Asc1 Supports Cell-Wall Integrity Near Bud Sites by a Pkc1 Independent Mechanism

Background: The yeast ribosomal protein Asc1 is a WD-protein family member. Its mammalian ortholog, RACK1 was initially discovered as a receptor for activated protein C kinase (PKC) that functions to maintain the active conformation of PKC and to support its movement to target sites. In the budding yeast though, a connection between Asc1p and the PKC signaling pathway has never been reported. Methodology/Principal Findings: In the present study we found that asc1-deletion mutant (asc1D) presents some of the hallmarks of PKC signaling mutants. These include an increased sensitivity to staurosporine, a specific Pkc1p inhibitor, and susceptibility to cell-wall perturbing treatments such as hypotonic- and heat shock conditions and zymolase treatment. Microscopic analysis of asc1D cells revealed cell-wall invaginations near bud sites after exposure to hypotonic conditions, and the dynamic of cells ’ survival after this stress further supports the involvement of Asc1p in maintaining the cell-wall integrity during the mid-to late stages of bud formation. Genetic interactions between asc1 and pkc1 reveal synergistic sensitivities of a double-knock out mutant (asc1D/pkc1D) to cell-wall stress conditions, and high basal level of PKC signaling in asc1D. Furthermore, Asc1p has no effect on the cellular distribution or redistribution of Pkc1p at optimal or at cell-wall stress conditions. Conclusions/Significance: Taken together, our data support the idea that unlike its mammalian orthologs, Asc1p act

Hypotonicity cause rapid reduction in viability of dividing <i>asc1Δ</i> cells.

<p>Cells were grown in YPD supplemented with 1 M NaCl either to logarithmic growth phase (A) or to stationary phase (B). Cells were then shifted to media without NaCl to create hypotonic shock. At the indicated time points, dead cells were counted by Methylene Blue (MB) staining.</p

<i>asc1Δ</i> is sensitive to a PKC inhibitor.

<p>Wild-type and asc1Δ cells were grown to mid-logarithmic phase in rich media (YPD) in the absence (A) or in the presence of 0.8 M NaCl as an osmotic stabilizer (B), and diluted to concentrations of 10⁵ cells/ml. At the indicated time points (marked by black arrows) each culture was divided to two, and the two halves were supplemented either with staurosporine (+St) to a final concentration of 40 µg/ml or with an equal volume of water. Cell growth was monitored before and after Staurosporine addition by counting the cells' number using a hemocytometer.</p

<i>asc1Δ</i> cells display aberrant cell-wall morphologies upon exposure to hypotonic conditions.

<p>Microscopic observations of wild-type and <i>asc1Δ</i> cells either under sustained growth in rich media (YPD) containing 1M NaCl (A, D), or following one hour exposure to hypotonic shock conditions (shift from 1 M to 0 M NaCl containing YPD media)(B, E), or in hypotonic conditions in the presence of 0.1 mg/ml Cycloheximide (CHX) (C, F). Arrows point to cell-wall deformations sites. Inset in E is a higher magnification of deformed cells.</p

Phenotypes of <i>asc1Δ/pkc1Δ</i>.

<p>A) The indicated strains were grown in rich media in the presence of 0.5 M NaCl to logarithmic growth phase, and diluted to an OD₆₀₀ of 0.1. Growth was monitored by measuring the OD₆₀₀ values at the indicated time points. Results of one representative experiment out of three are shown. B) Microscopic morphology of the indicated strains when grown in rich media plus 1 M NaCl. C) Hypotonic shock sensitivity of the indicated strains. Cells were grown to mid-log phase in liquid YPD media supplemented with 0.5 M NaCl, serially diluted and plated on YPD plates with or without 0.5 M NaCl. Pictures were taken after 48 hrs of incubation at 30°C. C) Cell were grown in liquid YPD +0.5 M NaCl to mid-log phase, serially diluted and spotted on YPD plates either with no added NaCl, or with 0.5 M NaCl. Plates were incubated at 30°C or at 37°C. D) Sensitivity to Zymolase. Cells were grown in YPD +1 M NaCl to mid-logarithmic phase and concentrated to an OD₆₀₀ of 5.0. Samples were then treated with 0.2 mg/ml Zymolase, and at the indicated time points cell-wall sensitivity was determined by adding SDS (final concentration 1%) and measuring the sample's turbidity.</p

<i>asc1Δ</i> sensitivities to Pkc1 mutations.

<p><i>asc1Δ</i> and its parental strain carrying plasmids that express either wild-type (wt), inactive (inact.) or constitutively-active (const.act.) forms of Pkc1p under galactose-inducible promoter were grown to mid logarithmic phase in non-inducing conditions (with glucose as a carbon source). The effect of each Pkc1p variant on survival was determined by seeding equal amounts of cells on plates containing either the inducing or the non-inducing carbon source, and calculating the induced/non-induced ratio for the colony forming units (CFU). All values were normalized to the wild-type Pkc1, which was set to 100%. Note that the Y-axis is in logarithmic scale.</p

Response of <i>asc1Δ</i> cells to cell wall stresses.

<p><b>The indicated</b> strains were grown for 24 hrs at 30°C in liquid YPD media containing 0 M, 0.4 M, 1 M or 1.4 M NaCl (A) or in the presence of 1 M NaCl, 1 M KCl, or 1.5 M Sorbitol (B), or 0.5 M NaCl (C). To impose hypotonic shock, cells were plated in a dilution series on YPD plates containing no supplemental osmolyte and grown either at 30°C (“30°C, Hypotonic shock”) or at 37°C (“37°C, Hypotonic shock”). For isotonic control, cells were spotted in a dilution series on YPD plates containing the same osmolyte concentrations as they grew in, and cultured at 30°C (“30°C Control”). Pictures of spotted colonies were taken after 24 hours (left panels) and 48 hours (right panels). To impose calcoflour white stress cells were plated on plates supplemented with 0.5 M NaCl and the indicated calcoflour white concentrations.</p

Asc1p has no effect on Pkc1p localization.

<p>A) Extracts of either wild-type (WT) or <i>asc1Δ</i> strains expressing HA-tagged Pkc1p were fractionated to membrane pellet (M) and cytosolic supernatant (C). The procedure for the WT strain was performed either in the absence or in the presence of 20 mM EDTA. Equivalent amounts of an unfractionated sample (T), cytosolic (C) and membranous (M) samples were subjected to Western analysis with antibodies recognizing the HA moiety of PKC1 or Asc1p. Pab1p (detected by α-pab1 antibody) was used as a marker for polysomes-associated factor, and Hxk1p (detected by α-Hxk1p antibody) was used as a cytosolic marker. B) Pkc1p-GFP was visualized in wild-type and <i>asc1Δ</i> cells grown to mid-log phase either without (“no stress”) or with one hour treatment with Zymolase (“Zymolase stress”).</p

RESEARCH Influence of Pneumococcal Vaccines and Respiratory Syncytial Virus on Alveolar Pneumonia, Israel

Postlicensure surveillance of pneumonia incidence can be used to estimate whether pneumococcal conjugate vaccines (PCVs) affect incidence. We used Poisson regression models that control for baseline seasonality to determine the impact of PCVs and the possible effects of variations in virus activity in Israel on these surveillance estimates. PCV was associated with significant declines in radiologically confirmed alveolar pneumonia (RCAP) among patients &lt;6 months, 6–17 months, and 18–35 months of age (–31% [95 % CI –51 % to –15%], –41 % [95 % CI –52 to –32%], and –34 % [95 % CI –42 % to –25%], respectively). Respiratory syncytial virus (RSV) activity was associated with strong increases in RCAP incidence, with up to 44 % of cases attributable to RSV among infants &lt;6 months of age and lower but significant impacts in older children. Seasonal variations, particularly in RSV activity, masked the impact of 7-valent PCVs, especially for young children in the first 2 years after vaccine introduction. Streptococcus pneumoniae is a major cause of pneumonia worldwide, but in only a small fraction of severe cases are bacteria detectable in blood or cerebrospinal fluid (1). Because of the limitations of the diagnostic tools, identifying pneumococcal pneumonia is difficult and insensitive. Thus, using an endpoint of radiologically confirmed alveolar pneumonia (RCAP) can provide a more sensitive