Absence of O antigen suppresses Shigella flexneri IcsA autochaperone region mutations

The Shigella flexneri IcsA (VirG) protein is a polarly distributed autotransporter protein. IcsA functions as a virulence factor by interacting with the host actin regulatory protein N-WASP, which in turn activates the Arp2/3 complex, initiating actin polymerization. Formation of F-actin comet tails allows bacterial cell-to-cell spreading. Although various accessory proteins such as periplasmic chaperones and the β-barrel assembly machine (BAM) complex have been shown to be involved in the export of IcsA, the IcsA translocation mechanism remains to be fully elucidated. A putative autochaperone (AC) region (amino acids 634–735) located at the C-terminal end of the IcsA passenger domain, which forms part of the self-associating autotransporter (SAAT) domain, has been suggested to be required for IcsA biogenesis, as well as for N-WASP recruitment, based on mutagenesis studies. IcsAi proteins with linker insertion mutations within the AC region have a significant reduction in production and are defective in N-WASP recruitment when expressed in smooth LPS (S-LPS) S. flexneri. In this study, we have found that the LPS O antigen plays a role in IcsAi production based on the use of an rmlD (rfbD) mutant having rough LPS (R-LPS) and a novel assay in which O antigen is depleted using tunicamycin treatment and then regenerated. In addition, we have identified a new N-WASP binding/interaction site within the IcsA AC region.Min Yan Teh, Elizabeth Ngoc Hoa Tran and Renato Moron

The bracteatus pineapple genome and domestication of clonally propagated crops

Domestication of clonally propagated crops such as pineapple from South America was hypothesized to be a 'one-step operation'. We sequenced the genome of Ananas comosus var. bracteatus CB5 and assembled 513 Mb into 25 chromosomes with 29,412 genes. Comparison of the genomes of CB5, F153 and MD2 elucidated the genomic basis of fiber production, color formation, sugar accumulation and fruit maturation. We also resequenced 89 Ananas genomes. Cultivars 'Smooth Cayenne' and 'Queen' exhibited ancient and recent admixture, while 'Singapore Spanish' supported a one-step operation of domestication. We identified 25 selective sweeps, including a strong sweep containing a pair of tandemly duplicated bromelain inhibitors. Four candidate genes for self-incompatibility were linked in F153, but were not functional in self-compatible CB5. Our findings support the coexistence of sexual recombination and a one-step operation in the domestication of clonally propagated crops. This work guides the exploration of sexual and asexual domestication trajectories in other clonally propagated crops

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Analysis of Shigella flexneri cell surface virulence factors.

The IcsA autotransporter (AT) is a key virulence protein of Shigella flexneri, a human pathogen that causes bacillary dysentery through invasion of colonic epithelium. IcsA is a polarly distributed, outer membrane protein that confers motility to intracellular bacteria by engaging the host actin regulatory protein, neural Wiskott-Aldrich syndrome protein (NWASP). The activated N-WASP in turn activates Arp2/3 complex, which initiates de novo actin nucleation and polymerisation to form F-actin comet tails and allow actin-based motility (ABM). The N-terminal surface-exposed IcsA passenger -domain (aa 53-758) is responsible for N-WASP interaction, where multiple IcsA regions: aa 185-312 (N-WASP interacting region [IR] I), aa 330-382 (N-WASP IR II) and aa 508-730 (N-WASP IR III), have been suggested to be interacting with N-WASP from previous linker-insertion mutagenesis (IcsAi). A putative autochaperone (AC) region (aa 634-735) located at the C-terminal end of IcsA passenger domain, which forms part of the self-associating AT (SAAT) domain, has been suggested to be required for IcsA biogenesis. IcsAi proteins with linker insertion mutations within the AC region had a significant reduction in production when expressed in smooth lipopolysaccharide (S-LPS) S. flexneri. This thesis investigated the biogenesis of IcsA, seeking to identify factors that affect IcsA AC mutant production in the S-LPS background. IcsAi AC mutant production was restored to a wild-type comparable levels in the rough LPS (R-LPS) S. flexneri (that lack the O-antigen component). The same phenotypes were observed in S. flexneri (both S-LPS and R-LPS) expressing site-directed mutagenised IcsA AC protein (aa 716-717). Various approaches were performed to identify the factors that caused different IcsA AC mutant production between SLPS and R-LPS S. flexneri. Both LPS Oag and DegP (a periplasmic chaperone/protease) were identified to affect IcsA AC mutant production in S-LPS strain, as the IcsA AC mutant production was restored in S. flexneri ΔdegP S-LPS strain. In addition, site-directed mutagenesis of residues Y716 and D717 within the AC region showed that these residues are critical for IcsA production and/or stability in the S-LPS background but not in the R-LPS background. Another aim of this work was to further define N-WASP IRs II and III via site-directed mutagenesis of specific amino acids. Mutant IcsA protein production level, N-WASP recruitment and F-actin comet tail formation by S-LPS and R-LPS S. flexneri were characterised. Residues 330-331, and residue 382 within N-WASP IR II, and residues 716- 717 within N-WASP IR III, were identified to be involved in N-WASP recruitment. It was shown for the first time that N-WASP activation involves interaction with different regions on different IcsA molecules and hence that oligomeric IcsA is needed for this interaction. Various “GFP-N-WASP” sub-domain proteins and IcsA protein were over-expressed, purified and used in protein binding assays. These provided preliminary data for protein binding assays which investigate the relationship between N-WASP and IcsA. Another S. flexneri virulence protein, IcsB, which is involved in preventing autophagy activation by intracellular bacteria was investigated. An S. flexneri 2457T ΔicsB mutant was created and characterised in this study. In this background, the icsB mutation had a less of an effect on plaque formation than reported for another S. flexneri background.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201

Expression of IcsA::BIO with mutations at Y716 D717 by S-LPS and R-LPS <i>S. flexneri</i>.

<p>(A) Whole cell lysates from mid-exponential phase <i>S. flexneri ΔicsA</i> (S-LPS) and <i>S. flexneri</i> Δ<i>icsA</i> Δ<i>rmlD</i> (R-LPS) expressing (A) IcsA::BIO or various IcsA::BIO Y716* D717* mutants; (B) IcsA::BIO, IcsA::BIO Y716F or IcsA::BIO Y716G; (C) IcsA::BIO or IcsA::BIO D717G; were prepared and analysed by Western immunoblotting using anti-IcsA antibody. <i>S. flexneri</i> carrying an empty vector was used as a negative control (IcsA⁻). Strain names are shown above each lane. The 120 kDa band corresponds to the full length IcsA; the 85 kDa band corresponds to the cleaved form (IcsA’). S = S-LPS; R = R-LPS.</p

Analysis of <i>S. flexneri</i> strains expressing IcsA::BIO Y716* and/or D717* in S-LPS and R-LPS backgrounds.

a<p>P, polar; NP, non-polar.</p>b<p>The “+++”, “++” and “+” symbols indicate relative band intensities of Western immunoblots of whole cell lysates.</p>c, d<p>“+++”, WT N-WASP recruitment/F-actin comet tail or capping formation; “++”, 20%–80% reduction in N-WASP recruitment/F-actin comet tail or capping formation; “+/−”, 90% reduction in N-WASP recruitment/F-actin comet tail or capping formation; “−”, N-WASP/F-actin tail not detected.</p>e<p>“+++”, WT plaques; “++”, small plaques; “−”, no plaques.</p

Summary of <i>S. flexneri</i> strains expressing IcsA::BIO T381*V382* and IcsA::BIO V382R.

a<p>P, polar; NP, non-polar.</p>b<p>The “+++” symbol indicates relative band intensities of Western immunoblots of whole cell lysates.</p>c, d<p>“+++”, WT N-WASP recruitment/F-actin comet tail formation; “++”, 20%–80% reduction in N-WASP recruitment/F-actin comet tail or capping formation; “−”, N-WASP/F-actin comet tail not detected.</p>e<p>“+++”, WT plaques; “++”, small plaques; “−”, no plaques.</p

N-WASP interacting region complementation assay.

<p>(A, B) IF microscopy to detect N-WASP, Arp3 recruitment and F-actin comet tail formation by intracellular <i>S. flexneri</i> strains. HeLa cells were infected with mid-exponential phase <i>S. flexneri ΔicsA ΔrmlD</i> (R-LPS) strains expressing either IcsA::BIO only or co-expressing IcsA::BIO V382R (N-WASP IR II) and IcsA::BIO Y716G D717G (N-WASP IR III), and formalin fixed. HeLa cells and bacteria nuclei were labelled with DAPI (blue), and N-WASP was labelled with anti-N-WASP antibody and either (A) Alex Fluor 488-conjugated donkey anti-rabbit antibody (green) or (B) Alex Fluor 594-conjugated donkey anti-rabbit antibody (red). (A) Arp3 was labelled with anti-Arp3 monoclonal antibody and an Alex Fluor 594-conjugated donkey anti-mouse antibody (red). (B) F-actin was labelled with Alexa Fluor-488-phalloidin (green). IF images were observed at 100×magnification. Arrows indicate N-WASP, Arp3 recruitment and F-actin tail formation. Enlargements of relevant region shown for clarity. Strains were assessed in two independent experiments. Scale bar = 10 µm. (C) Quantification of N-WASP/Arp3 recruitment, and (D) N-WASP/F-actin tail or capping formation, by intracellular <i>S. flexneri ΔicsA ΔrmlD</i> strains expressing IcsA::BIO only or co-expressing IcsA::BIO V382R (N-WASP IR II) and IcsA::BIO Y716G D717G (N-WASP IR III). Bacteria that either recruited both N-WASP and Arp3 (C), or recruited N-WASP and formed F-actin comet tail/capping (D), were scored from infected HeLa cells (<i>n</i> = 20 HeLa cells; ∼250–350 bacteria). Data are represented as percentage of N-WASP/Arp3 recruitment ± SEM of two independent experiments (C); and, as percentage of N-WASP recruitment/F-actin tail or capping formation ± SEM of two independent experiments (D). **, 0.001<<i>P</i><0.01; ***, <i>P</i><0.001 (determined by Student’s unpaired one-tailed <i>t</i> test). IR II & III = Interacting region II and III (<i>S. flexneri</i> co-expressing IcsA::BIO V382R and IcsA::BIO Y716G D717G).</p