Genomic and Functional Analysis of the Type VI Secretion System in <em>Acinetobacter</em>

<div><p>The genus <em>Acinetobacter</em> is comprised of a diverse group of species, several of which have raised interest due to potential applications in bioremediation and agricultural purposes. In this work, we show that many species within the genus <em>Acinetobacter</em> possess the genetic requirements to assemble a functional type VI secretion system (T6SS). This secretion system is widespread among Gram negative bacteria, and can be used for toxicity against other bacteria and eukaryotic cells. The most studied species within this genus is <em>A. baumannii,</em> an emerging nosocomial pathogen that has become a significant threat to healthcare systems worldwide. The ability of <em>A. baumannii</em> to develop multidrug resistance has severely reduced treatment options, and strains resistant to most clinically useful antibiotics are frequently being isolated. Despite the widespread dissemination of <em>A. baumannii</em>, little is known about the virulence factors this bacterium utilizes to cause infection. We determined that the T6SS is conserved and syntenic among <em>A. baumannii</em> strains, although expression and secretion of the hallmark protein Hcp varies between strains, and is dependent on TssM, a known structural protein required for T6SS function. Unlike other bacteria, <em>A. baumannii</em> ATCC 17978 does not appear to use its T6SS to kill <em>Escherichia coli</em> or other <em>Acinetobacter</em> species. Deletion of <em>tssM</em> does not affect virulence in several infection models, including mice, and did not alter biofilm formation. These results suggest that the T6SS fulfils an important but as-yet-unidentified role in the various lifestyles of the <em>Acinetobacter</em> spp.</p> </div

Genetic organization of T6SS loci.

<p>Selected genomes of sequenced <i>Acinetobacter</i> strains were probed for the presence of T6SS genes, with those genes predicted to be involved in T6SS colored and identified below the figure. Gene accession numbers are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055142#pone-0055142-t001" target="_blank">Table 1</a>.</p

The T6SS of 17978 is not used for killing of <i>E. coli</i> MG1655.

<p>Survival of <i>E. coli</i> was determined by plate counts after exposure to wild type17978, 17978 with vector control (17978/pWH1266), the 17978 Δ<i>tssM</i> T6SS mutant, and its complemented (pTssM) and vector control (pWH1266) derivatives. Wild type <i>V. cholerae (</i>V52), and the isogenic <i>tssM</i> mutant derivative (V52 Δ<i>tssM</i>), were used as positive and negative controls for bacterial killing, respectively. The data presented correspond to three independent experiments and are plotted as means ± SD. Comparison of the 17978 strains shows no significant differences in killing (n.s.; p>0.05; Tukey’s multiple comparison post-test).</p

Identification of conserved T6SS components in selected <i>Acinetobacter</i> spp.

<p>Locus tag identifiers are shown for the conserved <i>tss</i> components of several T6SS-containing <i>Acinetobacters</i>, as well as their homologs in <i>V. cholerae</i>, <i>P. aeruginosa</i>, and <i>B. pseudomallei</i>.</p

The T6SS is active in several species of <i>Acinetobacter</i>.

<p> A) Whole cell and supernatant samples prepared from cultures of several <i>A. baumannii</i> strains were probed with anti-Hcp (top panels) and the lysis control anti-RNA polymerase (RNAP; bottom panels). B) Whole cell and supernatant samples prepared from cultures of different species within the genus <i>Acinetobacter</i> probed as described above. C) Summary of growth and Hcp secretion characteristics, determined by Western blot and ELISA, of all T6SS-positive strains analyzed in this study. “Fast” growing strains (++) and “slow” growing strains (+) were defined as those which reached a high or low optical density, respectively, and set arbitrarily by the indicated line in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055142#pone.0055142.s002" target="_blank">Figure S2</a>. Hcp secretion is summarized as high (↑) or low (↓) based on Western blots and ELISA assays (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055142#pone.0055142.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055142#pone.0055142.s002" target="_blank">S2</a>).</p

<i>A. baumannii</i> ATCC 17978 requires the conserved TssM protein for T6SS activity.

<p>A) Whole cell and supernatant samples prepared from cultures of wild type 17978, the T6SS mutant 17978 Δ<i>hcp,</i> and its complemented (pHcp) or vector control (pWH1266) derivatives, were separated by SDS-PAGE and probed by Western blot with anti-Hcp (upper panel) or an anti-RNA polymerase (RNAP; lower panel) antibodies. B) Western blot of whole cell and supernatant samples prepared from cultures of wild type 17978, the T6SS mutant 17978 Δ<i>tssM,</i> and its complemented (pTssM) or vector control (pWH1266) derivatives probed for Hcp (upper panel) and RNAP (lower panel).</p

The T6SS is not required for virulence towards <i>G. mellonella</i> or in a mouse model of pneumonia.

<p>A) Groups of 10 larvae were injected with approximately 10⁶ or 10⁷ CFU of wild type 17978 or the <i>tssM</i> mutant, incubated at 37°C, and monitored for survival. No significant difference (p>0.05) in survival was observed (log-rank test). B) Bacterial burden of lung and liver tissue from mice infected intranasally with either wild type 17978 or Δ<i>tssM</i> 36h post infection. No significant difference (p>0.05; two-tailed, unpaired Student’s <i>t</i> test) in bacterial burden of the two strains was observed in either tissue.</p

Identification of an <em>Acinetobacter baumannii</em> Zinc Acquisition System that Facilitates Resistance to Calprotectin-mediated Zinc Sequestration

<div><p><em>Acinetobacter baumannii</em> is an important nosocomial pathogen that accounts for up to 20 percent of infections in intensive care units worldwide. Furthermore, <em>A. baumannii</em> strains have emerged that are resistant to all available antimicrobials. These facts highlight the dire need for new therapeutic strategies to combat this growing public health threat. Given the critical role for transition metals at the pathogen-host interface, interrogating the role for these metals in <em>A. baumannii</em> physiology and pathogenesis could elucidate novel therapeutic strategies. Toward this end, the role for calprotectin- (CP)-mediated chelation of manganese (Mn) and zinc (Zn) in defense against <em>A. baumannii</em> was investigated. These experiments revealed that CP inhibits <em>A. baumannii</em> growth <em>in vitro</em> through chelation of Mn and Zn. Consistent with these <em>in vitro</em> data, Imaging Mass Spectrometry revealed that CP accompanies neutrophil recruitment to the lung and accumulates at foci of infection in a murine model of <em>A. baumannii</em> pneumonia. CP contributes to host survival and control of bacterial replication in the lung and limits dissemination to secondary sites. Using CP as a probe identified an <em>A. baumannii</em> Zn acquisition system that contributes to Zn uptake, enabling this organism to resist CP-mediated metal chelation, which enhances pathogenesis. Moreover, evidence is provided that Zn uptake across the outer membrane is an energy-dependent process in <em>A. baumannii</em>. Finally, it is shown that Zn limitation reverses carbapenem resistance in multidrug resistant <em>A. baumannii</em> underscoring the clinical relevance of these findings. Taken together, these data establish Zn acquisition systems as viable therapeutic targets to combat multidrug resistant <em>A. baumannii</em> infections.</p> </div

CP inhibits <i>A. baumannii</i> growth <i>in vitro</i> through chelation of Mn and Zn.

<p>(<b>A</b>) <i>A. baumannii</i> growth in the presence of increasing concentrations of CP with (dashed lines) or without (solid lines) 25 µM Mn and 25 µM Zn added back. Data represent the average of three biological replicates. (<b>B</b>) Treatment with CP reduces intracellular Mn and Zn accumulation. ICP-MS analyses of intracellular Mn, Fe and Zn expressed as the relative ratio compared to intracellular Cu. ** <i>p</i><0.01, *** <i>p</i><0.001 by two-way ANOVA.</p

Contribution of the Znu system to pathogenesis <i>in vivo</i>.

<p>(<b>A–F</b>) <i>In vivo</i> GFP expression driven by the <i>znu</i> promoter. Frozen sections of lungs harvested at 36 hpi from wildtype (A–C) or S100A9^−/− (D–F) mice infected with WT::<i>p</i>znuGFP and stained with DAPI. Arrowheads in the merged images indicate bacteria expressing GFP. (<b>G–H</b>) Competition infection between wildtype and Δ<i>znuB</i>. Bacterial burden in lungs (<b>G</b>) and livers (<b>H</b>) of mice co-infected with wildtype <i>A. baumannii</i> and Δ<i>znuB</i>. Red symbols indicate CFU below the limit of detection. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 as determined by one-way ANOVA.</p