Proteomic Approach for Extracting Cytoplasmic Proteins from Streptococcus sanguinis using Mass Spectrometry

Streptococcus sanguinis is a commensal and early colonizer of oral cavity as well as an opportunistic pathogen of infectious endocarditis. Extracting the soluble proteome of this bacterium provides deep insights about the physiological dynamic changes under different growth and stress conditions, thus defining “proteomic signatures” as targets for therapeutic intervention. In this protocol, we describe an experimentally verified approach to extract maximal cytoplasmic proteins from Streptococcus sanguinis SK36 strain. A combination of procedures was adopted that broke the thick cell wall barrier and minimized denaturation of the intracellular proteome, using optimized buffers and a sonication step. Extracted proteome was quantitated using Pierce BCA Protein Quantitation assay and protein bands were macroscopically assessed by Coomassie Blue staining. Finally, a high resolution detection of the extracted proteins was conducted through Synapt G2Si mass spectrometer, followed by label-free relative quantification via Progenesis QI. In conclusion, this pipeline for proteomic extraction and analysis of soluble proteins provides a fundamental tool in deciphering the biological complexity of Streptococcus sanguinis

Remendando sueños

No sé que tenga que ver el cuerpo atlético y el movimiento de gambeta del rey Pele con el pasito acicalado y el sombrero ladeado de Gardel, pero donde los vi juntos, dejando caer la mirada en blando tobogán desde la altura de un balcón inexistente, una sutil luz azul neón y cierto aire imantado une las más inverosímiles situaciones, en un espacio donde la ley de la gravedad permite volar y la del accidente no deja que a uno se le parta una pata. Aquí también esta la boa, larga, amarilla y canela, con sus secretos diseños en la espalda, cuya carne rosada y fresca ya puedo oler y alcanzo a sentir su sabor, parecido al del crudo y delicioso salmón que reparten en los aviones, sin llegar a ponerla en mi boca. La culebra esta estirada en sentido vertical a mi mirada y tiene un corte longitudinal finamente trazado en la espalda. Tengo la incierta certeza de que no debo cortar la cabeza para que la relación de energía entre los ojos y el cuerpo no se pierda, pero debo  desvestirla

Bacterial strains, plasmids, and primers used in this study.

<p>Bacterial strains, plasmids, and primers used in this study.</p

SEM analysis further reveals altered biofilm morphology and an increase in filamentous structures.

<p>Biofilms formed by the wild-type SK36, the <i>brpT</i> mutant, Δ<i>brpT</i>, and the complemented mutant, Δ<i>brpT_C</i>, scanned under (A) 1000x magnification and (B) 20,000x magnification revealed an altered morphology and an increase in filamentous structures for Δ<i>brpT</i> compared to the wild-type and complemented mutant. White arrows indicate filamentous substances.</p

Efficiency of glucan accumulation in <i>S</i>. <i>sanguinis</i> biofilms.

<p><i>S</i>. <i>sanguinis</i> wild-type SK36, the <i>brpT</i> mutant Δ<i>brpT</i> and the complemented mutant Δ<i>brpT</i>_C were grown anaerobically for 24 h in BM medium containing 1% sucrose at 37°C. The amounts of (A) water soluble glucans and (B) water insoluble glucans in the biofilms were quantified using the phenol-sulfuric acid method and normalized to the concentration of genomic DNA.</p

Determination of glucans accumulated in <i>S</i>. <i>sanguinis</i> biofilm.

<p>Determination of glucans accumulated in <i>S</i>. <i>sanguinis</i> biofilm.</p

Deletion of <i>gtfP</i> in <i>S</i>. <i>sanguinis</i> decreases biofilm attachment and glucan synthesis.

<p>Wild-type, the <i>gtfP</i> mutant, Δ<i>gtfP</i>, and the double mutant, Δ<i>brpT</i>/Δ<i>gtfP</i>, were cultured in BM with 1% sucrose for 24 h anaerobically and analyzed. (A) Weak attachment of the Δ<i>gtfP</i> and the Δ<i>brpT</i>/Δ<i>gtfP</i> biofilm (pellicle) to the polystyrene surface and reduced biofilm biomass determined by CV staining. (B) Quantification of biofilm formation (OD₆₀₀). Quantification of (C) water soluble glucans, WSG and (D) water insoluble glucans, WIG accumulated within the biofilm. **, indicates significance with <i>P</i> <0.01.</p

Deletion of <i>brpT</i> alters the biofilm structure.

<p>(A) Wild-type <i>S</i>. <i>sanguinis</i>, SK36, the <i>brpT</i> mutant, Δ<i>brpT</i>, and the complemented mutant, Δ<i>brpT</i>_C were grown in BM as described in <i>Materials and Methods</i>. After 24-h growth, the biofilms were washed and stained with SYTO 9, and <i>z</i>-stacks of each were acquired by CLSM with a Plan-Neofluar ×10/0.3 objective lens. Representative orthogonal views from three independent experiments are displayed. (B) Quantification of biofilm thickness by CLSM for the wild-type, Δ<i>brpT</i> and Δ<i>brpT</i>_C. (C) Quantification of biofilm roughness for the wild-type, Δ<i>brpT</i> and Δ<i>brpT</i>_C. **, indicates significance with <i>P</i> <0.01.</p