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Acinetobacter baumannii Virulence Is Mediated by the Concerted Action of Three Phospholipases D.

Stahl J, Bergmann H, Göttig S, Ebersberger I, Averhoff B - PLoS ONE (2015)

Bottom Line: Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host.They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core.Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University Frankfurt, Frankfurt am Main, Germany.

ABSTRACT
Acinetobacter baumannii causes a broad range of opportunistic infections in humans. Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host. To date very little is known about the molecular basis of the latter. Here we demonstrate that A. baumannii can use phosphatidylcholine, an integral part of human cell membranes, as sole carbon and energy source. We report on the identification of three phospholipases belonging to the PLD superfamily. PLD1 and PLD2 appear restricted to the bacteria and display the general features of bacterial phospholipases D. They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core. The third candidate, PLD3, is found in bacteria as well as in eukaryotes and harbours only one PLDc_2 PFAM domain and one conserved HKD motif, which however do not overlap. Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants. Galleria mellonella infection studies as well as invasion experiments using A549 human lung epithelial cells revealed that the three PLDs act in a concerted manner as virulence factors and are playing an important role in host cell invasion.

No MeSH data available.


Related in: MedlinePlus

Bacterial competition between A. baumannii ATCC 19606T and E. coli or P. putida.A. baumannii ATCC 19606T wild-type or pld mutant cells and E. coli DH5α pET28a cells (A) or P. putida 548.C8 cells (B) were mixed at a ratio of 10:1, spotted onto an LB-agar plates and incubated for 4 hours at 37°C. To quantify the number of surviving E. coli or P. putida cells serial dilutions were plated onto kanamycin containing LB-agar and colony forming units were counted after incubation overnight at 37°C or 30°C, respectively. The standard deviation was calculated from three independent experiments. LB medium was used instead of A. baumannii as negative control.
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pone.0138360.g005: Bacterial competition between A. baumannii ATCC 19606T and E. coli or P. putida.A. baumannii ATCC 19606T wild-type or pld mutant cells and E. coli DH5α pET28a cells (A) or P. putida 548.C8 cells (B) were mixed at a ratio of 10:1, spotted onto an LB-agar plates and incubated for 4 hours at 37°C. To quantify the number of surviving E. coli or P. putida cells serial dilutions were plated onto kanamycin containing LB-agar and colony forming units were counted after incubation overnight at 37°C or 30°C, respectively. The standard deviation was calculated from three independent experiments. LB medium was used instead of A. baumannii as negative control.

Mentions: Recently, it was shown that PLDs of Pseudomonas aeruginosa are implicated in bacterial competition [47]. Therefore we addressed the question whether A. baumannii ATCC 19606T outcompetes other bacteria. We used E. coli as model strain in a bacterial competition assay as described earlier for competition between A. nosocomialis and E. coli [41,42]. A. baumannii ATCC 19606T wild-type cells were mixed with kanamycin resistant E. coli in a 10:1 ratio. A mixture of LB-medium and E. coli in the same ratio was used as negative control. After incubation of the mixture for 4 hours, the colony forming units of E. coli cells were determined by plating serial dilutions onto kanamycin containing agar plates. Incubation with A. baumannii ATCC 19606T wild-type revealed 1.9∙105 ± 0.8∙105 colony forming E. coli cells (Fig 5A). Incubation of E. coli cells with LB medium as control led to the detection of 4.9∙107 ± 0.9∙107 CFU. This provides clear evidence that A. baumannii ATCC 19606T outcompetes E. coli. To verify our results we repeated the competition analyses using a kanamycin resistant Pseudomonas putida strain 548.C8 [48]. 1.4x105 ± 0.5∙105P. putida CFU were determined when incubated with A. baumannii wild-type cells, which clearly shows that A. baumannii also outcompetes P. putida (Fig 5B).


Acinetobacter baumannii Virulence Is Mediated by the Concerted Action of Three Phospholipases D.

Stahl J, Bergmann H, Göttig S, Ebersberger I, Averhoff B - PLoS ONE (2015)

Bacterial competition between A. baumannii ATCC 19606T and E. coli or P. putida.A. baumannii ATCC 19606T wild-type or pld mutant cells and E. coli DH5α pET28a cells (A) or P. putida 548.C8 cells (B) were mixed at a ratio of 10:1, spotted onto an LB-agar plates and incubated for 4 hours at 37°C. To quantify the number of surviving E. coli or P. putida cells serial dilutions were plated onto kanamycin containing LB-agar and colony forming units were counted after incubation overnight at 37°C or 30°C, respectively. The standard deviation was calculated from three independent experiments. LB medium was used instead of A. baumannii as negative control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4574555&req=5

pone.0138360.g005: Bacterial competition between A. baumannii ATCC 19606T and E. coli or P. putida.A. baumannii ATCC 19606T wild-type or pld mutant cells and E. coli DH5α pET28a cells (A) or P. putida 548.C8 cells (B) were mixed at a ratio of 10:1, spotted onto an LB-agar plates and incubated for 4 hours at 37°C. To quantify the number of surviving E. coli or P. putida cells serial dilutions were plated onto kanamycin containing LB-agar and colony forming units were counted after incubation overnight at 37°C or 30°C, respectively. The standard deviation was calculated from three independent experiments. LB medium was used instead of A. baumannii as negative control.
Mentions: Recently, it was shown that PLDs of Pseudomonas aeruginosa are implicated in bacterial competition [47]. Therefore we addressed the question whether A. baumannii ATCC 19606T outcompetes other bacteria. We used E. coli as model strain in a bacterial competition assay as described earlier for competition between A. nosocomialis and E. coli [41,42]. A. baumannii ATCC 19606T wild-type cells were mixed with kanamycin resistant E. coli in a 10:1 ratio. A mixture of LB-medium and E. coli in the same ratio was used as negative control. After incubation of the mixture for 4 hours, the colony forming units of E. coli cells were determined by plating serial dilutions onto kanamycin containing agar plates. Incubation with A. baumannii ATCC 19606T wild-type revealed 1.9∙105 ± 0.8∙105 colony forming E. coli cells (Fig 5A). Incubation of E. coli cells with LB medium as control led to the detection of 4.9∙107 ± 0.9∙107 CFU. This provides clear evidence that A. baumannii ATCC 19606T outcompetes E. coli. To verify our results we repeated the competition analyses using a kanamycin resistant Pseudomonas putida strain 548.C8 [48]. 1.4x105 ± 0.5∙105P. putida CFU were determined when incubated with A. baumannii wild-type cells, which clearly shows that A. baumannii also outcompetes P. putida (Fig 5B).

Bottom Line: Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host.They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core.Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University Frankfurt, Frankfurt am Main, Germany.

ABSTRACT
Acinetobacter baumannii causes a broad range of opportunistic infections in humans. Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host. To date very little is known about the molecular basis of the latter. Here we demonstrate that A. baumannii can use phosphatidylcholine, an integral part of human cell membranes, as sole carbon and energy source. We report on the identification of three phospholipases belonging to the PLD superfamily. PLD1 and PLD2 appear restricted to the bacteria and display the general features of bacterial phospholipases D. They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core. The third candidate, PLD3, is found in bacteria as well as in eukaryotes and harbours only one PLDc_2 PFAM domain and one conserved HKD motif, which however do not overlap. Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants. Galleria mellonella infection studies as well as invasion experiments using A549 human lung epithelial cells revealed that the three PLDs act in a concerted manner as virulence factors and are playing an important role in host cell invasion.

No MeSH data available.


Related in: MedlinePlus