Publications

Several pathogenic Escherichia coli strains cause diarrhea. Enteroaggregative E. coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC cells form a "stacked-brick" arrangement over the intestinal epithelial cells. EAEC isolates express, among other virulence determinants, the AggR transcriptional activator and the aggregative adherence fimbriae (AAF). Overexpression of the aggR gene results in increased expression of virulence factors such as the aff genes, as well as several genes involved in specific metabolic pathways such as fatty acid degradation (fad) and arginine degradation (ast). To support the hypothesis that induction of the expression of some of these pathways may play a role in EAEC virulence, in this study we used a murine infection model to evaluate the impact of the expression of these pathways on infection parameters. Mice infected with a mutant derivative of the EAEC strain 042, characterized by overexpression of the aggR gene, showed increased disease symptoms compared to those exhibited by mice infected with the wild type (wt) strain 042. Several of these symptoms were not increased when the infecting mutant, which overexpressed aggR, lacked the fad and ast pathways. Therefore, our results support the hypothesis that different metabolic pathways contribute to EAEC virulence.

JTD Keywords: Adherence, Aggr, Burde, Chain fatty-acids, Children, Enteroaggregative e. coli, Escherichia-coli, Etiology, Infection, Mice, Microbiota, Persistent diarrhea, Protein, Scfa, Sex-differences

Gene duplications occur in prokaryotic genomes at a detectable frequency. In many instances, the biological function of the duplicates is unknown, and hence, the significance of the presence of multiple copies of these genes remains unclear.; Gene duplications significantly impact the gene repertoires of both eukaryotic and prokaryotic microorganisms. The genomes of pathogenic Escherichia coli strains share a group of duplicated genes whose function is mostly unknown. The irmA gene is one of the duplicates encoded in several pathogenic E. coli strains. The function of its gene product was investigated in the uropathogenic E. coli strain CFT073, which contains a single functional copy. The IrmA protein structure mimics that of human interleukin receptors and likely plays a role during infection. The enteroaggregative E. coli strain 042 contains two functional copies of the irmA gene. In the present work, we investigated their biological roles. The irmA_4509 allele is expressed under several growth conditions. Its expression is modulated by the global regulators OxyR and Hha, with optimal expression at 37 degrees C and under nutritional stress conditions. Expression of the irmA_2244 allele can only be detected when the irmA_4509 allele is knocked out. Differences in the promoter regions of both alleles account for their differential expression. Our results show that under several environmental conditions, the expression of the IrmA protein in strain 042 is dictated by the irmA_4509 allele. The irmA_2244 allele appears to play a backup role to ensure IrmA expression when the irmA_4509 allele loses its function. IMPORTANCE Gene duplications occur in prokaryotic genomes at a detectable frequency. In many instances, the biological function of the duplicates is unknown, and hence, the significance of the presence of multiple copies of these genes remains unclear. In pathogenic E. coli isolates, the irmA gene can be present either as a single copy or in two or more copies. We focused our work on studying why a different pathogenic E. coli strain encodes two functional copies of the irmA gene. We show that under several environmental conditions, one of the alleles dictates IrmA expression, and the second remains silent. The latter allele is only expressed when the former is silenced. The presence of more than one functional copy of the irmA gene in some pathogenic E. coli strains can result in sufficient expression of this virulence factor during the infection process.

JTD Keywords: 042, aec69, enteroaggregative e. coli, gene duplications, 042, Adaptation, Aec69, Aggregative adherence, Chromosomal genes, Coli, Duplication, Enteroaggregative e, Escherichia-coli, Evolution, Gene duplications, Hha/ymoa, Irma, Mechanism, Outer-membrane, Protein

The low efficacy of current conventional treatments for bacterial infections increases mortality rates worldwide. To alleviate this global health problem, we propose drug-free enzyme-based nanomotors for the treatment of bacterial urinary-tract infections. We develop nanomotors consisting of mesoporous silica nanoparticles (MSNPs) that were functionalized with either urease (U-MSNPs), lysozyme (L-MSNPs), or urease and lysozyme (M-MSNPs), and use them against nonpathogenic planktonic Escherichia coli. U-MSNPs exhibited the highest bactericidal activity due to biocatalysis of urea into NaHCO3 and NH3, which also propels U-MSNPs. In addition, U-MSNPs in concentrations above 200 μg/mL were capable of successfully reducing 60% of the biofilm biomass of a uropathogenic E. coli strain. This study thus provides a proof-of-concept, demonstrating that enzyme-based nanomotors are capable of fighting infectious diseases. This approach could potentially be extended to other kinds of diseases by selecting appropriate biomolecules.

JTD Keywords: biofilms, carbonate, e. coli, enzymatic nanomotors, infections, lysozyme, micromotors, nanomachines, proteins, self-propulsion, Anti-bacterial agents, Biocatalysis, Biofilms, Canavalia, Drug carriers, E. coli, Eliminate escherichia-coli, Enzymatic nanomotors, Escherichia coli, Escherichia coli infections, Humans, Infections, Muramidase, Nanomachines, Nanoparticles, Self-propulsion, Silicon dioxide, Urease, Urinary tract infections

Introduction: Application of lipopolysaccharide (LPS) is a widely employed model to mimic acute respiratory distress syndrome (ARDS). Available data regarding LPS-induced biomechanical changes on pulmonary epithelial cells are limited only to P. aeruginosa LPS. Considering that LPS from different bacteria could promote a specific mechanical response in epithelial cells, we aim to assess the effect of E. coli LPS, widely employed as a model of ARDS, in the biomechanics of alveolar epithelial cells. Methods: Young’s modulus (E) of alveolar epithelial cells (A549) was measured by atomic force microscopy every 5 min throughout 60 min of experiment after treatment with LPS from E. coli (100 μg/mL). The percentage of cells presenting actin stress fibers (F-actin staining) was also evaluated. Control cells were treated with culture medium and the values obtained were compared with LPS-treated cells for each time-point. Results: Application of LPS induced significant increase in E after 20 min (77%) till 60 min (104%) in comparison to controls. Increase in lung epithelial cell stiffness induced by LPS was associated with a higher number of cells presenting cytoskeletal remodeling. Conclusions: The observed effects of E. coli LPS on alveolar epithelial cells suggest that this widely-used LPS is able to promote a quick formation of actin stress fibers and stiffening cells, thereby facilitating the disruption of the pulmonary epithelial barrier.

JTD Keywords: Acute respiratory distress syndrome model, Alveolar epithelium, Biomechanics, E. coli, Lipopolysaccharide

Bacteria biohybrids employ the motility and power of swimming bacteria to carry and maneuver microscale particles. They have the potential to perform microdrug and cargo delivery in vivo, but have been limited by poor design, reduced swimming capabilities, and impeded functionality. To address these challenge, motile Escherichia coli are captured inside electropolymerized microtubes, exhibiting the first report of a bacteria microswimmer that does not utilize a spherical particle chassis. Single bacterium becomes partially trapped within the tube and becomes a bioengine to push the microtube though biological media. Microtubes are modified with "smart" material properties for motion control, including a bacteria-attractant polydopamine inner layer, addition of magnetic components for external guidance, and a biochemical kill trigger to cease bacterium swimming on demand. Swimming dynamics of the bacteria biohybrid are quantified by comparing "length of protrusion" of bacteria from the microtubes with respect to changes in angular autocorrelation and swimmer mean squared displacement. The multifunctional microtubular swimmers present a new generation of biocompatible micromotors toward future microbiorobots and minimally invasive medical applications.

JTD Keywords: Biohybrids, E. coli, Micromotors, Microswimmers, Polydopamine

Abstract Here we describe the fabrication of a highly sensitive and label-free ITO-based impedimetric immunosensor for the detection of pathogenic bacteria Escherichia coli O157:H7. Anti-E. coli antibodies were immobilized onto ITO electrodes using a simple, robust and direct methodology. First, the covalent attachment of epoxysilane on the ITO surface was demonstrated by Atomic Force Microscopy and cyclic voltammetry. The immobilization of antibody on the epoxysilane layer was quantified by Optical Waveguide Lightmode Spectroscopy, obtaining a mass variation of 12 ng cm− 2 (0.08 pmol cm− 2). Microcontact printing and fluorescence microscopy were used to demonstrate the specific binding of E. coli O157:H7 to the antibody-patterned surface. We achieved a ratio of 1:500 Salmonella typhimurium/E. coli O157:H7, thus confirming the selectivity of the antibodies and efficiency of the functionalization procedure. Finally, the detection capacity of the ITO-based immunosensor was evaluated by Electrochemical Impedance Spectroscopy. A very low limit of detection was obtained (1 CFU mL− 1) over a large linear working range (10–106 CFU mL− 1). The specificity of the impedimetric immunosensor was also examined. Less than 20% of non-specific bacteria (S. typhimurium and E. coli K12) was observed. Our results reveal the applicability of ITO for the development of highly sensitive and selective impedimetric immunosensors.

JTD Keywords: E. coli O157:H7, Electrochemical Impedance Spectroscopy, Immunosensor, Indium tin oxide, Label-free detection

Electric Fields are increasingly used to manipulate bacteria. However, there is no systematic and definitive study on how the different electric parameters change bacteria viability. Here we present a study on the effects of electric field intensity and temperature to bacterial cultures. Escherichia coli colonies have been exposed to different electric field intensities at 1MHz during 5 minutes by means of a microfluidic device specially designed for the experiment. From the analysis of the results it is possible to see that Escherichia coli survival rate diminishes when applying field intensities as low as 220V during 5 minutes. Death rates also increase when stronger fields are applied. However, viability of survived bacteria is maintained. Additionally, temperature shows a synergistic effect with voltage. When temperature was increased, results showed a stronger sensitivity of cells to the electric field. Moreover, the expression patterns of Outer Membrane Protein A and Ribosomal Proteins differ in control and treated samples, suggesting changes in bacterial metabolism and structure.

JTD Keywords: E. coli, Electric field, Temperature, Viability

Background The HilA protein is the master regulator of the Salmonella pathogenicity island 1 (SPI1). EilA and YgeH proteins show a moderate similarity to HilA and are encoded in pathogenicity islands from several E. coli strains, both pathogenic and non-pathogenic. In the present work we characterize the YgeH protein from the enteroaggregative E. coli strain 042 (locus tag EC042_3050). Results We show that both E. coli 042 YgeH and EilA proteins are able to functionally replace HilA in Salmonella. Interestingly, this is not the rule for all YgeH proteins: the YgeH protein from the enterohaemorragic E. coli strain O157 appears to be non-functional. ygeH expression is not influenced by growth osmolarity or temperature, and moderately increases in cells entering the stationary phase. H-NS represses ygeH expression under all growth conditions tested, and binds with specificity to the ygeH promoter region. As expected, expression of ETT2 (Escherichia coli type 3 secretion system 2) genes requires YgeH: ETT2 operons are downregulated in a ygeH mutant. Accordingly, since H-NS represses ygeH expression, ETT2 expression is significantly increased in an hns mutant. Conclusion E. coli 042 YgeH protein is functional and able to replace HilA in Salmonella. ETT2 gene expression requires YgeH activity which, in turn, is subjected to H-NS silencing.

JTD Keywords: HilA, YgeH, E. coli 042, H-NS

Electromagnetic Fields are increasingly used to manipulate bacteria. However, there is no systematic and definitive study on how the different electric parameters change bacteria viability. Here we present preliminary data on the effect of electric field intensity and temperature applica- tion. E. Coli colonies have been exposed to different voltages at 1MHz during 5 minutes by means of a custom-made micro- fluidic device. Results show that E.Coli survival rate is already reduced by applying field intensities as low as 220V/cm during 5 minutes. The use of stronger fields resulted in death rates increase also. Viability of survived bacteria was maintained. On the other hand, temperature has shown a synergistic effect with voltage. When temperature is increased results seem to indicate stronger sensitivity of cells to the electric field. It is necessary to continue studying the contribution of other para- meters as intensity, time, frequency or concentration, to study further synergies.

JTD Keywords: E. Coli, Electromagnetic Field, Temperature, Viability

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

Dielectrophoresis (DEP) represents a powerful approach to manipulate and study living cells. Hitherto, several approaches have used 2-D DEP chips. With the aim to increase sample volume, in this study we used a 3-D carbon-electrode DEP chip to trap and release bacterial cells. A continuous flow was used to plug an Escherichia coli cell suspension first, to retain cells by positive DEP, and thereafter to recover them by washing with peptone water washing solution. This approach allows one not only to analyze DEP behavior of living cells within the chip, but also to further recover fractions containing DEP-trapped cells. Bacterial concentration and flow rate appeared as critical parameters influencing the separation capacity of the chip. Evidence is presented demonstrating that the setup developed in this study can be used to separate different types of bacterial cells.

JTD Keywords: Bacteria, Carbon electrode, Dielectrophoresis, E. coli, Separation