Actinobacillus pleuropneumoniae exotoxin ApxI induces cell death via attenuation of FAK through LFA-1.

ApxI exotoxin is an important virulence factor derived from Actinobacillus pleuropneumoniae that causes pleuropneumonia in swine. Here, we investigate the role of lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), a member of the ß2 integrin family, and the involvement of the integrin signaling molecules focal adhesion kinase (FAK) and Akt in ApxI cytotoxicity. Using Western blot analysis, we found that ApxI downregulated the activity of FAK and Akt in porcine alveolar macrophages (AMs). Preincubation of porcine AMs with an antibody specific for porcine CD18 reduced ApxI-induced cytotoxicity as measured by a lactate dehydrogenase release assay and decreased ApxI-induced FAK and Akt attenuation, as shown by Western blot analysis. Pretreatment with the chemical compounds PMA and SC79, which activate FAK and Akt, respectively, failed to overcome the ApxI-induced attenuation of FAK and Akt and death of porcine AMs. Notably, the transfection experiments revealed that ectopic expression of porcine LFA-1 (pLFA-1) conferred susceptibility to ApxI in ApxI-insensitive cell lines, including human embryonic kidney 293T cells and FAK-deficient mouse embryonic fibroblasts (MEFs). Furthermore, ectopic expression of FAK significantly reduced ApxI cytotoxicity in pLFA-1-cotransfected FAK-deficient MEFs. These findings show for the first time that pLFA-1 renders cells susceptible to ApxI and ApxI-mediated attenuation of FAK activity via CD18, thereby contributing to subsequent cell death.

Molecular cloning, expression, and functional characterization of the ß-agarase AgaB-4 from Paenibacillus agarexedens.

In this study, a ß-agarase gene, agaB-4, was isolated for the first time from the agar-degrading bacterium Paenibacillus agarexedens BCRC 17346 by using next-generation sequencing. agaB-4 consists of 2652 bp and encodes an 883-amino acid protein with an 18-amino acid signal peptide. agaB-4 without the signal peptide DNA was cloned and expressed in Escherichia coli BL21(DE3). His-tagged recombinant AgaB-4 (rAgaB-4) was purified from the soluble fraction of E. coli cell lysate through immobilized metal ion affinity chromatography. The optimal temperature and pH of rAgaB-4 were 55 °C and 6.0, respectively. The results of a substrate specificity test showed that rAgaB-4 could degrade agar, high-melting point agarose, and low-melting point agarose. The Vmax and Km of rAgaB-4 for low-melting point agarose were 183.45 U/mg and 3.60 mg/mL versus 874.61 U/mg and 9.29 mg/mL for high-melting point agarose, respectively. The main products of agar and agarose hydrolysis by rAgaB-4 were confirmed to be neoagarotetraose. Purified rAgaB-4 can be used in the recovery of DNA from agarose gels and has potential application in agar degradation for the production of neoagarotetraose.

Improved subtilisin YaB production in Bacillus subtilis using engineered synthetic expression control sequences.

Alkaline elastase YaB, a favorable meat tenderizer, is an extracellular subtilisin-type protease produced by wild strain alkalophilic Bacillus YaB. The gene ale coding for subtilisin YaB with its own expression control sequence has been cloned and expressed in Bacillus subtilis, but at levels much lower than in the parental strain Bacillus YaB. This study investigates the influence of various expression control sequences including expression control sequences of cdd and veg from B. subtilis, a synthetic expression control sequence (SECS), and engineered synthetic expression control sequences (engineered SECSs) on the expression of subtilisin YaB in B. subtilis. The engineered SECSs were generated by using the Polymerase Chain Reaction; their UP element, Shine-Dargarno (SD) sequence, or both were different from those of the native SECS. The expression efficiencies of SECS and engineered SECSs were higher than those of expression control sequences of ale, cdd, and veg. Substitution of the SD sequence of SECS resulted in higher expression of subtilisin YaB than substitution of the UP element, whereas combined substitution of both gave the highest expression. These results demonstrate that engineering of SECSs is an approach for improving subtilisin YaB production in B. subtilis. Moreover, it is suggested that these enginnered SECSs could potentially be used to express homologous and heterologous proteins in B. subtilis at high level.

Involvement of NF-κB in regulation of Actinobacillus pleuropneumoniae exotoxin ApxI-induced proinflammatory cytokine production in porcine alveolar macrophages.

Actinobacillus pleuropneumoniae is a crucial respiratory pathogen that causes fibrinous, hemorrhagic, necrotizing pleuropneumonia in pigs. A. pleuropneumoniae exotoxins (ApxI to IV) are the major virulence factors contributing to A. pleuropneumoniae pathogenesis. Previously, we demonstrated that ApxI induces the expression of proinflammatory cytokines in porcine alveolar macrophages (PAMs) via the mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK). Nonetheless, the role of nuclear factor (NF)-κB-a transcription factor widely implicated in immune and inflammatory responses-in ApxI-elicited cytokine production has yet to be defined. In the present study, we examined the involvement of NF-κB in ApxI-elicited production of interleukin (IL)-1ß, IL-8, and tumor necrosis factor (TNF)-α in PAMs and investigated the correlation between NF-κB and MAPK (p38 and JNK) pathways in this event. The results of Western blot analysis, confocal microscopy, and a DNA binding activity assay revealed that the classical NF-κB pathway was activated by ApxI, as evidenced by the decreased levels of IκB and subsequent NF-κB translocation and activation in ApxI-stimulated PAMs. Moreover, the blocking of ApxI-induced NF-κB activation significantly attenuated the levels of mRNA and protein secretion of IL-1ß, IL-8, and TNF-α in PAMs. Notably, the attenuation of JNK activation by a specific inhibitor (SP600125) reduced ApxI-induced NF-κB activation, whereas a p38 blocker (SB203580) had no effect on the NF-κB pathway. Further examination revealed that the level of phosphorylation at serine 536 on the NF-κB p65 subunit was dependent on JNK activity. Collectively, this study, for the first time, demonstrates a pivotal role of NF-κB in ApxI-induced IL-1ß, IL-8, and TNF-α production; JNK, but not p38, may positively affect the activation of the classical NF-κB pathway.

Pathogenic microbiological baseline survey of pork carcasses in Taiwan.

From 2004 to 2010, pork carcass swabs from state-inspected slaughter plants in Taiwan were intermittently analyzed to determine the prevalence of selected pathogenic microorganisms associated with foodborne illness. The prevalences of Staphylococcus aureus each year from 2006 to 2010 were 6.6, 10.8, 5.1, 6.4, and 7.4%, respectively, while those of Listeria monocytogenes were 1.2% in 2004, 1.3% in 2005, and 3.5% in 2008. The prevalences of Clostridium perfringens were 0.9% in 2004, 3.2% in 2005, and 1.1% in 2008. Campylobacter jejuni and Campylobacter coli had a higher recovery rate than the other surveyed microorganisms, with prevalences during 2004, 2005, and 2008 of 21.1, 13.7, and 8.1%, respectively. Salmonella strains were analyzed each year, and their prevalences ranged between 3.0 and 6.9%. Derby, Typhimurium, Anatum, Choleraesuis, and Agona were the five serovars most frequently identified among the Salmonella isolates. Escherichia coli O157:H7 was not detected in 2004, 2005, or 2010. Routine baseline surveying of pork carcasses to determine the prevalence of selected pathogens of concern for food safety can provide valuable information regarding the effectiveness of the slaughtering procedures or the need for interventions.

Chromosomal integration of a synthetic expression control sequence achieves poly-gamma-glutamate production in a Bacillus subtilis strain.

Poly-gamma-glutamate (gamma-PGA) has applications in food, medical, cosmetic, animal feed, and wastewater industries. Bacillus subtilis DB430, which possesses the gamma-PGA synthesis ywsC-ywtAB genes in its chromosome, cannot produce gamma-PGA. An efficient synthetic expression control sequence (SECS) was introduced into the upstream region of the ywtABC genes, and this resulted in gamma-PGA-producing B. subtilis mutant strains. Mutant B. subtilis PGA6-2 stably produces high levels of gamma-PGA in medium A without supplementation of extra glutamic acid or ammonium chloride. The mutant B. subtilis PGA 6-2 is not only a gamma-PGA producer, but it is also a candidate for the genetic and metabolic engineering of gamma-PGA production.