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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.

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G.mellonella virulence assay.(A) Pathogenicity of Newman pEmpty, Newman ΔsecDF pEmpty and Newman ΔsecDF pSecDF in G. mellonella. Larvae were monitored every 24 hours. PBS was used as negative control. Three independent experiments were pooled and plotted as Kaplan-Meier survival curve, P<0.001. (B) Bacterial burden per (live) larvae was measured in triplicates 24, 48 and 72 h post infection (hpi). The symbols triangle, square, diamond and line correspond to replicates I, II, III and the average, Ø, respectively. The inoculum (CFU/larvae) is shown at time point zero.
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pone-0063513-g005: G.mellonella virulence assay.(A) Pathogenicity of Newman pEmpty, Newman ΔsecDF pEmpty and Newman ΔsecDF pSecDF in G. mellonella. Larvae were monitored every 24 hours. PBS was used as negative control. Three independent experiments were pooled and plotted as Kaplan-Meier survival curve, P<0.001. (B) Bacterial burden per (live) larvae was measured in triplicates 24, 48 and 72 h post infection (hpi). The symbols triangle, square, diamond and line correspond to replicates I, II, III and the average, Ø, respectively. The inoculum (CFU/larvae) is shown at time point zero.

Mentions: To assess the influence of SecDF on virulence, Newman strains were injected into last instar larval stage G. mellonella and survival of the larvae was monitored over time. Resuspension buffer for bacterial cultures was used as a negative control. The dimensions and masses of larvae in one group spanned representative and comparable ranges, as preparatory experiments did not show any correlation concerning larval weight and survival (data not shown). Pathogenicity of Newman ΔsecDF pEmpty in G. mellonella was significantly reduced (P<0.001) in comparison to the wild type Newman pEmpty and the complemented mutant Newman ΔsecDF pSecDF (Figure 5A). To ensure that the attenuated virulence was not due to growth deficiencies of the mutant, the bacterial burden per larvae was measured after 24, 48 and 72 hours. Variation of CFU per larvae was rather high within the strains, for instance at 24 hours post infection (hpi) the wild type showed a bacterial burden ranging from 1.54×105 to 4.62×107 (Figure 5B). However, all three strains were able to multiply in the larvae within a similar range during the first 48 hpi, with CFUs being even higher in the secDF mutant after 72 hpi than in the wild type and the complemented mutant. The different peak time points of CFU/larvae in the secDF mutant (72 hpi) compared to the wild type or complemented mutant (48 hpi) was not reflected in the survival of G. mellonella, further indicating a strongly reduced virulence in Newman ΔsecDF pEmpty, that was not compensated by increased CFU numbers.


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)

G.mellonella virulence assay.(A) Pathogenicity of Newman pEmpty, Newman ΔsecDF pEmpty and Newman ΔsecDF pSecDF in G. mellonella. Larvae were monitored every 24 hours. PBS was used as negative control. Three independent experiments were pooled and plotted as Kaplan-Meier survival curve, P<0.001. (B) Bacterial burden per (live) larvae was measured in triplicates 24, 48 and 72 h post infection (hpi). The symbols triangle, square, diamond and line correspond to replicates I, II, III and the average, Ø, respectively. The inoculum (CFU/larvae) is shown at time point zero.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643904&req=5

pone-0063513-g005: G.mellonella virulence assay.(A) Pathogenicity of Newman pEmpty, Newman ΔsecDF pEmpty and Newman ΔsecDF pSecDF in G. mellonella. Larvae were monitored every 24 hours. PBS was used as negative control. Three independent experiments were pooled and plotted as Kaplan-Meier survival curve, P<0.001. (B) Bacterial burden per (live) larvae was measured in triplicates 24, 48 and 72 h post infection (hpi). The symbols triangle, square, diamond and line correspond to replicates I, II, III and the average, Ø, respectively. The inoculum (CFU/larvae) is shown at time point zero.
Mentions: To assess the influence of SecDF on virulence, Newman strains were injected into last instar larval stage G. mellonella and survival of the larvae was monitored over time. Resuspension buffer for bacterial cultures was used as a negative control. The dimensions and masses of larvae in one group spanned representative and comparable ranges, as preparatory experiments did not show any correlation concerning larval weight and survival (data not shown). Pathogenicity of Newman ΔsecDF pEmpty in G. mellonella was significantly reduced (P<0.001) in comparison to the wild type Newman pEmpty and the complemented mutant Newman ΔsecDF pSecDF (Figure 5A). To ensure that the attenuated virulence was not due to growth deficiencies of the mutant, the bacterial burden per larvae was measured after 24, 48 and 72 hours. Variation of CFU per larvae was rather high within the strains, for instance at 24 hours post infection (hpi) the wild type showed a bacterial burden ranging from 1.54×105 to 4.62×107 (Figure 5B). However, all three strains were able to multiply in the larvae within a similar range during the first 48 hpi, with CFUs being even higher in the secDF mutant after 72 hpi than in the wild type and the complemented mutant. The different peak time points of CFU/larvae in the secDF mutant (72 hpi) compared to the wild type or complemented mutant (48 hpi) was not reflected in the survival of G. mellonella, further indicating a strongly reduced virulence in Newman ΔsecDF pEmpty, that was not compensated by increased CFU numbers.

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