Genetic Regulation of Virulence and Antibiotic Resistance in Acinetobacter baumannii

Multidrug resistant microorganisms are forecast to become the single biggest challenge to medical care in the 21st century. Over the last decades, members of the genus Acinetobacter have emerged as bacterial opportunistic pathogens, in particular as challenging nosocomial pathogens because of the rapid evolution of antimicrobial resistances. Although we lack fundamental biological insight into virulence mechanisms, an increasing number of researchers are working to identify virulence factors and to study antibiotic resistance. Here, we review current knowledge regarding the regulation of virulence genes and antibiotic resistance in Acinetobacter baumannii. A survey of the two-component systems AdeRS, BaeSR, GacSA and PmrAB explains how each contributes to antibiotic resistance and virulence gene expression, while BfmRS regulates cell envelope structures important for pathogen persistence. A. baumannii uses the transcription factors Fur and Zur to sense iron or zinc depletion and upregulate genes for metal scavenging as a critical survival tool in an animal host. Quorum sensing, nucleoid-associated proteins, and non-classical transcription factors such as AtfA and small regulatory RNAs are discussed in the context of virulence and antibiotic resistance

The small RNA RssR regulates myo-inositol degradation by Salmonella enterica

Small noncoding RNAs (sRNAs) with putative regulatory functions in gene expression have been identified in the enteropathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Two sRNAs are encoded by the genomic island GEI4417/4436 responsible for myo-inositol (MI) degradation, suggesting a role in the regulation of this metabolic pathway. We show that a lack of the sRNA STnc2160, termed RssR, results in a severe growth defect in minimal medium (MM) with MI. In contrast, the second sRNA STnc1740 was induced in the presence of glucose, and its overexpression slightly attenuated growth in the presence of MI. Constitutive expression of RssR led to an increased stability of the reiD mRNA, which encodes an activator of iol genes involved in MI utilization, via interaction with its 5'-UTR. SsrB, a response regulator contributing to the virulence properties of salmonellae, activated rssR transcription by binding the sRNA promoter. In addition, the absence of the RNA chaperone Hfq resulted in strongly decreased levels of RssR, attenuated S. Typhimurium growth with MI, and reduced expression of several iol genes required for MI degradation. Considered together, the extrinsic RssR allows fine regulation of cellular ReiD levels and thus of MI degradation by acting on the reiD mRNA stability

Transmission of an oxygen availability signal at the Salmonella enterica serovar Typhimurium fis promoter.

The nucleoid-associated protein FIS is a global regulator of gene expression and chromosome structure in Escherichia coli and Salmonella enterica. Despite the importance of FIS for infection and intracellular invasion, very little is known about the regulation of S. enterica fis expression. Under standard laboratory growth conditions, fis is highly expressed during rapid growth but is then silenced as growth slows. However, if cells are cultured in non-aerated conditions, fis expression is sustained during stationary phase. This led us to test whether the redox-sensing transcription factors ArcA and FNR regulate S. enterica fis. Deletion of FNR had no detectable effect, whereas deletion of ArcA had the unexpected effect of further elevating fis expression in stationary phase. ArcA required RpoS for induction of fis expression, suggesting that ArcA indirectly affects fis expression. Other putative regulators were found to play diverse roles: FIS acted directly as an auto-repressor (as expected), whereas CRP had little direct effect on fis expression. Deleting regions of the fis promoter led to the discovery of a novel anaerobically-induced transcription start site (Pfis-2) upstream of the primary transcription start site (Pfis-1). Promoter truncation also revealed that the shortest functional fis promoter was incapable of sustained expression. Moreover, fis expression was observed to correlate directly with DNA supercoiling in non-aerated conditions. Thus, the full-length S. enterica fis promoter region may act as a topological switch that is sensitive to stress-induced duplex destabilisation and up-regulates expression in non-aerated conditions

Gapless sequence alignment of the <i>E. coli</i> and <i>S</i>. Typhimurium <i>fis</i> promoter regions.

<p>CRP and FIS (grey boxes) and IHF (underline) binding sites identified in <i>E. coli</i> are shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084382#B5" target="_blank">5</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084382#B6" target="_blank">6</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084382#B40" target="_blank">40</a>]. Promoter elements, transcription start sites and <i>dusB</i> translation initiation codons are boxed. The angled arrows show the locations of P<i>fis</i>-1 (both species) and P<i>fis</i>-2 (<i>S</i>. Typhimurium). The locations of promoter truncations in <i>S</i>. Typhimurium are indicated by black triangles.</p

Effects of transcription factor mutations on <i>fis</i> expression in aerated and non-aerated conditions.

<p>A) Column graph comparing <i>dusB-fis</i>::<i>gfp</i>^TCD expression at 22 hours. All data are expressed relative to wild type at 22 hours in well-aerated conditions (dashed line). <b>B</b>) Average and standard deviation of four replicate culture densities after 22 hours. C-F) Time courses of <i>dusB-fis::gfp</i>^TCD expression in <i>S</i>. Typhimurium wild type and Δ<i>fnr</i> (C), Δ<i>arcA</i> (D), Δ<i>rpoS</i> (E), and Δ<i>arcA</i>/Δ<i>rpoS</i> (F) mutants. The blue arrow in D indicates the increased sustained expression observed in the Δ<i>arcA</i> mutant after 22 hours in non-aerated conditions. <b>A</b>, <b>C</b>-<b>F</b>) Mean and standard deviation of GFP fluorescence from three or four biological replicates are plotted in arbitrary units. All data are expressed relative to wild type levels at time point 0 in well-aerated conditions. The same wild type data are presented in panels C-F. <b>G</b>) Growth dynamics of strains in aerated and non-aerated conditions. Smoothed curves were generated by GraphPad Prism 5.0d from the average of four or more replicate growth curves. </p

Defining functional regions and transcription factor input at the <i>fis</i> promoter.

<p>A) Schematic of the <i>E. coli</i> and <i>S</i>. Typhimurium intergenic regions. The locations of truncations in <i>S</i>. Typhimurium are indicated. B) Time courses of expression of plasmid-borne P<i>fis</i>::<i>gfp</i> promoter truncates in aerated and non-aerated <i>S</i>. Typhimurium cultures. C and D) Expression of promoter truncates in <i>S</i>. Typhimurium transcription factor mutant backgrounds at 22 hours. In B-D, mean and standard deviation of GFP fluorescence from three or more biological replicates are plotted in arbitrary units that are reported relative to the chromosomal <i>dusB-fis::gfp</i>^TCD fluorescence output presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084382#pone-0084382-g002" target="_blank">Figure 2</a>. </p

DNA supercoiling control of <i>fis</i> expression.

<p>A) Median and interquartile ranges of DNA supercoiling from four biological replicates, plotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084382#pone-0084382-g004" target="_blank">Figure 4E</a>. Fluorescence data from P<i>fis</i>(-298) (green circles) and <i>dusB-fis::gfp</i>^TCD (blue diamonds) for each biological replicate is plotted on the right y-axis; units as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084382#pone-0084382-g002" target="_blank">Figure 2</a>. For both the DNA supercoiling and gene expression measurements, wild type cells were grown to stationary phase cells in the indicated volume of culture medium. The dashed lines are nonlinear curves fit to the expression data, with goodness-of-fit R² >0.85 in both cases. The degree of DNA supercoiling (σ) was determined by measuring the migration of topoisomers relative to fully relaxed DNA in chloroquine gels; each topoisomer represents a change of 1 in the linking number. B) DNA supercoiling states in <i>S</i>. Typhimurium wildtype and Δ<i>arcA</i> mutant cells at 22 hours in non-aerated conditions. Medians (black bar) and interquartile ranges of pUC18 topoisomer distributions in stationary phase in well-aerated cultures. For each strain, the average interquartile range from four biological replicates is plotted. C) SIDD profile of the <i>fis</i> promoter region. The energy required for DNA strand separation at a base pair, G(x), is a function of adjacent and distant DNA sequence [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084382#B30" target="_blank">30</a>], and G(x) values below 10 indicate positions prone to SIDD. G(x) values for linear DNA were calculated by WebSIDD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084382#B27" target="_blank">27</a>] using 3,500 bp of chromosomal DNA sequence on either side of the <i>dusB</i> start codon (7,000 bp total); only the 550 bp region containing P<i>fis</i>-1 and P<i>fis</i>-2 is shown. </p