Limits...
Secretome analysis defines the major role of SecDF in Staphylococcus aureus virulence.

Quiblier C, Seidl K, Roschitzki B, Zinkernagel AS, Berger-Bächi B, Senn MM - PLoS ONE (2013)

Bottom Line: Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the secDF mutant.Adhesion, invasion, and cytotoxicity of the secDF mutant were reduced in human umbilical vein endothelial cells.Virulence was significantly reduced using a Galleria mellonella insect model.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.

ABSTRACT
The Sec pathway plays a prominent role in protein export and membrane insertion, including the secretion of major bacterial virulence determinants. The accessory Sec constituent SecDF has been proposed to contribute to protein export. Deletion of Staphylococcus aureus secDF has previously been shown to reduce resistance, to alter cell separation, and to change the expression of certain virulence factors. To analyse the impact of the secDF deletion in S. aureus on protein secretion, a quantitative secretome analysis was performed. Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the secDF mutant. However, two Sec-dependent hydrolases were increased in comparison to the wild type, suggesting additional indirect, regulatory effects to occur upon deletion of secDF. Adhesion, invasion, and cytotoxicity of the secDF mutant were reduced in human umbilical vein endothelial cells. Virulence was significantly reduced using a Galleria mellonella insect model. Altogether, SecDF is a promising therapeutic target for controlling S. aureus infections.

Show MeSH

Related in: MedlinePlus

Complementation of SecDF-dependent changes.Western blot analysis of extracellular proteins in the SN of two different strain backgrounds. (A) Newman harbouring empty plasmid pEmpty (pCN34), the mutant strain Newman ΔsecDF harbouring empty plasmid pEmpty (pCN34) and the complemented mutant Newman ΔsecDF mutant harbouring plasmid pSecDF (pCQ27) containing the secDF gene with its endogenous promoter. (B) CHE482, the mutant strain CHE482 ΔsecDF and the complemented mutant strain CHE482 ΔsecDF harbouring plasmid pSecDF (pCQ27). Specific antibodies against CHIPS, FnBPA, Eap, LytM, SceD and SEA were used. Truncated FnBPA in Newman runs below the 70 kDa marker band. Putative degradation bands of FnBPA have been observed before [56], [80], [81], [82] and are indicated by a ring. Additional protein bands due to unspecific binding to protein A or Sbi are indicated by an asterisk.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3643904&req=5

pone-0063513-g002: Complementation of SecDF-dependent changes.Western blot analysis of extracellular proteins in the SN of two different strain backgrounds. (A) Newman harbouring empty plasmid pEmpty (pCN34), the mutant strain Newman ΔsecDF harbouring empty plasmid pEmpty (pCN34) and the complemented mutant Newman ΔsecDF mutant harbouring plasmid pSecDF (pCQ27) containing the secDF gene with its endogenous promoter. (B) CHE482, the mutant strain CHE482 ΔsecDF and the complemented mutant strain CHE482 ΔsecDF harbouring plasmid pSecDF (pCQ27). Specific antibodies against CHIPS, FnBPA, Eap, LytM, SceD and SEA were used. Truncated FnBPA in Newman runs below the 70 kDa marker band. Putative degradation bands of FnBPA have been observed before [56], [80], [81], [82] and are indicated by a ring. Additional protein bands due to unspecific binding to protein A or Sbi are indicated by an asterisk.

Mentions: As expected, the SN of Newman ΔsecDF showed reduced protein amounts of CHIPS, FnBPA, Eap and SEA and increased amounts of LytM and SceD in comparison to the wild type and the complemented mutant (Figure 2A). This phenotype was validated and confirmed for CHIPS, FnBPA and SceD in strain background CHE482 (Figure 2B).


Secretome analysis defines the major role of SecDF in Staphylococcus aureus virulence.

Quiblier C, Seidl K, Roschitzki B, Zinkernagel AS, Berger-Bächi B, Senn MM - PLoS ONE (2013)

Complementation of SecDF-dependent changes.Western blot analysis of extracellular proteins in the SN of two different strain backgrounds. (A) Newman harbouring empty plasmid pEmpty (pCN34), the mutant strain Newman ΔsecDF harbouring empty plasmid pEmpty (pCN34) and the complemented mutant Newman ΔsecDF mutant harbouring plasmid pSecDF (pCQ27) containing the secDF gene with its endogenous promoter. (B) CHE482, the mutant strain CHE482 ΔsecDF and the complemented mutant strain CHE482 ΔsecDF harbouring plasmid pSecDF (pCQ27). Specific antibodies against CHIPS, FnBPA, Eap, LytM, SceD and SEA were used. Truncated FnBPA in Newman runs below the 70 kDa marker band. Putative degradation bands of FnBPA have been observed before [56], [80], [81], [82] and are indicated by a ring. Additional protein bands due to unspecific binding to protein A or Sbi are indicated by an asterisk.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643904&req=5

pone-0063513-g002: Complementation of SecDF-dependent changes.Western blot analysis of extracellular proteins in the SN of two different strain backgrounds. (A) Newman harbouring empty plasmid pEmpty (pCN34), the mutant strain Newman ΔsecDF harbouring empty plasmid pEmpty (pCN34) and the complemented mutant Newman ΔsecDF mutant harbouring plasmid pSecDF (pCQ27) containing the secDF gene with its endogenous promoter. (B) CHE482, the mutant strain CHE482 ΔsecDF and the complemented mutant strain CHE482 ΔsecDF harbouring plasmid pSecDF (pCQ27). Specific antibodies against CHIPS, FnBPA, Eap, LytM, SceD and SEA were used. Truncated FnBPA in Newman runs below the 70 kDa marker band. Putative degradation bands of FnBPA have been observed before [56], [80], [81], [82] and are indicated by a ring. Additional protein bands due to unspecific binding to protein A or Sbi are indicated by an asterisk.
Mentions: As expected, the SN of Newman ΔsecDF showed reduced protein amounts of CHIPS, FnBPA, Eap and SEA and increased amounts of LytM and SceD in comparison to the wild type and the complemented mutant (Figure 2A). This phenotype was validated and confirmed for CHIPS, FnBPA and SceD in strain background CHE482 (Figure 2B).

Bottom Line: Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the secDF mutant.Adhesion, invasion, and cytotoxicity of the secDF mutant were reduced in human umbilical vein endothelial cells.Virulence was significantly reduced using a Galleria mellonella insect model.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.

ABSTRACT
The Sec pathway plays a prominent role in protein export and membrane insertion, including the secretion of major bacterial virulence determinants. The accessory Sec constituent SecDF has been proposed to contribute to protein export. Deletion of Staphylococcus aureus secDF has previously been shown to reduce resistance, to alter cell separation, and to change the expression of certain virulence factors. To analyse the impact of the secDF deletion in S. aureus on protein secretion, a quantitative secretome analysis was performed. Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the secDF mutant. However, two Sec-dependent hydrolases were increased in comparison to the wild type, suggesting additional indirect, regulatory effects to occur upon deletion of secDF. Adhesion, invasion, and cytotoxicity of the secDF mutant were reduced in human umbilical vein endothelial cells. Virulence was significantly reduced using a Galleria mellonella insect model. Altogether, SecDF is a promising therapeutic target for controlling S. aureus infections.

Show MeSH
Related in: MedlinePlus