Publications

This study investigates the synthesis and optimization of nanobots (NBs) loaded with pDNA using the layer-by-layer (LBL) method and explores the impact of their collective motion on the transfection efficiency. NBs consist of biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles and are powered by the urease enzyme, enabling autonomous movement and collective swarming behavior. In vitro experiments were conducted to validate the delivery efficiency of fluorescently labeled NBs, using two-dimensional (2D) and three-dimensional (3D) cell models: murine urothelial carcinoma cell line (MB49) and spheroids from human urothelial bladder cancer cells (RT4). Swarms of pDNA-loaded NBs showed enhancements of 2.2- to 2.6-fold in delivery efficiency and 6.8- to 8.1-fold in material delivered compared to inhibited particles (inhibited enzyme) and the absence of fuel in a 2D cell culture. Additionally, efficient intracellular delivery of pDNA was demonstrated in both cell models by quantifying and visualizing the expression of eGFP. Swarms of NBs exhibited a >5-fold enhancement in transfection efficiency compared to the absence of fuel in a 2D culture, even surpassing the Lipofectamine 3000 commercial transfection agent (cationic lipid-mediated transfection). Swarms also demonstrated up to a 3.2-fold enhancement in the amount of material delivered in 3D spheroids compared to the absence of fuel. The successful transfection of 2D and 3D cell cultures using swarms of LBL PLGA NBs holds great potential for nucleic acid delivery in the context of bladder treatments.

JTD Keywords: Animals, Barrier, Cell line, tumor, Dna, Drug delivery, Drug-delivery, Enzyme catalysis, Gene delivery, Gene transfer techniques, Humans, Lactic acid, Mice, Nanobots, Nanoparticles, Pdna, Plasmids, Polyglycolic acid, Polylactic acid-polyglycolic acid copolymer, Swarming, Transfectio, Transfection, Urease, Urinary bladder neoplasms

Transmission of a plasmid from one bacterial cell to another, in several instances, underlies the dissemination of antimicrobial resistance (AMR) genes. The process requires well-characterized enzymatic machinery that facilitates cell-to-cell contact and the transfer of the plasmid.

JTD Keywords: antimicrobial resistance, bacterial ig-like proteins, bacterial lg-like proteins, chromosomal genes, identification, inca/c, mutational analysis, plasmid conjugation, products, r-factors, resistance plasmids, salmonella-enterica, sequence, Antimicrobial resistance, Bacterial ig-like proteins, Escherichia-coli, Plasmid conjugation

Plaques of the amyloid beta (A beta) peptide are a pathological hallmark of Alzheimer's disease (AD), the most common form of dementia. Mutations in A beta also cause familial forms of AD (fAD). Here, we use deep mutational scanning to quantify the effects of >14,000 mutations on the aggregation of A beta. The resulting genetic landscape reveals mechanistic insights into fibril nucleation, including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation. Strikingly, unlike computational predictors and previous measurements, the empirical nucleation scores accurately identify all known dominant fAD mutations in A beta, genetically validating that the mechanism of nucleation in a cell-based assay is likely to be very similar to the mechanism that causes the human disease. These results provide the first comprehensive atlas of how mutations alter the formation of any amyloid fibril and a resource for the interpretation of genetic variation in A beta.

JTD Keywords: aggregation, kinetics, oligomers, onset, rates, state, Aggregation, Alzheimer disease, Alzheimer's, Amyloid, Amyloid beta-peptides, Computational biology, Deep mutagenesis, Dna mutational analysis, Genetics, Genomics, High-throughput nucleotide sequencing, Kinetics, Mutation, Nucleation, Oligomers, Onset, Plasmids, Precursor protein, Rates, S. cerevisiae, Saccharomyces cerevisiae, State, Systems biology

IncHI plasmids account for multiple antimicrobial resistance in Salmonella and other enterobacterial genera. These plasmids are generally very stable in their bacterial hosts. R27 is the archetype of IncHI1 plasmids. A high percentage of the R27-encoded open reading frames (ORFs) (66.7%) do not show similarity to any known ORFs. We performed a deletion analysis of all non-essential R27 DNA sequences to search for hitherto non-identified plasmid functions that might be required for plasmid stability. We report the identification of a short DNA sequence (incC) that is essential for R27 stability. That region contains several repeats (incC repeats), belongs to one of the three-plasmid replicons (R27 FIA-like) and is targeted by the R27 E protein. Deletion of the incC sequence drastically reduces R27 stability both in Escherichia coli and in Salmonella, the effect being more pronounced in this latter species. Interfering with incC-E protein interaction must lead to a reduced IncHI1 plasmid stability, and may represent a new approach to combat antimicrobial resistance.

JTD Keywords: Antimicrobial resistance, E protein, IncC, IncHI1 plasmids, Plasmid R27, Plasmid stability

R27 is the prototype of the IncHI group of conjugative plasmids, which are associated with multidrug resistance in several relevant pathogens. The transfer of this plasmid is thermodependent and all transfer-related genes are encoded in six operons (tra operons). Very little is known about the factors involved in the regulation of the R27 conjugation. This report describes transcriptional studies of the six tra operons. Our results indicate that HtdA, encoded in the R27 plasmid, is involved in the transcriptional repression of four tra operons (F, H, AC and Z). Although HtdA plays a pivotal role in the transcriptional regulation of those operons, it does not exert its effect as a classical repressor. The data indicate the existence of a crosstalk between HtdA and other unknown regulatory factors. The HtdA-mediated regulation of conjugation is independent of the R27 H-NS protein.

JTD Keywords: Plasmid conjugation, IncHI, R27, tra Operons regulation, HtdA

H-NS and Hha belong to the nucleoid-associated family of proteins and modulate gene expression in response to environmental stimuli. Genes coding for these proteins can be either chromosomally or plasmid-encoded. In this work, we analyse the regulatory role of the Hha protein encoded in the virulence plasmid of the enterohemorrhagic Escherichia coli O157:H7 (HhapO157). This plasmid is present in all clinical isolates of E. coli O157:H7 and contributes to virulence. Both, HhapO157 and E. coli O157:H7-chromosomal Hha (Hhachr) exhibit a significant degree of similarity. The hha gene from plasmid pO157 is transcribed from its own putative promoter and is overexpressed in a chromosomal hha mutant. As its chromosomal counterpart, HhapO157 is able to interact with H-NS. Remarkably, HhapO157 targets only a subset of the genes modulated by Hhachr. This has been evidenced by both assaying the ability of HhapO157 to complement expression of a specific operon (i.e., the haemolysin operon) and by comparing the global transcriptome of the wt strain and its hhap, hhac and hhapc mutant derivatives. HhapO157 and Hhachr share some common regulatory features, however they also display specific targeting of some genes and even a different modulatory role in some others.

JTD Keywords: E. coli O157:H7, Hha, H-NS, Plasmid, pO157, Nucleoid-associated proteins

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

JTD Keywords: 2A strain 2457T, Escherichia-Coli, Salmonella-Enterica, Protein, DNA, Expression, Binding, HHA, Shigella, Plasmid