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Tyx R.E.,State University of New York at Buffalo | Roche-Hakansson H.,State University of New York at Buffalo | Hakansson A.P.,State University of New York at Buffalo | Hakansson A.P.,Witebsky Center for Microbial Pathogenesis and Immunology | Hakansson A.P.,Buffalo Center of Excellence
Journal of Bacteriology | Year: 2011

Streptococcus pneumoniae strains lacking the enzyme dihydrolipoamide dehydrogenase (DLDH) show markedly reduced ability to grow on raffinose and stachyose as sole carbon sources. Import of these sugars occurs through the previously characterized raffinose ATP-binding cassette (ABC) transport system, encoded by the raf operon, that lacks the necessary ATP-binding protein. In this study, we identified the raffinose ATP-binding protein RafK and showed that it was directly involved in raffinose and stachyose import. RafK carries a C-terminal regulatory domain present in a subset of ATP-binding proteins that has been involved in both direct regulation of transporter activity (inducer exclusion) and transcription of transporter genes. Pneumococci lacking RafK showed a 50- to 80-fold reduction in expression of the raf operon genes aga (alpha-galactosidase) and rafEFG (raffinose substrate binding and permease genes), and both glucose and sucrose inhibited raffinose uptake through inducer exclusion. Like RafK, the presence of DLDH also activated the expression of raf operon genes, as DLDH-negative pneumococci showed a significantly decreased expression of aga and rafEFG, but DLDH did not regulate rafK or the putative regulatory genes rafR and rafS. DLDH also bound directly to RafK both in vitro and in vivo, indicating the possibility that DLDH regulates raffinose transport by a direct interaction with the regulatory domain of the transporter. Finally, although not as attenuated as DLDHnegative bacteria, pneumococci lacking RafK were significantly outcompeted by wild-type bacteria in colonization experiments of murine lung and nasopharynx, indicating a role for raffinose and stachyose transport in vivo. © 2011, American Society for Microbiology.

Clementi E.A.,State University of New York at Buffalo | Wilhelm K.R.,Umeå University | Schleucher J.,Umeå University | Morozova-Roche L.A.,Umeå University | And 3 more authors.
PLoS ONE | Year: 2013

HAMLET and ELOA are complexes consisting of oleic acid and two homologous, yet functionally different, proteins with cytotoxic activities against mammalian cells, with HAMLET showing higher tumor cells specificity, possibly due to the difference in propensity for oleic acid binding, as HAMLET binds 5-8 oleic acid molecules per protein molecule and ELOA binds 11-48 oleic acids. HAMLET has been shown to possess bactericidal activity against a number of bacterial species, particularly those with a respiratory tropism, with Streptococcus pneumoniae displaying the greatest degree of sensitivity. We show here that ELOA also displays bactericidal activity against pneumococci, which at lower concentrations shows mechanistic similarities to HAMLET's bactericidal activity. ELOA binds to S. pneumoniae and causes perturbations of the plasma membrane, including depolarization and subsequent rupture, and activates an influx of calcium into the cells. Selective inhibition of calcium channels and sodium/calcium exchange activity significantly diminished ELOA's bactericidal activity, similar to what we have observed with HAMLET. Finally, ELOA-induced death was also accompanied by DNA fragmentation into high molecular weight fragments - an apoptosis-like morphological phenotype that is seen during HAMLET-induced death. Thus, in contrast to different mechanisms of eukaryote cell death induced by ELOA and HAMLET, these complexes are characterized by rather similar activities towards bacteria. Although the majority of these events could be mimicked using oleic acid alone, the concentrations of oleic acid required were significantly higher than those present in the ELOA complex, and for some assays, the results were not identical between oleic acid alone and the ELOA complex. This indicates that the lipid, as a common denominator in both complexes, is an important component for the complexes' bactericidal activities, while the proteins are required both to solubilize and/or present the lipid at the bacterial membrane and likely to confer other and separate functions during the bacterial death. © 2013 Clementi et al.

Havel V.E.,Witebsky Center for Microbial Pathogenesis and Immunology | Wool N.K.,Witebsky Center for Microbial Pathogenesis and Immunology | Ayad D.,Witebsky Center for Microbial Pathogenesis and Immunology | Downey K.M.,Witebsky Center for Microbial Pathogenesis and Immunology | And 4 more authors.
Eukaryotic Cell | Year: 2011

Adaptation to host temperature is a prerequisite for any pathogen capable of causing deep infection in humans. Our previous studies demonstrated that a Cryptococcus neoformans ccr4{increment} mutant lacking the major deadenylase involved in regulated mRNA decay was defective in host temperature adaptation and therefore virulence. In this study, the ccr4{increment} mutant was found to exhibit characteristics of chronic unfolded-protein response (UPR) engagement in both the gene expression profile and phenotype. We demonstrate that host temperature adaptation in C. neoformans is accompanied by transient induction of the endoplasmic reticulum (ER) stress response and that Ccr4-dependent posttranscriptional gene regulation contributes to resolution of ER stress during host temperature adaptation. © 2011, American Society for Microbiology. All Rights Reserved.

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