agr-Dependent interactions of Staphylococcus aureus USA300 with human polymorphonuclear neutrophils

Abstract

The emergence of serious infections due to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has fueled interest in the contributions of specific staphylococcal virulence factors to clinical disease. To assess the contributions of agr-dependent factors to the fate of organisms in polymorphonuclear neutrophils (PMN), we examined the consequences for organism and host cells of feeding PMN with wild-type CA-MRSA (LAC) or CA-MRSA (LAC agr KO) at different multiplicities of infection (MOIs). Phagocytosed organisms rapidly increased the transcription of RNAIII in a time- and MOI-dependent fashion; extracellular USA300 (LAC) did not increase RNAIII expression despite having the capacity to respond to autoinducing peptide-enriched culture medium. HOCl-mediated damage and intracellular survival were the same in the wild-type and USA300 (LAC agr KO). PMN lysis by ingested USA300 (LAC) was time- and MOI-dependent and, at MOIs >1, required α-hemolysin (hla) as USA300 (LAC agr KO) and USA300 (LAC hla KO) promoted PMN lysis only at high MOIs. Taken together, these data demonstrate activation of the agr operon in human PMN with the subsequent production of α-hemolysin and PMN lysis. The extent to which these events in the phagosomes of human PMN contribute to the increased morbidity and mortality of infections with USA300 (LAC) merits further study.

Fig. 1

Viability of S. aureus USA300 (LAC), USA300 (LAC agr KO), USA300 (LAC hla KO), and USA300 (18811) recovered from PMN. PMN were fed opsonized S. aureus strains, either USA300 (LAC), USA300 (LAC agr KO), USA300 (LAC hla KO), or USA300 (18811), at an MOI of 1: 1. The viability of the intracellular bacteria for each strain was determined by colony plating. Samples were analyzed at 30, 60, and 120 min after phagocytosis. The data are presented as means ± SEM (n ≥ 3). The USA300 (LAC) strain was compared against each of the other strains using an unpaired t test. ∗ p < 0.05 between USA300 (LAC) and USA300 (LAC hla KO) at 30 and 120 min.

Fig. 2

Upregulation of RNAIII expression in S. aureus after ingestion by PMN. Normal PMN were fed opsonized S. aureus, either strain ALC1435, USA300 (LAC), or USA300 (LAC hla KO), at MOIs of 1: 1 (a), 2.5: 1 (b), and 5: 1 (c, e). In parallel, PMN pre-treated with DPI prior to microbial challenge were fed S. aureus (d) at an MOI of 5: 1. At 0, 60, and 120 min after phagocytosis, RNAIII expression in recovered S. aureus was quantified by real-time RTPCR using GAPDH as the reference gene. The fold change for each sample relative to the postopsonized S. aureus and prior to exposure to PMN was calculated and is shown as the mean ± SEM (n ≥ 3). The inset in b shows the data for the ALC1435 strain where the y-axis has been expanded between 0 and 30 for clarity.

Fig. 3

RNAIII expression by intracellular and extracellular USA300 (LAC). Opsonized S. aureus USA300 (LAC) were incubated at an MOI of 2.5: 1 with either PMN, in which phagocytosis was inhibited by pretreatment with DHCB, or normal PMN in the absence or presence of 10% AIP-rich culture supernatant (10% sup). RNAIII expression at 60 and 120 min after phagocytosis was quantified from bacteria recovered from the phagosomes of normal PMN, nonphagocytosed bacteria outside DHCB-treated PMN, and nonphagocytosed bacteria in the presence of AIP. A subculture of USA300 (LAC) grown in TSB was used as a positive control. GAPDH was used as the reference gene and the fold change relative to the postopsonized control was calculated. Data are presented as means ± SEM (n ≥ 3). The mean values for intraphagosomal S. aureus, extracellular S. aureus, extracellular S. aureus + 10% supernatant, and S. aureus in TSB at 60 min were 26.0, 11.4, 1,135.3, and 44.3, respectively; at 120 min the values were 50.0, 14.2, 1,440.1, and 182.9, respectively. sup = Supernatant; SA = S. aureus.

Fig. 4

PMN lysis after ingestion of USA300 (LAC) and USA300 (LAC agr KO). PMN were fed opsonized S. aureus, either strain USA300 (LAC) or USA300 (LAC agr KO), at an MOI of 1: 1 (a),5: 1 (b), or 10: 1 (c). The percent of cell lysis was calculated using the LDH release assay at 0, 60, 120, 180, and 240 min after phagocytosis and the results are presented as means ± SEM (n ≥ 4). ∗ p < 0.05, using a paired t test, between USA300 (LAC) and USA300 (LAC agr KO) at the indicated time points and MOIs.

Fig. 5

α – Hemolysin expression and activity in USA300 (LAC), the isogenic KO strains, and USA300 (18811). Clarified overnight culture medium was prepared from the following S. aureus strains: USA300 (LAC), USA300 (LAC hla KO), USA300 (LAC hla KO) complemented mutant, USA300 (LAC agr KO), USA300 (LAC agrA KO), USA300 (LAC agrA KO) complemented mutant, and USA300 (18811). a Hemolytic activity present in the culture medium from each strain was determined by the rabbit RBC lysis assay, and the results were normalized to the USA300 (LAC) strain and reported as mean fold changes ± SEM (n = 3). b Proteins in each sample were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an anti-α-hemolysin-HRP conjugated antibody. The USA300 (LAC hla KO) and USA300 (LAC hla KO) complemented strains serve as negative and positive reference controls, respectively. comp = Complemented.

Fig. 6

PMN lysis after ingestion of USA300 (LAC) and USA300 (LAC hla KO). As in figure 4, PMN were fed opsonized S. aureus and USA300 (LAC), and LDH release was monitored over time. Independently, PMN were fed USA300 (LAC hla KO) at an MOI of 1: 1 (a), 5: 1 (b), or 10: 1 (c). The percent of cell lysis was calculated using the LDH release assay at 0, 60, 120, 180, and 240 min after phagocytosis and the results are presented as means ± SEM (n ≥ 7). ∗ p < 0.05, using a paired t test, between strains USA300 (LAC) and USA300 (LAC hla KO) at the indicated time points and MOIs.

Fig. 7

PMN lysis after ingestion of USA300 (LAC) versus USA300 (18811). PMN were fed opsonized USA300 (LAC) or USA300 (18811) at MOIs of 1: 1 (a), 5: 1 (b), or 10: 1 (c), and LDH release was monitored over time. The percent of cell lysis was calculated using the LDH release assay at 0, 60, 120, 180, and 240 min after phagocytosis and the results are presented as means ± SEM (n = 3). No significant differences were observed between strains at the chosen time points using a paired t test.