Bacterial toxins can inhibit host cell autophagy through cAMP generation.

Autophagy plays a significant role in innate and adaptive immune responses to microbial infection. Some pathogenic bacteria have developed strategies to evade killing by host autophagy. These include the use of 'camouflage' proteins to block targeting to the autophagy pathway and the use of pore-forming toxins to block autophagosome maturation. However, general inhibition of host autophagy by bacterial pathogens has not been observed to date. Here we demonstrate that bacterial cAMP-elevating toxins from B. anthracis and V. cholera can inhibit host anti-microbial autophagy, including autophagic targeting of S. Typhimurium and latex bead phagosomes. Autophagy inhibition required the cAMP effector protein kinase A. Formation of autophagosomes in response to rapamycin and the endogenous turnover of peroxisomes was also inhibited by cAMP-elevating toxins. These findings demonstrate that cAMP-elevating toxins, representing a large group of bacterial virulence factors, can inhibit host autophagy to suppress immune responses and modulate host cell physiology.

A diacylglycerol-dependent signaling pathway contributes to regulation of antibacterial autophagy.

Autophagy mediates the degradation of cytoplasmic contents in the lysosome and plays a significant role in immunity. Lipid second messengers have previously been implicated in the regulation of autophagy. Here, we demonstrate a signaling role for diacylglycerol (DAG) in antibacterial autophagy. DAG production was necessary for efficient autophagy of Salmonella, and its localization to bacteria-containing phagosomes preceded autophagy. The actions of phospholipase D and phosphatidic acid phosphatase were required for DAG generation and autophagy. Furthermore, the DAG-responsive delta isoform of protein kinase C was required, as were its downstream targets JNK and NADPH oxidase. Previous studies have revealed a role for the ubiquitin-binding adaptor molecules p62 and NDP52 in autophagy of S. Typhimurium. We observed bacteria-containing autophagosomes colocalizing individually with either DAG or ubiquitinated proteins, indicating that both signals can act independently to promote antibacterial autophagy. These findings reveal an important role for DAG-mediated PKC function in mammalian antibacterial autophagy.

Salmonella-containing vacuoles display centrifugal movement associated with cell-to-cell transfer in epithelial cells.

Intracellular Salmonella enterica serovar Typhimurium (serovar Typhimurium) occupies a Salmonella-containing vacuole (SCV) where bacterial effector proteins are secreted into the host cell using type III secretion systems (T3SS). Cytoskeletal motor proteins and T3SS-delivered effector proteins facilitate SCV positioning to juxtanuclear positions where bacterial replication occurs. Here, we show that this characteristic SCV positioning is not maintained by all SCVs during infection of HeLa cells. Notably, juxtanuclear SCV localization that occurs by 8 to 14 h postinfection is followed by significant centrifugal displacement of a subset of SCVs toward the host cell periphery by 24 h postinfection. This novel phenotype requires bacterial protein synthesis, a functional Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS, intact microtubules, and kinesin-1 motor protein. Bacteria lacking PipB2, a kinesin-recruiting T3SS effector, did not exhibit centrifugal displacement and remained at juxtanuclear positions throughout 24 h of infection. While levels of the SPI-2 effectors PipB2 and SifA increased during 24 h postinfection, a corresponding decrease in levels of the SPI-1 T3SS effectors SipA and SopB, both known to mediate juxtanuclear SCV positioning, was observed. A fluorescence-based assay indicated that wild-type serovar Typhimurium transferred from infected to uninfected epithelial cells while strains deficient in SPI-2 T3SS secretion or PipB2 did not. Our results reveal a novel SCV phenotype implicated in the cell-to-cell spread of serovar Typhimurium during infection.