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. 2012;7(9):e45952.
doi: 10.1371/journal.pone.0045952. Epub 2012 Sep 24.

Induction of the staphylococcal proteolytic cascade by antimicrobial fatty acids in community acquired methicillin resistant Staphylococcus aureus

Benjamin Arsic  1 , Yue Zhu, David E Heinrichs, Martin J McGavin
Affiliations

Induction of the staphylococcal proteolytic cascade by antimicrobial fatty acids in community acquired methicillin resistant Staphylococcus aureus

Benjamin Arsic et al. PLoS One. 2012.

Abstract

Community acquired methicillin resistant Staphylococcus aureus (CA-MRSA), and the USA300 strain of CA-MRSA in particular, are known for their rapid community transmission, and propensity to cause aggressive skin and soft tissue infections. To assess factors that contribute to these hallmark traits of CA-MRSA, we evaluated how growth of USA300 and production of secreted virulence factors was influenced on exposure to physiologic levels of unsaturated free fatty acids that would be encountered on the skin or anterior nares, which represent the first sites of contact with healthy human hosts. There was a sharp threshold between sub-inhibitory and inhibitory concentrations, such that 100 µM sapienic acid (C16∶1) and linoleic acid (C18∶1) were sufficient to prevent growth after 24 h incubation, while 25 µM allowed unrestricted growth, and 50 µM caused an approximate 10-12 h lag, followed by unimpeded exponential growth. Conversely, saturated palmitic or stearic acids did not affect growth at 100 µM. Although growth was not affected by 25 µM sapienic or linoleic acid, these and other unsaturated C16 and C18 fatty acids, but not their saturated counterparts, promoted robust production of secreted proteases comprising the Staphylococcal proteolytic cascade. This trait was also manifested to varying degrees in other CA-MRSA, and in genetically diverse methicillin susceptible S. aureus strains. Therefore, induction of the Staphylococcal proteolytic cascade by unsaturated fatty acids is another feature that should now be evaluated as a potential contributing factor in the aggressive nature of skin and soft tissue infections caused by USA300, and as a general virulence mechanism of S. aureus.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Influence of fatty acids on growth of USA300.
Each point represents the mean of OD600 (A, C, D-G) or cfu/ml determination (B, H) from triplicate flasks of USA300 grown in TSB supplemented with the indicated amount of fatty acid; (○), TSB only; (▵), 25 µM; (□), 50 µM; (•), 100 µM; (⋄), 200 µM; (♦), 250 µM. Lauric acid (C12∶0) was provided in the form of triacylglycerol-monolaurate. Y-axes, OD600 or cfu/ml; X-axis, growth time (h).
Figure 2
Figure 2. SDS-PAGE of secreted proteins (A, C) and Western blot for detection of SspA and Hla (B), in culture supernatant of USA300 after growth for 18–24 h in the presence of C16 (A) or C18 (C) fatty acids.
Cultures were grown with the indicated amounts of C16∶1▵6 (sapienic acid), C16∶1▵9 (palmitoleic acid), C16∶0 (palmitic acid), C18∶2 (linoleic acid), C18∶1 (oleic acid), C18∶3 (linolenic acid) or C18∶0 (stearic acid) fatty acids. Proteins in the cell-free culture supernatant were precipitated in ice-cold TCA, and after solubilization in SDS-PAGE reducing buffer, protein equivalent to 2.0 OD600 units of culture supernatant was loaded in each lane (A and C). For Western blot (C), 0.02 OD600 units of cell free culture supernatant were subjected directly to SDS-PAGE, prior to detection with specific antisera (see Materials and Methods).
Figure 3
Figure 3. SDS-PAGE and Western blot analyses of secreted proteins produced by USA300 and isogenic variants after 8 h of growth in TSB, or TSB supplemented with 25 µM linoleic acid (A), and assay of total protease activity in culture supernatant (B).
For (A), protein loading was 2.0 OD600 units for Coomassie staining, and 0.02 OD600 units for Western blots, which were developed with primary antibody specific for Aur, and SspA as indicated. Arrows on the Coomassie stained gel indicate the selective induction of secreted proteases in response to linoleic acid. The arrow on the right margin indicates the position of proGeh. In (B), total protease activity in 8 h culture supernatant of USA300 and isogenic variants was determined with FITC-casein substrate. Cultures were grown with 25 µM linoleic acid as indicated, and assay buffer was supplemented with 10 mM EDTA where indicated, to inhibit metalloprotease. Data are reported as fluorescence emission at 535 nm (ε535), measured in arbitrary fluorescence units.
Figure 4
Figure 4. β-galactosidase reporter gene assay in cell lysate of USA300aur after growth for 5–8 h in TSB, or TSB supplemented with 25 µM palmitic (C16∶0) or palmitoleic (C16∶1) acid.
Figure 5
Figure 5. SDS-PAGE and Coomassie staining (A and C), or Western blot for detection of SspA (B, D and E), in cultures of S. aureus grown in TSB containing 0 or 25 µM linoleic acid (LA) as indicated.
Protein loading was 2.0 OD600 units for SDS-PAGE, and 0.02 OD600 units for Western blot. The S. aureus strains are defined in Table 1. Arrows and labels on the right margins of panels A and C indicate the location of 72 kDa glycerol ester hydrolase precursor (proGeh) and mature lipase (Geh), while arrows on the protein gels point to SspA protein that is induced in response to 25 µM LA. SspA exhibits some expected variation in size, being comprised of 327 amino acids in USA400 (MW_0932), 336 amino acids in USA300 (SAUSA300_0951), and 357 amino acids in MRSA252 (SAR_1022) and other CC30 strains, due to variation in a C-terminal disordered segment comprised of tripeptide repeats. Different isomers produced by the same strain as shown on Western blot (5E), and explained in the text, are attributed to varying degrees of processing of the N-terminal propeptide of the SspA precursor, proSspA.
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This work was supported by Natural Sciences and Engineering Research Council of Canada Discovery Grants (http://www.nserc-crsng.gc.ca) and Western University Schulich School of Medicine and Dentistry internal award (http://www.schulich.uwo.ca/research) to MJM and DEH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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