RNAP II produces capped 18S and 25S ribosomal RNAs resistant to 5'-monophosphate dependent processive 5' to 3' exonuclease in polymerase switched Saccharomyces cerevisiae.

BACKGROUND: We have previously found that, in the pathogenic yeast Candida albicans, 18S and 25S ribosomal RNA components, containing more than one phosphate on their 5'-end were resistant to 5'-monophosphate requiring 5' → 3″ exonuclease. Several lines of evidence pointed to RNAP II as the enzyme producing them. RESULTS: We now show the production of such 18S and 25S rRNAs in Saccharomyces cerevisiae that have been permanently switched to RNAP II (due to deletion of part of RNAP I upstream activator alone, or in combination with deletion of one component of RNAP I itself). They contain more than one phosphate at their 5'-end and an anti-cap specific antibody binds to them indicating capping of these molecules. These molecules are found in RNA isolated from nuclei, therefore are unlikely to have been modified in the cytoplasm. CONCLUSIONS: Our data confirm the existence of such molecules and firmly establish RNAP II playing a role in their production. The fact that we see these molecules in wild type Saccharomyces cerevisiae indicates that they are not only a result of mutations but are part of the cells physiology. This adds another way RNAP II is involved in ribosome production in addition to their role in the production of ribosome associated proteins.

Discovery and characterization of Hv1-type proton channels in reef-building corals.

Voltage-dependent proton-permeable channels are membrane proteins mediating a number of important physiological functions. Here we report the presence of a gene encoding Hv1 voltage-dependent, proton-permeable channels in two species of reef-building corals. We performed a characterization of their biophysical properties and found that these channels are fast-activating and modulated by the pH gradient in a distinct manner. The biophysical properties of these novel channels make them interesting model systems. We have also developed an allosteric gating model that provides mechanistic insight into the modulation of voltage-dependence by protons. This work also represents the first functional characterization of any ion channel in scleractinian corals. We discuss the implications of the presence of these channels in the membranes of coral cells in the calcification and pH-regulation processes and possible consequences of ocean acidification related to the function of these channels.

Exonuclease resistant 18S and 25S ribosomal RNA components in yeast are possibly newly transcribed by RNA polymerase II.

BACKGROUND: We have previously reported 18S and 25S ribosomal RNA molecules in Candida albicans resistant to processive 5' → 3' exonuclease, appearing as cells approached stationary growth phase. Initial analysis pointed to extra phosphate(s) at their 5'- end raising the possibility that they were newly transcribed. Here we report on additional experiments exploring this possibility and try to establish which of the RNA polymerases may be transcribing them. RESULTS: Oligo-ligation and primer extension again showed the presence of extra phosphate at the 5'-end of the reported processing sites for both 18S and 25S ribosomal RNA components. Inhibition of Pol I with BMH-21 increased the presence of the molecules. Quantitation with an Agilent Bioanalyzer showed that resistant 18S and 25S molecules are primarily produced in the nucleus. Utilizing an RNA cap specific antibody, a signal could be detected on these molecules via immunoblotting; such signal could be eliminated by decapping reaction. Both the cap specific antibody and eIF4E cap-binding protein, increased fold enrichment upon quantitative amplification. Antibodies specific for the RNA Polymerase II c-terminal domain and TFIIB initiator factor showed the presence of Pol II on DNA sequences for both 18S and 25S molecules in chromatin precipitation and qPCR assays. Rapamycin inhibition of TOR complex also resulted in an increase of resistant 18S and 25S molecules. CONCLUSIONS: These data raise the possibility of a role for RNA Polymerase II in the production of 18S and 25S molecules and indicate that efforts for more direct proof may be worthwhile. If definitively proven it will establish an additional role for RNA Polymerase II in ribosomal production.

Nutrient depletion and TOR inhibition induce 18S and 25S ribosomal RNAs resistant to a 5'-phosphate-dependent exonuclease in Candida albicans and other yeasts.

BACKGROUND: Messenger RNA (mRNA) represents a small percentage of RNAs in a cell, with ribosomal RNA (rRNA) making up the bulk of it. To isolate mRNA from eukaryotes, typically poly-A selection is carried out. Recently, a 5´-phosphate-dependent, 5´â†’3´ processive exonuclease called Terminator has become available. It will digest only RNA that has a 5´-monophosphate end and therefore it is very useful to eliminate most of rRNAs in cell. RESULTS: We have found that in the pathogenic yeast Candida albicans, while 18S and 25S components isolated from yeast in robust growth phase are easily eliminated by Terminator, those isolated from cells in the nutritionally diminished stationary phase, become resistant to digestion by this enzyme. Additional digestions with alkaline phosphatase, tobacco pyrophosphatase combined with Terminator point toward the 5'-prime end of 18S and 25S as the source of this resistance. Inhibition of TOR by rapamycin also induces resistance by these molecules. We also find that these molecules are incorporated into the ribosome and are not just produced incidentally. Finally, we show that three other yeasts show the same behavior. CONCLUSIONS: Digestion of RNA by Terminator has revealed 18S and 25S rRNA molecules different from the accepted processed ones seen in ribosome generation. The reason for these molecules and the underlying mechanism for their formation is unknown. The preservation of this behavior across these yeasts suggests a useful biological role for it, worthy of further inquiry.

Decrease in Ribosomal RNA in Candida albicans Induced by Serum Exposure.

Candida albicans is an important polymorphic human pathogen. It can switch from a unicellular yeast form to germinating hypha, which may play a role in making it the successful pathogen it is. This hyphal transformation can be triggered by various extracellular stimuli, the most potent one being serum from any source. We have previously reported that Candida albicans transiently polyadenylates portions of both the large and small subunits of ribosomal RNA, shortly after serum exposure. Northern blots at the same time suggested that serum might induce a decrease in total ribosomal RNA. We have carried out a number of experiments to carefully assess this possibility and now report that serum significantly reduces ribosomal RNA in Candida albicans. Fluorometric measurements, Northern blotting and quantitative RT-PCR, have all confirmed this decrease. Timed experiments show that serum induces this decrease rapidly, as it was seen in as early as five minutes. Cell mass is not decreased as total cellular protein content remains the same and metabolic activity does not appear to slow, as assessed by XTT assay, and by the observation that cells form hyphal structures robustly. Another hyphal inducer, N-acetylglucosamine, also caused RNA decrease, but to a lesser extent. We also observed it in non-germinating yeast, such as Candida glabrata. The reason for this decrease is unknown and overall our data suggests that decrease in rRNA does not play a causal role in hyphal transformation. Rapid and significant decrease in a molecule so central to the yeast's biology is of some importance, and further studies, such as its effect on protein metabolism, will be required to better understand its purpose.

Separately or combined, LukG/LukH is functionally unique compared to other staphylococcal bicomponent leukotoxins.

Staphylococcus aureus is a major human pathogen that elaborates several exotoxins. Among these are the bicomponent leukotoxins (BCLs), which include γ-hemolysin, Panton-Valentine leukocidin (PVL), and LukDE. The toxin components are classified as either F or S proteins, which are secreted individually and assemble on cell surfaces to form hetero-oligomeric pores resulting in lysis of PMNs and/or erythrocytes. F and S proteins of γ-hemolysin, PVL and LukDE have ∼ 70% sequence homology within the same class and several heterologous combinations of F and S members from these three bicomponent toxin groups are functional. Recently, an additional BCL pair, LukGH (also called LukAB) that has only 30% homology to γ-hemolysin, PVL and LukDE, has been characterized from S. aureus. Our results showed that LukGH was more cytotoxic to human PMNs than PVL. However, LukGH-induced calcium ion influx in PMNs was markedly attenuated and slower than that induced by PVL and other staphylococcal BCLs. In contrast to other heterologous BCL combinations, LukG in combination with heterologous S components, and LukH in combination with heterologous F components did not induce calcium ion entry or cell lysis in human PMNs or rabbit erythrocytes. Like PVL, LukGH induced IL-8 production by PMNs. While individual components LukG and LukH had no cytolytic or calcium influx activity, they each induced high levels of IL-8 transcription and secretion. IL-8 production induced by LukG or LukH was dependent on NF-κB. Therefore, our results indicate LukGH differs functionally from other staphylococcal BCLs.

Detection and quantification of Panton-Valentine leukocidin in Staphylococcus aureus cultures by ELISA and Western blotting: diethylpyrocarbonate inhibits binding of protein A to IgG.

Enzyme-linked immunosorbent assay (ELISA) and Western blotting are common techniques used to detect and quantify proteins in Staphylococcus aureus culture supernatants, such as Panton-Valentine leukocidin (PVL). However, protein A (Spa) secreted by most S. aureus strains may interfere with these assays by binding to the capturing and detecting antibodies. Here, we have shown that the addition of diethylpyrocarbonate (DEPC) inhibits the binding of Spa to rabbit anti-PVL used as the capturing antibody in ELISA. In Western blotting, the presence of DEPC prevented the binding of detecting antibody to Spa. These modified ELISA and Western blot techniques should prove useful for detecting and quantifying proteins in S. aureus culture supernatants.

Staphylococcus aureus Panton-Valentine leukocidin contributes to inflammation and muscle tissue injury.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) threatens public health worldwide, and epidemiologic data suggest that the Panton-Valentine Leukocidin (PVL) expressed by most CA-MRSA strains could contribute to severe human infections, particularly in young and immunocompetent hosts. PVL is proposed to induce cytolysis or apoptosis of phagocytes. However, recent comparisons of isogenic CA-MRSA strains with or without PVL have revealed no differences in human PMN cytolytic activity. Furthermore, many of the mouse studies performed to date have failed to demonstrate a virulence role for PVL, thereby provoking the question: does PVL have a mechanistic role in human infection? In this report, we evaluated the contribution of PVL to severe skin and soft tissue infection. We generated PVL mutants in CA-MRSA strains isolated from patients with necrotizing fasciitis and used these tools to evaluate the pathogenic role of PVL in vivo. In a model of necrotizing soft tissue infection, we found PVL caused significant damage of muscle but not the skin. Muscle injury was linked to induction of pro-inflammatory chemokines KC, MIP-2, and RANTES, and recruitment of neutrophils. Tissue damage was most prominent in young mice and in those strains of mice that more effectively cleared S. aureus, and was not significant in older mice and mouse strains that had a more limited immune response to the pathogen. PVL mediated injury could be blocked by pretreatment with anti-PVL antibodies. Our data provide new insights into CA-MRSA pathogenesis, epidemiology and therapeutics. PVL could contribute to the increased incidence of myositis in CA-MRSA infection, and the toxin could mediate tissue injury by mechanisms other than direct killing of phagocytes.

Human immunoglobulin G2 (IgG2) and IgG4, but not IgG1 or IgG3, protect mice against Cryptococcus neoformans infection.

The encapsulated yeast Cryptococcus neoformans is a significant cause of meningitis and death in patients with AIDS. Some murine monoclonal antibodies (MAbs) against the glucuronoxylomannan (GXM) component of the C. neoformans capsular polysaccharide can prolong the lives of infected mice, while others have no effect or can even shorten survival. To date, no one has systematically compared the efficacies of antibodies with the same variable regions and different human constant regions with their unique combination of effector functions in providing protection against murine C. neoformans infection. In the present study, we examined the efficacies of anti-GXM MAbs of the four human immunoglobulin G (IgG) subclasses, which have identical variable regions but differ in their capacities to bind the three types of Fc receptors for IgG (FcgammaR), their abilities to activate complement, and their half-lives. IgG2 and IgG4 anti-GXM prolonged the lives of infected BALB/c mice, IgG3 anti-GXM did not affect animal survival, while mice treated with IgG1 anti-GXM died earlier than mice treated with phosphate-buffered saline or irrelevant isotype-matched MAbs. All MAbs decreased serum GXM in infected animals. Effector pathways traditionally believed to be important in defense against microbes, such as opsonophagocytosis and complement binding, negatively correlated with antibody efficacy. It is generally accepted that human IgG1 has the most favorable combination of effector functions for therapeutic use against infections. Therefore, our findings have significant implications for humanization of the mouse IgG1 currently in clinical trials for cryptococcal meningitis and for the design of antibody therapeutics to treat other infectious diseases as well.