Genetic Requirements for Salmonella -Induced Cytopathology in Human Monocyte-Derived Macrophages

Abstract

Infection of human macrophages with Salmonella enterica serovar Typhimurium or Salmonella enterica serovar Dublin produces delayed cytotoxicity characterized by cell detachment and associated apoptosis. Using a site-specific mutant in the SpvB active site, we verify that the ADP-ribosylation activity of SpvB is required for delayed cytotoxicity in human macrophages infected with Salmonella. SipB and the type III protein secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI1) are not involved, whereas the SPI2 TTSS is absolutely required for SpvB-dependent cytotoxicity. Furthermore, we show that infection of macrophage cultures with wild-type or sipB mutant bacteria led to a complete loss of polymerized actin in over half of the cells after 24 h. In contrast, macrophages infected with the spvB or SPI2 (ssaV or ssaJ) mutant strain retained normal F-actin filaments, despite similar numbers of intracellular bacteria. We conclude that SpvB and a functional SPI2 TTSS are essential for Salmonella-induced delayed cytotoxicity of human macrophages.Salmonella enterica subspecies I strains are responsible for enteric fever, gastroenteritis, and bacteremia in humans and a broad array of illnesses in animals, including enteritis and septicemia in livestock. A number of subspecies I serovars carry virulence plasmids encoding the highly conserved spv locus associated with disease syndromes of greater severity (13). The spv genes are found more frequently in human extraintestinal isolates than in environmental or fecal isolates, and most cases of human nontyphoidal bacteremia are caused by serovars that contain the spv genes (12, 13).The plasmid-encoded spv locus consists of the spvR regulatory gene and four structural genes, spvABCD (26). spvR encodes a transcriptional regulator activating its own expression and the spvABCD operon (9, 27). Transcription of the spv operon also requires the stationary-phase alternate factor RpoS (5, 10). Since both rpoS and spv are upregulated after phagocytosis by macrophages (4), expression of the spv genes appears to be strongly induced by the intracellular environment of the host cell. The spv genes appear to promote the macrophage phase of the disease process, avoiding destruction by neutrophils and facilitating proliferation of Salmonella strains at extraintestinal sites of infection (13).In vivo experiments suggest that the spv genes enhance bacterial replication in macrophages (18). In human macrophages, spv gene expression is required for induction of cytotoxicity characterized by cell detachment and eventual apoptosis (29). The spvB gene within the spv operon is essential for the spv virulence phenotype, as shown by mutational analysis (35). The spvB gene encodes a 65.6-kDa protein (recently characterized as an ADP-ribosyl transferase) that modifies actin and blocks polymerization to F-actin filaments (28, 38). Site-directed mutagenesis of the conserved NAD-binding site sequence demonstrates that this ADP-ribosylation activity is essential for virulence in mice (28)...

Impact ofSalmonella entericaType III Secretion System Effectors on the Eukaryotic Host Cell

“…One report showed that SpvB could be secreted into a culture medium mimicking the intracellular iron concentrations of eukaryotic cells in a T3SS-independent manner [316]. However, several components of the T3SS2, have been shown to be essential for SpvB-induced actin depolymerization and delayed cytotoxicity, strongly supporting the idea that SpvB is translocated into the host cell through T3SS2 [314,317].…”

“…SpvB inhibits the formation of vacuole-associated actin polymerizations (VAPs) in HeLa cells [312] and negatively regulates the formation of SIFs [313]. The ADP-ribosylation activity of SpvB is also required for a delayed form of cell death that is observed in host cells 18-24 h after infection with noninvasive bacteria or T3SS1 mutants [67,85,314,315]. One report showed that SpvB could be secreted into a culture medium mimicking the intracellular iron concentrations of eukaryotic cells in a T3SS-independent manner [316].…”

ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death

“…Browne and coworkers demonstrated earlier that the infection of human macrophages with S. enterica resulted in delayed apoptosis at about 18 to 24 h following infection and that the underlying reason was most likely the ADP-ribosylation of actin by SpvB (9). Strains harboring inactive SpvB do not induce apoptosis (9).…”

Salmonella Rapidly Kill Dendritic Cells via a Caspase-1- Dependent Mechanism

“…Indeed, longterm residence and replication within phagocytic cells appear to be essential for Salmonella virulence because mutant bacteria that cannot survive or multiply inside macrophages are attenuated for systemic infection in mice (10). The complex nature of this hostpathogen interaction is further illustrated by recent evidence that Salmonella can kill macrophages via at least two distinct mechanisms, resulting in the rapid activation of host caspase-1 and caspase-2 (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Caspase-1 is a member of a family of cysteinecontaining aspartate-specific proteases that play an important role in apoptosis (22,23).…”