Staphylococcus aureus uses the ArlRS and MgrA cascade to regulate immune evasion during skin infection

Abstract

Skin is one of the most common sites of host immune response against Staphylococcus aureus infection. Here, through a combination of in vitro assays, mouse models, and intravital imaging, we find that S. aureus immune evasion in skin is controlled by a cascade composed of the ArlRS two-component regulatory system and its downstream effector, MgrA. S. aureus lacking either ArlRS or MgrA is less virulent and unable to form correct abscess structure due to de-repression of a giant surface protein, Ebh. These S. aureus mutants also have decreased expression of immune evasion factors (leukocidins, chemotaxis-inhibitory protein of S. aureus [CHIPS], staphylococcal complement inhibitor [SCIN], and nuclease) and are unable to kill neutrophils, block their chemotaxis, degrade neutrophil extracellular traps, and survive direct neutrophil attack. The combination of disrupted abscess structure and reduced immune evasion factors makes S. aureus susceptible to host defenses. ArlRS and MgrA are therefore the main regulators of S. aureus immune evasion and promising treatment targets.

Keywords: Staphylococcus aureus; abscess; gene regulation; immune evasion; innate immunity; intravital microscopy; neutrophil; skin infection; surface proteins.

Figure 1.. ArlRS and MgrA control S. aureus skin infection severity

(A) C57BL/6 mice were infected with S. aureus through subcutaneous injection, and developing skin infection was observed. The size of dermonecrotic lesions was measured daily. (B) On selected days, the infected areas were biopsied, and skin bacterial burden in homogenized biopsy specimens was measured. (C and D) Additionally, skin biopsy specimens were taken on day 1 of infection, and histopathological sections of the biopsy specimens were used to measure the size of abscesses formed in skin (C) and the presence of tightly clumped staphylococcal abscess communities (SACs) inside these abscesses (D). Scale bars, 300 μm. Data are shown as mean ± SEM. N = 9 (A), 5–8 (B), and 8 (C and D). *p < 0.05; **p < 0.01; ***p < 0.001. All p values are for comparisons to WT.

Figure 2.. ArlRS and MgrA control formation and immune evasion of model in vitro S. aureus abscess communities

(A–C) Three-dimensional SACs formed from individual S. aureus cells after culturing in collagen/fibrinogen/RPMI gels for 16 h. These were used to determine the role of fibrinogen present in the culture medium (A), the effects of mutations in the ArlRS-MgrA signaling system (B), and the role of giant surface proteins SraP and Ebh in causing the starburst phenotype in the ΔarlRS mutant strains (C). (D) Expression of ebh in mid-exponential S. aureus RPMI culture was measured with qPCR and normalized to gyrB expression. (E) Behavior of human neutrophils (stained green with CFDA-SE) 3 h after addition to the in vitro three-dimensional abscess models was also visualized, with propidium iodide (PI) added before imaging to stain extracellular DNA and lysed cells. (F) Survival of S. aureus after 1-h incubation with fresh human blood was quantified and normalized to WT survival. (G) Survival of S. aureus co-incubated with purified human neutrophils was measured. Representative images are shown. Image size: 350 × 350 μm. Data are shown as mean ± SEM. n = 6 (D and F) or 5 (G). *p < 0.05; ***p < 0.001; ****p < 0.0001. All p values are for comparisons to WT. See also Figures S2–S4.

Figure 3.. ArlRS and MgrA control innate immune evasion of S. aureus

(A) Expression of immune evasion genes in mid-exponential S. aureus RPMI culture was measured with qPCR and normalized to gyrB expression. (B and C) Nuclease activity in culture supernatants (B) and their ability to digest NETs, visualized with PI (C), were measured. (D and E) The ability of S. aureus culture supernatants to kill human neutrophils (D) and block neutrophil chemotaxis (E) was measured. Representative images are shown. Scale bar, 100 μm. Data are shown as mean ± SEM. n = 3 (A), 3–6 (B), 4 (D), and 6 (E). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. In (A), all p values for comparisons to WT. All significant p values between the groups are marked on graphs. See also Figure S5.

Figure 4.. ArlRS and MgrA control S. aureus survival after neutrophil phagocytosis

(A–C) Resistance of S. aureus to various compounds used by neutrophils to kill the bacteria, including human α-defensin HNP-1 (A), sodium hypochlorite (B), and hydrogen peroxide (C), was measured with agar diffusion assays. Additionally, survival of S. aureus 1 h after phagocytosis by human neutrophils was measured (D). Data are shown as mean ± SEM. n = 8 (A, C, and D) or 4 (B). *p < 0.05; **p < 0.01. All significant p values between the groups are marked on graphs.

Figure 5.. ArlRS and MgrA allow S. aureus to affect neutrophil movement during skin infection in vivo

Multiphoton intravital microscopy was used to image neutrophil/S. aureus interactions in vivo for 10 min at 24 h post-infection. (A) Representative image taken from time-lapse videos showing neutrophils at the infection site from WT, ΔarlRS, and ΔmgrA skin infections. (B) Quantification of neutrophil track displacement length in the x-y position in S. aureus skin infections. (C and D) Quantification of the mean displacement of neutrophils per minute (velocity). Data are shown as mean ± SEM. n = 5–7. *p < 0.05. See also Videos S1 and S2.

Figure 6.. ArlRS and MgrA are needed for immune evasion during skin infection in vivo

Multiphoton intravital microscopy was used to image neutrophil/S. aureus interactions in vivo at 24 h post-infection. (A) Representative intravital image showing a three-dimensional stitched image viewed from the x-z plane (side view) showing neutrophil localization at the infection site. (B and C) Image analysis quantification of total neutrophil spots at the infection site. (D and E) Total S. aureus surface volume at the infection site. (F and G) S.-aureus-positive neutrophils. (H and I) Percentage of S. aureus volume that was infiltrated by neutrophils. Data are shown as mean ± SEM. n = 6–11. *p < 0.05; **p < 0.01. See also Videos S3 and S4.

Figure 7.. Proposed model of innate immune evasion control by ArlRS and MgrA during S. aureus infection

With a functional ArlRS-MgrA cascade, the initial signal detected by the ArlRS two-component system induces expression of the global regulator MgrA, which in turn controls expression of various genes involved in virulence and immune evasion. By suppressing expression of large surface proteins with anti-adhesive properties (Ebh and SraP), the active cascade allows S. aureus to bind fibrinogen and form tight three-dimensional abscess communities where bacteria are shielded from phagocytes. Active cascade also causes S. aureus to secrete various immune evasion factors, such a leukocidins (LukAB and LukSF), CHIPS, SCIN, and nuclease, which together act to kill incoming neutrophils, prevent their chemotaxis and movement, and digest NETs used by neutrophils to ensnare bacteria. Finally, due to the cascade’s involvement with S. aureus resistance to antimicrobial peptides and, to a smaller degree, oxygen radicals, active ArlRS and MgrA promote bacterial survival inside neutrophils after phagocytosis. See also Figure S6.