Polímeros capsulares bacterianos en E. coli K92: ácido colánico y ácido polisiálico

228 p.Escherichia coli K92 es un bacilo gram negativo capaz de sintetizar una cápsula de ácidos polisiálicos cuando crece a temperaturas superiores a los 20ºC. En primer lugar, este trabajo se ha centrado en el aislamiento, caracterización y purificación de un nuevo polímero capsular, conocido como ácido colánico, que esta bacteria produce principalmente cuando crece a temperaturas restrictivas (inferiores a 25ºC). Una vez identificados ambos polisacáridos capsulares, hemos tratado de averiguar la influencia de la temperatura de crecimiento sobre la expresión de varios genes implicados en la biosíntesis de ambos tipos de cápsulas y en la regulación de polímeros capsulares en E. coli. En base a estos últimos resultados, la obtención de mutantes por deleción de varios genes reguladores, tales como rcsA, rcsB y rfaH, nos ha permitido establecer el papel de dichos genes sobre la termorregulación que rige la biosíntesis de las cápsulas de ácido colánico y/o ácido polisiálico en E. coli K92. Finalmente, dado que las cápsulas de ácido colánico son sintetizadas bajo dichas condiciones restrictivas, nuestros estudios se han centrado en tratar de averiguar el papel fisiológico de este tipo de cápsulas en E. coli K92, para lo que se realizaron experimentos de adhesión bacteriana y formación de biofilms, así como de resistencia a antibióticos y a determinadas condiciones estresantes tales como estrés osmótico y oxidativ

Multiserotype Protection Elicited by a Combinatorial Prime-Boost Vaccination Strategy against Bluetongue Virus

Bluetongue virus (BTV) belongs to the genus Orbivirus within the family Reoviridae. The development of vector-based vaccines expressing conserved protective antigens results in increased immune activation and could reduce the number of multiserotype vaccinations required, therefore providing a cost-effective product. Recent recombinant DNA technology has allowed the development of novel strategies to develop marker and safe vaccines against BTV. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2, VP7 and NS1 proteins from BTV-4. IFNAR(−/−) mice inoculated with DNA/rMVA-VP2,-VP7-NS1 in an heterologous prime boost vaccination strategy generated significant levels of antibodies specific of VP2, VP7, and NS1, including those with neutralizing activity against BTV-4. In addition, vaccination stimulated specific CD8+ T cell responses against these three BTV proteins. Importantly, the vaccine combination expressing NS1, VP2 and VP7 proteins of BTV-4, elicited sterile protection against a lethal dose of homologous BTV-4 infection. Remarkably, the vaccine induced cross-protection against lethal doses of heterologous BTV-8 and BTV-1 suggesting that the DNA/rMVA-VP2,-VP7,-NS1 marker vaccine is a promising multiserotype vaccine against BTV

Modeling Dynamics of Human Gut Microbiota Derived from Gluten Metabolism: Obtention, Maintenance and Characterization of Complex Microbial Communities

[EN] Western diets are rich in gluten-containing products, which are frequently poorly digested. The human large intestine harbors microorganisms able to metabolize undigested gluten fragments that have escaped digestion by human enzymatic activities. The aim of this work was obtaining and culturing complex human gut microbial communities derived from gluten metabolism to model the dynamics of healthy human large intestine microbiota associated with different gluten forms. For this purpose, stool samples from six healthy volunteers were inoculated in media containing predigested gluten or predigested gluten plus non-digested gluten. Passages were carried out every 24 h for 15 days in the same medium and community composition along time was studied via V3–V4 16S rDNA sequencing. Diverse microbial communities were successfully obtained. Moreover, communities were shown to be maintained in culture with stable composition for 14 days. Under non-digested gluten presence, communities were enriched in members of Bacillota, such as Lachnospiraceae, Clostridiaceae, Streptococcaceae, Peptoniphilaceae, Selenomonadaceae or Erysipelotrichaceae, and members of Actinomycetota, such as Bifidobacteriaceae and Eggerthellaceae. Contrarily, communities exposed to digested gluten were enriched in Pseudomonadota. Hence, this study shows a method for culture and stable maintenance of gut communities derived from gluten metabolism. This method enables the analysis of microbial metabolism of gluten in the gut from a community perspectiveSIThis research was funded by Junta de Castilla y León, grant number LE015P20, and Ministerio de Ciencia e Innovación, grant PID2020-119044GB-I00. Y.C.M. received a grant from Conserjería de Educación of Junta de Castilla y León, co-funded by the European Social Fund (Orden de 12 de diciembre de 2019

Identification of Synthetic Host Defense Peptide Mimics That Exert Dual Antimicrobial and Anti-Inflammatory Activities

A group of synthetic antimicrobial oligomers, inspired by naturally occurring antimicrobial peptides, were analyzed for the ability to modulate innate immune responses to Toll-like receptor (TLR) ligands. These synthetic mimics of antimicrobial peptides (SMAMPs) specifically reduced cytokine production in response to Staphylococcus aureus and the S. aureus component lipoteichoic acid (LTA), a TLR2 agonist. Anti-inflammatory SMAMPs prevented the induction of tumor necrosis factor (TNF), interleukin 6 (IL-6), and IL-10 in response to S. aureus or LTA, but no other TLR2 ligands. We show that these SMAMPs bind specifically to LTA in vitro and prevent its interaction with TLR2. Importantly, the SMAMP greatly reduced the induction of TNF and IL-6 in vivo in mice acutely infected with S. aureus while simultaneously reducing bacterial loads dramatically (4 log10). Thus, these SMAMPs can eliminate the damage induced by pathogen-associated molecular patterns (PAMPs) while simultaneously eliminating infection in vivo. They are the first known SMAMPs to demonstrate anti-inflammatory and antibacterial activities in vivo

Detection of BTV RNA in blood of immunized and non-immunized IFNAR^(−/−) mice after challenge with BTV-4 by RT-qPCR_S5.

<p>Results expressed as <i>C</i>t and transferred to negative (neg.) according to the cut-off <i>C</i>t≥38 described by Toussaint et al. (2007).</p><p>I, immunized mice. C, nonimmunized mice.</p>†<p>, death mice.</p

Detection of BTV RNA in blood of immunized and non-immunized IFNAR^(−/−) mice after challenge with BTV-8 or BTV-1 by RT-qPCR_S5.

<p>Results expressed as <i>C</i>t and transferred to negative (neg.) according to the cut-off <i>C</i>t≥38 described by Toussaint et al. (2007).</p><p>I, immunized mice. C, nonimmunized mice.</p>†<p>, death mice.</p

Cytokine responses in IFNAR^(−/−) mice after immunization.

<p>Sera from mice immunized with pcDNA3-VP2,-VP7,-NS1 (day 0) and rMVA-VP2,-VP7,-NS1 (day 14) (solid symbols) or pcDNA3 (day 0) and MVA (day 14) (symbols) were collected at days 0, 14, 21, and 28. The levels of cytokines were evaluated by a multiplex fluorescent bead immunoassay for quantitative detection of 5 mouse cytokines (Millipore's MILLIPLEX Mouse Cytokine kit). Samples were analyzed with a Luminex 2010 (Luminex Corporation). Means are presented (⁃).</p

Protection of VP2, VP5 and VP7 vaccinated IFNAR^(−/−) mice against lethal BTV-4, BTV-8, and BTV-1 challenges.

<p>Mice (8 weeks old, 6 per group) were immunized twice by heterologous prime boost vaccination with DNAs and rMVAs expressing VP2, VP7, and NS1 BTV-4 proteins (immunized, black line) or pcDNA3 and MVA (non-immunized, dotted line), administered 2 weeks apart. Two weeks after immunization mice were intravenously inoculated with 100 PFUs (lethal dose) of BTV-8 (▪) or BTV-1 (•). (A) Survival rates of immunized and non-immunized IFNAR^(−/−) mice after inoculation with BTV-8 or BTV-1. The mice were observed every 24 h for 12 days. (B) Titers of BTV-8 (▪) or BTV-1 (•) recovered in blood of immunized and non-immunized IFNAR^(−/−) mice after challenge. Virus was extracted from blood and determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034735#s4" target="_blank">Materials and Methods</a>. Each point represents the mean values of the viral titer of six animals, and standard deviations are shown as error bars.</p