A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors

Bacterial pathogens often subvert the innate immune system to establish a successful infection. The direct inhibition of downstream components of innate immune pathways is particularly well documented but how bacteria interfere with receptor proximal events is far less well understood. Here, we describe a Toll/interleukin 1 receptor (TIR) domain‐containing protein (PumA) of the multi‐drug resistant Pseudomonas aeruginosa PA7 strain. We found that PumA is essential for virulence and inhibits NF‐κB, a property transferable to non‐PumA strain PA14, suggesting no additional factors are needed for PumA function. The TIR domain is able to interact with the Toll‐like receptor (TLR) adaptors TIRAP and MyD88, as well as the ubiquitin‐associated protein 1 (UBAP1), a component of the endosomal‐sorting complex required for transport I (ESCRT‐I). These interactions are not spatially exclusive as we show UBAP1 can associate with MyD88, enhancing its plasma membrane localization. Combined targeting of UBAP1 and TLR adaptors by PumA impedes both cytokine and TLR receptor signalling, highlighting a novel strategy for innate immune evasion.     

The Presynaptic Cytomatrix Protein Bassoon: Sequence and Chromosomal Localization of the HumanBSNGene

Bassoon is a novel 420-kDa protein recently identified as a component of the cytoskeleton at presynaptic neurotransmitter release sites. Analysis of the rat and mouse sequences revealed a polyglutamine stretch in the C-terminal part of the protein. Since it is known for some proteins that abnormal amplification of such polyglutamine regions can cause late-onset neurodegeneration, we cloned and localized the human BASSOON gene (BSN). Phage clones spanning most of the open reading frame and the 3′ untranslated region were isolated from a human genomic library and used for chromosomal localization ofBSNto chromosome 3p21 by FISH. The localization was confirmed by PCR on rodent/human somatic cell hybrids; it is consistent with the localization of the murineBsngene at chromosome 9F. Sequencing revealed a polyglutamine stretch of only five residues in human, and PCR amplifications from 50 individuals showed no obvious length polymorphism in this region. Analysis of the primary structure of Bassoon and comparison to previous database entries provide evidence for a newly emerging protein family.     

The coral symbiont Candidatus Aquarickettsia is variably abundant in threatened Caribbean acroporids and transmitted horizontally

The symbiont “Candidatus Aquarickettsia rohweri” infects a diversity of aquatic hosts. In the threatened Caribbean coral, Acropora cervicornis, Aquarickettsia proliferates in response to increased nutrient exposure, resulting in suppressed growth and increased disease susceptibility and mortality of coral. This study evaluated the extent, as well as the ecology and evolution of Aquarickettsia infecting threatened corals, Ac. cervicornis, and Ac. palmata and their hybrid (“Ac. prolifera”). Aquarickettsia was found in all acroporids, with coral host and geographic location impacting the infection magnitude. Phylogenomic and genome-wide single-nucleotide variant analysis of Aquarickettsia found phylogenetic clustering by geographic region, not by coral taxon. Analysis of Aquarickettsia fixation indices suggests multiple sequential infections of the same coral colony are unlikely. Furthermore, relative to other Rickettsiales species, Aquarickettsia is undergoing positive selection, with Florida populations experiencing greater positive selection relative to other Caribbean locations. This may be due in part to Aquarickettsia proliferating in response to greater nutrient stress in Florida, as indicated by greater in situ replication rates in these corals. Aquarickettsia was not found to significantly codiversify with either the coral animal or the coral’s algal symbiont (Symbiodinium “fitti”). Quantitative PCR analysis showed that gametes, larvae, recruits, and juveniles from susceptible, captive-reared coral genets were not infected with Aquarickettsia. Thus, horizontal transmission of Aquarickettsia via coral mucocytes or an unidentified host is more likely. The prevalence of Aquarickettsia in Ac. cervicornis and its high abundance in the Florida coral population suggests that coral disease mitigation efforts focus on preventing early infection via horizontal transmission.Subject terms: Population genetics, Phylogenetics     

Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol

Cancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario.Subject terms: Cell biology, Molecular biology, Experimental models of disease     

Whiskers amiss, a new vibrissae and hair mutation near the Krt1 cluster on mouse Chromosome 11

Whiskers amiss (wam) is a new spontaneous recessive mutation in the SELH/Bc strain of mice that causes a phenotype of askew, sometimes kinked or curled, breakable whiskers and disheveled-appearing body hair, apparently owing to disoriented guard hairs. Heterozygotes on three genetic backgrounds are indistinguishable from normal. Using informative SSLPs in the F₂ generation after crosses to two normal strains, we have mapped wam to the region of the type I keratin cluster on Chromosome (Chr) 11, within an approximately 6-cM segment according to the current Mouse Genome Database (MGD) map position of flanking SSLPs. Although several other hair mutations also map to the Krt1 region (Re, Rim3, Bda, Bsk), none has a hair and whisker phenotype similar to that of wam, and, because all are transmitted as dominants, interpretable complementation tests could not be done. Scabbing and tissue loss occur on the rims of the pinnae and tail tip in some aging wam homozygotes, suggesting that wam may be an animal model of a genetic ectodermal disorder. The SELH/Bc strain background appears to have an unusually high rate of spontaneous mutation; wam is the sixth mutation to be described. Received: 13 October 1999 / accepted: 7 December 1999     

Lipid composition of somatic and zygotic embryos from Prunus avium. Effect of a cold treatment on somatic embryo quality

In order to evaluate the quality of Prunus avium somatic embryos, a comparison of lipid composition between somatic and zygotic embryos was undertaken. In both zygotic and somatic embryos, neutral glycerolipids (NL) and phosphatidylcholine (PC) were the 2 major lipid classes. The content of NL increased over the course of development in zygotic embryos and reached 490 μg per embryo, while the PC content reached 100 μg per embryo. However, the contents of NL and PC in somatic embryos were similar to immature zygotic embryos at stage 3. Fatty acid composition of NL from both zygotic and somatic embryos revealed more unsaturated than saturated fatty acids. In somatic embryos, the saturated/unsaturated fatty acid ratios of NL and phosphatidylinositol (PI) were similar to those observed in immature zygotic embryos up to stage 6. Conversely, in phosphatidylethanolamine (PE) the ratio was similar to the ratio observed in mature zygotic embryos, at stage 7. Histological studies confirmed the immaturity of somatic embryos: no protein or lipid reserves were observed in the vacuolated cotyledonary cells. Maturation of somatic embryos was improved by a 2-month cold period. In cold-treated somatic embryos, both NL and PC increased to levels comparable to those observed in mature zygotic embryos, and the PE content reached 10 times the level of that in mature zygotic embryos. The cold treatment induced a large increase in the saturated/unsaturated fatty acid ratio in phospholipids but only a slight increase in that of neutral glycerolipids. Histological studies revealed a lipid accumulation at cellular level. Lipid bodies surrounded by protein bodies were observed in cotyledonary cells of cold-treated somatic embryos. Furthermore, the cold-treated somatic embryos developed into plantlets with a frequency of 14%, whereas no development was obtained with the non-treated somatic embryos.     

Computational study,synthesis and evaluation of active peptides derived from Parasporin-2 and spike protein from Alphacoronavirus against colorectal cancer cells

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and β-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.     

Development of bovine embryos in single in vitro production (sIVP) systems

Single in vitro production (sIVP) of embryos enables the study of developmental parameters of individual oocytes or embryos. Because several previously published sIVP systems showed varying levels of success, we attempted to design a simple, semidefined sIVP system that resulted in developmental rates similar to those obtained through group production (gIVP). In a 5 × 3 × 4 factorial experiment, 4200 oocytes were randomly assigned to combinations of various maturation (sIVM), fertilization (sIVF), and culture (sIVC) treatments based on media TCM199 (5 treatments), TALP (3 treatments), and SOF/aa/BSA (4 treatments), respectively. All sIVP steps were carried out in 10–12 μl drops under oil. Embryo development to blastocyst on days 7 and 8 of culture was determined and blastocyst cell numbers measured as an indicator of embryo quality. No interaction was found within any combination of sIVM, sIVF and sIVC treatments. Also, there was no difference in percentage of development to various stages for embryos in any of the sIVM or sIVF treatments (over all treatment combinations). However, when treatment combinations included charcoal-treated serum addition on day 5 of culture, a significant increase in development (39.0% total blastocysts/total oocytes vs. 22.7, 23.8 and 23.5% for the other 3 sIVC treatments, respectively; P < 0.001) and decrease in mean cell number (114.2 vs. 149.1, 150.5 and 143.7 cells, respectively; P < 0.001) was observed. These results are comparable to those routinely obtained in this laboratory with gIVP and establish standard conditions for individual embryo production. Mol. Reprod. Dev. 51:143–147, 1998. © 1998 Wiley-Liss, Inc.