Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei

<p>Abstract</p> <p>Background</p> <p><it>Penicillium marneffei </it>is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37°C), a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in <it>P. marneffei </it>that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of <it>P. marneffei</it>.</p> <p>Results</p> <p>Whole cell proteins from the early stages of mould and yeast development in <it>P. marneffei </it>were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated <it>RanA</it>, was subsequently cloned and characterized. The <it>P. marneffei </it>RanA protein sequence, which contained the signature motif of Ran-GTPases, exhibited 90% homology to homologous <it>Aspergillus </it>proteins.</p> <p>Conclusion</p> <p>This study clearly demonstrates the utility of proteomic approaches to studying dimorphism in <it>P. marneffei</it>. Moreover, this strategy complements and extends current genetic methodologies directed towards understanding the molecular mechanisms of phase transition. Finally, the documented increased levels of RanA expression suggest that cellular development in this fungus involves additional signaling mechanisms than have been previously described in <it>P. marneffei</it>.</p

Rapid Titration of Measles and Other Viruses: Optimization with Determination of Replication Cycle Length

Background: Measles virus (MV) is a member of the Paramyxoviridae family and an important human pathogen causing strong immunosuppression in affected individuals and a considerable number of deaths worldwide. Currently, measles is a re-emerging disease in developed countries. MV is usually quantified in infectious units as determined by limiting dilution and counting of plaque forming unit either directly (PFU method) or indirectly from random distribution in microwells (TCID50 method). Both methods are time-consuming (up to several days), cumbersome and, in the case of the PFU assay, possibly operator dependent. Methods/Findings: A rapid, optimized, accurate, and reliable technique for titration of measles virus was developed based on the detection of virus infected cells by flow cytometry, single round of infection and titer calculation according to the Poisson’s law. The kinetics follow up of the number of infected cells after infection with serial dilutions of a virus allowed estimation of the duration of the replication cycle, and consequently, the optimal infection time. The assay was set up to quantify measles virus, vesicular stomatitis virus (VSV), and human immunodeficiency virus type 1 (HIV-1) using antibody labeling of viral glycoprotein, virus encoded fluorescent reporter protein and an inducible fluorescent-reporter cell line, respectively. Conclusion: Overall, performing the assay takes only 24–30 hours for MV strains, 12 hours for VSV, and 52 hours for HIV-1

Defining the titration time for different viruses.

<p>MV-Halle (panel A), wt MV-G954 (panel B), and VSVeGFP (panel C) stocks were used in 3-fold serial dilutions to infect Vero, Vero-SLAM and Vero cells, respectively. At different times post infection, cells were collected and analyzed by flow cytometry to determine infected cells for each virus input. The optimal time for virus titration reflects the first overlap between the peaks of the most concentrated dilution and the adjacent one and is marked with a “star” on the corresponding histogram. For each dilution, titers have been calculated by applying the Poisson law, and values expressed in IU/ml as function of the inoculation volume (see the charts below the histogram panel for each virus). The red curves reflect the time when a single replication cycle has occurred. The titer is determined by the average of the values on the level curve (red) and is compared to the titer obtained by TCID50 technique below each chart.</p

Adaptation of the titration protocol for a given virus.

<p>Adaptation of the titration protocol for a given virus.</p

Calculation of HIV-1 NL4-3 titer using GFP expressing indicator cells.

<p>HIV-1 (NL4-3 strain) stock was titrated both by serial dilutions in SupT1 cells and GHOST cells. End-point titration using the lymphocytic cell line SupT1 (data not shown) was based on the appearance of syncytia and took up to 2 weeks. The GHOST indicator cells were used for HIV-1 titration by flow cytometry. These cells express the GFP protein under the control of the HIV-1 LTR that can be activated by Tat from the incoming HIV-1 (A). GHOST cells seeded one hour before were infected with serial 3-fold dilutions of HIV-1 stock in the absence (C) or presence (D) of 1 µg/ml aphidicolin. At various times post-infection, cells were collected, fixed and GFP expression analyzed by flow cytometry. In parallel, at every time point, the cells were collected from non-infected wells to count the cell number for cell growth determination (B).</p

Experimental scheme and data analysis for virus titration based on flow cytometry numbering of infected cells – “Protocol at a glance”.

<p>Top panel from right to left, 3-fold dilution of unknown virus sample. Middle panel, example of histogram profiles of infected cells according to the virus inoculum size. Bottom panel: % of infected cells as graphically determined and equation used to calculate the titer for each inoculum volume. Underlined in grey, selected values for titer determination by averaging.</p

Virus titration by TCID50 is intrinsically discontinuous.

<p>Discontinuous titer values given by all possible combinations of percentage of infected wells above, at, and below 50% of eight replicates within one Log range. Note that (i) some mantissa are identical for two or four combinations (indicated in italics in x axis) and (ii) identical sets of mantissa of the logarithm are obtained at any place below and above the displayed titer range. Vertical bars indicate the variable interval separating one titer value from its nearest superior and inferior titer value.</p

Determination of the duration of a single virus replication cycle and the reading time for titration.

<p><b>(A) The rapid method for MV titration is based on a single infection cycle.</b> Vero cells were inoculated with MV-Halle strain for 6 hours. Then, they were washed with PBS to eliminate residual virus. Medium supplemented or not with 0.1 mg/ml of the fusion inhibitory peptide (FIP) was then added. At 18 h.p.i., cells were analyzed by flow cytometry and similar titers were found in both cases (i.e. with and without FIP) (Compare “a” 7.86 Log IU/ml and “b” 7.92 Log IU/ml. As a control for FIP efficiency, its addition just before the virus inoculation completely prevented MV infection “c”. <b>(B) Validation of estimated virus cycle duration as reading time for titration.</b> Similar titers were obtained after inhibition of secondary infection by anti-measles serum. Vero cells were infected with MV-eGFP for 6 hours, then washed with PBS and further incubated in the presence “a” or absence “b” of a potent neutralizing antiserum. “c” - neutralization efficiency of the anti-serum upon addition to the virus inoculum prior to infection. Note for the highest inoculum volume, a small GFP background signal was detected due to the low amount of GFP embarked into the input virus particles.</p

Correlation between titers obtained by the TCID50 technique and the rapid method for titration based on flow cytometry.

<p>Ninety five viral stocks from MV strains were titrated by the two techniques with highly correlated values (r = 0,930, 2α<0.001).</p

The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells

International audienceInterleukin 15 (IL-15) controls both the homeostasis and the peripheral activation of natural killer (NK) cells. The molecular basis for this duality of action remains unknown. Here we found that the metabolic checkpoint kinase mTOR was activated and boosted bioenergetic metabolism after exposure of NK cells to high concentrations of IL-15, whereas low doses of IL-15 triggered only phosphorylation of the transcription factor STAT5. mTOR stimulated the growth and nutrient uptake of NK cells and positively fed back on the receptor for IL-15. This process was essential for sustaining NK cell proliferation during development and the acquisition of cytolytic potential during inflammation or viral infection. The mTORC1 inhibitor rapamycin inhibited NK cell cytotoxicity both in mice and humans; this probably contributes to the immunosuppressive activity of this drug in different clinical settings