Reconocimiento selectivo sensor/operador en reguladores de respuesta a metales monovalentes en Salmonella enterica

Dos reguladores transcripcionales parálogos de la familia MerR, CueR y GolS, son responsables de la detección y la resistencia a iones de metales monovalentes en Salmonella enterica. A pesar de que presentan un grado de similitud alto en su secuencia proteica y en la secuencia nucleotídica de sus operadores blanco, estas proteínas difieren en la naturaleza de la señal que reconocen así como en el conjunto de genes cuya transcripción regulan. Recientemente, en el laboratorio donde se desarrolló esta Tesis se demostró que el reconocimiento selectivo de secuencias promotoras depende de la presencia de bases específicas localizadas en las posiciones 3´ y 3 de los operadores reconocidos por estos reguladores. En el primer Capítulo de la presente Tesis demostramos que el residuo de metionina en la posición 16 de GolS, absolutamente conservado entre proteínas homólogas a este regulador pero ausente en todos los xenólogos del tipo CueR, es clave para el reconocimiento selectivo de secuencias promotoras que presentan la marca distintiva de todos los operadores regulados por GolS, en tanto que el residuo en la posición 19 auxilia en la interacción específica sensor/operador; y que el reemplazo de estos residuos por los conservados en el regulador parálogo cambia el conjunto de genes reconocidos por estos factores transcripcionales. A su vez, demostramos que existe una actividad regulatoria diferencial entre CueR de Salmonella, CueR STM, y su ortólogo de E. coli, CueREC, probablemente debido a diferencias sutiles en el dominio N-terminal de unión al ADN. Estos resultados indican que la co-evolución de un regulador y sus operadores blanco dentro de la célula bacteriana provee de las condiciones necesarias para evitar el reconocimiento cruzado y garantizar la respuesta adecuada al daño provocado por un estresante dado, en este caso un ión metálico. Finalmente, iniciamos la caracterización de otro regulador transcripcional MerR, específico de Salmonella y homólogo a MlrA, implicado en la regulación de la motilidad en y la formación de biopelículas. Los resultados que se describen en el segundo Capítulo de esta Tesis demuestran que altos niveles de STM1266 disminuyen la motilidad, incrementan la producción y secreción de celulosa, confieren mayor resistencia a estreptomicina y estimulan la formación de biopelículas, posiblemente debido a la participación de este regulador en la modulación transcripcional de csgD.Fil: Humbert, María Victoria. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Structure of the Neisseria Adhesin Complex Protein (ACP) and its role as a novel lysozyme inhibitor

Pathogenic and commensal Neisseria species produce an Adhesin Complex Protein, which was first characterised in Neisseria meningitidis (Nm) as a novel surface-exposed adhesin with vaccine potential. In the current study, the crystal structure of a recombinant (r)Nm-ACP Type I protein was determined to 1.4 Å resolution: the fold resembles an eight-stranded β-barrel, stabilized by a disulphide bond between the first (Cys38) and last (Cys121) β-strands. There are few main-chain hydrogen bonds linking β4-β5 and β8-β1, so the structure divides into two four-stranded anti-parallel β-sheets (β1-β4 and β5-β8). The computed surface electrostatic charge distribution showed that the β1-β4 sheet face is predominantly basic, whereas the β5-β8 sheet is apolar, apart from the loop between β4 and β5. Concentrations of rNm-ACP and rNeisseria gonorrhoeae-ACP proteins ≥0.25 μg/ml significantly inhibited by ~80-100% (P<0.05) the in vitro activity of human lysozyme (HL) over 24 h. Specificity was demonstrated by the ability of murine anti-Neisseria ACP sera to block ACP inhibition and restore HL activity. ACP expression conferred tolerance to HL activity, as demonstrated by significant 3-9 fold reductions (P<0.05) in the growth of meningococcal and gonococcal acp gene knock-out mutants in the presence of lysozyme. In addition, wild-type Neisseria lactamica treated with purified ACP-specific rabbit IgG antibodies showed similar fold reductions in bacterial growth, compared with untreated bacteria (P<0.05). Nm-ACPI is structurally similar to the MliC/PliC protein family of lysozyme inhibitors. However, Neisseria ACP proteins show <20% primary sequence similarity with these inhibitors and do not share any conserved MliC/PliC sequence motifs associated with lysozyme recognition. These observations suggest that Neisseria ACP adopts a different mode of lysozyme inhibition and that the ability of ACP to inhibit lysozyme activity could be important for host colonization by both pathogenic and commensal Neisseria organisms. Thus, ACP represents a dual target for developing Neisseria vaccines and drugs to inhibit host-pathogen interactions

Pseudomonas aeruginosa host-pathogen interactions in human corneal infection models

In the whole tissue model (WTM), donor buttons were infected with PAO1 by scoring and intrastromal in-jection. Tissue was examined after 3, 9 and 24 h post challenge by transmission electron microscopy (TEM) andscanning electron microscopy (SEM).In the cell culture model (CCM), cornealfibroblasts (CF) were infected in vitro with PAO1. Bacterial ad-herence and internalization were assayed at 3, 6 and 9 h by SEM and TEM. Adherent and internalized bacteriawere measured by the gentamicin protection invasion assayPostprint (published version

Vaccine potential of bacterial macrophage infectivity potentiator (MIP)-like peptidyl prolyl cis/trans isomerase (PPIase) proteins

Peptidyl prolyl cis/trans isomerases (PPIases) are a superfamily of proteins ubiquitously distributed among living organisms, which function primarily to assist the folding and structuring of unfolded and partially folded polypeptide chains and proteins. In this review, we focus specifically on the Macrophage Infectivity Potentiator (MIP)-like PPIases, which are members of the immunophilin family of FK506-binding proteins (FKBP). MIP-like PPIases have accessory roles in virulence and are candidates for inclusion in vaccines protective against both animal and human bacterial pathogens. A structural vaccinology approach obviates any issues over molecular mimicry and potential cross-reactivity with human FKBP proteins and studies with a representative antigen, the Neisseria meningitidis-MIP, support this strategy. Moreover, a dual approach of vaccination and drug targeting could be considered for controlling bacterial infectious diseases of humans and animals.</p

Structure of rNm-ACPI and comparison with the MliC/PliC canonical fold.

<p><b>A)</b> Ribbon structure, showing the eight β-strands and disulphide bond. <b>B)</b> Topology diagram showing residue ranges in each β-strand, and the disulfide bond between Cys38 and Cys121 residues. <b>C)</b> Two orthogonal views of rNm-ACPI with the corresponding electrostatic surface in the same orientations below. <b>D)</b> Structural superposition of rNm-ACPI (blue) with PliC from <i>Brucella abortus</i> (Ba-PliC; PDB 4ML7; lilac), as a representative of the MliC/PliC lysozyme inhibitor family. Loop 4 is highlighted in red for rNm-ACPI and green for Ba-PliC.Images were constructed using CCP4MG [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006448#ppat.1006448.ref058" target="_blank">58</a>].</p

Measurement of membrane permeability using dye viability assay.

<p>Wild-type <i>Neisseria meningitidis</i> strains MC58 and MC161 and wild-type <i>Neisseria gonorrhoeae</i> strain FA1090 and the corresponding <i>Δacp</i> and complemented strains were grown overnight on supplemented GC agar medium and colonies were used to inoculate 5 mL of GC broth. Cultures were incubated at 37°C with shaking (200 rpm) until mid-logarithmic growth phase was reached (ODλ₆₀₀nm >0.4) and viability was examined with the LIVE/DEAD <i>Bac</i>light Bacterial Viability Kit (Molecular Probes). For each bacterium, approximately 5–6 independent fields of view were examined in duplicate, counting ~100 colonies per field. The percentage of bacteria positive for red propidium iodide (PI) staining, indicating bacteria with permeable membranes, was calculated by dividing PI-positive bacteria by total (red and Syto9 green) bacteria. The columns represent the mean percentages of PI-labelled bacteria (membrane permeant) and the error bars the standard error of the means from a minimum of n = 3 biological replicates per bacterium. A two-sample t-Test was used to compare statistical significance of ACP^- viability with ACP⁺ viability data.</p

Structure-based sequence alignment of Nm-ACPI with the MliC/PliC family of lysozyme inhibitor proteins.

<p>The sequence of ACP from <i>Neisseria meningitidis</i> serogroup B strain MC58 (E6MZT7) was aligned with those for St-PliC from <i>Salmonella typhimurium</i> (Q8ZPY8); Pa-MliC from <i>Pseudomonas aeruginosa</i> (Q91574) and Ba-PliC from <i>Brucella abortus</i> (Q57ES7) based on structural superpositions carried out using CCP4MG [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006448#ppat.1006448.ref058" target="_blank">58</a>]. β-strands β1 – β8 are indicated by the shaded arrows, and are based on Nm-ACPI structure. Conserved cysteine residues of the mature proteins are blocked in black. Numbering follows the Nm-ACPI sequence. Underlined residues had weak or no density and were not included in the crystal structures. Sequence motifs involved in the interaction of <i>P</i>. <i>aeruginosa</i> MliC with Hewl are in red and green, and conserved residues within these regions are marked in bold and identified by similarly coloured asterisks (*).</p

Sensitivity of pathogenic <i>Neisseria</i> spp. to HL: Colony Forming Units (CFU) used to plot Fig 8A.

<p>Sensitivity of pathogenic <i>Neisseria</i> spp. to HL: Colony Forming Units (CFU) used to plot <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006448#ppat.1006448.g008" target="_blank">Fig 8A</a>.</p

<i>Neisseria</i> spp. sensitivity to HL.

<p><b>A)</b> Bacterial killing (as measured by the ratio of colony-forming units (CFU) in the absence [N₀] and presence [N] of human lysozyme: N₀/N) after 8 h incubation with 10 μg/ml HL and 3 mg/ml lactoferrin, was determined using <i>N</i>. <i>meningitidis</i> MC58, MC161 and <i>N</i>. <i>gonorrhoeae</i> FA1090 wild type (black bars), <i>acp</i> knock-out (white bars) and complemented (grey bars) strains. <b>B)</b> Wild type <i>N</i>. <i>lactamica</i> sensitivity to HL was addressed by determining the N₀/N ratio at 8 h after contact with HL and lactoferrin, in the absence or presence of purified IgG antibodies directed against rNm-ACPI (100% identical to NlY92-1009-ACP), or control IgG obtained from pre-immunisation sera. The columns represent the mean and the error bars the standard error of the means calculated from n = 5 independent experiments. Data were compared with a two-sample t-Test, and lysozyme treatments resulting in significant differences (P<0.05) compared to the corresponding controls, are marked with an asterisk (*).</p

Minimum inhibitory concentration (MIC) of vancomycin and streptomycin antibiotics for wild-type, <i>Δacp and</i> <i>Δacp</i> complemented meningococci and gonococci.

<p>Minimum inhibitory concentration (MIC) of vancomycin and streptomycin antibiotics for wild-type, <i>Δacp and</i> <i>Δacp</i> complemented meningococci and gonococci.</p