Caspase-3 cleavage of Salmonella type III secreted effector protein SifA is required for localization of functional domains and bacterial dissemination

SifA is a bi-functional Type III Secretion System (T3SS) effector protein that plays an important role in Salmonella virulence. The N-terminal domain of SifA binds SifA-Kinesin-Interacting-Protein (SKIP), and via an interaction with kinesin, forms tubular membrane extensions called Sif filaments (Sifs) that emanate from the Salmonella Containing Vacuole (SCV). The C-terminal domain of SifA harbors a WxxxE motif that functions to mimic active host cell GTPases. Taken together, SifA functions in inducing endosomal tubulation in order to maintain the integrity of the SCV and promote bacterial dissemination. Since SifA performs multiple, unrelated functions, the objective of this study was to determine how each functional domain of SifA becomes processed. Our work demonstrates that a linker region containing a caspase-3 cleavage motif separates the two functional domains of SifA. To test the hypothesis that processing of SifA by caspase-3 at this particular site is required for function and proper localization of the effector protein domains, we developed two tracking methods to analyze the intracellular localization of SifA. We first adapted a fluorescent tag called phiLOV that allowed for type-III secretion system (T3SS) mediated delivery of SifA and observation of its intracellular colocalization with caspase-3. Additionally, we created a dual-tagging strategy that permitted tracking of each of the SifA functional domains following caspase-3 cleavage to different subcellular locations. The results of this study reveal that caspase-3 cleavage of SifA is required for the proper localization of functional domains and bacterial dissemination. Considering the importance of these events in Salmonella pathogenesis, we conclude that caspase-3 cleavage of effector proteins is a more broadly applicable effector processing mechanism utilized by Salmonella to invade and persist during infection

Study of second and third harmonic generation from an indium tin oxide nanolayer: Influence of nonlocal effects and hot electrons

We report comparative experimental and theoretical studies of the second and third harmonic generation from a 20 nm-thick indium tin oxide layer in proximity of the epsilon-near-zero condition. Using a tunable optical parametric amplifier, we record both spectral and angular dependence of the generated harmonic signals close to this particular point. In addition to the enhancement of the second harmonic efficiency close to the epsilon-near-zero wavelength, at oblique incidence, third harmonic generation displays an unusual behavior, predicted but not observed before. We implement a comprehensive, first-principles hydrodynamic approach able to simulate our experimental conditions. The model is unique, flexible, and able to capture all major physical mechanisms that drive the electrodynamic behavior of conductive oxide layers: nonlocal effects, which blueshift the epsilon-near-zero resonance by tens of nanometers; plasma frequency redshift due to variations of the effective mass of hot carriers; charge density distribution inside the layer, which determines the nonlinear surface and magnetic interactions; and the nonlinearity of the background medium triggered by bound electrons. We show that, by taking these contributions into account, our theoretical predictions are in very good qualitative and quantitative agreement with our experimental results. We expect that our results can be extended to other geometries where epsilon-near-zero nonlinearity plays an important role.Peer ReviewedPostprint (published version

Regulatory T cells control the dynamic and site-specific polarization of total CD4 T cells following Salmonella infection

FoxP3+ regulatory T cells (Tregs) control inflammation and maintain mucosal homeostasis, but their functions during infection are poorly understood. Th1, Th2, and Th17 cells can be identified by master transcription factors (TFs) T-bet, GATA3, and RORγT; Tregs also express these TFs. While T-bet+ Tregs can selectively suppress Th1 cells, it is unclear whether distinct Treg populations can alter Th bias. To address this, we used Salmonella enterica serotype Typhimurium to induce nonlethal colitis. Following infection, we observed an early colonic Th17 response within total CD4 T cells, followed by a Th1 bias. The early Th17 response, which contains both Salmonella-specific and non-Salmonella-specific cells, parallels an increase in T-bet+ Tregs. Later, Th1 cells and RORγT+ Tregs dominate. This reciprocal dynamic may indicate that Tregs selectively suppress Th cells, shaping the immune response. Treg depletion 1–2 days post-infection shifted the early Th17 response to a Th1 bias; however, Treg depletion 6–7 days post-infection abrogated the Th1 bias. Thus, Tregs are necessary for the early Th17 response, and for a maximal Th1 response later. These data show that Tregs shape the overall tissue CD4 T cell response and highlight the potential for subpopulations of Tregs to be used in targeted therapeutic approaches

Kin-Aggregations Explain Chaotic Genetic Patchiness, a Commonly Observed Genetic Pattern, in a Marine Fish

The phenomenon of chaotic genetic patchiness is a pattern commonly seen in marine organisms, particularly those with demersal adults and pelagic larvae. This pattern is usually associated with sweepstakes recruitment and variable reproductive success. Here we investigate the biological underpinnings of this pattern in a species of marine goby Coryphopterus personatus. We find that populations of this species show tell-tale signs of chaotic genetic patchiness including: small, but significant, differences in genetic structure over short distances; a non-equilibrium or “chaotic” pattern of differentiation among locations in space; and within locus, within population deviations from the expectations of Hardy-Weinberg equilibrium (HWE). We show that despite having a pelagic larval stage, and a wide distribution across Caribbean coral reefs, this species forms groups of highly related individuals at small spatial scales (metres). These spatially clustered family groups cause the observed deviations from HWE and local population differentiation, a finding that is rarely demonstrated, but could be more common than previously thought

Radio Properties of z>4 Optically-Selected Quasars

We report on two programs to address differential evolution between the radio-loud and radio-quiet quasar populations at high (z>4) redshift. Both programs entail studying the radio properties of optically-selected quasars. First, we have observed 32 optically-selected, high-redshift (z>4) quasars with the VLA at 6 cm (5 GHz). These sources comprise a statistically complete and well-understood sample. We detect four quasars above our 3-sigma limit of ~0.15 mJy, which is sufficiently sensitive to detect all radio-loud quasars at the probed redshift range. Second, we have correlated 134 z>4 quasars, comprising all such sources that we are aware of as of mid-1999, with FIRST and NVSS. These two recent 1.4 GHz VLA sky surveys reach 3-sigma limits of approximately 0.6 mJy and 1.4 mJy respectively. We identify a total of 15 z>4 quasars, of which six were not previously known to be radio-loud. The depth of these surveys does not reach the radio-loud/radio-quiet demarcation luminosity density (L(1.4 GHz) = 10^32.5 h(50)^(-2) ergs/s/Hz) at the redshift range considered; this correlation therefore only provides a lower limit to the radio-loud fraction of quasars at high-redshift. The two programs together identify eight new radio-loud quasars at z>4, a significant increase over the seven currently in the published literature. We find no evidence for radio-loud fraction depending on optical luminosity for -25 > M_B > -28 at z~2, or for -26>M_B>-28 at z>4. Our results also show no evolution in the radio-loud fraction between z~2 and z>4 (-26>M_B>-28).Comment: 19 pages, 7 figures; to appear in The Astronomical Journal (April 2000

Draft Genome Sequence of the Tumor-Targeting Salmonella enterica Serovar Typhimurium Strain SL7207

Salmonella enterica serovar Typhimurium strain SL7207 is a genetically modified derivative of strain SL1344, which preferentially accumulates in tumors and can be used as a vehicle for tis..

Draft Genome Sequence of the Commensal Escherichia coli Strain F-18

Here, we report the draft genome sequence of Escherichia coli strain F-18, originally isolated from the feces of a healthy individual in 1977. The draft genome is 5,246,829 bp, with a G+C content of 50.50%, and it encodes 4,933 predicted coding sequences (CDSs), 10 rRNAs, and 84 tRNAs

Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohn’s disease associated adherent-invasive Escherichia coli infection

Bacterial infection is a dynamic process resulting in a heterogenous population of infected and uninfected cells. These cells respond differently based on their bacterial load and duration of infection. In the case of infection of macrophages with Crohn’s disease (CD) associated adherent-invasive Escherichia coli (AIEC), understanding the drivers of pathogen success may allow targeting of cells where AIEC replicate to high levels. Here we show that stratifying immune cells based on their bacterial load identifies novel pathways and therapeutic targets not previously associated with AIEC when using a traditional homogeneous infected population approach. Using flow cytometry-based cell sorting we stratified cells into those with low or high intracellular pathogen loads, or those which were bystanders to infection. Immune cells transcriptomics revealed a diverse response to the varying levels of infection while pathway analysis identified novel intervention targets that were directly related to increasing intracellular AIEC numbers. Chemical inhibition of identified targets reduced AIEC intracellular replication or inhibited secretion of tumour necrosis factor alpha (TNFα), a key cytokine associated with AIEC infection. Our results have identified new avenues of intervention in AIEC infection that may also be applicable to CD through the repurposing of already available inhibitors. Additionally, they highlight the applicability of immune cell stratification post-infection as an effective approach for the study of microbial pathogens

A level playing ‘field’? A Bourdieusian analysis of the career aspirations of further education students on sports courses

There is currently a distinct dearth of research into how sports students’ career aspirations are formed during their post-compulsory education. This article, based on an ethnographic study of sport students in tertiary education, draws on data collected from two first-year cohorts (n = 34) on two different courses at a further education college in England. The study draws on ethnographic observations, and semi-structured group interviews, to examine in-depth the contrasting occupational perspectives emergent within these two groups of mainly working-class students, and how specific cultural practices affect students’ career aspirations. Utilising a Bourdieusian framework, the paper analyses the internalised, often latent cultural practices that impact upon these students’ diverse career aspirations. The hitherto under-researched dimension of inter-habitus interaction and also the application of doxa are outlined. The article reveals how the two student cohorts are situated within a complex field of relations, where struggles for legitimisation, academic accomplishment and numerous forms of lucrative capital become habituated. The study offers salient Bourdieusian-inspired insights into the career aspirations of these predominantly working-class students and the ways in which certain educational practices contribute to the production and reproduction of class inequalities