Autophagy defends the mammalian cytosol against bacterial infection.1-3 Efficient pathogen engulfment is mediated by cargo-selecting autophagy adaptors that rely on unidentified pattern-recognition or danger receptors to label invading pathogens as autophagy cargo, typically by poly-ubiquitin coating.4-9 Here we show that galectin-8, a cytosolic lectin, is a danger receptor that restricts Salmonella proliferation. Galectin-8 monitors endo-lysosomal integrity and detects bacterial invasion by binding host glycans exposed on damaged Salmonella-containing vacuoles. By recruiting NDP52 galectin-8 activates anti-bacterial autophagy. Galectin-8-dependent recruitment of NDP52 to Salmonella-containing vesicles is transient and followed by ubiquitin-dependent NDP52 recruitment. Since galectin-8 also detects sterile damage to endosomes or lysosomes, as well as invasion by Listeria or Shigella, we suggest galectin-8 serves as a versatile receptor for vesicle-damaging pathogens. Our results illustrate how cells deploy the danger receptor galectin-8 to combat infection by monitoring endo-lysososomal integrity based on the specific lack of complex carbohydrates in the cytosol.

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in 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, whereas 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 anti-bacterial defense.

SummaryInterferon exposure boosts cell-autonomous immunity for more efficient pathogen control. But how interferon-enhanced immunity protects the cytosol against bacteria and how professionally cytosol-dwelling bacteria avoid clearance are insufficiently understood. Here we demonstrate that the interferon-induced GTPase family of guanylate-binding proteins (GBPs) coats Shigella flexneri in a hierarchical manner reliant on GBP1. GBPs inhibit actin-dependent motility and cell-to-cell spread of bacteria but are antagonized by IpaH9.8, a bacterial ubiquitin ligase secreted into the host cytosol. IpaH9.8 ubiquitylates GBP1, GBP2, and GBP4 to cause the proteasome-dependent destruction of existing GBP coats. This ubiquitin coating of Shigella favors the pathogen as it liberates bacteria from GBP encapsulation to resume actin-mediated motility and cell-to-cell spread. We conclude that an important function of GBP recruitment to S. flexneri is to prevent the spread of infection to neighboring cells while IpaH9.8 helps bacterial propagation by counteracting GBP-dependent cell-autonomous immunity.

SummaryHigh levels of copper are toxic and therefore bacteria must limit free intracellular levels to prevent cellular damage. In this study, we show that a number of pneumococcal genes are differentially regulated by copper, including an operon encoding a CopY regulator, a protein of unknown function (CupA) and a P1-type ATPase, CopA, which is conserved in all sequenced Streptococcus pneumoniae strains. Transcriptional analysis demonstrated that the cop operon is induced by copper in vitro, repressed by the addition of zinc and is autoregulated by the copperresponsive CopY repressor protein. We also demonstrate that the CopA ATPase is a major pneumococcal copper resistance mechanism and provide the first evidence that the CupA protein plays a role in copper resistance. Our results also show that copper homeostasis is important for pneumococcal virulence as the expression of the cop operon is induced in the lungs and nasopharynx of intranasally infected mice, and a copA -mutant strain, which had decreased growth in high levels of copper in vitro, showed reduced virulence in a mouse model of pneumococcal pneumonia. Furthermore, using the copA -mutant we observed for the first time in any bacteria that copper homeostasis also appears to be required for survival in the nasopharynx.

Intramembrane proteolytic cleavage by signal peptide peptidase is required for the turnover of some ER-resident, tail-anchored membrane proteins.