During sepsis, activation of phagocytes leads to the overproduction of proinflammatory cytokines, causing systemic inflammation. Despite substantial information regarding the underlying molecular mechanisms that lead to sepsis, several elements in the pathway remain to be elucidated. We found that the enzyme sphingosine kinase 1 (SphK1) is up-regulated in stimulated human phagocytes and in peritoneal phagocytes of patients with severe sepsis. Blockade of SphK1 inhibited phagocyte production of endotoxin-induced proinflammatory cytokines. We observed protection against sepsis in mice treated with a specific SphK1 inhibitor that was enhanced by treatment with a broad-spectrum antibiotic. These results demonstrated a critical role for SphK1 in endotoxin signaling and sepsis-induced inflammatory responses and suggest that inhibition of SphK1 is a potential therapy for septic shock.

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Cyclic diguanylic acid (c-di-GMP) is an intracellular signaling molecule involved in regulation of cellular functions such as motility, biofilm formation and virulence. Intracellular level of c-di-GMP is controlled through opposing diguanylate cyclase (DGC) and phosphodiesterase (PDE) activities of GGDEF and EAL domain proteins, respectively. We report the identification and characterization of cdpA, a gene encoding a protein containing an EAL domain in the Gram-negative soil bacillus and human pathogen Burkholderia pseudomallei KHW. Purified recombinant CdpA protein exhibited PDE activity in vitro. Evidence that CdpA is a major c-di-GMP-specific PDE in B. pseudomallei KHW was shown by an 8-fold-higher c-di-GMP level in the cdpA-null mutant as compared to the wild type and the complemented cdpA mutant. The presence of higher intracellular c-di-GMP levels in the cdpA-null mutant was associated with increased production of exopolysaccharides, increased cell-to-cell aggregation, absence of flagella and swimming motility, and increased biofilm formation. The relevance of CdpA in B. pseudomallei virulence was demonstrated by a 3-fold reduction in invasion of human lung epithelial cells and a 6-fold reduction in cytotoxicity on human macrophage cells infected with the cdpA mutant.Burkholderia pseudomallei, a motile Gram-negative soil bacillus, is endemic to Southeast Asia and northern Australia. It causes melioidosis in humans, a potentially fatal septicemic infection which often manifests as acute pulmonary infection, localized skin infection, or acute septicemia (41). Its polar tuft of flagella is involved in swimming motility and is also a virulence determinant of B. pseudomallei during intranasal infection of mice (6). As an environmental saprophyte, it inhabits the soil at the root zone of plants where the bacteria readily form biofilm at the solid-liquid interface. B. pseudomallei can also be internalized within amoebic cysts or in the cytoplasm of arbuscular mycorrhizal fungi in order to survive hostile environmental conditions for a prolonged period (17,21).Pathogenicity of B. pseudomallei is dependent on a number of virulence factors and the expression of some virulence factors, such as phospholipase C and siderophores, is regulated by quorum sensing (29). B. pseudomallei mutants lacking components of the quorum-sensing systems exhibit reduced organ colonization of aerosolized BALB/c mice and increased time to death, while those lacking the BpsIR quorum sensing system were also impaired in biofilm formation and produced less MprA protease (29,36,37). Recent studies have linked the mechanisms of intercellular quorum sensing to intracellular signaling mediated by cyclic di-GMP (c-di-GMP) (39,42,43).The sequenced genome of B. pseudomallei K96243 reveals several putative GGDEF-EAL domain proteins. These proteins use a combination of diguanylate cyclase (DGC) activities in the conserved GG(D/E)EF domains and phosphodiesterase (PDE) activities in the EAL domains to adjust intracellular c-di-GMP levels. Proteins con...

Triple-negative breast cancer (TNBC) is characterized by unique aggressive behavior and lack of targeted therapies. Among the various molecular subtypes of breast cancer, it was observed that TNBCs express elevated levels of sphingosine kinase 1 (SPHK1) compared to other breast tumor subtypes. High levels of SPHK1 gene expression correlated with poor overall and progression- free survival, as well as poor response to Doxorubicin-based treatment. Inhibition of SPHK1 was found to attenuate ERK1/2 and AKT signaling and reduce growth of TNBC cells in vitro and in a xenograft SCID mouse model. Moreover, SPHK1 inhibition by siRNA knockdown or treatment with SKI-5C sensitizes TNBCs to chemotherapeutic drugs. Our findings suggest that SPHK1 inhibition, which effectively counteracts oncogenic signaling through ERK1/2 and AKT pathways, is a potentially important anti-tumor strategy in TNBC. A combination of SPHK1 inhibitors with chemotherapeutic agents may be effective against this aggressive subtype of breast cancer.

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