Impact of glucose levels on expression of hypha-associated secreted aspartyl proteinases in Candida albicans.

BACKGROUND: Ten secreted aspartyl proteinase (Sap) genes were identified in Candida albicans. The products of SAP genes are considered to be virulent factors of C. albicans that participated in causing mucocutaneous and systemic candidiasis in humans. Depending on environmental conditions, C. albicans may stay in yeast-form or convert into invasive hypha-form, and these issues may affect the expression of SAP genes. In this study we explored the component(s) of culture media that may affect the expression of hypha-associated SAP genes. RESULTS: We demonstrate that glucose levels modulate both the hyphae development and the expression strength of hypha-associated SAP genes (SAP4-6). In contrast to high glucose concentration (2%), lower glucose level (0.1%) is more potent to promote hyphae development and to promptly elicit the expression of hypha-associated Sap proteins during yeast-to-hypha transition of C. albicans. Both Cph1-mediated MAP kinase cascade and Efg1-mediated cAMP/PKA pathway, although the latter seemed dominant, participate in convey the glucose signaling to regulate the expression of hypha-associated SAP genes and this glucose level effect may perform at very early stage of yeast-to-hypha transition. In addition, when C. albicans was co-cultured with THP-1 human monocytes, the engulfed C. albicans was developing hypha efficiently within 1 hr and the expression of hypha-associated Sap proteins could be detected on the distal surface of hyphae. CONCLUSION: We propose that the glucose level of bloodstream (approximately 0.1%) may be facilitated for stimulation of C. albicans to develop invasive hypha-form and to elicit promptly production of high-level hypha-associated Sap proteins.

Sap6, a secreted aspartyl proteinase, participates in maintenance the cell surface integrity of Candida albicans.

BACKGROUND: The polymorphic species Candida albicans is the major cause of candidiasis in humans. The secreted aspartyl proteinases (Saps) of C. albicans, encoded by a family of 10 SAP genes, have been investigated as the virulent factors during candidiasis. However, the biological functions of most Sap proteins are still uncertain. In this study, we applied co-culture system of C. albicans and THP-1 human monocytes to explore the pathogenic roles and biological functions of Sap proteinases. RESULTS: After 1 hr of co-culture of C. albicans strains and THP-1 human monocytes at 37°C, more than 60% of the THP-1-engulfed wild type and Δsap5 Candida cells were developing long hyphae. However, about 50% of THP-1-engulfed Δsap6 Candida cells were generating short hyphae, and more dead Candida cells were found in Δsap6 strain that was ingested by THP-1 cells (about 15% in Δsap6 strain vs. 2 ~ 2.5% in SC5314 and Δsap5 strains). The immunofluorescence staining demonstrated that the Sap6 is the major hyphal tip located Sap protein under THP-1 phagocytosis. The sap6-deleted strains (Δsap6, Δsap4/6, and Δsap5/6) appeared slower growth on Congo red containing solid medium at 25°C, and the growth defect was exacerbated when cultured at 37°C in Congo red or SDS containing medium. In addition, more proteins were secreted from Δsap6 strain and the ß-mercaptoethanol (ß-ME) extractable surface proteins from Δsap6 mutant were more abundant than that of extracted from wild type strain, which included the plasma membrane protein (Pma1p), the ER-chaperone protein (Kar2p), the protein transport-related protein (Arf1p), the cytoskeleton protein (Act1), and the mitochondrial outer membrane protein (porin 1). Moreover, the cell surface accessibility was increased in sap6-deleted strains. CONCLUSION: From these results, we speculated that the cell surface constitution of C. albicans Δsap6 strain was defect. This may cause the more accessible of ß-ME to disulfide-bridged cell surface components and may weaken the resistance of Δsap6 strain encountering phagocytosis of THP-1 cells. Sap6 protein displays a significant function involving in maintenance the cell surface integrity.

Differential regulation and activity against oxidative stress of Dps proteins in Bacillus cereus.

In this study, the sequence similarity, structure, ferroxidase activity and efficacy in antagonizing oxidative stress of three Dps-like proteins, Dps1, Dps2 and Dps3, encoded by Bacillus cereus were comparatively analyzed. The three Dps-like proteins are homologous to other bacterial Dps proteins that exhibit ferroxidase activity. Both Dps1 and Dps2 have a typical Dps spherical structure, but Dps3 has a unique filamentous structure. Several dps mutant strains were generated to investigate the functional role of dps genes in cell protection. The dps1 null strain was the most labile to oxidative stress in the stationary phase, and the loss of dps2 resulted in greater sensitivity to peroxide exposure compared with the other mutant strains in the log phase. Interestingly, after simultaneous deletion of dps1 and dps2, the survival rate was dramatically reduced by approximately 5 log in the stationary phase. Immunoblotting analysis demonstrated that Dps1 and Dps2 in the wild-type strain were induced by oxidative stress, and Dps3 responded to general stress in the log phase. Constitutively high expression of Dps2 in a perR null mutant and PerR-specific binding of the promoter region of dps2 confirmed Dps2 as a member of the PerR regulon. In addition, the expression of Dps1 and Dps2, absent any stress, was initiated in the log phase and was abundant in the stationary phase, suggesting that the expression of Dps1 and Dps2 was dependent on the bacterial growth stage. In summary, the three Dps proteins conferred cellular protection, particularly from oxidative stress, and were differentially regulated in response to varied stress conditions.

Burkholderia multivorans acts as an antagonist against the growth of Burkholderia pseudomallei in soil.

In this study, it was demonstrated, by using agar diffusion tests and a Transwell system, that Burkholderia multivorans NKI379 has an antagonistic effect against the growth of B. pseudomallei. Bacterial representatives were isolated from agricultural crop soil and mixed to construct a partial bacterial community structure that was based on the results of reproducible patterns following PCR-denaturing gradient gel electrophoresis analysis of total soil chromosomes. The antagonistic effect of B. multivorans on B. pseudomallei was observed in this imitate community. In a field study of agricultural crop soil, the presence of B. pseudomallei was inversely related to the presence of the antagonistic strains B. multivorans or B. cenocepacia. B. multivorans NKI379 can survive in a broader range of pH, temperatures and salt concentrations than B. pseudomallei, suggesting that B. multivorans can adapt to extreme environmental changes and therefore predominates over B. pseudomallei in natural environments.

Determinants of Rbp1p localization in specific cytoplasmic mRNA-processing foci, P-bodies.

Rbp1p, a yeast RNA-binding protein, decreases the level of mitochondrial porin mRNA by enhancing its degradation, but the intracellular location of the Rbp1p-mediated degradation complex remains unknown. We show here that Rbp1p in xrn1Delta mutant yeast localizes in specific cytoplasmic foci that are known as P-bodies. The N-terminal and RNA recognition motif (RRM) 1 domains of Rbp1p are necessary but not sufficient for its localization in P bodies. Rbp1p forms oligomers through its C-terminal domain in vivo; N-terminal-delete, or RRM1-mutated Rbp1p can be more efficiently recruited to P-bodies in an xrn1Delta strain, expressing a full-length Rbp1p. Although POR1 mRNA is localized to P bodies in an xrn1Delta strain, this localization does not depend on Rbp1p. Decapping activator Dhh1p directly interacts with Rbp1p. However, the recruitment of Rbp1p to P-bodies does not require Dhh1p or Ccr4p. In wild-type cells, Rbp1p can localize to P-bodies under glucose deprivation or treatment with KCl. In addition, Rbp1p-mediated porin mRNA decay is elicited by Xrn1p, a 5 ' to 3 ' exonuclease. These results provide new insight into the mechanism of Rbp1p function.

The yeast RNA-binding protein Rbp1p modifies the stability of mitochondrial porin mRNA.

The Saccharomyces cerevisiae RNA-binding protein Rbp1p was initially identified as a negative growth regulator; however, its function is still obscure. Here, we show that Rbp1p in cells is associated with structures that sediment at 10,000 as well as 100,000 x g. It appears microscopically as punctate signals partially localized to the perinuclear region. Over-expression of Rbp1p in yeast resulted in growth defects on nonfermentable carbon sources, suggesting a function for Rbp1p in mitochondrial biogenesis. Absence of Rbp1p increased the level of mitochondrial porin, whereas over-expression of Rbp1p, but not an N-terminally truncated form, decreased porin levels. Over-expression of Rbp1p also decreased the level of mitochondrial porin mRNA by enhancing its degradation, an effect that was dependent on all three of the Rbp1p RNA recognition motifs. In cells, the porin mRNA is associated with Rbp1p.RNP (ribonucleoprotein) complexes. In vitro binding assays showed that Rbp1p most likely interacts with a (C/G)U-rich element in the porin mRNA 3'-UTR. Based on these observations, we infer that Rbp1p has a role in negatively regulating mitochondrial porin expression post-transcriptionally.

Functional characterization and localization of acetyl-CoA hydrolase, Ach1p, in Saccharomyces cerevisiae.

Acetyl-CoA hydrolase (Ach1p), catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating intracellular acetyl-CoA or CoASH pools; however, its intracellular functions and distribution remain to be established. Using site-directed mutagenesis analysis, we demonstrated that the enzymatic activity of Ach1p is dependent upon its putative acetyl-CoA binding sites. The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis. Overexpression of Ach1p, but not constructs containing acetyl-CoA binding site mutations, in ach1-1 complemented the defect of acetate utilization. By subcellular fractionation, most of the Ach1p in yeast was distributed with mitochondria and little Ach1p in the cytoplasm. By immunofluorescence microscopy, we show that Ach1p and acetyl-CoA binding site-mutated constructs, but not its N-terminal deleted construct, are localized in mitochondria. Moreover, the onset of pseudohyphal development in homozygote ach1-1 diploids was abolished. We infer that Ach1p may be involved in a novel acetyl-CoA biogenesis and/or acetate utilization in mitochondria and thereby indirectly affect pseudohyphal development in yeast.

The yeast ADP-ribosylation factor GAP, Gcs1p, is involved in maintenance of mitochondrial morphology.

Membrane trafficking is regulated, in part, by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family. ARF function depends on the controlled binding and hydrolysis of GTP. In vitro, the GTPase activity of yeast ARF proteins can be stimulated by Gcs1p. Although Gcs1p was implicated in the regulation of retrograde transport from the Golgi to the ER and in actin cytoskeletal organization, its intracellular functions and distribution remain to be established. Following subcellular fractionation of yeast grown in rich medium, Gcs1p was localized in denser fractions than it was in cells grown in minimal medium. In yeast grown in rich or minimal medium, Gcs1p was distributed over the cytoplasm in a fine punctate pattern with more concentrated staining in the perinuclear regions. Overexpressed Gcs1p in yeast was localized partially with mitochondria and partially in perinuclear structures close to mitochondria. The Gcs1p PH-domain was required for localization in mitochondria but not for the perinuclear region. Transport of carboxypeptidase Y and invertase was not significantly altered by disruption of the gcs1 gene. This mutation did, however, reduce mitochondrial lateral distribution and branching when yeast were grown in rich medium. In yeast overexpressing Gcs1p, mitochondrial morphology was aberrant, with unbranched tubules and large spherical structures. We suggest that Gcs1p may be involved in the maintenance of mitochondrial morphology, possibly through organizing the actin cytoskeleton in Saccharomyces.