Staphylococcus aureus remains a common cause of nosocomial bacterial infections and are often antibiotic resistant. The role of NK cells and IL-15 and their relationship in host defense against extracellular bacterial pathogens including S. aureus remain unclear. We have undertaken several approaches to address this issue using wild type (WT), IL-15 gene knock-out (KO), and NK cell-depleted mouse models. Upon pulmonary staphylococcal infection WT mice had markedly increased activated NK cells, but not NKT or γδ T cells, in the airway lumen that correlated with IL-15 production in the airway and with alveolar macrophages. In vitro exposure to staphylococcal products and/or coculture with lung macrophages directly activated NK cells. In contrast, lung macrophages better phagocytosed S. aureus in the presence of NK cells. In sharp contrast to WT controls, IL-15 KO mice deficient in NK cells were found to be highly susceptible to pulmonary staphylococcal infection despite markedly increased neutrophils and macrophages in the lung. In further support of these findings, WT mice depleted of NK cells were similarly susceptible to staphylococcal infection while they remained fully capable of IL-15 production in the lung at levels similar to those of NK-competent WT hosts. Our study thus identifies a critical role for NK cells in host defense against pulmonary extracellular bacterial infection and suggests that IL-15 is involved in this process via its indispensable effect on NK cells, but not other innate cells. These findings hold implication for the development of therapeutics in treating antibiotic-resistant S. aureus infection.

Kaiso is the first member of the POZ family of zinc finger transcription factors reported to bind DNA with dual-specificity in both a sequence- and methyl-CpG-specific manner. Here, we report that Kaiso associates with and regulates the cyclin D1 promoter via the consensus Kaiso binding site (KBS), and also via methylated CpG-dinucleotides. The methyl-CpG sites appear critical for Kaiso binding to the cyclin D1 promoter, while a core KBS in close proximity to the methyl-CpGs appears to stabilize Kaiso DNA binding. Kaiso’s binding to both sites was demonstrated in vitro using electrophoretic mobility shift assays (EMSA) and in vivo using Chromatin immunoprecipitation (ChIP). To elucidate the functional relevance of Kaiso’s binding to the cyclin D1 promoter, we assessed Kaiso overexpression effects on a minimal cyclin D1 promoter-reporter that contains both KBS and CpG sites. Kaiso repressed this minimal cyclin D1 promoter-reporter in a dose-dependent manner and transcriptional repression occurred in a KBS-specific and methyl-CpG-dependent manner. Collectively our data validates cyclin D1 as a Kaiso target gene and demonstrates a mechanism for Kaiso binding and regulation of the cyclin D1 promoter. Our data also provides a mechanistic basis for how Kaiso may regulate other target genes whose promoters possess both KBS and methyl-CpG sites.

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Genetic variability in a putative virulence factor, the neutral trehalase ( Ntl) gene, was examined in strains of the insect pathogenic fungi Metarhizium anisopliae and Metarhizium flavoviride by restriction fragment length polymorphism (RFLP). The Ntl gene was sequenced from four of these strains that showed dissimilar RFLP patterns. Enzyme kinetic experiments were also performed on the partially purified neutral trehalase in order to assess whether nucleotide changes in these strains related to differences in enzyme catalytic function (i.e., Km, Vmax, and Kcat). Finally, the Metarhizium strains were assessed in bioassays against waxworm larvae in order to relate nucleotide variation with Ntl enzyme kinetics and insect virulence. The greatest RFLP variation was observed with Rsa1. M. flavoviride was found to be most dissimilar in RFLP patterns when compared with the M. anisopliae strains. RFLP patterns for Ntl were diagnostic markers for previously studied genetic groups of M. anisopliae. Comparisons of Ntl sequences showed that the introns were found to be more variable (6.2%) than the exons (3.1%). Comparisons of the translated nucleotide codons showed high levels (91%) of synonymous sequence variation between strains. Another fraction of the remaining mutations was neutral, resulting in amino acid substitutions with similar functions. The neutral trehalase was partially purified by preparative isoelectric focus, revealing a single band of enzyme activity as assessed by analytical isoelectric focusing (pI ca. 5). Kinetic properties of the neutral trehalases revealed no differences between the M. anisopliae strains, while the M. flavovoride had a lower Kcat/Km. However, there was lower virulence in one strain that showed Ntl enzyme kinetic properties that were similar to the other strains, suggesting that factors other than neutral trehalase may be responsible for delimiting virulence in this insect pathogenic fungi. Although there is nucleotide variation in genes involved in pathogenicity, this variation is mostly neutral in nature, and there is strong stabilizing selection to maintain enzyme function.

Znf131 is a member of the BTB/POZ family of transcription factors with roles in development and carcinogenesis. Like many members of this protein family, Znf131 displays robust nuclear localization in cultured cells, but the mechanism(s) of Znf131 nuclear trafficking is unknown. Here, we report the mechanism of Znf131 nuclear localization. Visual inspection of the Znf131 amino acid sequence revealed three basic regions (BR-1, -2 and -3) with the potential to serve as nuclear localization signals (NLS). Of the three basic regions, only BR-1 functioned independently to efficiently target heterologous beta-gal-GFP fusion proteins to HeLa cell nuclei. However, a Znf131 truncation mutant containing BR-2 and BR-3 efficiently targeted heterologous beta-gal-GFP fusion proteins to HeLa cell nuclei. Mutational analysis of full-length GFP-tagged Znf131 revealed that loss of any one BR alone did not prevent Znf131 nuclear localization. This apparent redundancy in NLS activity was due to the fact that intact BR-1 or BR-2 alone could target full-length Znf131 to nuclei. Consequently, simultaneous mutation of BR-1 and BR-2 abolished full-length Znf131 nuclear localization. Therefore, BR-1 and BR-2 are functional NLSs for Znf131 and as such are designated NLS-1 and NLS-2. Finally, wild type Znf131, and not a Znf131 NLS-defective mutant (NLS-1m/NLS-2m) interacted preferentially with the nuclear import receptor Importin-alpha3 in vitro.