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

Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes.

Glucocorticoids and FoxO3 exert similar metabolic effects in skeletal muscle. FoxO3 gene expression was increased by dexamethasone (Dex), a synthetic glucocorticoid, both in vitro and in vivo. In C2C12 myotubes the increased expression is due to, at least in part, the elevated rate of FoxO3 gene transcription. In the mouse FoxO3 gene, we identified three glucocorticoid receptor (GR) binding regions (GBRs): one being upstream of the transcription start site, -17kbGBR; and two in introns, +45kbGBR and +71kbGBR. Together, these three GBRs contain four 15-bp glucocorticoid response elements (GREs). Micrococcal nuclease (MNase) assay revealed that Dex treatment increased the sensitivity to MNase in the GRE of +45kbGBR and +71kbGBR upon 30- and 60-min Dex treatment, respectively. Conversely, Dex treatment did not affect the chromatin structure near the -17kbGBR, in which the GRE is located in the linker region. Dex treatment also increased histone H3 and/or H4 acetylation in genomic regions near all three GBRs. Moreover, using chromatin conformation capture (3C) assay, we showed that Dex treatment increased the interaction between the -17kbGBR and two genomic regions: one located around +500 bp and the other around +73 kb. Finally, the transcriptional coregulator p300 was recruited to all three GBRs upon Dex treatment. The reduction of p300 expression decreased FoxO3 gene expression and Dex-stimulated interaction between distinct genomic regions of FoxO3 gene identified by 3C. Overall, our results demonstrate that glucocorticoids activated FoxO3 gene transcription through multiple GREs by chromatin structural change and DNA looping

Effects of Cluster Sets and Rest-Redistribution on Mechanical Responses to Back Squats in Trained Men

Eight resistance-trained men completed three protocols separated by 48-96 hours. Each protocol included 36 repetitions with the same rest duration, but the frequency and length of rest periods differed. The cluster sets of four (CS4) protocol included 30 s of rest after the 4th, 8th, 16th, 20th, 28th, and 32nd repetition in addition to 120 s of rest after the 12th and 24th repetition. For the other two protocols, the total 420 s rest time of CS4 was redistributed to include nine sets of four repetitions (RR4) with 52.5 s of rest after every four repetitions, or 36 sets of single repetitions (RR1) with 12 s of rest after every repetition. Mean (MF) and peak (PF) force, velocity (MV and PV), and power output (MP and PP) were measured during 36 repetitions and were collapsed into 12 repetitions for analysis. Repeated measures ANOVA 3 (protocol) x 12 (repetition) showed a protocol x repetition interaction for PF, MV, PV, MP, and PP (p-values fro

International Graduate Student Labor as Mergers and Acquisitions

This study critically examines the self-reported experiences of international graduate students using a framework understanding internationalization as acquisitions and mergers. Students reported positive experiences with their advisors. However, students’ accounts of laboratories and other research settings were diverse, ranging from co-contributors to knowledge and respected collaborators to employed cheap labor that their advisors depended upon for their own gains. In some cases, these students feared that their funding would be cut off or dismissed from the program (and consequently deported from the US) if they challenged their advisors. Whether such apprehensions were valid is unknown as this study focused on perceptions of the students only. The findings do lead to important future directions for research and practice

Angular Dependence in Proton-Proton Correlation Functions in Central 40Ca+40Ca^{40}Ca+^{40}Ca and 48Ca+48Ca^{48}Ca+^{48}Ca Reactions

The angular dependence of proton-proton correlation functions is studied in central $^{40}Ca+^{40}Ca$ and $^{48}Ca+^{48}Ca$ nuclear reactions at E=80 MeV/A. Measurements were performed with the HiRA detector complemented by the 4$\pi$ Array at NSCL. A striking angular dependence in the laboratory frame is found within p-p correlation functions for both systems that greatly exceeds the measured and expected isospin dependent difference between the neutron-rich and neutron-deficient systems. Sources measured at backward angles reflect the participant zone of the reaction, while much larger sources observed at forward angles reflect the expanding, fragmenting and evaporating projectile remnants. The decrease of the size of the source with increasing momentum is observed at backward angles while a weaker trend in the opposite direction is observed at forward angles. The results are compared to the theoretical calculations using the BUU transport model.Comment: 8 pages, 3 figures, submitted to PR

Investigations of three, four, and five-particle exit channels of levels in light nuclei created using a 9C beam

The interactions of a E/A=70-MeV 9C beam with a Be target was used to populate levels in Be, B, and C isotopes which undergo decay into many-particle exit channels. The decay products were detected in the HiRA array and the level energies were identified from their invariant mass. Correlations between the decay products were examined to deduce the nature of the decays, specifically to what extent all the fragments were created in one prompt step or whether the disintegration proceeded in a sequential fashion through long-lived intermediate states. In the latter case, information on the spin of the level was also obtained. Of particular interest is the 5-body decay of the 8C ground state which was found to disintegrate in two steps of two-proton decay passing through the 6Beg.s. intermediate state. The isobaric analog of 8Cg.s. in 8B was also found to undergo two-proton decay to the isobaric analog of 6Beg.s. in 6Li. A 9.69-MeV state in 10C was found to undergo prompt 4-body decay to the 2p+2alpha exit channel. The two protons were found to have a strong enhancementin the diproton region and the relative energies of all four p-alpha pairs were consistent with the 5Lig.s. resonance