RESUMEN
Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested bacteria. In this study, we performed random mutagenesis of Dictyostelium cells and isolated a mutant defective for growth on bacteria. This mutant is characterized by the genetic inactivation of the lrrkA gene, which encodes a protein with a kinase domain and leucine-rich repeats. LrrkA knockout (KO) cells kill ingested Klebsiella pneumoniae bacteria inefficiently. This defect is not additive to the killing defect observed in kil2 KO cells, suggesting that the function of Kil2 is partially controlled by LrrkA. Indeed, lrrkA KO cells exhibit a phenotype similar to that of kil2 KO cells: Intraphagosomal proteolysis is inefficient, and both intraphagosomal killing and proteolysis are restored upon exogenous supplementation with magnesium ions. Bacterially secreted folate stimulates intracellular killing in Dictyostelium cells, but this stimulation is lost in cells with genetic inactivation of kil2, lrrkA, or far1. Together, these results indicate that the stimulation of intracellular killing by folate involves Far1 (the cell surface receptor for folate), LrrkA, and Kil2. This study is the first identification of a signalling pathway regulating intraphagosomal bacterial killing in Dictyostelium cells.
Asunto(s)
Dictyostelium/enzimología , Ácido Fólico/metabolismo , Fagosomas/microbiología , Fosfotransferasas/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal , Dictyostelium/genética , Dictyostelium/microbiología , Regulación Bacteriana de la Expresión Génica , Espacio Intracelular/microbiología , Klebsiella pneumoniae/metabolismo , Leucina/química , Fagocitosis , Fosfotransferasas/genética , Dominios Proteicos , Proteínas Protozoarias/genéticaRESUMEN
Dictyostelium STATa is a homologue of metazoan signal transducers and activators of transcription (STATs) and is important for morphogenesis. STATa is activated by phosphorylation on Tyr702 when cells are exposed to extracellular cAMP. Although two tyrosine kinase-like (TKL) proteins, Pyk2 and Pyk3, have been definitively identified as STATc kinases, no kinase is known for STATa activation. Based on homology to the previously identified tyrosine-selective TKLs, we identified DrkA, a member of the TKL family and the Dictyostelium receptor-like kinase (DRK) subfamily, as a candidate STATa kinase. The drkA gene is almost exclusively expressed in prestalk A (pstA) cells, where STATa is activated. Transient over-expression of DrkA increased STATa phosphorylation, although over-expression of the protein causes a severe growth defect and cell death. Furthermore, recombinant DrkA protein is auto-phosphorylated on tyrosine and threonine residues, and an in vitro kinase assay shows that DrkA can phosphorylate STATa on Tyr702 in a STATa-SH2 (phosphotyrosine binding) domain-dependent manner. These observations strongly suggest that DrkA is one of the key regulators of STATa tyrosine phosphorylation and is consistent with it being the kinase that directly activates STATa.
Asunto(s)
Dictyostelium/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción STAT/metabolismo , Animales , Dictyostelium/citología , Dictyostelium/genética , Morfogénesis , Mutación , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/fisiología , Tirosina/metabolismoRESUMEN
Dictyostelium harbors multiple expansin-like genes with generally unknown functions. Thus, we analyzed the expansin-like 3 (expL3) gene and found that its expression was reduced in a null mutant for a STATa gene encoding a transcription factor. The expression of expL3 was developmentally regulated and its transcript was spliced only in the multicellular stages. The expL3 promoter was activated in the anterior prestalk region of the parental strain and downregulated in the STATa null slug, although the expL3 promoter was still expressed in the prestalk region. The expL3 overexpressing strain exhibited delayed development and occasionally formed an aberrant structure, i.e., a fruiting body-like structure with a short stalk. The ExpL3-myc protein bound cellulose.