Induction of Guanylate Binding Protein 5 by Gamma Interferon Increases Susceptibility to Salmonella enterica Serovar Typhimurium-Induced Pyroptosis in RAW 264.7 Cells▿

The regulation of caspase-1 activation in macrophages plays a central role in host defense against bacterial pathogens. The activation of caspase-1 by the detection of bacterial products through Nod-like receptors leads to the secretion of mature interleukin-1β (IL-1β) and IL-18 and the induction of rapid host cell death (pyroptosis). Here, we report that pyroptosis induced by Salmonella enterica serovar Typhimurium can be positively regulated by prior gamma interferon (IFN-γ) stimulation of RAW 264.7 cells. This increase in cell death is dependent on both caspase-1 activation and, in part, Salmonella pathogenicity island 1 (SPI-1) expression by Salmonella. Furthermore, the exogenous expression of the IFN-γ-induced protein guanylate binding protein 5 (GBP-5) is sufficient to induce a heightened susceptibility of RAW 264.7 cells to Salmonella-induced pyroptosis, and the endogenous expression of GBP-5 is important for this phenomenon. RAW 264.7 cells with decreased expression of GBP-5 mRNA (inhibited by short hairpin RNA against GBP-5) release twofold less lactate dehydrogenase (a marker of membrane permeability) upon infection by invasive S. enterica serovar Typhimurium than do infected control cells. Importantly, 3× FLAG-tagged GBP-5 is localized to membrane ruffles, which contact invasive Salmonella, and is found on the membranes of spacious phagosomes containing Salmonella (although it is also found in the cytoplasm and on other cellular membranes), placing 3× FLAG GBP-5 at the interface of secreted SPI-1 effectors and host protein machinery. The regulation of pyroptosis by the IFN-γ-induced protein GBP-5 may play an important role in the host defense against Salmonella enterica serovar Typhimurium and perhaps other invasive bacterial pathogens

Dictyostelium LvsB Mutants Model the Lysosomal Defects Associated with Chediak-Higashi Syndrome

Chediak-Higashi syndrome is a genetic disorder caused by mutations in a gene encoding a protein named LYST in humans (“lysosomal trafficking regulator”) or Beige in mice. A prominent feature of this disease is the accumulation of enlarged lysosome-related granules in a variety of cells. The genome of Dictyostelium discoideum contains six genes encoding proteins that are related to LYST/Beige in amino acid sequence, and disruption of one of these genes, lvsA (large volume sphere), results in profound defects in cytokinesis. To better understand the function of this family of proteins in membrane trafficking, we have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD, lvsE, and lvsF. Of all these, only lvsA and lvsB mutants displayed interesting phenotypes in our assays. lvsA-null cells exhibited defects in phagocytosis and contained abnormal looking contractile vacuole membranes. Loss of LvsB, the Dictyostelium protein most similar to LYST/Beige, resulted in the formation of enlarged vesicles that by multiple criteria appeared to be acidic lysosomes. The rates of endocytosis, phagocytosis, and fluid phase exocytosis were normal in lvsB-null cells. Also, the rates of processing and the efficiency of targeting of lysosomal α-mannosidase were normal, although lvsB mutants inefficiently retained α-mannosidase, as well as two other lysosomal cysteine proteinases. Finally, results of pulse-chase experiments indicated that an increase in fusion rates accounted for the enlarged lysosomes in lvsB-null cells, suggesting that LvsB acts as a negative regulator of fusion. Our results support the notion that LvsB/LYST/Beige function in a similar manner to regulate lysosome biogenesis