Although progress has been made, the basis for the enhanced virulence phenotype of CA-MRSA is incompletely defined. We previously demonstrated that prominent CA-MRSA strains have significant capacity to survive after phagocytosis by neutrophils and cause eventual host cell lysis [5
]. Thus, the ability of USA300 and USA400 strains to cause relatively rapid destruction of neutrophils after phagocytosis may be linked in part to the observed high-virulence phenotype of CA-MRSA. As such, we investigated the mechanism used by USA300 to cause neutrophil destruction after phagocytosis.
CA-MRSA strains such as USA300 secrete multiple cytolytic toxins, including PVL, γ-hemolysin and α-type phenol-soluble modulins, which have cytolytic activity toward neutrophils [30
]. However, the neutrophil lysis described herein appears distinct from the mechanism used by pore-forming toxins such as PVL to kill neutrophils. First, PMN lysis after phagocytosis did not always correlate with permeabilization of the neutrophil plasma membrane by molecules present in USA300 culture supernatants (fig. ). For example, culture supernatant from USA300 isolate 18811 had limited capacity to cause permeabilization of the neutrophil plasma but retained capacity to cause lysis after phagocytosis. Also, pore-forming toxins produced within phagosomes would need access to the plasma membrane and our TEM analyses suggest phagosome membranes remained intact to the point of host cell lysis (fig. ). This finding is not compatible with cytolysis that requires disruption of the phagosome membrane by pore-forming toxins. In accordance with this idea, we demonstrated previously that PMN lysis after phagocytosis of USA300 or USA400 is PVL independent [20
]. Further studies with additional isogenic gene deletion strains are needed to determine whether cytolytic toxins other than PVL contribute to the PMN lysis described herein.
Another notable finding was that lysis of PMNs was significantly increased following uptake of serum opsonized of LAC compared with unopsonized bacteria (fig. ). Although differential kinetics of phagocytosis may account for some of the observed differences in PMN lysis, it is also possible that ligation of specific receptors during opsonophagocytosis of USA300 ultimately contributes to PMN lysis. This observation merits further investigation.
Within 4 h after phagocytosis of USA300, neutrophils underwent morphological changes consistent with apoptosis, including plasma membrane blebbing and nuclear condensation (fig. , ; online suppl. video
). However, neither FAS-mediated nor phagocytosis-induced apoptosis caused significant neutrophil lysis within the same time period (fig. ). Therefore, although phagocytosis-induced apoptosis may be occurring in PMNs containing ingested USA300, the rapid neutrophil lysis is likely unrelated to this process.
Inasmuch as PMN lysis occurred following phagocytosis of strains that produce little or no cytolytic activity in culture supernatants (cytolysis mediated by pore-forming toxins) and required new transcription and protein synthesis within the first 3 h after uptake, the process appears to be a form of programmed cell death. Pyroptosis is a type of programmed cell death that culminates with host cell lysis, inflammation of the surrounding tissue, and phagocyte recruitment to the site of infection [33
]. This process is caspase 1 dependent and involves fragmentation of DNA, activation of IL-1β and IL-18, and formation of pores in the plasma membrane [36
]. Pyroptosis is mediated by the inflammasome, which assembles after phagocytosed bacteria escape from phagosomes and bacterial products are detected by pattern-recognition receptors in the cytosol [35
]. Since USA300 failed to escape from neutrophil phagosomes in our current studies and PMN lysis was caspase independent (fig. ), S. aureus
-mediated PMN destruction reported here was not pyroptosis.
Our data [current study, [5
]] indicate S. aureus
causes a form of neutrophil necrosis, albeit there are clearly strain-dependent differences in the magnitude of lysis (for example, lysis was high for USA300 and low for strain COL). This notion is strongly supported by the recommendations of the Nomenclature Committee on Cell Death in 2009, as the characteristics of neutrophil death after phagocytosis of S. aureus
fit those described for necrosis [39
]. Vandenabeele and colleagues [40
] recently provided compelling evidence that cell necrosis can be a form of programmed cell death involving multiple signal transduction pathways. Programmed necrotic cell death may involve alteration of calcium ion levels [41
], findings compatible with our results. Although we did not measure alterations in intracellular calcium levels during S. aureus
-mediated PMN lysis, transcripts encoding proteins involved in calcium homeostasis (CACNA1A
were significantly upregulated in PMNs 2 h following phagocytosis of community-associated S. aureus
strains (fig. ). Further studies are required to elucidate the signal transduction pathways underlying necrotic neutrophil death after ingestion of S. aureus
. Such studies will provide insight into the basis of enhanced PMN necrosis caused by CA-MRSA strains and thus lead to a better understanding of the enhanced virulence phenotype of CA-MRSA.