Srv Mediated Dispersal of Streptococcal Biofilms Through SpeB Is Observed in CovRS+ Strains

Group A Streptococcus (GAS) is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1) and MGAS315 (serotype M3), both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong

E64 treatment reduced lesion size in RGAS053Δ<i>srv</i> infected mice but increased lesion size in MGAS315Δ<i>srv</i> infected mice.

<p>The infecting dose (∼2×10⁸ CFU) of MGAS315, MGAS315Δ<i>srv,</i> RGAS053 or RGAS053Δ<i>srv</i> was resuspended in 333 µM E64 (0.1 ml), and 333 µM E64 (0.1 ml) was injected directly into the abscess each day following infection (n = 3 mice/strain). Lesion development (mm²) and weight were monitored over 8 days. Representative images of subcutaneous infections are shown from 1, 3, and 8 dpi for each strain. (A) No difference was observed between E64 treated (open circles) and untreated (closed circles) RGAS053 infections over 8 dpi. (B) A trend was observed where lesion formation was decreased following E64 treatment of RGAS053Δ<i>srv</i> (open circles) compared to inoculation with RGAS053Δ<i>srv</i> alone (closed circles). (C) No difference was observed between E64 treated (open triangles) and untreated (closed triangles) MGAS315 infections over 8 dpi. (D) Lesion formation was significantly increased following E64 treatment of MGAS315Δ<i>srv</i> (open triangles) compared to untreated infections (closed triangles) (*p≤0.05; unpaired t-test).</p

Detection of active SpeB in GAS biofilms.

<p>Western immunoblot analysis was used to detect the presence of SpeB in supernatants collected from (A) RGAS053, (B) MGAS315 (C) MGAS5005 wild-type and Δ<i>srv</i> static biofilms at 12, 24, 36 and 48 h post seeding. The Mean Pixel Intensity (MPI) of active SpeB (28 kDa) was measured with Carestream Image Software. MPI of SpeB for both RGAS053 and MGAS315 increased over 48 h, and SpeB production was increased in Δ<i>srv</i> mutants compared to wild-type for both strains. Low/no SpeB was detected in MGAS5005 biofilms, but was detected in MGAS5005Δ<i>srv</i> biofilms at all time points.</p

Allelic replacement of <i>srv</i> lead to increased lesion size in a murine subcutaneous infection model.

<p>Groups of 10 mice (Crl:SKH1-hrBR) were challenged subcutaneously with ∼2.0×10⁸ CFU (0.1 ml) of either MGAS315, MGAS315Δ<i>srv</i>, RGAS053 or RGAS053Δ<i>srv</i>. The area of the lesion formed (mm²) was measured with a caliper daily and the percentage of weight lost was monitored for 8 dpi. (A) A trend of increased lesion area formed by RGAS053Δ<i>srv</i> (open circles) than those formed by RGAS053 (closed circles) was observed. Over the course of the infection, there was no difference in weight loss except at 8 dpi. (B) A trend of larger lesions was also observed for MGAS315Δ<i>srv</i> infected mice (open triangles) when compared to MGAS315 infected mice (closed triangles). Beginning at 5 dpi, mice infected with MGAS315 (closed triangles) had increased weight loss compared to those infected with MGAS315Δ<i>srv</i> (open triangles) (*p≤0.05, **p≤0.01, ***<i>p</i>≤.001; unpaired t-test).</p

Inactivation of <i>srv</i> in RGAS053 and MGAS315 resulted in decreased biofilm formation.

<p>Log-phase cultures of (A) RGAS053 (solid line) and RGAS053Δ<i>srv</i> (dashed line), (B) MGAS315 (solid line) and MGAS315Δ<i>srv</i> (dashed line), or (C) MGAS5005 (solid line) MGAS5005Δ<i>srv</i> (dashed line) were grown in 6-well plates and adherence was measured over a course of 48h using a CV staining assay. All Δ<i>srv</i> mutants were significantly reduced in forming biofilms compared to wild-type strains. Each reported value for the CV assay is an average of 6 replicates and is adjusted by the dilution factor required to obtain a spectrophometric reading (OD_{600 nm}) (*<i>p</i>≤0.01, **<i>p</i>≤0.001, ***<i>p</i>≤.0001; unpaired t-test).</p

COMSTAT analysis of MGAS315 and MGAS315Δ<i>srv in vitro</i> static biofilms.

<p>Static biofilms were stained with a LIVE/DEAD reagent and imaged using CLSM for COMSTAT analysis. 12 individual fields of view were used for each strain from 12, 24 and 48h biofilms. (A) Total biomass of RGAS053 was significantly greater than RGAS0535Δ<i>srv</i> at all timepoints, and formed significantly thicker biofilms at 48 h. (B, C) Total biomass and average thickness were significantly increased in wild-type strains compared to Δ<i>srv</i> strains for both MGAS315 and MGAS5005, respectively. (**<i>p</i>≤.01, ***<i>p</i>≤0.001; unpaired t-test).</p

Enzymic inhibition and disruption of <i>in vitro</i> wild-type and Δ<i>srv</i> biofilms.

<p>DNase I (200 µg/ml) or proteinase K (0.1 or 1 mg/ml) were added to <i>in vitro</i> biofilms at the time of seeding (inhibition) or after 24h growth (disruption). (A) RGAS053 and RGAS053Δ<i>srv</i> biofilm formation were both disrupted and inhibited by DNase I and proteinase K. (B) MGAS315 and MGAS315Δ<i>srv</i> were not inhibited or disrupted by DNase I. Proteinase K inhibited and disrupted MGAS315 biofilm formation. MGAS315Δ<i>srv</i> biofilm formation was inhibited by 1 mg/ml proteinase K, but biofilm disruption was not observed with either concentration of proteinase K. (C) Proteinase K and DNase I both inhibited and disrupted MGAS5005 and MGAS5005Δ<i>srv</i> biofilm formation. Each reported value for the CV assay is an average of 6 replicates and is adjusted by the dilution factor required to obtain a spectrophometric reading (OD_{600 nm}) (*<i>p</i>≤.05, **<i>p</i>≤.01, ***<i>p</i>≤0.001; unpaired t-test).</p