Search bioRxiv⌕ Search

Biology subjects

Merino, S.

Publications and source records attributed to Merino, S..

6 recordsLinked to original sources

Engineering Nanobody Conformational Switches

Conformational signaling is widely used in biology, but remains difficult to engineer into proteins. Here, we explore ligand-induced folding of nanobodies as a general strategy to generate conformational receptors for a broad range of target ligands. More specifically, we have generated partially unfolded nanobody variants that fold exclusively upon binding to their target ligand. To do so, we have introduced destabilizing mutations in the framework region, carefully tuning their stability while preserving molecular recognition. Given the high conservation of the framework across different nanobodies, this approach is broadly applicable to many other target ligands, which has enabled us to engineer conformational switches for three different important biomarkers and drugs: the pregnancy hormone chorionic gonadotropin, the cytokine tumor necrosis factor alpha, and the chemotherapy and immunosuppressant drug methotrexate. Another useful property of conformational signaling is that it can directly gate signal transduction, which we have leveraged to build an optical sensor for the quantitative detection of the pregnancy hormone in urine. Finally, conformational signaling introduces new mechanisms for the allosteric modulation of binding, which we have explored to control the affinity and dynamic range of the sensor. Our work thus establishes a rational strategy for engineering conformational signaling receptors with customizable properties and generalizable for many different target ligands.

biophysics↗

Deciphering the microbial contributors to methane cycling in coastal wetlands

Although wetlands are increasingly recognized as important contributors to the global methane budget, the microorganisms and processes involved methane cycling are poorly characterized, particularly in coastal brackish and saline systems. Here, we investigated microbial and geochemical factors contributing to methane dynamics in three coastal wetlands with different salinities, dominant vegetation types, and soil chemical characteristics. These included a freshwater flotant marsh, a cypress swamp, and a mesohaline salt marsh. Specifically, we paired methane porewater concentrations, surface fluxes, geochemistry, and 16S rRNA gene sequencing to address how microbial community composition links to porewater concentrations and its potential effects on emissions. We found that porewater methane concentrations across sites were the highest in the swamp, followed by the salt marsh and the flotant marsh, and were explained by methanogen richness and abundance. While methane-cycling microbial communities were significantly structured by salinity, two microbial taxa (Methanosaeta and Methanomicrobiaceae) were present across all sites. Hydrogenotrophs were the most abundant methanogen functional group, with Methanomicrobiaceae and Methanobacterium discriminant among wetlands. In contrast, methanotroph functional types varied among wetlands. Type I dominated the freshwater flotant marsh, while the anaerobic methanotrophic archaea the saltwater marsh. These findings contribute to an enhanced understanding of the microbiological contributions to methane emissions from coastal wetlands. Scientific Significance Statement TopicThis study provides critical insights into the microbial and geochemical controls on methane emissions across coastal wetlands along a salinity gradient. Challenging the prevailing paradigm, methane porewater concentrations did not inversely correlate with salinity, as the swamp site with intermediate salinity exhibited the highest concentrations. Methanogen richness and abundance emerged as strong predictors of methane concentrations, while methanotroph richness had no predictive value. Two core methanogens, Methanosaeta and Methanomicrobiaceae, were consistently present across all wetland types. The findings highlight potential role of the water column as a biological methane filter, especially in saline environments. This study significantly advances the understanding of methane cycling in coastal wetlands by decoupling methane emissions from salinity gradients and emphasizing the role of microbial communities and local environmental factors. These insights are essential for refining biogeochemical models to forecast greenhouse gas emissions under sea-level rise and saltwater intrusion scenarios. Scientific Significance Statement OutletThis work integrates microbial ecology, geochemistry, and ecosystem structure to address interdisciplinary questions relevant to the limnological community. Here, we reveal how microbial community composition, rather than salinity alone, predicts methane emissions, offering a fresh perspective into carbon cycling in estuarine and coastal environments. The findings also provide critical insights for improving greenhouse gas emission models to predict climate change feedback from coastal areas.

microbiology↗

Characterizing putative glycosyltransferases within the flagella glycosylation island (FGI) of Aeromonas hydrophila ATCC 7966T

Motility is an important virulence factor for many pathogenic bacteria, enabling locomotion towards favourable conditions and away from hostile environments. Flagellar-mediated motility is driven by one or more flagellar filaments that extend outside of the cell and rapidly rotate to generate movement. These filaments are assembled through the polymerization of thousands of copies of structural flagellin proteins. It has been shown that flagellin glycosylation is often a prerequisite for proper flagella structure and function. Aeromonas hydrophila ATCC 7966T, a clinical and environmental pathogen, elaborates a single polar flagellum. The polar flagellin structural proteins FlaA and FlaB are glycosylated with a heterologous collection of complex penta- and hexa-saccharide chains. This study characterized the involvement of four genes with homology to known glycosyltransferases located within the A. hydrophila ATCC 7966T flagellar glycosylation island (FGI) in the biosynthesis of the complex polysaccharide glycans modifying the polar flagellins. Deletion of genes AHA_4167, AHA_4169, AHA_4170, and AHA_4171 were observed to have truncated glycans with sequentially shorter chain length, and all of these mutant strains had reduced motility compared to wild type bacteria.

biochemistry↗

Polar flagellin glycan metaheterogeneity of Aeromonas hydrophila strain ATCC 7966T

Motile pathogens often rely upon flagellar motility as an essential virulence factor and in many species the structural flagellin protein is glycosylated. This post-translational modification has been shown to be necessary for proper folding of the flagellin structural proteins and proper function of the flagellar filament in a number of bacterial species. Aeromonas hydrophila is a ubiquitous aquatic pathogen with a constitutively expressed polar flagellum. Using a suite of mass spectrometry techniques, the flagellin FlaA and FlaB structural proteins of A. hydrophila strain ATCC 7966T were shown to be glycosylated with significant metaheterogeneity: heterologous glycans were observed with variable site occupancy. The penta- and hexa-saccharide glycan chains contained a previously unreported pseudaminic acid derivative with a mass of 422 Da as the linking sugar, followed in sequence by two hexoses, an N-acetylglucosamine derivative, a deoxy N-acetylglucosamine derivative, and sometimes an additional N-acetylglucosamine.

biochemistry↗

Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance.

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

microbiology↗

Roles of proteins containing immunoglobulin-like domains in the conjugation of bacterial plasmids.

Horizontal transfer of bacterial plasmids generates genetic variability and contributes to the dissemination of the genes that enable bacterial cells to develop antimicrobial resistance (AMR). Several aspects of the conjugative process have long been known, namely, those related to the proteins that participate in the establishment of cell-to-cell contact and to the enzymatic processes associated with the processing of plasmid DNA and its transfer to the recipient cell. In this work, we describe the role of newly identified proteins that influence the conjugation of several plasmids. Genes encoding high-molecular-weight proteins that contain one or several immunoglobulin-like domains (Big) are located in the transfer regions of several plasmids that usually harbor AMR determinants. These Big proteins are exported to the external medium and target two extracellular organelles: the flagella and the conjugative pili. The plasmid-encoded Big proteins facilitate conjugation by reducing cell motility and facilitating cell-to-cell contact by binding both to the flagella and to the conjugative pilus. They use the same export machinery as that used by the conjugative pilus components. In the examples characterized in this paper, these proteins influence conjugation at environmental temperatures (i.e., 25{degrees}C). This suggests that they may play relevant roles in the dissemination of plasmids in natural environments. Taking into account that they interact with outer surface organelles, they could be targeted to control the dissemination of different bacterial plasmids carrying AMR determinants.

microbiology↗