WIPF2 promotes Shigella flexneri actin‐based motility and cell‐to‐cell spread

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin‐based motility and formation of membrane protrusions at cell–cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell‐to‐cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation‐promoting factor N‐WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N‐WASP exists as a complex with WIP. The involvement of WIP in N‐WASP‐dependent actin‐based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT‐29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non‐motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N‐WASP to the bacterial pole, and among N‐WASP‐positive bacteria, a decrease in actin tail‐positive bacteria, suggesting that WIPF2 is required for N‐WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.     

Comparison of the genetic structures surrounding qnrA1 in Korean Enterobacter cloacae and Chinese Escherichia coli strains isolated in the early 2000s: evidence for qnrA mobilization via Inc HI2 type plasmid

The flanking genetic structure of qnrA1 in Korean Enterobacter loacae was identical to that of the Chinese Escherichia coli strain, the first qnrA1-carrying strain reported in Asia. Analysis of restriction enzyme sites and Southern blot hybridization results showed that qnrA1 was transferred between E. cloacae and E. coli via Inc HI2 type plasmid.     

Proteomic analysis on acetate metabolism in Citrobacter sp. BL-4

Mass production of glucosamine (GlcN) using microbial cells is a worthy approach to increase added values and keep safety problems in GlcN production process. Prior to set up a microbial cellular platform, this study was to assess acetate metabolism in Citrobacter sp. BL-4 (BL-4) which has produced a polyglucosamine PGB-2. The LC-MS analysis was conducted after protein separation on the 1D-PAGE to accomplish the purpose of this study. 280 proteins were totally identified and 188 proteins were separated as acetate-related proteins in BL-4. Acetate was converted to acetyl-CoA by acetyl-CoA synthetase up-regulated in the acetate medium. The glyoxylate bypass in the acetate medium was up-regulated with over-expression of isocitrate lyases and 2D-PAGE confirmed this differential expression. Using (1)H-NMR analysis, the product of isocitrate lyases, succinate, increased about 15 times in the acetate medium. During acetate metabolism proteins involved in the lipid metabolism and hexosamine biosynthesis were over-expressed in the acetate medium, while proteins involved in TCA cycle, pentose phosphate cycle and purine metabolism were down-regulated. Taken together, the results from the proteomic analysis can be applied to improve GlcN production and to develop metabolic engineering in BL-4.     

Electroantennogram and field responses of Korean population of the rice leaf folder,Cnaphalocrocis medinalis (Lepidoptera: Crambidae), to sex attractant candidates

A series of studies was conducted to determine the optimum sex attractant for monitoring the rice leaf folder, Cnaphalocrocis medinalis, in Korea, based on the previously reported pheromone composition in this species. (Z)-13-Octadecenal (Z13-18:Al) was most active in electroantennogram (EAG) and field tests. (Z)-11-Hexadecenal (Z11-16:Al), (Z)-11-octadecenal (Z11-18:Al), (Z)-11-hexadecenyl acetate (Z11-16:Ac), (Z)-13-octadecenyl acetate (Z13-18:Ac) and (Z)-13-octadecenol (Z13-18:OH) individually elicited significant EAG responses, but were not individually attractive in field trials. (Z)-11-Octadecenol (Z11-18:OH) alone was inactive in both EAG and field tests. The addition of Z11-18:OH and Z13-18:OH to the binary mixture of Z11-18:Al and Z13-18:Al, as the previously reported composition of the Japanese blend, significantly increased the trap catches of males in field trials. In contrast, the Philippine and Indian blends were not attractive to this species. Interestingly, when Z13-18:Ac alone was added to the binary mixture of Z11-18:Al and Z13-18:Al, the trap catch number was the same as that of the Japanese blend. The present study indicates that the four-component blend (Z11-18:Al/Z13-18:Al/Z11-18:OH/Z13-18:OH = 11/100/24/36) and the three-component blend (Z11-18:Al/Z13-18:Al/Z13-18:Ac = 11/100/11) can be used as sex attractants for monitoring the Korean populations of C. medinalis.     

Antagonist-mediated down-regulation of toll-like receptors increases the prevalence of human papillomavirus infection in systemic lupus erythematosus

IntroductionPrevalence of an abnormal Papanicolaou smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk human papillomavirus (HPV) infection. The nucleic acid-specific Toll-like receptors (TLRs) locate at the endolysosomal compartments and trigger the induction of cytokines for the innate immune response. This study evaluated whether abnormal host innate immune response in lupus patients may enhance HPV persistence.MethodsProtein levels of TLRs 3, 7, 8 and 9 in cervical epithelial cells of lupus patients and controls with or without HPV infection were assessed using flow cytometry. Characteristics associated with the differential expression of TLRs in systemic lupus erythematosus (SLE) were elucidated. The effect and interferon-stimulated genes (ISGs) (ISG15 and Mx-1) gene expressions were then measured in oncogenic HeLa (HPV18), CaSki (HPV) and C33A (HPV negative) cell lines using flow cytometry and quantitative real-time PCR. Ex vivo productions of cytokines and interferon-gamma (IFN-γ) upon TLR ligands stimulations were subsequently measured using cytometric bead array and ELISA.ResultsFor subjects with HPV infection, levels of TLR3 and TLR7 were significantly lower in lupus patients compared with controls. Significantly decreased TLRs 7, 8 and 9 levels were observed in HPV-negative SLE compared to healthy controls. For SLE with and without HPV infection, TLR7 and 9 levels were significantly lower in infected SLE than those in HPV-negative patients. Independent explanatory variables associated with down-regulation of TLR7 level included HPV infection and a higher cumulative dose of prednisolone; while a higher cumulative dose of hydroxychloroquine and HPV infection were associated with down-regulation of TLR9 level. In cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral ISG15 and Mx-1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands (R837, ssRNA and ODN2395) was greatly impaired in CaSki and HeLa than C33A cells.ConclusionsIn conclusion, prednisolone and TLR antagonist (hydroxychloroquine) may down-regulate protein levels of TLR7 and TLR9 in lupus patients, thereby decreasing the innate immune response against HPV infection. Upon infection, HPV further down-regulate TLR7 and 9 levels for viral persistence. Furthermore, reduction of nucleic acid-sensing TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs (ISG15 and Mx-1) on a biologically relevant antiviral response.     

Genes Encoding the N-Acyl Homoserine Lactone-Degrading Enzyme Are Widespread in Many Subspecies of Bacillus thuringiensis

Gram-negative bacteria can communicate with each other by N-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers. Recently, the aiiA gene (encoding an enzyme catalyzing the degradation of AHL) has been cloned from Bacillus sp. strain 240B1. During investigations in the course of the ongoing Bacillus thuringiensis subsp. morrisoni genome project, an aiiA homologue gene in the genome sequence was found. These results led to consideration of the possibility of the widespread existence of the gene in B. thuringiensis. aiiA homologue genes were found in 16 subspecies of B. thuringiensis, and their sequences were determined. Comparison of the Bacillus sp. strain 240B1 aiiA gene with the B. thuringiensis aiiA homologue genes showed high homologies of 89 to 95% and 90 to 96% in the nucleotide sequence and deduced amino acid sequence, respectively. Among the subspecies of B. thuringiensis having an aiiA gene, the subspecies aizawai, galleriae, kurstaki, kyushuensis, ostriniae, and subtoxicus were shown to degrade AHL. It was observed that recombinant Escherichia coli producing AiiA proteins also had AHL-degrading activity and could also attenuate the plant pathogenicity of Erwinia carotovora. These results indicate that insecticidal B. thuringiensis strains might have potential to compete with gram-negative bacteria in natural ecosystems by autoinducer-degrading activity.