Global transcriptome analysis of Staphylococcus aureus biofilms in response to innate immune cells

Abstract

The potent phagocytic and microbicidal activities of neutrophils and macrophages are among the first lines of defense against bacterial infections. Yet Staphylococcus aureus is often resistant to innate immune defense mechanisms, especially when organized as a biofilm. To investigate how S. aureus biofilms respond to macrophages and neutrophils, gene expression patterns were profiled using Affymetrix microarrays. The addition of macrophages to S. aureus static biofilms led to a global suppression of the biofilm transcriptome with a wide variety of genes downregulated. Notably, genes involved in metabolism, cell wall synthesis/structure, and transcription/translation/replication were among the most highly downregulated, which was most dramatic at 1 h compared to 24 h following macrophage addition to biofilms. Unexpectedly, few genes were enhanced in biofilms after macrophage challenge. Unlike coculture with macrophages, coculture of S. aureus static biofilms with neutrophils did not greatly influence the biofilm transcriptome. Collectively, these experiments demonstrate that S. aureus biofilms differentially modify their gene expression patterns depending on the leukocyte subset encountered.

Fig 1

S. aureus biofilm growth states. (A and B) USA300 LAC-GFP was inoculated into sterile 2-well glass chamber slides and incubated at 37°C under static aerobic conditions for a period of 4 (A) or 6 (B) days in RPMI 1640 supplemented with 10% FBS with daily medium replacement. Biofilms were visualized using confocal microscopy (magnification, ×63; 1-μm slices), and representative three-dimensional images were constructed. (C) Quantification of 4- and 6-day-old biofilm thickness. Significant differences are denoted with asterisks (***, P < 0.001 using an unpaired two-tailed Student t test; n = 30 biofilms/time point).

Fig 2

S. aureus biofilm-leukocyte coculture paradigm. USA300 LAC-GFP static biofilms (green) were grown for 6 days, whereupon bone marrow-derived macrophages (A) (orange) or bone marrow-isolated neutrophils (B) (blue) were incubated with biofilms for 24 h and 4 h, respectively. Biofilm cocultures were visualized using confocal microscopy (magnification, ×63; 1-μm slices), and representative three-dimensional images were constructed. Insets show a higher magnification to highlight the absence of macrophage phagocytosis (A) and the presence of neutrophil phagocytosis (B) of staphylococcal biofilms. Results are representative of two independent experiments examining three individual biofilms each.

Fig 3

Differential responses of innate immune cells to S. aureus biofilms. A total of 10⁷ bone marrow-derived macrophages (Mϕ) or 5 × 10⁶ bone marrow-isolated neutrophils (PMN) were incubated with 6 day-old USA300 LAC-GFP biofilms for 18 to 24 h, whereupon the percentage of leukocytes exhibiting phagocytosis or death was enumerated using confocal microscopy. Significant differences are denoted with asterisks (**, P < 0.01 using an unpaired two-tailed Student t test; n = 3).

Fig 4

Acute macrophage addition to S. aureus biofilms leads to the transcriptional repression of numerous genes. The total number of genes significantly up- or downregulated in response to macrophage (A) or neutrophil (B) coculture in immature (4-day-old) or mature (6-day-old) S. aureus biofilms is shown, including those encoding hypothetical proteins.

Fig 5

Classification of genes significantly altered by leukocyte addition in S. aureus biofilms. S. aureus USA300 LAC-GFP static biofilms were grown for 4 or 6 days, whereupon macrophages (A and B) or neutrophils (C and D) were incubated with biofilms for 1 h. The numbers of genes with defined functions (grouped into cell wall/membrane, virulence/defense, regulation, metabolism, transcription/translation/replication, and miscellaneous categories) that were significantly altered after macrophage or neutrophil challenge are shown. Genes encoding hypothetical proteins were not included.

Fig 6

qRT-PCR validation of downregulated genes in S. aureus biofilms identified by microarray analysis. A subset of genes identified by microarray analysis was confirmed by qRT-PCR following either a 1- or 24-h coculture period of macrophages with 4-day-old USA300 LAC static biofilms. Results are presented as the relative gene expression after macrophage-biofilm coculture compared to that in biofilms that were not incubated with macrophages as a reference standard.

Fig 7

agr promotes S. aureus biofilm resistance to neutrophil challenge. S. aureus USA300 LAC-GFP wild-type (WT) and isogenic Δagr static biofilms were grown for 6 days and visualized using confocal microscopy. (A) Quantification of 4- and 6-day-old biofilm thickness. Significant differences are denoted with asterisks (***, P < 0.001 using an unpaired two-tailed Student t test; n = 60). (B) Neutrophils (5 × 10⁶) were incubated with biofilms for 20 h, whereupon the percentages of phagocytic and dead neutrophils were enumerated. Significant differences are denoted with asterisks (*, P < 0.05 using an unpaired two-tailed Student t test; n = 3).

Fig 8

Differential responses of macrophages and neutrophils to S. aureus biofilms are cell autonomous. Confocal microscopy quantification of invasion or phagocytosis of macrophages and neutrophils (5 × 10⁶ each) cultured either together (A and B) (**, P < 0.01 using an unpaired two-tailed Student t test [n = 4]; ***, P < 0.001 using an unpaired two-tailed Student t test [n = 4]) or separately (C and D) (*, P < 0.05 using an unpaired two-tailed Student t test [n = 2]; **, P < 0.01 using an unpaired two-tailed Student t test [n = 2]) with 6-day-old S. aureus USA300 LAC-GFP static biofilms at 4 and 24 h.