Guanylate-binding proteins coordinately regulate oxidative and autophagic responses to intracellular bacteria.

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in the membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ-stimulated cells guanylate-binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, and GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D-dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP–caspase-4 pathway in antibacterial defense.

Autophagy is a major innate immune defense pathway in both plants and animals. In mammals, this cascade can be elicited by cytokines (IFN-γ) or pattern recognition receptors (TLRs and nucleotide-binding oligomerization domain-like receptors). Many signaling components in TLR- and nucleotide-binding oligomerization domain-like receptor-induced autophagy are now known; however, those involved in activating autophagy via IFN-γ remain to be elucidated. In this study, we engineered macrophages encoding a tandem fluorescently tagged LC3b (tfLC3) autophagosome reporter along with stably integrated short hairpin RNAs to demonstrate IFN-γ–induced autophagy required JAK 1/2, PI3K, and p38 MAPK but not STAT1. Moreover, the autophagy-related guanosine triphosphatase Irgm1 proved dispensable in both stable tfLC3-expressing RAW 264.7 and tfLC3-transduced Irgm1−/− primary macrophages, revealing a novel p38 MAPK-dependent, STAT1-independent autophagy pathway that bypasses Irgm1. These unexpected findings have implications for understanding how IFN-γ–induced autophagy is mobilized within macrophages for inflammation and host defense.

The formation of cyclopropane fatty acid (CFA) and its role in the acid shock response inSalmonella entericaserovar Typhimurium (S. typhimurium) was investigated. Data obtained by GC/MS demonstrated that the CFA level inS. typhimuriumincreased upon its entry to the stationary phase, as in other bacteria. Thecfagene encoding CFA synthase was cloned, and mutants of thecfagene were constructed by allelic exchange. Acfamutant could not produce CFA and was sensitive to low pH. Introduction of a functionalcfagene into acfamutant cell made the mutant convert all unsaturated fatty acids to CFAs and partially restored resistance to low pH. Interestingly, the alternative sigma factor RpoS, which was induced during the stationary phase, affected the production of C₁₉CFA but not C₁₇CFA. Western blotting analysis showed that the increase in expression of CFA synthase at early stationary phase was due to the alternative sigma factor RpoS.