Role of EscU auto-cleavage in promoting type III effector translocation into host cells by enteropathogenic Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Type III secretion systems (T3SS) of bacterial pathogens coordinate effector protein injection into eukaryotic cells. The YscU/FlhB group of proteins comprises members associated with T3SS which undergo a specific auto-cleavage event at a conserved NPTH amino acid sequence. The crystal structure of the C-terminal portion of EscU from enteropathogenic <it>Escherichia coli </it>(EPEC) suggests this auto-cleaving protein provides an interface for substrate interactions involved in type III secretion events.</p> <p>Results</p> <p>We demonstrate EscU must be auto-cleaved for bacteria to efficiently deliver type III effectors into infected cells. A non-cleaving EscU(N262A) variant supported very low levels of <it>in vitro </it>effector secretion. These effector proteins were not able to support EPEC infection of cultured HeLa cells. In contrast, EscU(P263A) was demonstrated to be partially auto-cleaved and moderately restored effector translocation and functionality during EPEC infection, revealing an intermediate phenotype. EscU auto-cleavage was not required for inner membrane association of the T3SS ATPase EscN or the ring forming protein EscJ. In contrast, in the absence of EscU auto-cleavage, inner membrane association of the multicargo type III secretion chaperone CesT was altered suggesting that EscU auto-cleavage supports docking of chaperone-effector complexes at the inner membrane. In support of this interpretation, evidence of novel effector protein breakdown products in secretion assays were linked to the non-cleaved status of EscU(N262A).</p> <p>Conclusions</p> <p>These data provide new insight into the role of EscU auto-cleavage in EPEC. The experimental data suggests that EscU auto-cleavage results in a suitable binding interface at the inner membrane that accommodates protein complexes during type III secretion events. The results also demonstrate that altered EPEC genetic backgrounds that display intermediate levels of effector secretion and translocation can be isolated and studied. These genetic backgrounds should be valuable in deciphering sequential and temporal events involved in EPEC type III secretion.</p

Hydration in Deep Eutectic Solvents Induces Non-monotonic Changes in the Conformation and Stability of Proteins

The preservation of labile biomolecules presents a major challenge in chemistry, and deep eutectic solvents (DESs) have emerged as suitable environments for this purpose. However, how the hydration of DESs impacts the behavior of proteins is often neglected. Here, we demonstrate that the amino acid environment and secondary structure of two proteins (bovine serum albumin and lysozyme) and an antibody (immunoglobulin G) in 1:2 choline chloride:glycerol and 1:2 choline chloride:urea follow a re-entrant behavior with solvent hydration. A dome-shaped transition is observed with a folded or partially folded structure at very low (40 wt % H2O) DES hydration, while protein unfolding increases between those regimes. Hydration also affects protein conformation and stability, as demonstrated for bovine serum albumin in hydrated 1:2 choline chloride:glycerol. In the neat DES, bovine serum albumin remains partially folded and unexpectedly undergoes unfolding and oligomerization at low water content. At intermediate hydration, the protein begins to refold and gradually retrieves the native monomer–dimer equilibrium. However, ca. 36 wt % H2O is required to recover the native folding fully. The half-denaturation temperature of the protein increases with decreasing hydration, but even the dilute DESs significantly enhance the thermal stability of bovine serum albumin. Also, protein unfolding can be reversed by rehydrating the sample to the high hydration regime, also recovering protein function. This correlation provides a new perspective to understanding protein behavior in hydrated DESs, where quantifying the DES hydration becomes imperative to identifying the folding and stability of proteinsA.S.F. acknowledges the Spanish Ministerio de Universidades for the awarded Maria Zambrano fellowship. Also, the research in this study was performed with financial support from Vinnova─Swedish Governmental Agency for Innovation Systems within the NextBioForm Competence Centre and from The Crafoord Foundation (grant #20190750). The authors thank the Institute Laue-Langevin for the awarded beamtime (8-03-1049)S

The sex-specific transcriptome of the hermaphrodite sparid sharpsnout seabream (Diplodus puntazzo)

Background: Teleosts are characterized by a remarkable breadth of sexual mechanisms including various forms of hermaphroditism. Sparidae is a fish family exhibiting gonochorism or hermaphroditism even in closely related species. The sparid Diplodus puntazzo (sharpsnout seabream), exhibits rudimentary hermaphroditism characterized by intersexual immature gonads but single-sex mature ones. Apart from the intriguing reproductive biology, it is economically important with a continuously growing aquaculture in the Mediterranean Sea, but limited available genetic resources. Our aim was to characterize the expressed transcriptome of gonads and brains through RNA-Sequencing and explore the properties of genes that exhibit sex-biased expression profiles. Results: Through RNA-Sequencing we obtained an assembled transcriptome of 82,331 loci. The expression analysis uncovered remarkable differences between male and female gonads, while male and female brains were almost identical. Focused search for known targets of sex determination and differentiation in vertebrates built the sex-specific expression profile of sharpsnout seabream. Finally, a thorough genetic marker discovery pipeline led to the retrieval of 85,189 SNPs and 29,076 microsatellites enriching the available genetic markers for this species. Conclusions: We obtained a nearly complete source of transcriptomic sequence as well as marker information for sharpsnout seabream, laying the ground for understanding the complex process of sex differentiation of this economically valuable species. The genes involved include known candidates from other vertebrate species, suggesting a conservation of the toolkit between gonochorists and hermaphrodites

Inverse tuning of metal binding affinity and protein stability by altering charged coordination residues in designed calcium binding proteins

Ca2+ binding proteins are essential for regulating the role of Ca2+ in cell signaling and maintaining Ca2+ homeostasis. Negatively charged residues such as Asp and Glu are often found in Ca2+ binding proteins and are known to influence Ca2+ binding affinity and protein stability. In this paper, we report a systematic investigation of the role of local charge number and type of coordination residues in Ca2+ binding and protein stability using de novo designed Ca2+ binding proteins. The approach of de novo design was chosen to avoid the complications of cooperative binding and Ca2+-induced conformational change associated with natural proteins. We show that when the number of negatively charged coordination residues increased from 2 to 5 in a relatively restricted Ca2+-binding site, Ca2+ binding affinities increased by more than 3 orders of magnitude and metal selectivity for trivalent Ln3+ over divalent Ca2+ increased by more than 100-fold. Additionally, the thermal transition temperatures of the apo forms of the designed proteins decreased due to charge repulsion at the Ca2+ binding pocket. The thermal stability of the proteins was regained upon Ca2+ and Ln3+ binding to the designed Ca2+ binding pocket. We therefore observe a striking tradeoff between Ca2+/Ln3+ affinity and protein stability when the net charge of the coordination residues is varied. Our study has strong implications for understanding and predicting Ca2+-conferred thermal stabilization of natural Ca2+ binding proteins as well as for designing novel metalloproteins with tunable Ca2+ and Ln3+ binding affinity and selectivity

Self-Renewal Pathways in Acute Myeloid Leukemia Stem Cells

Acute myeloid leukemia (AML) is a difficult-to-treat blood cancer. A major challenge in treating patients with AML is relapse, which is caused by the persistence of leukemia stem cells (LSCs). Self-renewal is a defining property of LSCs and its deregulation is crucial for re-initiating a new leukemia after chemotherapy. Emerging therapeutic agents inhibiting aberrant self-renewal pathways, such as anti-RSPO3 monoclonal antibody discovered in our recent study, present significant clinical potential that may extend beyond the scope of leukemogenesis. In this chapter, we provide an overview of normal and malignant hematopoietic stem cells, discuss current treatments and limitations, and review key self-renewal pathways and potential therapeutic opportunities in AML

Cumulative effects matter: multi-brood responses of Daphnia to hypoxia

Periods of hypoxia lasting up to weeks are now anticipated in fresh waters, owing to anthropogenic influences. However, the cumulative effects of hypoxia on Daphnia, over multiple broods, have received virtually no attention, and to establish and evaluate such responses there is a need to make measurements over a wide range of oxygen concentrations, potentially revealing non-linear patterns. We predict that the effect of hypoxia on growth, survival and fecundity (i.e., production of new individuals) of Daphnia will increase over multiple broods, and with increasing oxygen these responses will approach asymptotic maxima, following a rectangular hyperbolic response. Daphnia similoides were exposed to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 8.0 mg oxygen L−1. To determine effects on the first brood we examined: number of offspring; time to the first eggs; time to the first brood and size of the female at the first eggs and the first brood. To determine cumulative effects of oxygen over multiple broods (up to 8 broods over 21 days), we measured: total number of offspring produced by a female; survival time and total number of moults and broods. To investigate how the cumulative effects arose over the multiple broods, we examined the number of offspring per brood in each brood over eight broods. To assess treatment effects and indicate responses, functions were fit to data using the most parsimonious function that reflected trends in the data. Measurements associated with a single brood responded linearly, or not at all, with changing oxygen concentration, whereas measurements made over the 21 days followed a rectangular hyperbolic response, increasing to an asymptote as oxygen increased. For the first brood, as oxygen concentration was raised from 1 to 8 mg L−1 the number of offspring produced and the time required to produce the brood were not affected; the time required to produce eggs decreased ˜0.3-fold; and the size of individuals at the time when the eggs and the brood were produced increased ˜0.1-fold. Over the 21 days, between 1 and 8 mg L−1 the total number of offspring increased ˜3.4-fold; individual survival and the number of moults increased ˜2-fold, and the number of broods increased ˜1-fold. For single-brood responses, there was no effect of decreasing oxygen levels on the number of offspring in the first brood, but there were negative effects on the second-to-fourth broods; the number of offspring in the remaining broods was not significantly related to oxygen levels, as there were fewer data at low levels due to poor survival. We conclude that assessments of Daphnia demographics should not rely on estimates of the effect of oxygen concentration on single broods. Instead, studies should consider cumulative changes over multiple broods. Following our approach, researchers may now explore the impacts of hypoxia on congeners and other zooplankton, and investigate the mechanisms associated with multi-brood responses

Foliar endophyte diversity in Eastern Asian-Eastern North American disjunct tree species – influences of host identity, environment, phylogeny, and geographic isolation

IntroductionThe well-known eastern Asian (EA) and eastern North American (ENA) floristic disjunction provides a unique system for biogeographic and evolutionary studies. Despite considerable interest in the disjunction, few studies have investigated the patterns and their underlying drivers of allopatric divergence in sister species or lineages isolated in the two areas. Endophyte diversity and assembly in disjunct sister taxa, as an ecological trait, may have played an important role in the processes of allopatric evolution, but no studies have examined endophytes in these lineages. Here we compared foliar endophytic fungi and bacteria-archaea (FEF and FEB) in 17 EA-ENA disjunct species or clade pairs from genera representing conifers and 10 orders of five major groups of angiosperms and 23 species of Cornus from EA and North America. MethodsMetagenomic sequencing of fungal ITS and bacterial-archaeal 16S rDNA was used to capture the foliar endophytic communities. Alpha and beta diversity of fungi and bacteria were compared at multiple scales and dimensions to gain insights into the relative roles of historical geographic isolation, host identity, phylogeny, and environment from samples at different sites in shaping endophytic diversity patterns. ResultsWe found that beta diversity of endophytes varied greatly among plant individuals within species and between species among genera at the same sampling site, and among three sampling sites, but little variation between region-of-origin of all plant species (EA vs ENA) and between EA-ENA disjunct counterparts within genera. Various numbers of indicator fungal species differing in abundance were identified for each plant genus and Cornus species. An overall significant correlation between endophyte community dissimilarity and phylogenetic distance of plants was detected among the disjunct genera but not among species of Cornus. However, significant correlations between beta diversities at different taxonomic scales of endophytes and phylogenetic distances of Cornus species were observed. DiscussionOur results suggest important roles of host identity and environment (sampling sites), and a likely minor role of phylogenetic divergence and historical biogeographic isolation in shaping the pattern of foliar endophyte diversity and assembly in the EA-ENA disjunct genera and Cornus. The results lead to a hypothesis that the sister taxa in EA and ENA likely differ in FEF and FEB when growing in native habitats due to differences in local environments, which may potentially drive allopatric divergence of the functional features of species