Analysis of the Staphylococcus aureus abscess proteome identifies antimicrobial host proteins and bacterial stress responses at the host-pathogen interface

Abstract

Abscesses are a hallmark of invasive staphylococcal infections and the site of a dynamic struggle between pathogen and host. However, the precise host and bacterial factors that contribute to abscess formation and maintenance have not been completely described. In this work, we define the Staphylococcus aureus abscess proteome from both wild-type and neutropenic mice to elucidate the host response to staphylococcal infection and uncover novel S. aureus virulence factors. Among the proteins identified, the mouse protein histone H4 was enriched in the abscesses of wild-type compared with neutropenic animals. Histone H4 inhibits staphylococcal growth in vitro demonstrating a role for this protein in the innate immune response to staphylococcal infection. These analyses also identified staphylococcal proteins within the abscess, including known virulence factors and proteins with previously unrecognized roles in pathogenesis. Within the latter group was the universal stress protein Usp2, which was enriched in kidney lesions from neutropenic mice and required for the S. aureus response to stringent stress. Taken together, these data describe the S. aureus abscess proteome and lay the foundation for the identification of contributors to innate immunity and bacterial pathogenesis.

Keywords: Staphylococcus aureus; innate immunity; pathogenesis; proteomics.

Figure 1. Neutrophil depletion results in large, disorganized renal lesions

(A and B) 10x magnification of immunohistochemistry for myeloperoxidase from a mouse treated with (A) the control antibody (SRF-3) or (B) the anti-Gr1 RB6 antibody. A substantial decrease in the number of neutrophils infiltrating and surrounding the bacteria in the neutropenic mice is observed and marked (*). (C) 4x magnification of H & E stained sections of the wildtype mouse where the inflammatory infiltrate consists of both neutrophils and macrophages forming densely packed sheets that encircle small bacterial aggregates (arrows) while the neutropenic mouse (D) has large, disorganized lesions and the inflammatory infiltrate consists predominantly of macrophages and abundant bacterial growth is visible (arrows). (E and F) 60x magnification of the sections described in (C) and (D), respectively.

Figure 2. Histone H4 is decreased upon neutrophil depletion

(A and B) Immunohistochemistry for histone H4. (A) In the SRF3-treated mouse (wildtype), there is increased extracellular immunoreactivity for histones (arrows) in proximity to the bacterial cells (*). (B) Anti-Gr1(RB6)-treated mice (neutropenic) have fewer infiltrating cells, very little extracellular histone H4, and large bacterial aggregates (*).

Figure 3. Histone H4 inhibits S. aureus growth in vitro

S. aureus was cultured in the presence of increasing concentrations of purified histone H4. The growth of the bacterial cells was monitored by measuring the optical density of the cultures at 600 nm. The data presented are the average of two independent experiments, each performed in triplicate. Error bars represent the standard error. * Denotes p < 0.01 versus buffer control as calculated by Student’s t test.

Figure 4. Expression of usp2 is growth phase-dependent

XylE assay for usp2 promoter activity. S. aureus containing a promoterless xylE construct (xylE) or xylE downstream of the usp2 promoter (usp2-xylE) were grown to the indicated growth phases and specific XylE activity was determined. The data presented are the average of three independent experiments, each one performed in triplicate, and the error bars represent the standard error. Student’s t test was used to calculate p values.

Figure 5. Expression of usp2 is induced during the stringent response

(A) The usp2 promoter was fused to GFP and fluorescence was used as a measurement of usp2 expression when exposed to mupirocin at a final concentration of 60 μg ml-1. The data are presented as fold increase in fluorescence as compared to the reporter exposed to the vehicle control. The data presented are the average of three independent experiments each performed in triplicate, and the error bars represent the standard error. B) Bacterial survival assay: wildtype NW/pOS1-lgt, NWΔusp2/pOS1-lgt and NWΔusp2/pusp2-c-myc were incubated with or without 120 μg ml-1 mupirocin and the percent survival was calculated by considering the number of bacteria present in the absence of mupirocin as 100%. The data presented are the average of three independent experiments each performed in triplicate, and the error bars represent the standard error. Student’s t test was used to calculate p values.

Figure 6. Usp2 does not contribute to S. aureus pathogenesis in the systemic model of infection

Bacterial colony forming units (CFU) measured in kidneys from mice intravenously infected with (A) wildtype NW/pOS1-lgt (circles), NWΔusp2/pOS1-lgt (squares), and NWΔusp2/plgt-usp2-c-myc (triangles). (B) wildtype USA300 (circles) or Δusp2 (squares). Each mouse is represented by a data point in the figure and the horizontal bar represents the mean of the log₁₀CFU.