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

Characterizing the Hexose-6-Phosphate Transport System of Vibrio cholerae, a Utilization System for Carbon and Phosphate Sources

ABSTRACT The facultative human pathogen Vibrio cholerae transits between the gastrointestinal tract of its host and aquatic reservoirs. V. cholerae adapts to different situations by the timely coordinated expression of genes during its life cycle. We recently identified a subclass of genes that are induced at late stages of infection. Initial characterization demonstrated that some of these genes facilitate the transition of V. cholerae from host to environmental conditions. Among these genes are uptake systems lacking detailed characterization or correct annotation. In this study, we comprehensively investigated the function of the VCA0682-to-VCA0687 gene cluster, which was previously identified as in vivo induced. The results presented here demonstrate that the operon encompassing open reading frames VCA0685 to VCA0687 encodes an ABC transport system for hexose-6-phosphates with K m values ranging from 0.275 to 1.273 μM for glucose-6P and fructose-6P, respectively. Expression of the operon is induced by the presence of hexose-6P controlled by the transcriptional activator VCA0682, representing a UhpA homolog. Finally, we provide evidence that the operon is essential for the utilization of hexose-6P as a C and P source. Thereby, a physiological role can be assigned to hexose-6P uptake, which correlates with increased fitness of V. cholerae after a transition from the host into phosphate-limiting environments. </jats:p

Identification of genes induced in Vibrio cholerae in a dynamic biofilm system

AbstractThe facultative human pathogen Vibrio cholerae, the causative agent of the severe secretory diarrheal disease cholera, persists in its aquatic reservoirs in biofilms during interepidemic periods. Biofilm is a likely form in which clinically relevant V. cholerae is taken up by humans, providing an infective dose. Thus, a better understanding of biofilm formation of V. cholerae is relevant for the ecology and epidemiology of cholera as well as a target to control the disease. Most previous studies have investigated static biofilms of V. cholerae and elucidated structural prerequisites like flagella, pili and a biofilm matrix including extracellular DNA, numerous matrix proteins and exopolysaccharide, as well as the involvement of regulatory pathways like two-component systems, quorum sensing and c-di-GMP signaling. However, aquatic environments are more likely to reflect an open, dynamic system. Hence, we used a biofilm system with constant medium flow and a temporal controlled reporter-system of transcription to identify genes induced during dynamic biofilm formation. We identified genes known or predicted to be involved in c-di-GMP signaling, motility and chemotaxis, metabolism, and transport. Subsequent phenotypic characterization of mutants with independent mutations in candidate dynamic biofilm-induced genes revealed novel insights into the physiology of static and dynamic biofilm conditions. The results of this study also reinforce the hypotheses that distinct differences in regulatory mechanisms governing biofilm development are present under dynamic conditions compared to static conditions

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

Oligonucleotide primers.

a<p>Restriction sites are underlined.</p>b<p>Bold letters indicate codons changed to obtain desired amino acid mutations.</p>c<p>Oligonucleotides for <i>rpoB</i> are according to reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047756#pone.0047756-Quinones1" target="_blank">[73]</a>.</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>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

<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