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. 2012 Apr 20;287(17):13889-98.
doi: 10.1074/jbc.M112.349860. Epub 2012 Feb 27.

Novel phenol-soluble modulin derivatives in community-associated methicillin-resistant Staphylococcus aureus identified through imaging mass spectrometry

David J Gonzalez  1 , Cheryl Y Okumura, Andrew Hollands, Roland Kersten, Kathryn Akong-Moore, Morgan A Pence, Cheryl L Malone, Jaclyn Derieux, Bradley S Moore, Alexander R Horswill, Jack E Dixon, Pieter C Dorrestein, Victor Nizet
Affiliations

Novel phenol-soluble modulin derivatives in community-associated methicillin-resistant Staphylococcus aureus identified through imaging mass spectrometry

David J Gonzalez et al. J Biol Chem. .

Abstract

Staphylococcus aureus causes a wide range of human disease ranging from localized skin and soft tissue infections to potentially lethal systemic infections. S. aureus has the biosynthetic ability to generate numerous virulence factors that assist in circumventing the innate immune system during disease pathogenesis. Recent studies have uncovered a set of extracellular peptides produced by community-associated methicillin-resistant S. aureus (CA-MRSA) with homology to the phenol-soluble modulins (PSMs) from Staphylococcus epidermidis. CA-MRSA PSMs contribute to skin infection and recruit and lyse neutrophils, and truncated versions of these peptides possess antimicrobial activity. In this study, novel CA-MRSA PSM derivatives were discovered by the use of microbial imaging mass spectrometry. The novel PSM derivatives are compared with their parent full-length peptides for changes in hemolytic, cytolytic, and neutrophil-stimulating activity. A potential contribution of the major S. aureus secreted protease aureolysin in processing PSMs is demonstrated. Finally, we show that PSM processing occurs in multiple CA-MRSA strains by structural confirmation of additional novel derivatives. This work demonstrates that IMS can serve as a useful tool to go beyond genome predictions and expand our understanding of the important family of small peptide virulence factors.

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Figures

FIGURE 1.
FIGURE 1.
Visualization of dPSM by imaging mass spectrometry. A, imaging mass spectrometry of S. aureus TCH1516 USA300, a representative CA-MRSA strain. Full-length PSMs are displayed through various false coloring, indicating a colony-bound spatial distribution. Truncated peptides dPSM1 and dPSM4 display a spatial distribution extending beyond the colony, indicating their release into the agar medium. B, amino acid sequences of PSMs identified by MALDI-TOF/TOF (spectra provided in the supplemental material). Characterized derivative peptide regions are underlined. C, location of PSMs in CA-MRSA TCH1516 genome. PSMα1 to -4 are adjacent, small ribosomally encoded peptides. PSMγ (also known as delta-toxin) is located within the agr gene cluster embedded within the rnaIII gene.
FIGURE 2.
FIGURE 2.
Antimicrobial activity exhibited by PSMs and identified derivatives. A, bacterial-bacterial interaction between CA-MRSA and S. epidermidis. The optical image of the interaction shows a perturbed morophology of the S. epidermidis colony (black arrow) presumably caused by the metabolic output of the adjacent CA-MRSA colony. IMS demonstrated dPSMα peptides localized within the zone of growth inhibition. B, IMS of the interaction between CA-MRSA and GAS. The optical image shows that CA-MRSA has the ability to inhibit the growth (black arrow) of the competing GAS colony. IMS showed that dPSMα is released and therefore potentially contributes to growth inhibition. Shown is the spatial distribution of dPSMα4 (dPSMα1 is omitted).
FIGURE 3.
FIGURE 3.
Antimicrobial activity exhibited by PSMs and derivatives. A, inhibition of GAS growth on solid agar. GAS was grown on agar plates in the presence of synthetic PSMs. **, p < 0.01; ***, p < 0.001; ns, no significant difference. B, in vivo growth inhibition of GAS on mammalian skin in the presence of PSMs. Inhibition of bacterial growth was calculated as a percentage of the initial inoculum. *, p < 0.05; ***, p < 0.001; ns, no significant difference. C, MIC of the synthetic peptides dPSMα1 and dPSM4. Derivative peptides displayed antimicrobial activity in contrast to their respective parent forms. Numerical values displayed in the table indicate the concentrations tested (μm).
FIGURE 4.
FIGURE 4.
Neutrophil and erythrocyte lysis caused by PSMs. A, neutrophils were incubated with 10 μg/ml synthetic PSMs and their respective derivatives for 180 min. Lysis was assessed by monitoring the release of lactate dehydrogenase by colorimetric spectroscopy. Absorbances were normalized by the use of a lysis reagent. B, human (white bars) and 5% sheep (black bars) blood was incubated with 10 μg/ml synthetic PSMs and their respective derivatives for 60 min. Lysis was then assessed by monitoring the release of heme spectroscopically at A450. C, human blood samples incubated with PSMs were further assessed with a Riveal contrast microscope to confirm erythrocyte lysis.
FIGURE 5.
FIGURE 5.
Immunostimulation of NETs. A, for visualization of NETs, neutrophils were placed on poly-l-lysine-coated glass slides and treated with PSMs or vehicle controls. NETs were visualized using a rabbit antimyeloperoxidase antibody followed by a secondary goat anti-rabbit Alexa 488 antibody; samples were embedded in DAPI to counterstain DNA in blue. B, in parallel with microscopy, the Quant-iT Picogreen assay was used to quantify the extracellular DNA upon stimulation by PSMs and the controls by colorimetric spectroscopy. Error bars, S.D.
FIGURE 6.
FIGURE 6.
Aureolysin PSM processing. A, dried droplet MALDI-TOF analysis was used to examine processed extracts originating from CA-MRSA LAC, LAC Δaur, and LAC Δaur + pAur for the presence and absence of dPSM1 and dPSM4. B, MALDI-TOF data showing the detection of dPSM4 in wild type CA-MRSA LAC, the loss of the dPSM4 in the LAC Δaur background, and its restoration in the complemented strain.
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References

    1. Pizarro-Cerdá J., Cossart P. (2006) Bacterial adhesion and entry into host cells. Cell 124, 715–727 - PubMed
    1. Kobayashi S. D., DeLeo F. R. (2009) An update on community-associated MRSA virulence. Curr. Opin. Pharmacol. 9, 545–551 - PubMed
    1. Molloy E. M., Cotter P. D., Hill C., Mitchell D. A., Ross R. P. (2011) Streptolysin S-like virulence factors. The continuing sagA. Nat. Rev. Microbiol. 9, 670–681 - PMC - PubMed
    1. Maree C. L., Daum R. S., Boyle-Vavra S., Matayoshi K., Miller L. G. (2007) Community-associated methicillin-resistant Staphylococcus aureus isolates causing healthcare-associated infections. Emerg. Infect. Dis. 13, 236–242 - PMC - PubMed
    1. Gorak E. J., Yamada S. M., Brown J. D. (1999) Clin. Infect. Dis. 29, 29797–29800 - PubMed

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