In silico identification of potential drug targets in swine pathogen Haemophilus parasuis

Gram-negative bacterium Haemophilus parasuis has recently become one of the most important etiological agents causing serious systemic disease (Gl?sser??s disease) in pigs. Antibiotic therapy has played a crucial role in the treatment of this disease. Antibiotic resistance observed from the clinical isolates of this pathogen urges us to discover novel drug targets for antimicrobial agents. In this study, we used a combined strategy including exploration of the gene essentiality and comparison of metabolic pathways to infer drug targets of H. parasuis. We identified 931 gene products essential for bacterial growth according to the DEG database. One hundred and ninety-nine enzyme-coding genes were found in the genome of H. parasuis but were absent in that of the swine host. Lastly, we determined 117 enzymes exhibiting essentiality and specificity to H. parasuis as a candidate set of drug targets. Comparison of metabolic pathways between the pathogen and host showed that 25 targeting enzymes belonged to nine unique pathways of the pathogen. The profile of promising targets identified in our study will provide a useful basis for developing more effective antibiotics against the severe swine disease caused by H. parasuis.     

Haemophilus parasuis infection in 3D4/21 cells induces autophagy through the AMPK pathway

Haemophilus parasuis (H. parasuis) is a common commensal in the upper respiratory tract of pigs, but causes Glässer's disease in stress conditions. To date, many studies focused on the immune evasion and virulence of H. parasuis; very few have focused on the role autophagy played in H. parasuis infection, particularly in porcine alveolar macrophages (PAMs). In this study, a PAM cell line, 3D4/21 cells were used to study the role of autophagy in H. parasuis infection. 3D4/21 cells tandemly expressing GFP, mCherry, and LC3 were infected with H. parasuis serovar 5 (Hps5). Western blot analysis and confocal and transmission electron microscopy showed that H. parasuis infection effectively induces autophagy. Using Hps strains of varying virulence (Hps4, Hps5, and Hps7) and UV‐inactivated Hps5, we demonstrated that autophagy is associated with the internalisation of living virulent strains into cells. In 3D4/21 cells pretreated with rapamycin and 3‐MA then infected by Hps4, Hps5, and Hps7, we demonstrated that autophagy affects invasion of H. parasuis in cells. AMPK signal results showed that Hps5 infection can upregulate the phosphorylation level of AMPK, which is consistent with the autophagy development. 3D4/21 cells pretreated with AICAR or Compound C then infected by Hps5 revealed that the autophagy induced by Hps5 infection is associated with the AMPK pathway. Our study contributes to the theoretical basis for the study of H. parasuis pathogenesis and development of novel drugs target for prevention Glässer's disease.     

Comparison of real-time PCR and culture isolation in colostrum-deprived pigs immunized and challenged with Haemophilus parasuis

Aims: A real‐time PCR (RT‐PCR) based on the detection of the infB gene of Haemophilus parasuis is compared with culture isolation (Frandoloso et al., (2011) Clin Vaccine Immunol 18 , 50–58.), evaluating different subunit or commercial vaccines. Methods and Results: Samples from different tissues of 24 experimentally infected and challenged colostrum‐deprived piglets were tested. The RT‐PCR gave globally a 23·3% more of positive results than culture, and all samples being positive by culture were positive by RT‐PCR also. H. parasuis could not be cultured from any of the samples of the piglets included in the three vaccinated groups resulting in a strong protection, but it could be detected by RT‐PCR in six samples in the group immunized with the commercial vaccine, in three in that vaccinated with native proteins with affinity to porcine transferrin (NPAPT) administered intramuscularly and in only two in that immunized with NPAPT intratracheally. Conclusions: The RT‐PCR was more sensitive than culture for H. parasuis detection in the organs compared. Significance and Impact of the Study: The RT‐PCR evidenced that NPAPT vaccines were those yielding the best protection results in terms of H. parasuis clearance.     

Assessment of genomic imprinting of <Emphasis Type="Italic">PPP1R9A</Emphasis>, <Emphasis Type="Italic">NAP1L5</Emphasis> and <Emphasis Type="Italic">PEG3</Emphasis> in pigs

Imprinted genes play significant roles in the regulation of fetal growth and development, function of the placenta, and maternal nurturing behaviour in mammals. At present, few imprinted genes have been reported in pigs compared to human and mouse. In order to increase understanding of imprinted genes in swine, a polymorphism-based approach was used to assess the imprinting status of three porcine genes in 12 tissue types, obtained from F1 pigs of reciprocal crosses between Rongchang and Landrace pure breeds. In contrast to human and mouse homologues, porcine PPP1R9A was not imprinted, and was found to be expressed in all tissues examined. The expression of porcine NAP1L5 was detected in pituitary, liver, spleen, lung, kiduey, stomach, small intestine, skeletal muscle, fat, ovary, and uterus, but undetectable in heart. Furthermore, porcine NAP1L5 was paternally expressed in the tissues where it’s expression was observed. For PEG3, pigs expressed the paternal allele in skeletal muscle, liver, spleen, kidney, and uterus, but biallele in heart, lung, fat, stomach, small intestine, and ovary. Our data indicate that tissue distribution of the three gene differs among mammals, and the imprinting of NAP1L5 and PEG3 is well conserved.     

Multiplex PCR assay for detection of <Emphasis Type="Italic">Actinobacillus pleuropneumoniae,Pasteurella multocida</Emphasis> and <Emphasis Type="Italic">Haemophilus parasuis</Emphasis> in lungs of pigs from a slaughterhouse

Multiplex PCR has been developed for parallel identification of Actinobacillus pleuropneumoniae, Pasteurella multocida and Haemophilus parasuis, important pathogens of swine, responsible for considerable economic losses in swine industry. Multiplex PCR and bacteriological cultivation was used to analyze lung samples from slaughterhouse pigs. From a total of 219 lung samples, 164 (74.9 %) were positive for P. multocida, 45 (20.5 %) for A. pleuropneumoniae and 4 (1.83 %) for H. parasuis. Bacteriological examination revealed that 145 samples (66.2 %) were positive for P. multocida, 31 (14.2 %) for A. pleuropneumoniae and 2 (0.91 %) for H. parasuis.     

HISTONE DEACETYLASE 6 represses pathogen defence responses in Arabidopsis thaliana

Plant defence mechanisms are suppressed in the absence of pathogen attack to prevent wasted energy and growth inhibition. However, how defence responses are repressed is not well understood. Histone deacetylase 6 (HDA6) is a negative regulator of gene expression, and its role in pathogen defence response in plants is not known. In this study, a novel allele of hda6 (designated as shi5) with spontaneous defence response was isolated from a forward genetics screening in Arabidopsis. The shi5 mutant exhibited increased resistance to hemibiotrophic bacterial pathogen Pst DC3000, constitutively activated expression of pathogen‐responsive genes including PR1, PR2, etc. and increased histone acetylation levels at the promoters of most tested genes that were upregulated in shi5. In both wild type and shi5 plants, the expression and histone acetylation of these genes were upregulated by pathogen infection. HDA6 was found to bind to the promoters of these genes under both normal growth conditions and pathogen infection. Our research suggests that HDA6 is a general repressor of pathogen defence response and plays important roles in inhibiting and modulating the expression of pathogen‐responsive genes in Arabidopsis.     

A rapid pulsed-field gel electrophoresis method of genotyping Haemophilus parasuis isolates

Aims: To develop a modified pulsed‐field gel electrophoresis (PFGE) method for characterizing Haemophilus parasuis isolates. Methods and Results: A modified PFGE procedure was designed using CpoI to generate restriction maps of H. parasuis genomic DNA. This approach was used to characterize 47 H. parasuis clinical isolates and 15 reference strains. All strains could be typed by this method, and the procedure was completed in 36 h. A total of 39 different PFGE patterns were identified among 47 epidemiologically unrelated clinical isolates. Conclusions: The modified PGFE described in this report efficiently characterized H. parasuis isolates. This method can be adopted for studying the epidemiology of Glässer’s disease outbreaks in addition to differentiating and classifying previously untypeable H. parasuis isolates. Significance and Impact of the Study: The modified PFGE method described is a novel means of characterizing H. parasuis isolates. It is also a highly discriminatory molecular typing method (discriminatory index of 0·98) that can overcome the limitations of serotyping.     

In vivo evidence of enhanced di-methylation of histone H3 K4 on upregulated genes in adipose tissue of diabetic db/db mice

Di-methylation of histone H3 lysine (K) 4, a component of the epigenetic memory, is associated with gene transactivation. In this study, we examined whether the development of diabetes induces di-methylation of histone H3 K4 on the upregulated genes. We searched for upregulated genes in mesenteric adipose tissue of insulin-resistant/diabetic db/db mice compared with non-diabetic db/m mice using microarray analysis. We also performed chromatin immunoprecipitation assays for di-methylation of histone H3 K4 in the upregulated genes in mesenteric adipose tissue of db/m and db/db mice. Di-methylation of histone H3 K4 was enhanced at the upstream and/or transcribed regions of upregulated genes including Atp6v0d2, Mmp12, Trem2 and Clec4d genes in mesenteric adipose tissue of db/db mice, as compared with db/m mice. These results suggest that di-methylation of histone H3 K4 is involved in the induction of Atp6v0d2, Mmp12, Trem2 and Clec4d in mesenteric adipose tissue in db/db mice.     

The infection cushion of Botrytis cinerea: a fungal ‘weapon’ of plant-biomass destruction

The necrotrophic plant-pathogen fungus Botrytis cinerea produces multicellular appressoria dedicated to plant penetration, named infection cushions (IC). A microarray analysis was performed to identify genes upregulated in mature IC. The expression data were validated by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and biochemical assays. 1231 upregulated genes and 79 up-accumulated proteins were identified. The data support the secretion of effectors by IC: phytotoxins, ROS, proteases, cutinases, plant cell wall–degrading enzymes and plant cell death–inducing proteins. Parallel upregulation of sugar transport and sugar catabolism–encoding genes would indicate a role of IC in nutrition. The data also reveal a substantial remodelling of the IC cell wall and suggest a role for melanin and chitosan in IC function. Lastly, mutagenesis of two upregulated genes in IC identified secreted fasciclin-like proteins as actors in the pathogenesis of B. cinerea. These results support the role of IC in plant penetration and also introduce other unexpected functions for this fungal organ, in colonization, necrotrophy and nutrition of the pathogen.     

Nuclease A (Gbs0661), an extracellular nuclease of Streptococcus agalactiae,attacks the neutrophil extracellular traps and is needed for full virulence

Most bacteria of the genus Streptococcus are opportunistic pathogens, and some of them produce extracellular DNases, which may be important for virulence. Genome analyses of Streptococcus agalactiae (GBS) neonate isolate NEM316 revealed the presence of seven genes putatively encoding secreted DNases, although their functions, if any, are unknown. In this study, we observed that respiration growth of GBS led to the extracellular accumulation of a putative nuclease, identified as being encoded by the gbs0661 gene. When overproduced in Lactococcus lactis, the protein was found to be a divalent cation‐requiring, pH‐stable and heat‐stable nuclease that we named Nuclease A (NucA). Substitution of the histidine¹⁴⁸ by alanine reduced nuclease activity of the GBS wild‐type strain, indicating that NucA is the major nuclease ex vivo. We determined that GBS is able to degrade the DNA matrix comprising the neutrophil extracellular trap (NET). The nucA^H148A mutant was impaired for this function, implicating NucA in the virulence of GBS. In vivo infection studies confirmed that NucA is required for full infection, as the mutant strain allowed increased bacterial clearance from lung tissue and decreased mortality in infected mice. These results show that NucA is involved in NET escape and is needed for full virulence.     

Virulence and Draft Genome Sequence Overview of Multiple Strains of the Swine Pathogen Haemophilus parasuis

Haemophilus parasuis is the cause of Glässer''s disease in swine, which is characterized by systemic infection resulting in polyserositis, meningitis, and arthritis. Investigation of this animal disease is complicated by the enormous differences in the severity of disease caused by H. parasuis strains, ranging from lethal systemic disease to subclinical carriage. To identify differences in genotype that could account for virulence phenotypes, we established the virulence of, and performed whole genome sequence analysis on, 11 H. parasuis strains. Virulence was assessed by evaluating morbidity and mortality following intranasal challenge of Caesarean-derived, colostrum-deprived (CDCD) pigs. Genomic DNA from strains Nagasaki (serotype 5), 12939 (serotype 1), SW140 (serotype 2), 29755 (serotype 5), MN-H (serotype 13), 84-15995 (serotype 15), SW114 (serotype 3), H465 (serotype 11), D74 (serotype 9), and 174 (serotype 7) was used to generate Illumina paired-end libraries for genomic sequencing and de novo assembly. H. parasuis strains Nagasaki, 12939, SH0165 (serotype 5), SW140, 29755, and MN-H exhibited a high level of virulence. Despite minor differences in expression of disease among these groups, all pigs challenged with these strains developed clinical signs consistent with Glässer''s disease between 1–7 days post-challenge. H. parasuis strains 84-15995 and SW114 were moderately virulent, in that approximately half of the pigs infected with each developed Glässer''s disease. H. parasuis strains H465, D74, and 174 were minimally virulent or avirulent in the CDCD pig model. Comparative genomic analysis among strains identified several noteworthy differences in coding regions. These coding regions include predicted outer membrane, metabolism, and pilin or adhesin related genes, some of which likely contributed to the differences in virulence and systemic disease observed following challenge. These data will be useful for identifying H. parasuis virulence factors and vaccine targets.     

低温促进毛竹叶片类黄酮合成及相关基因的表达模式分析

类黄酮在植物耐低温胁迫方面发挥着重要作用,为揭示低温对毛竹(Phyllostachys edulis)叶片中类黄酮合成的影响,采用分光光度法测定了不同生长时期和低温胁迫下毛竹幼苗叶片中的类黄酮含量,通过生物信息学方法对毛竹类黄酮早期生物合成关键酶基因进行了鉴定,并用q PCR方法分析了其表达模式。结果表明,随着叶片的生长,类黄酮含量呈现先升高后降低的趋势,而低温下,功能叶片中类黄酮含量则呈现上调趋势,且在8 h时达极显著水平。在毛竹基因组中鉴定了类黄酮早期生物合成3个酶基因家族共29个成员,包括20个查尔酮合酶基因(Pe CHSs)、8个查尔酮异构酶基因(Pe CHIs)和1个黄酮-3-烃化酶基因(Pe F3H1),这些基因的启动子中均含有响应低温及其他非生物胁迫的调控元件。Pe CHSs倾向在根和叶中表达,而PeCHIs为组成型表达。在不同生长时期的叶片中,仅PeCHS1表达与类黄酮的含量变化趋势一致;而低温胁迫下,3个PeCHSs、2个PeCHIs和PeF3H1在功能叶片中呈上调表达,与类黄酮含量变化趋势一致。因此,毛竹可能通过提高类黄酮早期生物合成酶基因的表达量促进类黄酮的合成来...