Deciphering the Multifactorial Nature of Acinetobacter baumannii Pathogenicity

Background: Acinetobacter baumannii is an emerging bacterial pathogen that causes a broad array of infections, particularly in hospitalized patients. Many studies have focused on the epidemiology and antibiotic resistance of A. baumannii, but little is currently known with respect to its virulence potential. Methodology/Principal Findings: The aim of this work was to analyze a number of virulence-related traits of four A. baumannii strains of different origin and clinical impact for which complete genome sequences were available, in order to tentatively identify novel determinants of A. baumannii pathogenicity. Clinical strains showed comparable virulence in the Galleria mellonella model of infection, irrespective of their status as outbreak or sporadic strains, whereas a non-human isolate was avirulent. A combined approach of genomic and phenotypic analyses led to the identification of several virulence factors, including exoproducts with hemolytic, phospholipase, protease and iron-chelating activities, as well as a number of multifactorial phenotypes, such as biofilm formation, surface motility and stress resistance, which were differentially expressed and could play a role in A. baumannii pathogenicity. Conclusion/Significance: This work provides evidence of the multifactorial nature of A. baumannii virulence. While A. baumannii clinical isolates could represent a selected population of strains adapted to infect the human host, subpopulations of highly genotypically and phenotypically diverse A. baumannii strains may exist outside the hospita

A História da Alimentação: balizas historiográficas

Os M. pretenderam traçar um quadro da História da Alimentação, não como um novo ramo epistemológico da disciplina, mas como um campo em desenvolvimento de práticas e atividades especializadas, incluindo pesquisa, formação, publicações, associações, encontros acadêmicos, etc. Um breve relato das condições em que tal campo se assentou faz-se preceder de um panorama dos estudos de alimentação e temas correia tos, em geral, segundo cinco abardagens Ia biológica, a econômica, a social, a cultural e a filosófica!, assim como da identificação das contribuições mais relevantes da Antropologia, Arqueologia, Sociologia e Geografia. A fim de comentar a multiforme e volumosa bibliografia histórica, foi ela organizada segundo critérios morfológicos. A seguir, alguns tópicos importantes mereceram tratamento à parte: a fome, o alimento e o domínio religioso, as descobertas européias e a difusão mundial de alimentos, gosto e gastronomia. O artigo se encerra com um rápido balanço crítico da historiografia brasileira sobre o tema

WalH and WalI are membrane-anchored extracellular proteins.

<p>(A) <i>E</i>. <i>coli</i> DH5α cells producing ‘PhoA’-‘LacZ’ fusion proteins (WalH_1-40-PhoA_21-471-LacZ_4-60, WalI_1-40-PhoA_21-471-LacZ_4-60, or WalJ_1-40-PhoA_21-471-LacZ_4-60) were plated on indicator medium with two chromogenic substrates, Red-Gal (for β-galactosidase activity) and X-Pho (for phosphatase activity). Blue coloring of the colonies (high phosphatase activity) indicates a membrane or extracellular localization of the fusion point. Red coloring of the colonies (high β-galactosidase activity) indicates cytosolic localization of the fusion point. (B) Schematic representation of Wal protein localization and topology with respect to the cell membrane. In the case of WalK and WalR, topology and localization were deduced from primary sequence analysis using the Phobius Hidden Markov Model (indicated by stars).</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

The <i>S</i>. <i>aureus wal</i> locus consists of two separate transcription units.

<p>(A) <i>S</i>. <i>aureus</i> total RNA was used to synthesize cDNAs and amplify intergenic regions with oligonucleotides hybridizing specifically within the upstream and downstream genes (indicated by arrows, not to scale). DNA fragments were separated by electrophoresis on ethidium bromide stained 1.5% agarose gels and their size is indicated. Lanes 1: positive PCR control using HG001 genomic DNA as the template; lanes 2: PCR using cDNA as the template; lanes 3: control PCR reaction using total RNA as the template (without reverse transcriptase treatment). M: 100 bp molecular mass ladder (Eurogentec, Angers, France). (B) Representation of the <i>wal</i> locus and the corresponding transcripts (not to scale).</p

Biofilm formation is decreased in the Δ<i>walH</i>, Δ<i>walI</i> and Δ<i>walHI</i> mutants.

<p>Biofilm assays were performed in microtiter plates after growth at 37°C for 24 h. Adherent biomass was quantified, normalized to the OD_{600 nm} of each cell culture and represented as n-fold variation compared to the parental strain. Dark grey bars indicate biomass levels in the parental and mutant strains and light grey bars correspond to values in the complemented strains carrying pMK4-Pprot<i>walHI</i>. Experiments were carried out in quadruplicate and standard deviations are indicated. ** <i>P</i><0.01 as determined using Student’s <i>t</i>-test.</p

Triton-induced autolysis is decreased in the absence of WalH.

<p>Bacteria were grown in TSB at 37°C with shaking until OD_{600 nm} ≈ 1, pelleted (10 min; 5,400 x <i>g</i>), resuspended in phosphate buffered saline (PBS) with Triton X-100 (0.1%), and incubated at 37°C with shaking. Lysis was determined as the decrease in OD_{600 nm} over time and indicated as a percentage of the initial OD (measured OD_{600 nm} / initial OD_{600 nm}). Results are shown as the mean and standard deviation of three independent experiments. Strains: HG001 (■); ST1397 Δ<i>walH</i> (○); ST1410 Δ<i>walHI</i> (△); ST1415 Δ<i>walH</i> pMK4Pprot-<i>walHI</i> (●); ST1417 Δ<i>walHI</i> pMK4Pprot-<i>walHI</i> (▲).</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Expression of <i>atlA</i> is increased in the Δ<i>walH</i>, Δ<i>walI</i> and Δ<i>walHI</i> mutants.

<p>The HG001 parental strain and the Δ<i>walH</i>, Δ<i>walI</i> and Δ<i>walHI</i> mutant strains were grown in TSB rich medium until OD_{600 nm} = 1. Total RNA was extracted and quantitative real time PCR was used to compare gene expression. Expression levels were normalized using 16S rRNA as an internal standard and are indicated as the <i>n</i>-fold change with respect to the HG001 parental strain, expressed as means and standard deviations. Dark grey bars indicate expression levels in the parental and mutant strains and light grey bars correspond to values in the complemented strains carrying pMK4-Pprot<i>walHI</i>. * <i>P</i><0.05 as determined using Student’s <i>t</i>-test.</p

Phylogenetic relationships within the Bacilli class and inferred losses of <i>wal</i> genes.

<p>(A) Maximum likelihood phylogeny of Bacilli based on a concatenation of 47 ribosomal proteins comprising 5,945 amino acid positions. The conservation of <i>wal</i> genes in each genome is indicated by circles: <i>walR</i> (red), <i>walK</i> (green), <i>walH</i> (blue), <i>walI</i> (yellow), <i>walJ</i> (purple). Red crosses indicate the loss of <i>walH</i> and <i>walI</i> in Streptococcaceae and of <i>walJ</i> in <i>Leuconostoc</i>. (B) Maximum likelihood phylogeny based on a concatenation of WalR, WalK, WalH, WalI and WalJ protein sequences comprising 1,102 amino acid positions. For both analyses, values at nodes represent bootstrap proportions calculated on 1,000 resamplings of the original data set. For clarity, only the values corresponding to monophyly of families and their evolutionary relationships are shown. The scale bar represents the average number of substitutions per site. For details on analyses, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151449#sec002" target="_blank">Materials and Methods</a>.</p