Limits...
Differential Role of the T6SS in Acinetobacter baumannii Virulence.

Repizo GD, Gagné S, Foucault-Grunenwald ML, Borges V, Charpentier X, Limansky AS, Gomes JP, Viale AM, Salcedo SP - PLoS ONE (2015)

Bottom Line: The T6SS genomic locus is present and was actively transcribed in all of the above strains.In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections.Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.

View Article: PubMed Central - PubMed

Affiliation: Bases Moléculaires et Structurales des Systèmes Infectieux, CNRS UMR 5086, Université Lyon 1, Institut de Biologie et Chimie des Protéines, Lyon, France.

ABSTRACT
Gram-negative bacteria, such as Acinetobacter baumannii, are an increasing burden in hospitals worldwide with an alarming spread of multi-drug resistant (MDR) strains. Herein, we compared a type strain (ATCC17978), a non-clinical isolate (DSM30011) and MDR strains of A. baumannii implicated in hospital outbreaks (Ab242, Ab244 and Ab825), revealing distinct patterns of type VI secretion system (T6SS) functionality. The T6SS genomic locus is present and was actively transcribed in all of the above strains. However, only the A. baumannii DSM30011 strain was capable of killing Escherichia coli in a T6SS-dependent manner, unlike the clinical isolates, which failed to display an active T6SS in vitro. In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections. Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.

No MeSH data available.


Related in: MedlinePlus

T6SS locus expression.A) RT-PCR transcriptional analysis of hcp and tssM expression in strains grown in L-broth; rpoB gene expression was used as endogenous control. The RNA not subjected to RT was also run in PCR (bottom panel, negative control) to ensure that PCR positive reactions were due to the presence of transcripts and not contaminating genomic DNA. B) The presence of Hcp (arrows) in concentrated culture supernatants of the indicated A. baumannii strains grown up to exponential phase in L-Broth was determined by 18% SDS-PAGE and Coomasie Blue staining. C) Control immunoblottings showing the presence of Hcp in the supernatant (first panel) and whole lysates fractions (second panel). A cell-lysis control using antibodies directed against EF-Tu (standard cytoplasmic marker) was performed using supernatant (third panel) or whole lysate (fourth panel) fractions. The final positions of the molecular mass markers (in kDa) are indicated on the right margin.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4581634&req=5

pone.0138265.g002: T6SS locus expression.A) RT-PCR transcriptional analysis of hcp and tssM expression in strains grown in L-broth; rpoB gene expression was used as endogenous control. The RNA not subjected to RT was also run in PCR (bottom panel, negative control) to ensure that PCR positive reactions were due to the presence of transcripts and not contaminating genomic DNA. B) The presence of Hcp (arrows) in concentrated culture supernatants of the indicated A. baumannii strains grown up to exponential phase in L-Broth was determined by 18% SDS-PAGE and Coomasie Blue staining. C) Control immunoblottings showing the presence of Hcp in the supernatant (first panel) and whole lysates fractions (second panel). A cell-lysis control using antibodies directed against EF-Tu (standard cytoplasmic marker) was performed using supernatant (third panel) or whole lysate (fourth panel) fractions. The final positions of the molecular mass markers (in kDa) are indicated on the right margin.

Mentions: As the different A. baumannii strains tested displayed very distinct abilities to outcompete E. coli we investigated if the T6SS was functional in these strains. RT-PCR analysis confirmed that hcp and tssM, two key T6SS genes, are being actively transcribed under growth conditions used for bacterial competition assays (L-broth) in all our strains (Fig 2A). In addition, immunoblots performed with an anti-Hcp antibody [22], showed that this protein is present in whole cell lysates for all the strains (Fig 2C, second panel). The identity of the Hcp bands (19 kDa) excised from Coomassie stained gels was further verified by LC-MS analysis.


Differential Role of the T6SS in Acinetobacter baumannii Virulence.

Repizo GD, Gagné S, Foucault-Grunenwald ML, Borges V, Charpentier X, Limansky AS, Gomes JP, Viale AM, Salcedo SP - PLoS ONE (2015)

T6SS locus expression.A) RT-PCR transcriptional analysis of hcp and tssM expression in strains grown in L-broth; rpoB gene expression was used as endogenous control. The RNA not subjected to RT was also run in PCR (bottom panel, negative control) to ensure that PCR positive reactions were due to the presence of transcripts and not contaminating genomic DNA. B) The presence of Hcp (arrows) in concentrated culture supernatants of the indicated A. baumannii strains grown up to exponential phase in L-Broth was determined by 18% SDS-PAGE and Coomasie Blue staining. C) Control immunoblottings showing the presence of Hcp in the supernatant (first panel) and whole lysates fractions (second panel). A cell-lysis control using antibodies directed against EF-Tu (standard cytoplasmic marker) was performed using supernatant (third panel) or whole lysate (fourth panel) fractions. The final positions of the molecular mass markers (in kDa) are indicated on the right margin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581634&req=5

pone.0138265.g002: T6SS locus expression.A) RT-PCR transcriptional analysis of hcp and tssM expression in strains grown in L-broth; rpoB gene expression was used as endogenous control. The RNA not subjected to RT was also run in PCR (bottom panel, negative control) to ensure that PCR positive reactions were due to the presence of transcripts and not contaminating genomic DNA. B) The presence of Hcp (arrows) in concentrated culture supernatants of the indicated A. baumannii strains grown up to exponential phase in L-Broth was determined by 18% SDS-PAGE and Coomasie Blue staining. C) Control immunoblottings showing the presence of Hcp in the supernatant (first panel) and whole lysates fractions (second panel). A cell-lysis control using antibodies directed against EF-Tu (standard cytoplasmic marker) was performed using supernatant (third panel) or whole lysate (fourth panel) fractions. The final positions of the molecular mass markers (in kDa) are indicated on the right margin.
Mentions: As the different A. baumannii strains tested displayed very distinct abilities to outcompete E. coli we investigated if the T6SS was functional in these strains. RT-PCR analysis confirmed that hcp and tssM, two key T6SS genes, are being actively transcribed under growth conditions used for bacterial competition assays (L-broth) in all our strains (Fig 2A). In addition, immunoblots performed with an anti-Hcp antibody [22], showed that this protein is present in whole cell lysates for all the strains (Fig 2C, second panel). The identity of the Hcp bands (19 kDa) excised from Coomassie stained gels was further verified by LC-MS analysis.

Bottom Line: The T6SS genomic locus is present and was actively transcribed in all of the above strains.In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections.Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.

View Article: PubMed Central - PubMed

Affiliation: Bases Moléculaires et Structurales des Systèmes Infectieux, CNRS UMR 5086, Université Lyon 1, Institut de Biologie et Chimie des Protéines, Lyon, France.

ABSTRACT
Gram-negative bacteria, such as Acinetobacter baumannii, are an increasing burden in hospitals worldwide with an alarming spread of multi-drug resistant (MDR) strains. Herein, we compared a type strain (ATCC17978), a non-clinical isolate (DSM30011) and MDR strains of A. baumannii implicated in hospital outbreaks (Ab242, Ab244 and Ab825), revealing distinct patterns of type VI secretion system (T6SS) functionality. The T6SS genomic locus is present and was actively transcribed in all of the above strains. However, only the A. baumannii DSM30011 strain was capable of killing Escherichia coli in a T6SS-dependent manner, unlike the clinical isolates, which failed to display an active T6SS in vitro. In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections. Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.

No MeSH data available.


Related in: MedlinePlus