Effect of Ethanol on Differential Protein Production and Expression of Potential Virulence Functions in the Opportunistic Pathogen <em>Acinetobacter baumannii</em>

<div><p><em>Acinetobacter baumannii</em> persists in the medical environment and causes severe human nosocomial infections. Previous studies showed that low-level ethanol exposure increases the virulence of <em>A. baumannii</em> ATCC 17978. To better understand the mechanisms involved in this response, 2-D gel electrophoresis combined with mass spectrometry was used to investigate differential protein production in bacteria cultured in the presence or absence of ethanol. This approach showed that the presence of ethanol significantly induces and represses the production of 22 and 12 proteins, respectively. Although over 25% of the ethanol-induced proteins were stress-response related, the overall bacterial viability was uncompromised when cultured under these conditions. Production of proteins involved in lipid and carbohydrate anabolism was increased in the presence of ethanol, a response that correlates with increased carbohydrate biofilm content, enhanced biofilm formation on abiotic surfaces and decrease bacterial motility on semi-solid surfaces. The presence of ethanol also induced the acidification of bacterial cultures and the production of indole-3-acetic acid (IAA), a ubiquitous plant hormone that signals bacterial stress-tolerance and promotes plant-bacteria interactions. These responses could be responsible for the significantly enhanced virulence of <em>A. baumannii</em> ATCC 17978 cells cultured in the presence of ethanol when tested with the <em>Galleria mellonella</em> experimental infection model. Taken together, these observations provide new insights into the effect of ethanol in bacterial virulence. This alcohol predisposes the human host to infections by <em>A. baumannii</em> and could favor the survival and adaptation of this pathogen to medical settings and adverse host environments.</p> </div

Differential production of carbon and lipid metabolism-related proteins in response to ethanol.

<p>Table legends same as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051936#pone-0051936-t001" target="_blank">Table 1</a>.</p

Differential production of porin synthesis, regulation, nucleotide and protein synthesis-related proteins and a protein of unknown function in response to ethanol.

<p>Table legends same as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051936#pone-0051936-t001" target="_blank">Table 1</a>.</p

Putative genes in proposed ethanol-induced IAA biosynthesis pathway in <i>A. baumannii</i> ATCC 17978.

a<p>Enzyme numbers correspond to numbers shown on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051936#pone-0051936-g008" target="_blank">Figure 8</a>.</p>b<p>References to homologous genes that have been demonstrated to be involved in ethanol assimilation, tryptophan catabolism and/or IAA biosynthesis.</p>c<p>Not applicable.</p

Proposed pathways for ethanol-induced IAA production in <i>A. baumannii</i>.

<p>The pathways were developed taking into account information previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051936#pone.0051936-Camarena1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051936#pone.0051936-Spaepen1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051936#pone.0051936-Idris1" target="_blank">[68]</a> and experimental data collected in this work. Numbers next to arrows refer to the enzymes listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051936#pone-0051936-t006" target="_blank">Table 6</a>. The asterisk (*) symbol denotes a suggested dual role for aldehyde dehydrogenase in the conversion of indole-3-acetaldehyde to indole-3-acetic acid and the conversion of acetaldehyde to acetic acid as previously demonstrated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051936#pone.0051936-Basse1" target="_blank">[51]</a>.</p

Effects of ethanol on biofilm formation and carbohydrate production.

<p>Biofilms formed by <i>A. baumannii</i> ATCC 17978 cells cultured in LB supplemented with 0%, 1% or 2% ethanol were visualized by crystal violet staining (A) and further quantified by colorimetry, after elution of the retained stain, in relation to the total biomass of each tested sample (B). A tube incubated with sterile medium (SM) was used as a negative control. (C) Carbohydrate content of biofilms formed on plastic by <i>A. baumannii</i> ATCC 17978 cells statically cultured in LB broth containing 0%, 1% or 2% ethanol determined with the L-cysteine monomeric carbohydrate assay. The carbohydrate concentration of biofilms was normalized to the protein content of each analyzed sample. Error bars shown in panels B and C represent 1 standard error. (D) Scanning electron microscopy of biofilms formed on plastic above, at and below the meniscus of LB broth static cultures containing 0%, 1% or 2% ethanol incubated overnight statically at 37°C. All micrographs were taken at a X25,000 magnification. The bar shown in the left bottom panel represents 1 µm.</p

Effect of ethanol on the pH of <i>A. baumannii</i> ATCC 17978 cultures.

<p>Sterile and inoculated LB broth cultures supplemented with 0%, 1%, or 2% ethanol were incubated in an orbital shaker overnight at 37°C. The pH of the sterile samples and cultured supernatants obtained after centrifugation at 10,000 rpm for 10 min was determined immediately. Error bars represent 1 standard error. Horizontal bars with the asterisk (*) symbol identify samples with statistically significant differences (<i>P</i><0.05) in pH values.</p

Effect of ethanol and/or tryptophan on IAA production.

<p>(A) The concentration of IAA in LB or SB <i>A. baumannii</i> ATCC 17978 cultures supplemented with 0%, 1% or 2% ethanol was determined after 24 h incubation at 37°C. (B) The effect of 5 mM L-tryptophan and/or 1% ethanol supplementation on the production of IAA bacteria cultured in SB after 24 h incubation 37°C. The plus (+) and minus (−) signs denote the addition or not of ethanol or tryptophan. IAA concentration values were normalized to an OD₆₀₀ of 1.0 of each tested sample. Error bars represent 1 standard error. Horizontal bars with the asterisk (*) symbol identify samples with statistically significant differences (<i>P</i><0.05) in IAA concentration values. All differences in IAA concentrations shown in panel B are statistically significant (<i>P</i><0.05).</p

Differential production of energy and general metabolism-related proteins in response to ethanol.

<p>Table legends same as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051936#pone-0051936-t001" target="_blank">Table 1</a>.</p