PCR-tips for rapid diagnosis of bacterial pathogens.

Micropipette tips are currently among the most used disposable devices in bioresearch and development laboratories. Their main application is the fractionation of solutions. New functionalities have recently been added to this device, widening their applications. This paper analyzed disposable micropipette tips as reagent holders of PCR reagents. PCR has become a prevalent and often indispensable technique in biological laboratories for various applications, such as the detection of coronavirus and other infectious diseases. A functional micropipette tip was implemented to simplify PCR analysis and reduce the contamination chances of deoxynucleotides and specific primers. This disposable device is prepared by tip coating processes of reagents, using polyvinyl alcohol polymer and additives. The coated layer is optimized to load and release PCR reagents efficiently. As a proof of concept, we show that the detection of Bordetella pertussis, the etiological agent of whooping cough whose diagnostic relies on PCR, can be quickly done using practical-functional tips. This device is an excellent example of testing the functionality and contribution of molecular diagnostic PCR tips. KEY POINTS: • Functional micropipette tips are prepared by coating with dNTPs and primers. • Functional tips are used to replace dNTPs and primers in the PCR master mix. • PCR diagnostic of Bordetella pertussis is performed using functional tips.

Human macrophage polarization shapes B. pertussis intracellular persistence.

We previously demonstrated that Bordetella pertussis, the etiologic agent of whooping cough, is able to survive inside human macrophages. The aim of this study was to examine the influence of macrophage polarization in the development of B. pertussis intracellular infections. To this end, primary human monocytes were differentiated into M1, M2a, or M2c macrophages and further infected with B. pertussis. Infected M1 macrophages showed a proinflammatory response evidenced by the production of TNF-α, IL-12p70, and IL-6. Conversely, infection of M2a and M2c macrophages did not induce TNF-α, IL-12p70, nor IL-6 at any time postinfection but showed a significant increase of M2 markers, such as CD206, CD163, and CD209. Interestingly, anti-inflammatory cytokines, like IL-10 and TGF-ß, were induced after infection in the 3 macrophage phenotypes. B. pertussis phagocytosis by M1 macrophages was lower than by M2 phenotypes, which may be ascribed to differences in the expression level of B. pertussis docking molecules on the surface of the different phenotypes. Intracellular bactericidal activity was found to be significantly higher in M1 than in M2a or M2c cells, but live bacteria were still detected within the 3 phenotypes at the late time points after infection. In summary, this study shows that intracellular B. pertussis is able to survive regardless of the macrophage activation program, but its intracellular survival proved higher in M2 compared with the M1 macrophages, being M2c the best candidate to develop into a niche of persistence for B. pertussis.

The targeting of starch binding domains from starch synthase III to the cell wall alters cell wall composition and properties.

KEY MESSAGE: Starch binding domains of starch synthase III from Arabidopsis thaliana (SBD123) binds preferentially to cell wall polysaccharides rather than to starch in vitro. Transgenic plants overexpressing SBD123 in the cell wall are larger than wild type. Cell wall components are altered in transgenic plants. Transgenic plants are more susceptible to digestion than wild type and present higher released glucose content. Our results suggest that the transgenic plants have an advantage for the production of bioethanol in terms of saccharification of essential substrates. The plant cell wall, which represents a major source of biomass for biofuel production, is composed of cellulose, hemicelluloses, pectins and lignin. A potential biotechnological target for improving the production of biofuels is the modification of plant cell walls. This modification is achieved via several strategies, including, among others, altering biosynthetic pathways and modifying the associations and structures of various cell wall components. In this study, we modified the cell wall of A. thaliana by targeting the starch-binding domains of A. thaliana starch synthase III to this structure. The resulting transgenic plants (E8-SDB123) showed an increased biomass, higher levels of both fermentable sugars and hydrolyzed cellulose and altered cell wall properties such as higher laxity and degradability, which are valuable characteristics for the second-generation biofuels industry. The increased biomass and degradability phenotype of E8-SBD123 plants could be explained by the putative cell-wall loosening effect of the in tandem starch binding domains. Based on these results, our approach represents a promising biotechnological tool for reducing of biomass recalcitrance and therefore, the need for pretreatments.

Improving the glycosyltransferase activity of Agrobacterium tumefaciens glycogen synthase by fusion of N-terminal starch binding domains (SBDs).

Glycogen and starch, the major storage carbohydrate in most living organisms, result mainly from the action of starch or glycogen synthases (SS or GS, respectively, EC 2.4.1.21). SSIII from Arabidopsis thaliana is an SS isoform with a particular modular organization: the C-terminal highly conserved glycosyltransferase domain is preceded by a unique specific region (SSIII-SD) which contains three in tandem starch binding domains (SBDs, named D1, D2 and D3) characteristic of polysaccharide degrading enzymes. N-terminal SBDs have a probed regulatory role in SSIII activity, showing starch binding ability and modulating the catalytic properties of the enzyme. On the other hand, GS from Agrobacterium tumefaciens has a simple primary structure organization, characterized only by the highly conserved glycosyltransferase domain and lacking SBDs. To further investigate the functional role of A. thaliana SSIII-SD, three chimeric proteins were constructed combining the SBDs from A. thaliana with the GS from A. tumefaciens. Recombinant proteins were expressed in and purified to homogeneity from Escherichia coli cells in order to be kinetically characterized. Furthermore, we tested the ability to restore in vivo glycogen biosynthesis in transformed E. coli glgA(-) cells, deficient in GS. Results show that the D3-GS chimeric enzyme showed increased capacity of glycogen synthesis in vivo with minor changes in its kinetics parameters compared to GS.

An enzyme-coupled continuous spectrophotometric assay for glycogen synthases.

The metabolic pathways leading to the synthesis of bacterial glycogen involve the action of several enzymes, among which glycogen synthase (GS) catalyzes the elongation of the α-1,4-glucan. GS from Agrobacterium tumefaciens uses preferentially ADPGlc, although UDPGlc can also be used as glycosyl donor with less efficiency. We present here a continuous spectrophotometric assay for the determination of GS activity using ADP- or UDPGlc. When ADPGlc was used as the substrate, the production of ADP is coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH). With UDPGlc as substrate, UDP was converted to ADP via adenylate kinase and subsequent coupling to PK and LDH reactions. Using this assay, we determined the kinetic parameters of GS and compared them with those obtained with the classical radiochemical method. For this purpose, we improved the expression procedure of A. tumefaciens GS using Escherichia coli BL21(DE3)-RIL cells. This assay allows the continuous monitoring of glycosyltransferase activity using ADPGlc or UDPGlc as sugar-nucleotide donors.

The starch-binding capacity of the noncatalytic SBD2 region and the interaction between the N- and C-terminal domains are involved in the modulation of the activity of starch synthase III from Arabidopsis thaliana.

Starch synthase III from Arabidopsis thaliana contains an N-terminal region, including three in-tandem starch-binding domains, followed by a C-terminal catalytic domain. We have reported previously that starch-binding domains may be involved in the regulation of starch synthase III function. In this work, we analyzed the existence of protein interactions between both domains using pull-down assays, far western blotting and co-expression of the full and truncated starch-binding domains with the catalytic domain. Pull-down assays and co-purification analysis showed that the D(316-344) and D(495-535) regions in the D2 and D3 domains, respectively, but not the individual starch-binding domains, are involved in the interaction with the catalytic domain. We also determined that the residues W366 and Y394 in the D2 domain are important in starch binding. Moreover, the co-purified catalytic domain plus site-directed mutants of the D123 protein lacking these aromatic residues showed that W366 was key to the apparent affinity for the polysaccharide substrate of starch synthase III, whereas either of these amino acid residues altered ADP-glucose kinetics. In addition, the analysis of full-length and truncated proteins showed an almost complete restoration of the apparent affinity for the substrates and V(max) of starch synthase III. The results presented here suggest that the interaction of the N-terminal starch-binding domains, particularly the D(316-344) and D(495-535) regions, with the catalytic domains, as well as the full integrity of the starch-binding capacity of the D2 domain, are involved in the modulation of starch synthase III activity.

Role of the N-terminal starch-binding domains in the kinetic properties of starch synthase III from Arabidopsis thaliana.

Starch synthase III (SSIII), one of the SS isoforms involved in plant starch synthesis, has been reported to play a regulatory role in the synthesis of transient starch. SSIII from Arabidopsis thaliana contains 1025 amino acid residues and has an N-terminal transit peptide for chloroplast localization which is followed by three repeated starch-binding domains (SBDs; SSIII residues 22-591) and a C-terminal catalytic domain (residues 592-1025) similar to bacterial glycogen synthase. In this work, we constructed recombinant full-length and truncated isoforms of SSIII, lacking one, two, or three SBDs, and recombinant proteins, containing three, two, or one SBD, to investigate the role of these domains in enzyme activity. Results revealed that SSIII uses preferentially ADPGlc, although UDPGlc can also be used as a sugar donor substrate. When ADPGlc was used, the presence of the SBDs confers particular properties to each isoform, increasing the apparent affinity and the V max for the oligosaccharide acceptor substrate. However, no substantial changes in the kinetic parameters for glycogen were observed when UDPGlc was the donor substrate. Under glycogen saturating conditions, the presence of SBDs increases progressively the apparent affinity and V max for ADPGlc but not for UDPGlc. Adsorption assays showed that the N-terminal region of SSIII, containing three, two, or one SBD module have increased capacity to bind starch depending on the number of SBD modules, with the D23 protein (containing the second and third SBD module) being the one that makes the greatest contribution to binding. The results presented here suggest that the N-terminal SBDs have a regulatory role, showing a starch binding capacity and modulating the catalytic properties of SSIII.

Functional and structural characterization of the catalytic domain of the starch synthase III from Arabidopsis thaliana.

Glycogen and starch are the major energy storage compounds in most living organisms. The metabolic pathways leading to their synthesis involve the action of several enzymes, among which glycogen synthase (GS) or starch synthase (SS) catalyze the elongation of the alpha-1,4-glucan backbone. At least five SS isoforms were described in Arabidopsis thaliana; it has been reported that the isoform III (SSIII) has a regulatory function on the synthesis of transient plant starch. The catalytic C-terminal domain of A. thaliana SSIII (SSIII-CD) was cloned and expressed. SSIII-CD fully complements the production of glycogen by an Agrobacterium tumefaciens glycogen synthase null mutant, suggesting that this truncated isoform restores in vivo the novo synthesis of bacterial glycogen. In vitro studies revealed that recombinant SSIII-CD uses with more efficiency rabbit muscle glycogen than amylopectin as primer and display a high apparent affinity for ADP-Glc. Fold class assignment methods followed by homology modeling predict a high global similarity to A. tumefaciens GS showing a fully conservation of the ADP-binding residues. On the other hand, this comparison revealed important divergences of the polysaccharide binding domain between AtGS and SSIII-CD.