Regulation of the Staphylococcal Superantigen-Like Protein 1 Gene of Community-Associated Methicillin-Resistant Staphylococcus aureus in Murine Abscesses

Abstract

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes substantial skin and soft tissue infections annually in the United States and expresses numerous virulence factors, including a family of toxins known as the staphylococcal superantigen-like (SSL) proteins. Many of the SSL protein structures have been determined and implicated in immune system avoidance, but the full scope that these proteins play in different infection contexts remains unknown and continues to warrant investigation. Analysis of ssl gene regulation may provide valuable information related to the function of these proteins. To determine the transcriptional regulation of the ssl1 gene of CA-MRSA strain MW2, an ssl1 promoter::lux fusion was constructed and transformed into S.aureus strains RN6390 and Newman. Resulting strains were grown in a defined minimal medium (DSM) broth and nutrient-rich brain-heart infusion (BHI) broth and expression was determined by luminescence. Transcription of ssl1 was up-regulated and occurred earlier during growth in DSM broth compared to BHI broth suggesting expression is regulated by nutrient availability. RN6390 and Newman strains containing the ssl1::lux fusion were also used to analyze regulation in vivo using a mouse abscess model of infection. A marked increase in ssl1 transcription occurred early during infection, suggesting SSL1 is important during early stages of infection, perhaps to avoid the immune system.

Keywords: defined minimal medium; gene regulation; lux fusion; methicillin-resistant Staphylococcus aureus; mouse abscess; superantigen-like protein.

Figure 1

Expression of staphylococcal superantigen-like (ssl1) in strain WS0501 (RN6390 background) grown in (A) brain-heart infusion (BHI) broth or (B) defined minimal medium (DSM) broth. One milliliter aliquots were removed every hour for 8 h and at 24 h to measure luminescence (white column) and optical density (600 nm) (black diamond). Relative luminescence units (RLU) were divided by the viable colony forming units and multiplying the per cell luminescence by 10⁶ to obtain the final RLU shown. The data represents the RLU means + standard deviation of at least three trials.

Figure 2

Expression of ssl1 in strain NS3513 (Newman background) grown in BHI broth (black column) and DSM broth (white column). One milliliter aliquots were removed every 2 h for 8 h and at 24 h to measure luminescence. Luminescence (RLU) was determined as was presented before. The data represents the RLU means + standard deviation of at least three trials.

Figure 3

Quantitative reverse transcribed-polymerase chain reaction results of S. aureus strain RN6390 ssl5 and ssl8 transcription grown in BHI broth (white column) compared to DSM broth (black column). Transcription following growth in BHI broth was set as the baseline values. The data represents the mean + standard deviation from three separate runs.

Figure 4

Expression of ssl1 in strains WS0501 (RN6390 background, white column) and WS2601 (RN6390 agr mutant; black column) grown in (A) BHI broth or (B) DSM broth. One milliliter aliquots were removed every 2 h for 8 h and at 24 h to measure luminescence. RLU were determined as was presented before. The data represents the RLU means + standard deviation of at least three trials.

Figure 5

Expression of ssl1 in strains WS0501 (RN6390 background, white column) and WS0604 (RN6390 saeS mutant; black column) grown in (A) BHI broth or (B) DSM broth. One milliliter aliquots were removed every 2 h for 8 h and at 24 h to measure luminescence. RLU were determined as was presented before. The data represents the RLU means + standard deviation of at least three trials.

Figure 6

Expression of ssl1 in a murine abscess model of infection. (A) Murine thighs were inoculated with either strain Newman (black column) or RN6390 (white column) and the thighs were collected at time points 0, 8, 24, 48, and 72 h post-inoculation. Each mouse is represented as the luminescence divided by the colony forming units (CFU) and then multiplied by 10⁶ (RLU). The black lines represent the median values of at least six mice for each strain per time point. (B) The CFU/g of abscess for each mouse infected with WS0501 (RN6390 background, white circles) or NS3513 (Newman background, black squares) at 0, 8, 24, 48, and 72-h post-inoculation. Black lines represent the median values for each strain at each time point.