Carlos Gamazo

Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose

We report here a new screening method based on the fluorescence of colonies on calcofluor agar plates to identify transposon insertion mutants of Salmonella enteritidis that are defective in biofilm development. The results not only confirmed the requirement of genes already described for the modulation of multicellular behaviour in Salmonella typhimurium and other species, but also revealed new aspects of the biofilm formation process, such as two new genetic elements, named as bcsABZC and bcsEFG operons, required for the synthesis of an exopolysaccharide, digestible with cellulase. Non‐polar mutations of bcsC and bcsE genes and complementation experiments demonstrated that both operons are respon‐sible for cellulose biosynthesis in both S. enteritidis and S. typhimurium. Using two different growth media, ATM and LB, we showed that the biofilm produced by S. enteritidis is made of different constituents, suggesting that biofilm composition and regulation depends on environmental conditions. Bacterial adherence and invasion assays of eukaryotic cells and in vivo virulence studies of cellulose‐deficient mutants indicated that, at least under our experimental conditions, the production of cellulose is not involved in the virulence of S. enteritidis. However, cellulose‐deficient mutants were more sensitive to chlorine treatments, suggesting that cellulose production and biofilm formation may be an important factor for the survival of S. enteritidis on surface environments.

BapA, a large secreted protein required for biofilm formation and host colonization of Salmonella enterica serovar Enteritidis.

In environmental settings, biofilms represent the common way of life of microorganisms. Salmonella enterica serovar Enteritidis, the most frequent cause of gastroenteritis in developed countries, produces a biofilm whose matrix is mainly composed of curli fimbriae and cellulose. In contrast to other bacterial biofilms, no proteinaceous compound has been reported to participate in the formation of this matrix. Here, we report the discovery of BapA, a large cell‐surface protein required for biofilm formation by S. Enteritidis. Deletion of bapA caused the loss of the capacity to form a biofilm whereas the overexpression of a chromosomal copy of bapA increased the biofilm biomass formation. We provide evidence that overproduction of curli fimbriae and not cellulose can compensate for the biofilm deficiency of a bapA mutant strain. BapA is secreted through a type I protein secretion system (BapBCD) situated downstream of the bapA gene and was found to be loosely associated with the cell surface. Experiments with mixed bacterial populations positive or negative for BapA showed that BapA minus cells are not recruited into the biofilm matrix. The expression of bapA is coordinated with that of genes encoding curli fimbriae and cellulose, through the action of csgD. Studies on the contribution of BapA to S. Enteritidis pathogenesis revealed that orally inoculated animals with a bapA‐deficient strain survived longer than those inoculated with the wild‐type strain. Also, a bapA mutant strain showed a significantly lower colonization rate at the intestinal cell barrier and consequently a decreased efficiency for organ invasion compared with the wild‐type strain. Taken together, these data demonstrate that BapA contributes both to biofilm formation and invasion through the regular Salmonella infection route.

Role of the GGDEF protein family in Salmonella cellulose biosynthesis and biofilm formation.

Salmonella enterica serovar Typhimurium is capable of producing cellulose as the main exopolysaccharide compound of the biofilm matrix. It has been shown for Gluconacetobacter xylinum that cellulose biosynthesis is allosterically regulated by bis‐(3′,5′) cyclic diguanylic acid, whose synthesis/degradation depends on diguanylate cyclase/phosphodiesterase enzymatic activities. A protein domain, named GGDEF, is present in all diguanylate cyclase/phosphodiesterase enzymes that have been studied to date. In this study, we analysed the molecular mechanisms responsible for the failure of Salmonella typhimurium strain SL1344 to form biofilms under different environmental conditions. Using a complementation assay, we were able to identify two genes, which can restore the biofilm defect of SL1344 when expressed from the plasmid pBR328. Based on the observation that one of the genes, STM1987, contains a GGDEF domain, and the other, mlrA, indirectly controls the expression of another GGDEF protein, AdrA, we proceeded on a mutational analysis of the additional GG[DE]EF motif containing proteins of S. typhimurium. Our results demonstrated that MlrA, and thus AdrA, is required for cellulose production and biofilm formation in LB complex medium whereas STM1987 (GGDEF domain containing protein A, gcpA) is critical for biofilm formation in the nutrient‐deficient medium, ATM. Insertional inactivation of the other six members of the GGDEF family (gcpB‐G) showed that only deletion of yciR (gcpE) affected cellulose production and biofilm formation. However, when provided on plasmid pBR328, most of the members of the GGDEF family showed a strong dominant phenotype able to bypass the need for AdrA and GcpA respectively. Altogether, these results indicate that most GGDEF proteins of S. typhimurium are functionally related, probably by controlling the levels of the same final product (cyclic di‐GMP), which include among its regulatory targets the cellulose production and biofilm formation of S. typhimurium.

Mannose-targeted systems for the delivery of therapeutics

Background: The specific targeting of nanomedicines to mannose receptors, highly expressed in cells of the immune system, performs a useful strategy for improving the efficacy of vaccines and chemotherapy. Objective: This review discusses the potential of mannose-targeted drug/antigen delivery systems for vaccination and treatment of diseases localized in macrophages and other antigen-presenting cells. Methods: The first part of the review describes the characteristics, localization and functions of mannose receptors. The following sections are devoted to the description of different methods used to deliver therapeutic agents, including mannose conjugates and mannosylated carriers or particulates (i.e., liposomes, nanoparticles and niosomes). Results/conclusions: A general overview of published reports confirms the effectiveness of mannosylation strategies, although the optimization and full exploitation of mannose-targeted drug delivery systems would require a deeper understanding of the structure–activity relationship. In the near future, these nanomedicines have the potential to treat a number of diseases (including cancer) and improve the quality of life of patients.

In vitro phagocytosis and monocyte-macrophage activation with poly(lactide) and poly(lactide-co-glycolide) microspheres

Treatment of many intracellular infections in the mononuclear phagocytic system (MPS), requires targeting of antibiotics by a drug delivery system. The objective of this study was to examine whether the particular nature of microspheres, made of end-group capped and uncapped poly(lactide) [PLA] and poly(lactide-co-glycolide) [PLGA 50:50 and PLGA 75:25], affect the uptake into and also the activation of monocyte-macrophages. Placebo and gentamicin sulfate containing microspheres were incubated with J774 murine monocyte-macrophages and fresh human blood monocytes. Phagocytosis became more efficient with increasing polymer hydrophobicity, whereas opsonization of the particles in serum exerted inconsistent effects. Monocyte activation was determined by flow cytometry and measured as oxidative burst. The cellular oxidative burst induced by the particles was higher for end-group uncapped polymers. Opsonization increased significantly the oxidative activity of J774 monocytes, but affected inconsistently that of human blood monocytes. The results demonstrate that PLA and PLGA microspheres loaded with gentamicin sulfate were efficiently phagocytosed in vitro. The end-group uncapped polymer-type microspheres promoted significantly cell activation, which may be of importance for drug delivery and targeting to intracellular infections.

Gentamicin encapsulation in PLA:PLGA microspheres in view of treating Brucella infections

In view of treating intracellular Brucella infections, microspheres made of poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) were developed as delivery system for the cationic and highly hydrophilic antibiotic gentamicin sulphate. Drug microencapsulation by spray drying yielded microspheres with regular morphology, an average particle size of approximately 3 micrometer and encapsulation efficiencies of up to 45%. Different copolymers of similar molecular weights gave varying encapsulation efficiencies and particle size distributions. The encapsulation efficiency generally increased with polymer hydrophilicity, except for the hydrophilic copolymer PLGA50:50H carrying carboxylic end groups. Encapsulation also depended on the pH value of the aqueous drug solution to be encapsulated. Moreover, increasing nominal gentamicin sulphate loading yielded lower efficiencies. For comparison, some formulations were also prepared by a (W(1)/O)W(2)-solvent evaporation method, which yielded lower encapsulation efficiencies, in the order of 13%. Finally, drug bioactivity was found to remain intact after microencapsulation, MS storage and MS incubation in aqueous medium. The results suggest that PLA/PLGA microspheres prepared by spray drying may be an appropriate delivery system for gentamicin sulphate to be used in the treatment of intracellular Brucella infections.

Nanomedicine: Novel approaches in human and veterinary therapeutics

Nanomedicine can be defined as the application of nanotechnology to the prevention and treatment of diseases as well as for diagnosis purposes. In this context, the development of various types of drug-carrier nanodevices offers new strategies for targeted drug delivery, minimising the secondary effects and the toxicity associated to drug widespread to healthy organs or cells. This review is divided in two different parts. The first one summarizes the main types of nanomedicines developed in the past few decades, including drug nanocrystals, polymer therapeutics, lipid-nanosized and polymeric-nanosized drug delivery systems. The second part of our review is devoted, more specifically, to the presentation of polymeric nanoparticles. Here, we discuss various aspects of nanoparticle formulation, characterization, behaviour in the body and some of their potential applications. More particularly we present some approaches for the treatment of cancer, treatment of infectious diseases and the potential of these nanoparticles as adjuvants for vaccination purposes.

Poly(Anhydride) Nanoparticles Act as Active Th1 Adjuvants through Toll-Like Receptor Exploitation

ABSTRACT The mechanisms that underlie the potent Th1-adjuvant capacity of poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (NPs) were investigated. Traditionally, polymer NPs have been considered delivery systems that promote a closer interaction between antigen and antigen-presenting cells (APCs). Our results revealed that poly(anhydride) NPs also act as agonists of various Toll-like receptors (TLRs) (TLR2, -4, and -5), triggering a Th1-profile cytokine release (gamma interferon [IFN-γ], 478 pg/ml versus 39.6 pg/ml from negative control; interleukin-12 [IL-12], 40 pg/ml versus 7.2 pg/ml from negative control) and, after incubation with dendritic cells, inducing a 2.5- to 3.5-fold increase of CD54 and CD86 costimulatory molecule expression. Furthermore, in vivo studies suggest that NPs actively elicit a CD8+ T-cell response. Immunization with empty NPs resulted in a significant delay in the mean survival date (from day 7 until day 23 postchallenge) and a protection level of 30% after challenge against a lethal dose of Salmonellaenterica serovar Enteritidis. Taken together, our results provide a better understanding of how NPs act as active Th1 adjuvants in immunoprophylaxis and immunotherapy through TLR exploitation.

Bioadhesive properties of pegylated nanoparticles

The design of bioadhesive nanoparticles (NPs) for targeting specific sites within the gut remains a major challenge. One possible strategy to solve this problem may be the use of pegylated NPs. In general, these carriers display different bioadhesive properties to nondecorated NPs. Thus, pegylated NPs show a higher ability to interact with the small intestine mucosa rather than with the stomach. However, the type of surface conformation of polyethylene glycol chains appears to have a great influence on the behaviour of these NPs. Theoretically, the traditional ‘brush’ polyethylene glycol corona would facilitate the penetration of the pegylated particles through the mucus layer and the subsequent adhesive interaction with the mucosa, which would promote their absorption by intestinal enterocytes. On the contrary, pegylated NPs with a ‘loop’ conformation would increase the time of residence of the adhered fraction of particles in the mucosa.

Poly(ε-caprolacton) nanospheres as an alternative way to reduce amphotericin B toxicity

Abstract A new stable amphotericin B nanosphere-based delivery system was obtained by a solvent displacement process. A reproducible and monodisperse size distribution centered on 220 nm was obtained, when AmB and poly(e-caprolacton) were dissolved in an appropriate solvent mixture. UV-Visible and circular dichroism spectroscopy suggested that nanoparticles modified the aggregation state of AmB, probably due to a weak interaction between the drug and the polymer of the nanospheres. Zeta potential measurements indicated that amphiphilic amphotericin B could be adsorbed onto the nanospheres. Fourier transformed infrared spectroscopy (FT-IR) seemed to confirm the absence of drug incorporation into the core of these carriers and that no chemical interaction between the drug and the polymer occurred. The reduction of the acute toxicity of AmB in healthy mice by association of the antibiotic with nanospheres warrants further investigation of the antifungal activity of these systems.