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Identification of genes essential for pellicle formation in Acinetobacter baumannii.

ABSTRACT: Acinetobacter baumannii is an opportunistic pathogen, which has the ability to persist in the clinical environment, causing acute and chronic infections. A possible mechanism contributing to survival of A. baumannii is its ability to form a biofilm-like structure at the air/liquid interface, known as a pellicle. This study aimed to identify and characterise the molecular mechanisms required for pellicle formation in A. baumannii and to assess a broad range of clinical A. baumannii strains for their ability to form these multicellular structures.Random transposon mutagenesis was undertaken on a previously identified hyper-motile variant of A. baumannii ATCC 17978 designated 17978hm. In total three genes critical for pellicle formation were identified; cpdA, a phosphodiesterase required for degradation of cyclic adenosine monophosphate (cAMP), and A1S_0112 and A1S_0115 which are involved in the production of a secondary metabolite. While motility of the A1S_0112::Tn and A1S_0115::Tn mutant strains was abolished, the cpdA::Tn mutant strain displayed a minor alteration in its motility pattern. Determination of cAMP levels in the cpdA::Tn strain revealed a ~24-fold increase in cellular cAMP, confirming the role CpdA plays in catabolising this secondary messenger molecule. Interestingly, transcriptional analysis of the cpdA::Tn strain showed significant down-regulation of the operon harboring the A1S_0112 and A1S_0115 genes, revealing a link between these three genes and pellicle formation. Examination of our collection of 54 clinical A. baumannii strains revealed that eight formed a measurable pellicle; all of these strains were motile.This study shows that pellicle formation is a rare trait in A. baumannii and that a limited number of genes are essential for the expression of this phenotype. Additionally, an association between pellicle formation and motility was identified. The level of the signalling molecule cAMP was found to be controlled, in part, by the cpdA gene product, in addition to playing a critical role in pellicle formation, cellular hydrophobicity and motility. Furthermore, cAMP was identified as a novel regulator of the operon A1S_0112-0118.

SUBMITTER: Giles SK

PROVIDER: S-EPMC4457973 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Identification of genes essential for pellicle formation in Acinetobacter baumannii.

Giles Sarah K SK Stroeher Uwe H UH Eijkelkamp Bart A BA Brown Melissa H MH

BMC microbiology 20150606

<h4>Background</h4>Acinetobacter baumannii is an opportunistic pathogen, which has the ability to persist in the clinical environment, causing acute and chronic infections. A possible mechanism contributing to survival of A. baumannii is its ability to form a biofilm-like structure at the air/liquid interface, known as a pellicle. This study aimed to identify and characterise the molecular mechanisms required for pellicle formation in A. baumannii and to assess a broad range of clinical A. bauma ...[more]

PMID: 26047954

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Project description:BACKGROUND: Interestingly, Acinetobacter baumannii presents an enhanced capacity to form biofilms (also named pellicles) at the air-liquid interface as compared to the other Acinetobacter species. This characteristic questions the contribution of this phenotype to an increased risk of clinical infections by this pathogen. METHODOLOGY/PRINCIPAL FINDINGS: By a proteomic approach using 2-D gel electrophoresis-LC-MS/MS mass spectrometry, we compared the membrane protein patterns of A. baumannii 77, a pellicle-forming clinical isolate, grown in planktonic and in sessile modes. We identified 52 proteins with a differential expression, including 32 up-regulated and 20 down-regulated in the pellicle state. Several proteins, differentially expressed during pellicle development, were of particular interest. We determined the over-expression of four siderophore iron uptake systems including the acinetobactin and enterobactin receptors and confirmed that the development of this type of biofilm is promoted by ferric ions. Two over-expressed proteins, CarO and an OprD-homologue, putative carbapenem-resistance associated porins, would be involved in the transport of specific compounds, like ornithine, a biosynthesis precursor of a siderophore from the hydroxamate family. We evidenced the overexpression of a lipase and a transporter of LCFA that may be involved in the recycling of lipids inside the pellicle matrix. Finally, we demonstrated both by proteomic and by AFM studies that this particular type of biofilm required multiple pili systems to maintain this cohesive structure at the air-liquid interface; two of these systems have never been described in A. baumannii. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated that several proteins, overexpressed at a late state of pellicle development, could be potentially involved in virulence processes. Therefore, regarding the number of potential virulence factors that are over-expressed in this growth mode, the pellicle-forming clinical isolates should be kept under survey.

Project description:Bacterial persistence is a feature that allows susceptible bacteria to survive extreme concentrations of antibiotics and it has been verified in a number of species, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus spp., Mycobacterium spp. However, even though Acinetobacter baumannii is an important nosocomial pathogen, data regarding its persistence phenotype are still lacking. Therefore, the aim of this study was to evaluate the persistence phenotype in A. baumannii strains, as well as its variation among strains after treatment with polymyxin B and tobramycin. Stationary cultures of 37 polymyxin B-susceptible clinical strains of A. baumannii were analyzed for surviving cells after exposure to 15 µg/mL of polymyxin B for 6 h, by serial dilutions and colony counting. Among these, the 30 tobramycin-susceptible isolates also underwent tobramycin treatment at a concentration of 160 µg/mL and persister cells occurrence was evaluated equally. A high heterogeneity of persister cells formation patterns among isolates was observed. Polymyxin B-treated cultures presented persister cells corresponding from 0.0007% to 10.1% of the initial population and two isolates failed to produce detectable persister cells under this condition. A high variability could also be observed when cells were treated with tobramycin: the persister fraction corresponded to 0.0003%-11.84% of the pre-treatment population. Moreover, no correlation was found between persister subpopulations comparing both antibiotics among isolates, indicating that different mechanisms underlie the internal control of this phenotype. This is the first report of persister cells occurrence in A. baumannii. Our data suggest that distinct factors regulate the tolerance for unrelated antibiotics in this species, contrasting the multi-drug tolerance observed in other species (eg. dormancy-mediated tolerance). Supporting this observation, polymyxin B--an antibiotic that is believed to act on non-dividing cells as well--failed to eradicate persister cells in the majority of the isolates, possibly reflecting a disconnection between persistence and dormancy.

Project description:UNLABELLED:Acinetobacter baumannii is a Gram-negative bacterium that causes diseases such as pneumonia, bacteremia, and soft tissue infections in hospitalized patients. Relatively little is known about how A. baumannii causes these infections. Thus, we used insertion sequencing (INSeq), a combination of transposon mutagenesis and massively parallel next-generation sequencing, to identify novel virulence factors of A. baumannii. To this end, we generated a random transposon mutant library containing 150,000 unique insertions in A. baumannii strain ATCC 17978. The INSeq analysis identified 453 genes required for growth in rich medium. The library was then used in a murine pneumonia model, and the relative levels of abundance of mutants before and after selection in the mouse were compared. When genes required for growth in rich medium were removed from the analysis, 157 genes were identified as necessary for persistence in the mouse lung. Several of these encode known virulence factors of A. baumannii, such as OmpA and ZnuB, which validated our approach. A large number of the genes identified were predicted to be involved in amino acid and nucleotide metabolism and transport. Other genes were predicted to encode an integration host factor, a transmembrane lipoprotein, and proteins involved in stress response and efflux pumps. Very few genes, when disrupted, resulted in an increase in A. baumannii numbers during host infection. The INSeq approach identified a number of novel virulence determinants of A. baumannii, which are candidate targets for therapeutic interventions. IMPORTANCE:A. baumannii has emerged as a frequent cause of serious infections in hospitals and community settings. Due to increasing antibiotic resistance, alternative approaches, such as antivirulence strategies, are desperately needed to fight A. baumannii infections. Thorough knowledge of A. baumannii pathogenicity is essential for such approaches but is currently lacking. With the increasingly widespread use of massively parallel sequencing, a class of techniques known as transposon insertion sequencing has been developed to perform comprehensive virulence screens of bacterial genomes in vivo. We have applied one of these approaches (INSeq) to uncover novel virulence factors in A. baumannii. We identified several such factors, including those predicted to encode amino acid and nucleotide metabolism proteins, an integration host factor protein, stress response factors, and efflux pumps. These results greatly expand the number of A. baumannii virulence factors and uncover potential targets for antivirulence treatments.

Project description:UNLABELLED:A critical feature of a potential antimicrobial target is the characteristic of being essential for growth and survival during host infection. For bacteria, genome-wide essentiality screens are usually performed on rich laboratory media. This study addressed whether genes detected in that manner were optimal for the identification of antimicrobial targets since the in vivo milieu is fundamentally different. Mutant derivatives of a clinical isolate of Acinetobacter baumannii were screened for growth on human ascites, an ex vivo medium that reflects the infection environment. A subset of 34 mutants with unique gene disruptions that demonstrated little to no growth on ascites underwent evaluation in a rat subcutaneous abscess model, establishing 18 (53%) of these genes as in vivo essential. The putative gene products all had annotated biological functions, represented unrecognized or underexploited antimicrobial targets, and could be grouped into five functional categories: metabolic, two-component signaling systems, DNA/RNA synthesis and regulation, protein transport, and structural. These A. baumannii in vivo essential genes overlapped poorly with the sets of essential genes from other Gram-negative bacteria catalogued in the Database of Essential Genes (DEG), including those of Acinetobacter baylyi, a closely related species. However, this finding was not due to the absence of orthologs. None of the 18 in vivo essential genes identified in this study, or their putative gene products, were targets of FDA-approved drugs or drugs in the developmental pipeline, indicating that a significant portion of the available target space within pathogenic Gram-negative bacteria is currently neglected. IMPORTANCE:The human pathogen Acinetobacter baumannii is of increasing clinical importance, and a growing proportion of isolates are multiantimicrobial-resistant, pan-antimicrobial-resistant, or extremely resistant strains. This scenario is reflective of the general problem of a critical lack of antimicrobials effective against antimicrobial-resistant Gram-negative bacteria, such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter sp., and Escherichia coli. This study identified a set of A. baumannii genes that are essential for growth and survival during infection and demonstrated the importance of using clinically relevant media and in vivo validation while screening for essential genes for the purpose of developing new antimicrobials. Furthermore, it established that if a gene is absent from the Database of Essential Genes, it should not be excluded as a potential antimicrobial target. Lastly, a new set of high-value potential antimicrobial targets for pathogenic Gram-negative bacteria has been identified.

Dataset Information

Identification of genes essential for pellicle formation in Acinetobacter baumannii.

Publications

Identification of genes essential for pellicle formation in Acinetobacter baumannii.

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