Identification of a General O-linked Protein Glycosylation System in Acinetobacter baumannii and Its Role in Virulence and Biofilm Formation

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening “superbugs” for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics

Disulfide Bond Formation and ToxR Activity in <em>Vibrio cholerae</em>

<div><p>Virulence factor production in <em>Vibrio cholerae</em> is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation.</p> </div

<i>dsbA</i> knockout mutant and ToxR forms.

<p>Immunoblot analyses are shown using anti-FLAG antibodies to detect FLAG-tagged ToxR produced in <i>V. cholerae</i> P27459-S Δ<i>toxRS</i> and Δ<i>toxRS</i> Δ<i>dsbA</i> mutant strain (as indicated in the figure). Bacterial cultures harboring pFLAGtoxRS were grown to mid-log phase in M9 glycerol and in LB broth and induced with IPTG. ToxR mobility in the different samples was monitored and differences for intrachain disulfide bond formation were detected. Immunoblot analysis was performed at least three times, and results were reproducible.</p

Bacteria strains and plasmids used in this study.

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

In vitro and in vivo competition of chromosomal encoded FLAG-tagged <i>toxR</i> versus FLAG-tagged <i>toxR^CC</i> mutant in El Tor P27459-S.

a<p>median and interquartile range of 12 independent experiments.</p>b<p>median and interquartile range of 5 independent experiments.</p

<i>toxRS</i> coexpression in <i>V. cholerae</i> P27459-S Δ<i>toxRS</i> mutant strain acts negatively on ToxR disulfide bond homodimer and oligomers.

<p>Shown is an immunoblot analysis derived from SDS-PAGE analysis performed under non-reducing conditions, utilizing anti-FLAG antibodies and <i>V. cholerae</i> cells harboring various pFLAGtoxRS expressing plasmids, grown in LB medium to mid-log phase and induced with IPTG. Molecular markers are indicated on the left side. Two different IPTG concentrations are indicated, showing different ToxR levels. Immunoblot analysis was performed at least three times, and results were reproducible.</p

Virulence factor production of chromosomal encoded FLAG-tagged <i>toxR</i> and FLAG-tagged <i>toxR^CC</i> mutants.

a<p>median and interquartile range of at least 7 independent experiments.</p>b<p>median and interquartile range of 9 independent experiments.</p>*<p>significant by Kruskal-Wallis test followed by Dunn's test of selected pairs of columns with <i>P</i><0.05.</p><p>−8.</p

<i>dsb</i> knockout mutations and porin production in <i>V. cholerae</i> P27459-S.

<p>Panel A, B shown are OMP profiles on SDS-PAGE of WT, Δ<i>toxR</i>, Δ<i>dsbA</i>, Δ<i>dsbA</i> (pBAD18), Δ<i>dsbA</i> (pdsbA), Δ<i>dsbB</i>, Δ<i>dsbB</i> (pBAD18), Δ<i>dsbB</i> (pdsbB) and <i>dsbC</i>::pGP704 (only panel B) strains derived from cells grown for 24 h and 72 h in M9 glycerol, respectively. Arrows mark OmpU and OmpT. As a negative control, Δ<i>toxR</i> mutant strain showed no production of OmpU and derepressed OmpT protein level. The arrowhead on the right indicates a ToxR independent protein band used as loading control. Panel C, shown are qRT-PCR analyses of WT and Δ<i>dsbA</i> strain for <i>ompU</i>, <i>ompT</i> and <i>toxR</i> transcripts. Fold change ratios were calculated by comparing cDNA levels of genes of interest and the reference gene <i>rpoB</i>, derived from cells grown in M9 glycerol for 72 h. Data are presented as median fold change and the error bars indicate the interquartile range of each data set. Experiments were performed with at least six independent samples, utilizing the Mann-Whitney U test, <i>P</i><0.05.</p

<i>toxRS</i> coexpression in <i>E. coli</i> XL1-Blue strain.

<p>Shown are immunoblot analyses utilizing anti-FLAG antibodies to monitor FLAG-tagged ToxR production of pFLAGtoxRS constructs, performed under reducing (panel A) and non-reducing conditions (panel B). pFLAGtoxRS was expressed in <i>E. coli</i> cells grown in LB broth to mid-log phase (OD₆₀₀ of 0.5) and subsequently induced with IPTG for 1 h. From left to right, shown are pFLAGtoxRS(Δ264), pFLAGtoxRS and pFLAGtoxR^CCS, either containing no ToxR operator sequence or <i>ompU</i> or <i>toxT</i> operator sequences, respectively. A 55 kDa ToxR cross-reacting protein band, associated with pFLAGtoxRS and pFLAGtoxR^CCS, is indicated by an arrow. To note, cysteinyl dependent homodimer and oligomer ToxR bands occurred diminished as observed for pFLAGtoxRS in comparison to pFLAGtoxRS(Δ264). Molecular size markers are indicated on the left. Immunoblot analysis was performed at least three times, and results were reproducible.</p

Transcriptional analysis of <i>toxR</i> and porin genes <i>ompU</i> and <i>ompT</i> in <i>V. cholerae</i> P27459-S.

<p>Using qRT-PCR analysis, transcriptional activity of chromosomal encoding FLAG-tagged <i>toxR</i> and <i>toxR^CC</i> strains was monitored for the porin genes <i>ompU</i> and <i>ompT</i> and also for <i>toxR</i> and <i>toxR^CC</i>. mRNA levels of <i>rpoB</i> (used as a reference gene) were determined and correlated with the mRNA level of the genes of interest. Data are presented as median fold change and the error bars indicate the interquartile range of each data set. Experiments were performed with six independent samples, the Mann-Whitney U test was used, <i>P</i><0.05.</p