Staphylococcus aureus hyaluronidase is a CodY-regulated virulence factor

Abstract

Staphylococcus aureus is a Gram-positive pathogen that causes a diverse range of bacterial infections. Invasive S. aureus strains secrete an extensive arsenal of hemolysins, immunomodulators, and exoenzymes to cause disease. Our studies have focused on the secreted enzyme hyaluronidase (HysA), which cleaves the hyaluronic acid polymer at the β-1,4 glycosidic bond. In the study described in this report, we have investigated the regulation and contribution of this enzyme to S. aureus pathogenesis. Using the Nebraska Transposon Mutant Library (NTML), we identified eight insertions that modulate extracellular levels of HysA activity. Insertions in the sigB operon, as well as in genes encoding the global regulators SarA and CodY, significantly increased HysA protein levels and activity. By altering the availability of branched-chain amino acids, we further demonstrated CodY-dependent repression of HysA activity. Additionally, through mutation of the CodY binding box upstream of hysA, the repression of HysA production was lost, suggesting that CodY is a direct repressor of hysA expression. To determine whether HysA is a virulence factor, a ΔhysA mutant of a community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 strain was constructed and found to be attenuated in a neutropenic, murine model of pulmonary infection. Mice infected with this mutant strain exhibited a 4-log-unit reduction in bacterial burden in their lungs, as well as reduced lung pathology and increased levels of pulmonary hyaluronic acid, compared to mice infected with the wild-type, parent strain. Taken together, these results indicate that S. aureus hyaluronidase is a CodY-regulated virulence factor.

FIG 1

Generation and initial characterization of the CA-MRSA USA300 ΔhysA mutant and complemented strains. (A) Location of hysA on the CA-MRSA USA300 LAC chromosome. Numbers indicate SAUSA300 locus numbers. The hysA gene is surrounded by genes with unknown function and is transcribed by itself on the chromosome. (B and C) HA plate and hyaluronidase (HL) specific activity assays of LAC-WT, LAC ΔhysA, and complemented (pHysA) strains. As shown, HysA activity is abolished for the ΔhysA mutant and restored to WT levels when it is complemented in both assays.

FIG 2

Transposon mutagenesis library screen for altered HysA activity. (A) Summary of results of screen for HysA activity on HA agarose plates. Values of HysA activity are reported as diameters (cm) of zones of clearing, measured with a caliper, and represent the means ± standard deviations of five technical replicates for each strain plated on HA agarose. Pictures are representative images of each strain. (B) Hyaluronidase specific activity assay of strains containing mutations that significantly altered HysA activity on HA agarose plates. The data are presented as the fold changes compared to the activity of USA300 WT strain JE2. Values represent the means and standard deviations of four technical replications for three independent biological determinations. Statistical significance (****, P < 0.0001) was determined by Student's t test.

FIG 3

Role of the agr system in hysA gene regulation. Cultures of various LAC strains were grown for 8 h, spent medium was prepared, and the hyaluronidase specific activity in each sample was measured. (A) To assess the agr and SigB interaction, activity was measured in the WT, agr and sigB single mutant, and agr sigB double mutant strains and plotted. (B) To assess the agr and CodY interaction, activity was measured in the WT, agr and codY single mutant, and codY agr double mutant strains. For panels A and B, statistical significance (****, P < 0.0001) was determined by Student's t test. (C) Hyaluronidase specific activity for the WT containing the empty vector (black bars) or RNAIII under the control of a tetracycline-inducible promoter (gray bars). Student's t test was used to determine statistical significance (**, P < 0.01; ****, P < 0.0001). (D) Model for SigB and CodY regulation of HysA through the agr system.

FIG 4

The CodY binding box is required for hysA regulation. (A) Location of the putative CodY binding box in the hysA promoter region. An alignment of the consensus CodY binding box and the proposed hysA box is shown. (B) Immunoblotting for HysA performed with a LAC-WT strain. Spent medium was concentrated 20- or 50-fold from the WT and the codY and codY hysA mutant strains, as indicated, and immunoblotted for HysA. In each strain, a Δspa deletion was engineered to remove background antibody binding. (C) Mutations generated in the CodY box in plasmids pCR04, pCR05, and pCR06. (D) Hyaluronidase specific activity assay at 8 h for the WT (LAC ΔhysA with one of the indicated plasmid constructs) or a codY mutant (the LAC ΔhysA codY double mutant with one of the indicated plasmid constructs) containing the pHysA, pCR04, pCR05, or pCR06 construct. Statistical analysis was performed using a Student's t test (****, P < 0.0001).

FIG 5

Effect of nutrient availability on hysA regulation. (A) Growth curve of LAC-WT grown in CDM or in CDM lacking isoleucine for the first 5 h. Cells were harvested by centrifugation, washed 1 time in PBS, and suspended in either fresh CDM or CDM containing 5× BCAAs. Arrows, times when samples were taken or where medium was switched, as indicated. (B) Hyaluronidase specific activity of samples taken at the time points indicated in panel A. Statistical analysis was performed using Student's t test (****, P < 0.0001). (C) Immunoblot for the LukS subunit of PVL at the time points indicated in panel A. (D) Quantification of LukS immunoblot using ImageJ software. post, samples taken at time points after the medium switch.

FIG 6

HysA is required for USA300 virulence. Mice were intranasally inoculated with 1.0 × 10⁷ CFU of either LAC-WT, its ΔhysA mutant strain, or PBS. (A) Mice (n = 27) were monitored daily for signs of morbidity and mortality. **, P = 0.0022, Mantel-Cox test. (B) Groups of mice (n = 21) were euthanized after 48 h, and the bacterial burden in the lungs was quantified by CFU enumeration. **, P = 0.0095. (C) Histopathology of the lungs from mice treated with PBS or infected with either LAC-WT or the ΔhysA mutant. Black arrowheads, areas shown in the enlarged images in the insets. Data are representative of those from three independent experiments.

FIG 7

HysA is required for the breakdown of hyaluronic acid in vivo. (A and B) Micrographs of immunofluorescently labeled HA in the lungs of mice inoculated with 1.0 × 10⁷ CFU of either strain LAC-WT or its ΔhysA mutant strain. (C) The mean fluorescence intensity was quantified for each sample (40 fields per section). A sample treated with hyaluronidase from Streptomyces hyalurolyticus (Strep Hys) was also included as a control. NS, not significant; ****, P < 0.0001.