Maize seeds as a production and delivery platform for HIV microbicides

La infecció pel virus de la immunodeficiència humana (VIH), és un dels problemes sanitaris i de desenvolupament més greus del món actual. Evitar la infecció és una de les estratègies que podria ajudar a reduir l’expansió del virus. Els microbicides són una nova classe de fàrmacs que podrien ajudar a realitzar-ho. Alguns dels reptes a superar que podrien frenar el desenvolupament de microbicides efectius son: que les molècules resultants siguin efectives, que la producció es pugui ampliar ràpidament i que els costos de producció puguin ser reduïts. Un dels sistemes de producció més prometedors son les plantes, al ser un sistema econòmic i segur. La producció de proteïnes recombinants farmacèutiques en plantes s’anomena “molecular pharming”. El principal objectiu d’aquesta tesis és la producció, individual o combinada, de molècules microbicides en llavors de blat de moro per tal de desenvolupar una nova estratègia de producció mes econòmica aplicable als països en vies de desenvolupament. La bioseguretat i els temes relacionats amb la legislació relativa a cultius millorats genèticament també són una part important d’aquesta tesi, ja que frenen el desenvolupament i la comercialització global d’aquest tipus de cultius.El virus de la inmunodeficiencia humana (VIH) es en la actualidad uno de los problemas de salud pública más graves en el mundo. La aplicación de medidas preventivas para evitar la infección por VIH es la principal vía para reducir la expansión del virus. Los microbicidas son una nueva clase de productos antivirales de bajo coste que podrían ayudar a conseguirlo. Algunos de los obstáculos que pueden frenar el desarrollo de microbicidas son la viabilidad, el aumento de área de cultivo y los altos costes de producción. Se están evaluando muchas plataformas para la producción de microbicidas. Una de las más prometedoras son las plantas, ya que constituyen un sistema económico y seguro. A la producción de proteínas recombinantes farmacéuticas en plantas se le denomina “molecular pharming”. El principal objetivo de esta tesis es el estudio de la producción de moléculas microbicidas tanto a nivel individual (una sola molécula) como combinada (simultaneamente varias moléculas) en semillas de maíz con el fin de desarrollar una nueva estrategia mas económica capaz de evitar la infección por VIH en países en vías de desarrollo. La bioseguridad y los temas relacionados con la legislación relativa a cultivos mejorados genéticamente también son una parte importante de esta tesis, ya que limitan el desarrollo y la comercialización global de este tipo de cultivos.HIV remains one of the world’s most serious health and development challenges. Preventing HIV infection is one of the strategies that could slow down the spread of the virus. Microbicides are a new class of products that could address this need. However, the feasibility of manufacturing, scalability, and cost are some of the challenges that can undermine the development of cost effective microbicides. One of the most promising production systems is plants because of cost benefits and biological safety. Production of medically important recombinant proteins in plants is known as molecular pharming. The focus of this thesis is the production of microbicide components individually or in combination in maize seeds in order to develop a novel and inexpensive strategy for the prevention of HIV infections in the developing world. I also discuss non-technical barriers to the adoption of GE crops in the European Union and their serious consequences

Lack of the PGA exopolysaccharide in Salmonella as an adaptive trait for survival in the host

Many bacteria build biofilm matrices using a conserved exopolysaccharide named PGA or PNAG (poly-β-1,6-N-acetyl-D-glucosamine). Interestingly, while E. coli and other members of the family Enterobacteriaceae encode the pgaABCD operon responsible for PGA synthesis, Salmonella lacks it. The evolutionary force driving this difference remains to be determined. Here, we report that Salmonella lost the pgaABCD operon after the divergence of Salmonella and Citrobacter clades, and previous to the diversification of the currently sequenced Salmonella strains. Reconstitution of the PGA machinery endows Salmonella with the capacity to produce PGA in a cyclic dimeric GMP (c-di-GMP) dependent manner. Outside the host, the PGA polysaccharide does not seem to provide any significant benefit to Salmonella: resistance against chlorine treatment, ultraviolet light irradiation, heavy metal stress and phage infection remained the same as in a strain producing cellulose, the main biofilm exopolysaccharide naturally produced by Salmonella. In contrast, PGA production proved to be deleterious to Salmonella survival inside the host, since it increased susceptibility to bile salts and oxidative stress, and hindered the capacity of S. Enteritidis to survive inside macrophages and to colonize extraintestinal organs, including the gallbladder. Altogether, our observations indicate that PGA is an antivirulence factor whose loss may have been a necessary event during Salmonella speciation to permit survival inside the host.[Author summary] During bacterial evolution, specific traits that optimize the organism’s fitness are selected. The production of exopolysaccharides is widespread among bacteria in which they play a protective shielding role as main constituents of biofilms. In contrast to closely related siblings, Salmonella has lost the capacity to produce the exopolysaccharide PGA. Our study reveals that Salmonella lost pga genes, and that the driving force for such a loss may have been the detrimental impact that PGA has during Salmonella invasion of internal organs where it augments the susceptibility to bile salts and oxygen radicals, reducing bacterial survival inside macrophages and rendering Salmonella avirulent. These results suggest that gene-loss has played an important role during Salmonella evolution.This work was supported by the Spanish Ministry of Economy and Competitiveness grants BIO2014-53530-R and SAF2014-56716-REDT (http://www.mineco.gob.es/portal/site/mineco/?lang_choosen=en). JV was supported by Ramon y Cajal (RYC-2009-03948) contract from the Spanish Ministry of Economy and Competitiveness

High-value products from transgenic maize

Maize (also known as corn) is a domesticated cereal grain that has been grown as food and animal feed for tens of thousands of years. It is currently the most widely grown crop in the world, and is used not only for food/feed but also to produce ethanol, industrial starches and oils. Maize is now at the beginning of a new agricultural revolution, where the grains are used as factories to synthesize high-value molecules. In this article we look at the diversity of high-value products from maize, recent technological advances in the field and the emerging regulatory framework that governs how transgenic maize plants and their products are grown, used and traded. (C) 2010 Elsevier Inc. All rights reserved

PGA provides, at the most, similar protection to that conferred by cellulose against chlorine treatment, UV light irradiation, heavy metal stress and phage infection.

<p><b>(A)</b> The cellulose overproducing strain, WT PcL::<i>adrA</i>, the PGA overproducing strain, <i>ΔbcsA</i> PcL::<i>adrA</i> pJET::<i>pga</i>, and the control strain, <i>ΔbcsA</i> PcL::<i>adrA</i>, were incubated on LB agar or LB agar Cb media for 48h at 28°C. Macrocolonies were then exposed to 200 p.p.m. sodium hypochlorite for 40 min. Surviving bacteria were enumerated by viable plate counts, and their numbers were compared with that of control bacteria that had not been incubated with NaOCl. Note the logarithmic scale in the y axis. The data represent the mean of two independent experiments performed in triplicate. <b>(B)</b> Serial dilutions of the cellulose overproducing strain, the PGA overproducing strain and the exopolysaccharide minus strain were plated on N minimal medium containing carbenicillin and after 24 hours of incubation at 28°C, plates were exposed to five minutes of UV light irradiation. Replica plates were not subjected to treatment and served as controls. After 48 hours of incubation at 28°C, numbers of surviving bacteria were counted. Results are shown as % survival relative to untreated samples. <i>ΔbcsA</i> PcL::<i>adrA</i> and WT PcL::<i>adrA</i> carried an empty plasmid so that the three strains could be incubated on the same plates. Note the logarithmic scale in the y axis. The data represent the mean of three independent experiments. <b>(C)</b> Macrocolony biofilms were grown on sterile polymer membrane filters and then exposed to 0.5 mM CdCl₂ for 3 h. Surviving bacteria were enumerated by viable plate counts, and their numbers were compared with that of control bacteria that had not been incubated with CdCl₂. Note the logarithmic scale in the y axis. The data represent the mean of three independent experiments performed in duplicate. <b>(D)</b> Macrocolony biofilms were grown on sterile polymer membrane filters and then subjected to phage infection with a P22 phage lysate generated from a streptomycin resistant strain. Transductants were enumerated by plate counts on LB Sm media and their numbers were compared with those of the exopolysaccharide minus strain, <i>ΔbcsA</i> PcL::<i>adrA</i>, which defined 100% transduction. The data represent the mean of three independent experiments. Statistical analysis in all assays was carried out using a Mann-Whitney <i>U</i> test. ns = no significant difference; * P < 0.05; ** P < 0.01.</p

Heterologous expression of the <i>pgaABCD</i> operon in <i>Salmonella</i> drives PGA synthesis in response to c-di-GMP levels and makes <i>Salmonella</i> able to build a PGA mediated biofilm.

<p><b>(A)</b> Dot blot analysis of the PGA accumulated by <i>S</i>. Enteritidis wild type, ΔXII and WT PcL::<i>adrA</i> and their corresponding transformed strains with plasmid pJET::<i>pga</i> after 48 hours of incubation in LB or LB Cb broth, at room temperature, under static conditions. Serial dilutions (1/10) of the samples were spotted onto nitrocellulose membranes and PGA production was detected with specific anti PIA/PNAG antibodies. UD; undiluted sample. <b>(B)</b> Biofilm phenotypes of wild type, WT PcL::<i>adrA</i> and their corresponding transformed strains with plasmid pJET::<i>pga</i> after incubation in LB or LB Cb broth, at 28°C for 16 hours under shaking conditions. A ring of cells adhered to the glass wall at the air–liquid interface corresponds with a PGA based biofilm. <b>(C)</b> Scanning electron microscopy analysis of the biofilms developed by the PGA overproducing strain, <i>ΔbcsA</i> PcL::<i>adrA</i> pJET::<i>pga</i>, after incubation in LB broth, at 28°C for 16 hours under shaking conditions and by the cellulose overproducing strain, WT PcL::<i>adrA</i>, after incubation in LB broth, at room temperature under static conditions for 72 hours. A control strain, <i>ΔbcsA</i> PcL::<i>adrA</i>, that produces neither PGA nor cellulose was also analyzed after incubation in LB broth, at 28°C for 16 hours under shaking conditions.</p