Host interferon-γ inducible protein contributes to Brucella survival.

Brucella spp. are highly adapted intracellular pathogens of mammals that cause chronic infections while surving and replicating in host monocytes and macrophages. Although monocytes are normally susceptible to infection, pretreatment with pro-inflammatory cytokine interferon-γ (IFN-γ) activates cellular defense mechanisms that increase intracellular killing of Brucella and prevents bacterial replication. We examined the contribution of the IFN-γ inducible GTPase, LRG-47, to B. abortus 2308 infection in in vitro and in vivo murine models. Infecting non-activated macrophages from LRG-47(-/-) mice revealed that loss of this host protein negatively effected the intracellular survival and replication of IgG opsonized B. abortus. In contrast, survival and replication of non-opsonized B. abortus was the same in both C57/B6 and LRG-47(-/-) peritoneal macrophages. Following IFN-γ activation of LRG-47(-/-) monocytes, IgG opsonized B. abortus survived better than non-opsonized bacteria. The differential fate of opsonized and non-opsonized B. abortus was only observed in macrophages collected from LRG-47(-/-) mice. Given the specific nature of the relationship between this host protein and the mechanism of Brucella internalization, LRG-47(-/-) mice were infected with B. abortus to assess whether the loss of the lrg47 protein would affect the ability of the bacteria to colonize or persist within the host. B. abortus were able to establish and maintain similar numbers of bacteria in both C57/B6 mice and LRG-47(-/-) through 3 weeks post intraperitoneal infection. By 9 weeks p.i. fewer B. abortus were recovered from LRG-47(-/-) mice than controls, suggesting that the host protein has a positive role in maintaining long term persistence of the bacteria within the host. These observations demonstrating a positive role for a host IFN-γ induced protein defense protein has yet to be reported. These results provide interesting insight into the complex interaction between Brucella and their host.

Large-scale purification of latex bead phagosomes from mouse macrophage cell lines and subsequent preparation for high-throughput quantitative proteomics.

Phagocytosis involves the engagement of a diverse array of cell surface receptors whose signal must be integrated on the membrane of the forming phagosomal cup. This method enables the quantitative proteomic analysis of phagosome fractions derived from phagocytes stimulated under two different conditions, thus allowing the complexity of phagosomal signaling to be analyzed in terms of the quantitative changes in phagosomal fraction protein content.

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-1beta (IL-1beta) 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-gamma) 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-gamma-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, 3x 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 3x FLAG GBP-5 at the interface of secreted SPI-1 effectors and host protein machinery. The regulation of pyroptosis by the IFN-gamma-induced protein GBP-5 may play an important role in the host defense against Salmonella enterica serovar Typhimurium and perhaps other invasive bacterial pathogens.