Allosteric mechanism controls traffic in the chaperone/usher pathway

Many virulence organelles of Gram-negative bacterial pathogens are assembled via the chaperone/ usher pathway. The chaperone transports organelle subunits across the periplasm to the outer membrane usher, where they are released and incorporated into growing fibers. Here, we elucidate the mechanism of the usher-targeting step in assembly of the Yersinia pestis F1 capsule at the atomic level. The usher interacts almost exclusively with the chaperone in the chaperone:subunit complex. In free chaperone, a pair of conserved proline residues at the beginning of the subunit-binding loop form a ‘‘proline lock’’ that occludes the usher-binding surface and blocks usher binding. Binding of the subunit to the chaperone rotates the proline lock away from the usher-binding surface, allowing the chaperone-subunit complex to bind to the usher. We show that the proline lock exists in other chaperone/usher systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of the free chaperone.

Within-host evolution of Staphylococcus aureus during asymptomatic carriage

Background Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus. Principal Findings We characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA). Significance This investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease.

Prediction of Staphylococcus aureus antimicrobial resistance by whole-genome sequencing

Whole-genome sequencing (WGS) could potentially provide a single platform for extracting all the information required to predict an organism’s phenotype. However, its ability to provide accurate predictions has not yet been demonstrated in large independent studies of specific organisms. In this study, we aimed to develop a genotypic prediction method for antimicrobial susceptibilities. The whole genomes of 501 unrelated Staphylococcus aureus isolates were sequenced, and the assembled genomes were interrogated using BLASTn for a panel of known resistance determinants (chromosomal mutations and genes carried on plasmids). Results were compared with phenotypic susceptibility testing for 12 commonly used antimicrobial agents (penicillin, methicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, vancomycin, trimethoprim, gentamicin, fusidic acid, rifampin, and mupirocin) performed by the routine clinical laboratory. We investigated discrepancies by repeat susceptibility testing and manual inspection of the sequences and used this information to optimize the resistance determinant panel and BLASTn algorithm. We then tested performance of the optimized tool in an independent validation set of 491 unrelated isolates, with phenotypic results obtained in duplicate by automated broth dilution (BD Phoenix) and disc diffusion. In the validation set, the overall sensitivity and specificity of the genomic prediction method were 0.97 (95% confidence interval [95% CI], 0.95 to 0.98) and 0.99 (95% CI, 0.99 to 1), respectively, compared to standard susceptibility testing methods. The very major error rate was 0.5%, and the major error rate was 0.7%. WGS was as sensitive and specific as routine antimicrobial susceptibility testing methods. WGS is a promising alternative to culture methods for resistance prediction in S. aureus and ultimately other major bacterial pathogens.

Bovine muc1 inhibits binding of enteric bacteria to caco-2 cells

Inhibition of bacterial adhesion to intestinal epithelial receptors by the consumption of natural food components is an attractive strategy for the prevention of microbial related gastrointestinal illness. We hypothesised that Muc1, a highly glycosylated mucin present in cows’ milk, may be one such food component. Purified bovine Muc1 was tested for its ability to inhibit binding of common enteric bacterial pathogens to Caco-2 cells grown in vitro. Muc1 caused dose-dependent binding inhibition of Escherichia coli, Salmonella enterica serovar Typhimurium (S. Typhimurium), Staphylococcus aureus and Bacillus subtilis. This inhibition was more pronounced for the Gram negative compared with Gram positive bacteria. It was also demonstrated that Muc1, immobilised on a membrane, bound all these bacterial species in a dose-dependent manner, although there was greater interaction with the Gram negative bacteria. A range of monosaccharides, representative of the Muc1 oligosaccharide composition, were tested for their ability to prevent binding of E. coli and S. Typhimurium to Caco-2 cells. Inhibition was structure dependent with sialic acid, L(-) fucose and D(+) mannose significantly inhibiting binding of both Gram negative species. N-acetylglucosamine and N-acetylgalactosamine significantly inhibited binding of E. coli whilst galactose, one of the most abundant Muc1 monosaccharides, showed the strongest inhibition against S. Typhimurium. Treatment with sialidase significantly decreased the inhibitory properties of Muc1, demonstrating the importance of sialic acid in adhesion inhibition. It is concluded that bovine Muc1 prevents binding of bacteria to human intestinal cells and may have a role in preventing the binding of common enteropathogenic bacteria to human intestinal epithelial surfaces.

Extensible motif and aberrant nucleotide extraction in genomic sequences

The project focusses on the discovery of conserved DNA sequences in bacterial genomes and comparative analysis of bacterial genomes to elicit evolutionary trends. The outcomes have produced novel techniques for modelling motifs in DNA and the characterisation of evolutionary processes in medically significant bacterial pathogens.

Transcriptomics-based analysis using RNA-Seq of the coconut (Cocos nucifera) leaf in response to yellow decline phytoplasma infection

Invasive phytoplasmas wreak havoc on coconut palms worldwide, leading to high loss of income, food insecurity and extreme poverty of farmers in producing countries. Phytoplasmas as strictly biotrophic insect-transmitted bacterial pathogens instigate distinct changes in developmental processes and defence responses of the infected plants and manipulate plants to their own advantage; however, little is known about the cellular and molecular mechanisms underlying host–phytoplasma interactions. Further, phytoplasma-mediated transcriptional alterations in coconut palm genes have not yet been identified. This study evaluated the whole transcriptome profiles of naturally infected leaves of Cocos nucifera ecotype Malayan Red Dwarf in response to yellow decline phytoplasma from group 16SrXIV, using RNA-Seq technique. Transcriptomics-based analysis reported here identified genes involved in coconut innate immunity. The number of down-regulated genes in response to phytoplasma infection exceeded the number of genes up-regulated. Of the 39,873 differentially expressed unigenes, 21,860 unigenes were suppressed and 18,013 were induced following infection. Comparative analysis revealed that genes associated with defence signalling against biotic stimuli were significantly overexpressed in phytoplasma-infected leaves versus healthy coconut leaves. Genes involving cell rescue and defence, cellular transport, oxidative stress, hormone stimulus and metabolism, photosynthesis reduction, transcription and biosynthesis of secondary metabolites were differentially represented. Our transcriptome analysis unveiled a core set of genes associated with defence of coconut in response to phytoplasma attack, although several novel defence response candidate genes with unknown function have also been identified. This study constitutes valuable sequence resource for uncovering the resistance genes and/or susceptibility genes which can be used as genetic tools in disease resistance breeding.

Norbornene-based anion receptors as D-alanine binders

Vancomycin is currently used as last-line therapy against many Gram-positive bacterial pathogens. Herein, we report a series of peptidomimetic norbornene-based anion receptors that were designed as simple vancomycin mimics New hosts were evaluated for their affinity to both acetate and acetyl D-Ala by 1H NMR titration. Modest binding to both anions was observed in DMSO-d6 (Log Ka 1-2 for TBA Acetyl D-Alanine) in the anticipated 1:1 mode of binding.

Norbornane-based peptidomimetics as antibacterial agents

 A library of norbornane-based peptide mimetics have been synthesised and shown to exhibit antibacterial activity against a range of troublesome Gram-positive and Gram-negative bacterial pathogens.

Análise por sequências multilocus de Xanthomonas fuscans subsp. aurantifolii

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Diversidade genética de espécies de Xanthomonas patogênicas a citros baseada em genes avr e leucine protein

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação da suplementação alimentar com bactéria probiótica no crescimento e sanidade de Arapaima gigas em sistema de recirculação de água

As bactérias acido lácticas tem se destacado por desempenhar efeitos benéficos ao hospedeiro, entre eles destacam-se a ativação no sistema imunológico, desempenho zootécnico e eficiência alimentar. O objetivo deste trabalho foi selecionar e a avaliar bactérias potencialmente probiótica no crescimento e sanidade de Arapaima gigas. A primeira etapa foi o isolamento e seleção de bactérias com potencial probiótico a partir de 10 juvenis de A. gigas sendo submetidas a testes in vitro de inibição de patógenos bacterianos conhecidos para aquicultura, e a segunda etapa consistiu em testes in vivo avaliando a colonização do trato intestinal pela cepa bacteriana e sua relação com a hematologia. A cepa com melhor halo de inibição foi identificada com o kit API50 CH como Lactobacillus paracasei (99.9%). Para avaliação in vivo, os animais foram alimentados durante 120 dias com dieta contendo somente leite estéril (T1), dieta controle sem bactéria (T2), dieta com 105 L. paracasei (T3) e dieta com 106 L. paracasei(T4). Após este período os peixes foram submetidos três tratamentos: injeção intraperitoneal com Aeromonas hydrophila, injeção de solução salina e peixes não injetados. Ao final de 24 horas de desafio foi observado elevada mortalidade dos peixes pertencentes ao grupo T3, T4 em relação ao T1. Os resultados nos permite concluir que a cepa utilizada nos testes in vivo não influenciou o desempenho zootécnico, além de ser ineficaz na proteção contra a infecção por Aeromonas.

Effect of a Silver Nanoparticles Solution on Staphylococcus aureus and Candida spp.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Synergistic Anti-Inflammatory Activity of the Antimicrobial Peptides Human Beta-Defensin-3 (hBD-3) and Cathelicidin (LL-37) in a Three-Dimensional Co-Culture Model of Gingival Epithelial Cells and Fibroblasts

Given the spread of antibiotic resistance in bacterial pathogens, antimicrobial peptides that can also modulate the immune response may be a novel approach for effectively controlling periodontal infections. In the present study, we used a three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) to investigate the anti-inflammatory properties of human beta-defensin-3 (hBD-3) and cathelicidin (LL-37) and to determine whether these antimicrobial peptides can act in synergy. The 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to LPS stimulation compared to fibroblasts and epithelial cells alone. The 3D co-culture model was stimulated with non-cytotoxic concentrations of hBD-3 (10 and 20 mu M) and LL-37 (0.1 and 0.2 mu M) individually and in combination in the presence of A. actinomycetemcomitans LPS. A multiplex ELISA assay was used to quantify the secretion of 41 different cytokines. hBD-3 and LL-37 acted in synergy to reduce the secretion of GRO-alpha, G-CSF, IP-10, IL-6, and MCP-1, but only had an additive effect on reducing the secretion of IL-8 in response to A. actinomycetemcomitans LPS stimulation. The present study showed that hBD-3 acted in synergy with LL-37 to reduce the secretion of cytokines by an LPS-stimulated 3D model of gingival mucosa. This combination of antimicrobial peptides thus shows promising potential as an adjunctive therapy for treating inflammatory periodontitis.

Validação de técnicas moleculares para o diagnóstico de bactérias em peixes, visando redução de tempo e custo

Pós-graduação em Microbiologia Agropecuária - FCAV

Anti-inflammatory effect of MAPK phosphatase-1 local gene transfer in inflammatory bone loss

Alveolar bone loss associated with periodontal diseases is the result of osteoclastogenesis induced by bacterial pathogens. The mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) is a critical negative regulator of immune response as a key phosphatase capable of dephosphorylating activated MAPKs. In this study, rat macrophages transduced with recombinant adenovirus (Ad.)MKP-1 specifically dephosphorylated activated MAPKs induced by lipopolysaccharide (LPS) compared with control cells. Bone marrow macrophages from MKP-1 knockout (KO) mice exhibited higher interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and select chemokine compared with wild-type (WT) mice when stimulated by LPS. In addition, bone marrow cultures from MKP-1 KO mice exhibited significantly more osteoclastogenesis induced by LPS than when compared with WT mice. Importantly, MKP-1 gene transfer in bone marrow cells of MKP-1 KO mice significantly decreased IL-6, IL-10, TNF-α and chemokine levels, and formed fewer osteoclasts induced by LPS than compared with control group of cells. Furthermore, MKP-1 gene transfer in an experimental periodontal disease model attenuated bone resorption induced by LPS. Histological analysis confirmed that periodontal tissues transduced with Ad. MKP-1 exhibited less infiltrated inflammatory cells, less osteoclasts and less IL-6 than compared with rats of control groups. These studies indicate that MKP-1 is a key therapeutic target to control of inflammation-induced bone loss.