Intracellular Staphylococcus aureus Escapes the Endosome and Induces Apoptosis in Epithelial Cells

We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a Staphylococcus aureus mastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections. S. aureus cells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of S. aureus, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalization S. aureus escapes the endosome and induces apoptosis in nonprofessional phagocytes.

Staphylococcus aureus is a pathogen with a broad host range and is a leading cause of infections in humans and domesticated animals worldwide. The rapidly increasing frequency of methicillin-resistant isolates and the looming threat of resistance to vancomycin by this organism have recently caused considerable alarm within the medical community. Infections associated with this organism are extremely common and often life threatening; therefore, there is serious potential for S. aureus to cause increased morbidity and mortality. S. aureus is also the leading cause of intramammary infection in ruminants, which is the most economically important disease to the dairy industry in the United States, with approximately one-half of dairy cows afflicted with some form of mastitis. This disease accounts for approximately 70% of the total expenses for dairy farmers (14) and results in the loss of billions of dollars each year (3). Thus, a thorough understanding of the pathogenesis of staphylococcal disease could have a profound impact on public health and agriculture in the United States and worldwide.The mechanism of persistence of staphylococci in its hosts, despite the induction of seemingly sufficient levels of humoral and mucosal antibodies, remains unexplained. This is an important issue for both humans and animals, which can have repeated occurrences of staphylococcal infections and toxigenic diseases such as toxic shock syndrome (7). While there is some evidence that immunosuppression induced by superantigens is partially responsible for both the persistence of infection and reduced antibody levels against some staphylococcal products, not all human and animal cases are attributed to superantigen-producing organisms. For example, less than half of bovine mastitis staphylococcal isolates produce known superantigen exotoxins (26).One confirmed mechanism employed by some bacteria to evade humoral immunity is to become internalized in host cells. For example, organisms such as Listeria monocytogenes and Mycobacterium tuberculosis are facultative intracellular pathogens that require cell-mediated immunity to be eliminated most efficiently; the presence of antibodies alone is ineffective during infection by these pathogens. S. aureus is generally not considered to be an intracellular pathogen of the magnitude associated with classical facultative intracellular pathogens (i.e., Listeria, Mycobacterium, Salmonella, and Shigella spp.). However, it is well documented that S. aureus can be internalized in epithelial cells (1) and endothelial cells (18, 37). Little is known regarding the mechanisms involved in internalization, the potential role of internalization of S. aureus by the host cell, or host cell responses.The objective of this study was to investigate the invasion of mammary epithelial cells by a strain of S. aureus known to cause bovine mastitis. We now report that S. aureus cells successfully invade epithelial cells, exist free in the cytoplasm after effecting release from the endosome, and induce the host cells to undergo apoptosis.