We infer that protection derives from stimulation of local innate immune mechanisms, and that activated lung epithelium is the most likely cellular effector of this response. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value.

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

Pneumonia is a serious problem worldwide. We recently demonstrated that innate defense mechanisms of the lung are highly inducible against pneumococcal pneumonia. To determine the breadth of protection conferred by stimulation of lung mucosal innate immunity, and to identify cells and signaling pathways activated by this treatment, mice were treated with an aerosolized bacterial lysate, then challenged with lethal doses of bacterial and fungal pathogens. Mice were highly protected against a broad array of Gram-positive, Gram-negative, and class A bioterror bacterial pathogens, and the fungal pathogen, Aspergillus fumigatus. Protection was associated with rapid pathogen killing within the lungs, and this effect was recapitulated in vitro using a respiratory epithelial cell line. Gene expression analysis of lung tissue showed marked activation of NF-kB, type I and II IFN, and antifungal Card9-Bcl10-Malt1 pathways. Cytokines were the most strongly induced genes, but the inflammatory cytokines TNF and IL-6 were not required for protection. Lung-expressed antimicrobial peptides were also highly up-regulated. Taken together, stimulated innate resistance appears to occur through the activation of multiple host defense signaling pathways in lung epithelial cells, inducing rapid pathogen killing, and conferring broad protection against virulent bacterial and fungal pathogens. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value for protection of patients with neutropenia or impaired adaptive immunity against opportunistic pneumonia, and for defense of immunocompetent subjects against a bioterror threat or epidemic respiratory infection.

No abstract

Developing Dictyostelium cells aggregate to form fruiting bodies containing typically 2 ؋ 10 4 cells. To prevent the formation of an excessively large fruiting body, streams of aggregating cells break up into groups if there are too many cells. The breakup is regulated by a secreted complex of polypeptides called counting factor (CF). Countin and CF50 are two of the components of CF. Disrupting the expression of either of these proteins results in cells secreting very little detectable CF activity, and as a result, aggregation streams remain intact and form large fruiting bodies, which invariably collapse. We find that disrupting the gene encoding a third protein present in crude CF, CF45-1, also results in the formation of large groups when cells are grown with bacteria on agar plates and then starve. However, unlike countin ؊ and cf50 ؊ cells, cf45-1 ؊ cells sometimes form smaller groups than wild-type cells when the cells are starved on filter pads. The predicted amino acid sequence of CF45-1 has some similarity to that of lysozyme, but recombinant CF45-1 has no detectable lysozyme activity. In the exudates from starved cells, CF45-1 is present in a ϳ450-kDa fraction that also contains countin and CF50, suggesting that it is part of a complex. Recombinant CF45-1 decreases group size in colonies of cf45-1 ؊ cells with a 50% effective concentration (EC 50 ) of ϳ8 ng/ml and in colonies of wild-type and cf50؊ cells with an EC 50 of ϳ40 ng/ml. Like countin ؊ and cf50 ؊ cells, cf45-1 ؊ cells have high levels of cytosolic glucose, high cell-cell adhesion, and low cell motility. Together, the data suggest that CF45-1 participates in group size regulation in Dictyostelium.