdown of p53 expression remarkably impaired hGBP1 expression induced by 5-fluorouracil, type I interferon treatment, or influenza A virus infection. Among three deductive p53REs present in the hGBP1 promoter region, two p53REs were found to be transactivated by p53.

Tumor suppressor p53 protects mice against Listeria monocytogenes infection

Tumor suppressor p53 is involved in regulating immune responses, which contribute to antitumor and antiviral activity. However, whether p53 has anti-bacterial functions remains unclear. Listeria monocytogenes (LM) causes listeriosis in humans and animals, and it is a powerful model for studying innate and adaptive immunity. In the present study, we illustrate an important regulatory role of p53 during LM infection. p53 knockout (p53KO) mice were more susceptible to LM infection, which was manifested by a shorter survival time and lower survival rate. p53KO mice showed significant impairments in LM eradication. Knockdown of p53 in RAW264.7 and HeLa cells resulted in increased invasion and intracellular survival of LM. Furthermore, the invasion and intracellular survival of LM was inhibited in p53-overexpressing RAW264.7 and HeLa cells. LM-infected p53KO mice exhibited severe clinical symptoms and organ injury, presumably because of the abnormal production of the pro-inflammatory cytokines TNF-α, IL-6, IL-12, and IL-18. Decreased IFN-γ and GBP1 productions were observed in LM-infected p53-deficient mice or cells. The combination of these defects likely resulted in the overwhelming LM infection in the p53KO mice. These observations indicate that p53 serves as an important regulator of the host innate immune that protects against LM infection.

Molecular Characterization of Enterohemorrhagic E. coli O157 Isolated from Animal Fecal and Food Samples in Eastern China

Objective. To elucidate the extent of food contamination by enterohemorrhagic Escherichia coli (EHEC) O157 in Eastern China. Methods. A total of 1100 food and animal fecal samples were screened for EHEC O157. Then, molecular characterization of each isolate was determined. Results. EHEC O157 was isolated as follows: pig feces, 4% (20/500); cattle feces, 3.3% (2/60); chicken feces, 1.43% (2/140); pork, 2.14% (3/140), milk, 1.67% (1/60); and chicken meat, 1.67% (1/60). The stx1, stx2, eae, and hlyA genes were present in 26.7% (8/30), 40% (12/30), 63.3% (19/30), and 50% (15/30) of the O157 isolates, respectively. Molecular typing showed that strains from fecal and food samples were clustered into the same molecular typing group. Furthermore, the isolates from pork and pig feces possessed the same characterization as the clinical strains ATCC35150 and ATCC43889. Biofilm formation assays showed that 53.3% of the EHEC O157 isolates could produce biofilm. However, composite analyses showed that biofilm formation of EHEC O157 was independent of genetic background. Conclusions. Animal feces, especially from pigs, serve as reservoirs for food contamination by EHEC O157. Thus, it is important to control contamination by EHEC O157 on farms and in abattoirs to reduce the incidence of foodborne infections in humans.