Photoswitchable glycoligands targeting Pseudomonas aeruginosa LecA.

Biofilm formation is one of main causes of bacterial antimicrobial resistance infections. It is known that the soluble lectins LecA and LecB, produced by Pseudomonas aeruginosa, play a key role in biofilm formation and lung infection. Bacterial lectins are therefore attractive targets for the development of new antibiotic-sparing anti-infective drugs. Building synthetic glycoconjugates for the inhibition and modulation of bacterial lectins have shown promising results. Light-sensitive lectin ligands could allow the modulation of lectins activity with precise spatiotemporal control. Despite the potential of photoswitchable tools, few photochromic lectin ligands have been developed. We have designed and synthesized several O- and S-galactosyl azobenzenes as photoswitchable ligands of LecA and evaluated their binding affinity with isothermal titration calorimetry. We show that the synthesized monovalent glycoligands possess excellent photophysical properties and strong affinity for targeted LecA with K d values in the micromolar range. Analysis of the thermodynamic contribution indicates that the Z-azobenzene isomers have a systematically stronger favorable enthalpy contribution than the corresponding E-isomers, but due to stronger unfavorable entropy, they are in general of lower affinity. The validation of this proof-of-concept and the dissection of thermodynamics of binding will help for the further development of lectin ligands that can be controlled by light.

Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury-Time-Dependent Changes in the Cortex, Striatum, Thalamus and Hippocampus in a Mouse Model.

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein-GFAP), microglia/macrophages (allograft inflammatory factor 1-IBA-1), and microglia (transmembrane protein 119-TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.

Chemical control over Asialo-GM1: A dual ligand for pili and Lectin A that activates swarming motility and facilitates adherence of Pseudomonas aeruginosa.

Glycolipid, ganglio-N-tetraosylceramide (asialo-GM1), on the mammalian cells are known to be recognized by type IV pili of Pseudomonas aeruginosa. In this work, we show that asialo-GM1 can also be recognized by Lectin A (LecA), another adhesin protein of the P. aeruginosa, by a fluorescent polarization assay, a label-free bacterial motility enabled binding assay, and bacterial mutant studies. On hydrated semi-solid gel surfaces, asialo-GM1 enables swarming and twitching motilities, while on solid surfaces facilitates the bacterial adherence of P. aeruginosa. These results indicate that asialo-GM1 can modulate bioactivities, adherence, and motilities, that are controlled by opposite signaling pathways. We demonstrate that when a solution of pilin monomers or LecA proteins are spread on hydrated gel surfaces, the asialo-GM1 mediated swarming motility is inhibited. Treatment of artificial liposomes containing asialo-GM1 as a component of lipid bilayer with pilin monomers or LecA proteins caused transient leakage of encapsulated dye from liposomes. These results suggest that pili and LecA proteins not only bind to asialo-GM1 but can also cause asialo-GM1 mediated leakage. We also show that both pili and LecA mutants of P. aeruginosa adhere to asialo-GM1 coated solid surfaces, and that a class of synthetic ligands for pili and LecA inhibits both pili and LecA-mediated adherence of P. aeruginosa on asialo-GM1-coated surfaces.

Single nucleotide variants in innate immune genes associated with Salmonella shedding and colonization in swine on commercial farms.

Foodborne human salmonellosis is an important food safety concern worldwide. Food-producing animals are one of the major sources of human salmonellosis, and thus control of Salmonella at the farm level could reduce Salmonella spread in the food supply system. Genetic selection of pigs with resistance to Salmonella infection may be one way to control Salmonella on swine farms. The objective of this study was to investigate the association between genetic variants in the porcine innate immune system with on-farm Salmonella shedding and Salmonella colonization tested at slaughter. Fourteen groups of pigs (total 809) were followed from birth to slaughter. Fecal samples collected five times at different stages of production and tissue samples obtained from tonsil and lymph nodes at slaughter were cultured for Salmonella. Genomic DNA was extracted and analyzed for 40 single nucleotide variants and two indels within porcine innate immune genes that were previously associated with Salmonella infection or other infectious diseases. A survey was used to collect information on farm management practices. A multilevel mixed-effects logistic regression modelling method was used to identify SNVs that are associated with Salmonella shedding and/or Salmonella colonization. One single nucleotide variant in the C-type lectin MBL1 and one single nucleotide variant in the cytosolic pattern recognition receptor NOD1 was associated with increased risk of on-farm shedding (p = 0.010) and internal colonization tested at slaughter (p = 0.018), respectively. These findings indicate the potential of these variants for genetic selection programs aimed at controlling Salmonella shedding and colonization in pigs.