Autophagy Controls Salmonella Infection in Response to Damage to the Salmonella-containing Vacuole (original) (raw)

2006, Journal of Biological Chemistry

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes disease in a variety of hosts. S. Typhimurium actively invade host cells and typically reside within a membrane-bound compartment called the Salmonella-containing vacuole (SCV). The bacteria modify the fate of the SCV using two independent type III secretion systems (TTSS). TTSS are known to damage eukaryotic cell membranes and S. Typhimurium has been suggested to damage the SCV using its Salmonella pathogenicity island (SPI)-1 encoded TTSS. Here we show that this damage gives rise to an intracellular bacterial population targeted by the autophagy system during in vitro infection. Approximately 20% of intracellular S. Typhimurium colocalized with the autophagy marker GFP-LC3 at 1 h postinfection. Autophagy of S. Typhimurium was dependent upon the SPI-1 TTSS and bacterial protein synthesis. Bacteria targeted by the autophagy system were often associated with ubiquitinated proteins, indicating their exposure to the cytosol. Surprisingly, these bacteria also colocalized with SCV markers. Autophagy-deficient (atg5 ؊/؊) cells were more permissive for intracellular growth by S. Typhimurium than normal cells, allowing increased bacterial growth in the cytosol. We propose a model in which the host autophagy system targets bacteria in SCVs damaged by the SPI-1 TTSS. This serves to retain intracellular S. Typhimurium within vacuoles early after infection to protect the cytosol from bacterial colonization. Our findings support a role for autophagy in innate immunity and demonstrate that Salmonella infection is a powerful model to study the autophagy process. Salmonella enterica serovar Typhimurium (S. Typhimurium) 4 is a Gram-negative bacterial pathogen that invades and multiplies within the cells of its host. It is responsible for a variety of host-specific diseases, including gastroenteritis in humans and a systemic disease resembling typhoid fever in permissive mouse models (1, 2). Inside host cells, S. Typhimurium replicates within a vacuolar compartment termed the Salmonella-containing vacuole (SCV). Maturation of this phagosome-like compartment is altered by the bacteria to create a niche favorable for replication (3-5). For example, delivery of NADPH oxidase and inducible nitric-oxide synthase to the SCV is blocked, along with fusion of the SCV with lysosomes (2, 6-10). Late stages of infection in vitro are characterized by the formation of tubular membranous extensions emanating from the SCV called Salmonella-induced filaments (Sifs) (11-13). To establish an intracellular niche, S. Typhimurium delivers bacterial effector proteins into the host cell via needle-like appendages on the bacterial surface termed type III secretion systems (TTSSs) (5, 14, 15). The TTSS encoded in Salmonella pathogenicity island (SPI)-1 is necessary for the invasion of non-phagocytic cells and the establishment of gastroenteritis in animal models (1, 14, 16). After the early stages of infection, the SPI-1 system is down-regulated (17, 18), and expression of the SPI-2 TTSS is induced to continue modification of the SCV and intracellular growth (17, 19-22). SifA is an effector protein of the SPI-2 TTSS and is necessary for both maintenance of the SCV and for Sif formation. ⌬sifA S. Typhimurium lose the SCV at late times postinfection (6-8 h) and enter the cytosol (11, 23-27). TTSSs are used by many Gram-negative pathogens to deliver bacterial effector proteins into host cells (14), and these needle-like structures have been shown to cause damage to host cell membranes. For example, TTSSs form pores in membranes to allow protein delivery (28) and can cause contact-dependent lysis of sheep red blood cells (28, 29). Roy et al. (30) have suggested that the S. Typhimurium SPI-1 TTSS can damage the SCV early after invasion, and have proposed a vacuole repair mechanism in which lysosomes are recruited to the damaged vacuoles in a calcium-dependent manner. Recently, we and others (23, 24, 31) have observed that a small but significant proportion of wild-type S. Typhimurium escape from the SCV early after invasion in vitro, though the mechanism(s) of this escape are currently unclear. The existence of cytosolic S. Typhimurium suggests that the mechanism proposed by Roy et al. (30) is not sufficient to retain the entire bacterial population within vacuoles. We have previously shown that bacteria that enter the cytosol in such a manner are targeted by the ubiquitin system (31). The consequences of this are still unknown, though it may play a role in MHC class I presentation in macrophages. Macroautophagy (hereafter referred to as autophagy) is a cellular process that mediates the degradation of long-lived proteins and unwanted organelles in the cytosol by delivering them to the lysosome (32-34). Autophagy is regulated by a multitude of factors, including nutritional status, hormones and intracellular signaling pathways (34-36). Recent evidence has suggested that the autophagy pathway inter-* This work was supported by an Investigators in Pathogenesis of Infectious Disease