Staphylococcus aureus Biofilm and Planktonic cultures differentially impact gene expression, mapk phosphorylation, and cytokine production in human keratinocytes - PubMed (original) (raw)

Staphylococcus aureus Biofilm and Planktonic cultures differentially impact gene expression, mapk phosphorylation, and cytokine production in human keratinocytes

Patrick R Secor et al. BMC Microbiol. 2011.

Abstract

Background: Many chronic diseases, such as non-healing wounds are characterized by prolonged inflammation and respond poorly to conventional treatment. Bacterial biofilms are a major impediment to wound healing. Persistent infection of the skin allows the formation of complex bacterial communities termed biofilm. Bacteria living in biofilms are phenotypically distinct from their planktonic counterparts and are orders of magnitude more resistant to antibiotics, host immune response, and environmental stress. Staphylococcus aureus is prevalent in cutaneous infections such as chronic wounds and is an important human pathogen.

Results: The impact of S. aureus soluble products in biofilm-conditioned medium (BCM) or in planktonic-conditioned medium (PCM) on human keratinocytes was investigated. Proteomic analysis of BCM and PCM revealed differential protein compositions with PCM containing several enzymes involved in glycolysis. Global gene expression of keratinocytes exposed to biofilm and planktonic S. aureus was analyzed after four hours of exposure. Gene ontology terms associated with responses to bacteria, inflammation, apoptosis, chemotaxis, and signal transduction were enriched in BCM treated keratinocytes. Several transcripts encoding cytokines were also upregulated by BCM after four hours. ELISA analysis of cytokines confirmed microarray results at four hours and revealed that after 24 hours of exposure, S. aureus biofilm induced sustained low level cytokine production compared to near exponential increases of cytokines in planktonic treated keratinocytes. The reduction in cytokines produced by keratinocytes exposed to biofilm was accompanied by suppressed phosphorylation of MAPKs. Chemical inhibition of MAPKs did not drastically reduce cytokine production in BCM-treated keratinocytes suggesting that the majority of cytokine production is mediated through MAPK-independent mechanisms.

Conclusions: Collectively the results indicate that S. aureus biofilms induce a distinct inflammatory response compared to their planktonic counterparts. The differential gene expression and production of inflammatory cytokines by biofilm and planktonic cultures in keratinocytes could have implications for the formation and persistence of chronic wounds. The formation of a biofilm should be considered in any study investigating host response to bacteria.

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Figures

Figure 1

Figure 1

1D SDS - PAGE and Total Protein Concentration in BCM and PCM. The total protein concentration in BCM and PCM did not differ drastically (A), but several differences in the extracellular proteome of planktonic and biofilm cultures of S. aureus were revealed by 1D SDS-PAGE (B). The presence of a smear and low molecular weight peptides in the BCM indicates the presence of a bacterial protease. Bands in (B) marked with an arrow were excised and analyzed by HPLC-MS/MS (Table 1).

Figure 2

Figure 2

Functional clustering of BCM induced genes. Functional terms significantly associated (p < 0.05, Benjamini correction for multiple testing) with BCM induced genes relative to PCM induced genes. Functional annotation clusters with an enrichment score greater than 1.5 were considered significant. (A) Analysis of significantly upregulated genes (fold change ≥1.5) revealed functional annotation clusters associated with response to bacteria and external stimuli, apoptosis, immune response and inflammation, and signal transduction. (B) Analysis of significantly downregulated genes (fold change ≤1.5) revealed functional annotation clusters associated with chromatin modification, transcription, and metabolism.

Figure 3

Figure 3

BCM induces apoptosis and cell detachment in HKs. (A) Percentage of HKs staining positive for TUNEL. BCM induces significant levels of apoptosis in HKs after 4 and 24 hours of exposure while PCM does not. TUNEL data represents positive TUNEL cell counts over total cell counts. (B) Total cell counts obtained from propidium iodide stained HKs. After 24 hours of exposure to BCM, roughly half of the BCM treated HKs were still adhering to the culture well. Results represented as mean ± SD, n = 4, ** p < 0.01.

Figure 4

Figure 4

Cytokine production in adherent, non - apoptotic HKs exposed to BCM or PCM. BCM induces more cytokines per adherent, non-apoptotic cell after four hours while PCM induces more cytokines per adherent, non-apoptotic cell after 24 hours. Cytokine levels in HKs after 4 (A) and 24 hours (B) of exposure to PCM, BCM, or Control. Data normalized to pg protein/100,000, TUNEL negative, adherent cells. Results represented as mean ± SD, n = 3, *p < 0.05, **p < 0.01.

Figure 5

Figure 5

MAPK phosphorylation in HKs exposed to BCM or PCM. MAPK phosphorylation in HKs exposed to PCM or BCM for 4 or 24 hours. p38 (A) and JNK (B) phosphorylation levels were decreased in BCM treated HKs after 4 and 24 hours of exposure to BCM while PCM induced p38 and JNK phosphorylation after 24 hours. ERK phosphorylation (C) was unchanged in PCM treated HKs and increased in BCM treated HKs. Results represented as mean ± SD, n = 6, *p < 0.05, **p < 0.01 relative to control cells.

Figure 6

Figure 6

MAPK inhibition and cytokine production in BCM and PCM treated HKs. Cytokine production in MAPK inhibited HKs after 4 hours of exposure to PCM or BCM relative to non-inhibited control (BCM or PCM supplemented with DMSO). In general, MAPK inhibition resulted in a greater reduction of cytokine production in PCM treated HKs compared to BCM treated HKs. Results represented as mean ± SD, n = 3, *p < 0.05, **p < 0.01.

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