Lignin-degrading peroxidases in white-rot fungus <i>Trametes hirsuta</i> 072. Absolute expression quantification of full multigene family

<div><p>Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete <i>Trametes hirsuta</i> 072, an efficient lignin degrader. The <i>T</i>. <i>hirsuta</i> genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle.</p></div

Total relative expression of PODs complex at different growth stages and under different culture conditions of <i>T</i>. <i>hirsuta</i>.

<p>I, lag phase; II, exponential growth phase; III, stationary and lysis phase. The samples were taken in 12 h interval. Each dot represents arithmetic mean from three biological replicates on each cultivation media (GP, BR and AL). Polynomial regression line is superimposed on the mean scatter plot. Error bars represent standard deviations.</p

Relative expression of PODs genes under different culture conditions. Gene wise expression of PODs.

<p>Each doted square represents absolute expression normalized on internal control gene expression (RQ). The expression correlates both with the size of a dot and the color of a box. Samples in which expression was not detected depicted as small unsquared dots. GP, BR and AL stand for glucose-peptone medium and glucose-peptone medium with addition of bromocresol green and alkali lignin respectively.</p

Properties of the predicted protein sequences for <i>T</i>. <i>hirsuta</i> peroxidases.

<p>Genes with high expression rate/RQ (according to ddPCR data, discussed below) are marked in dark grey.</p

Maximum likelihood phylogenetic tree of <i>T</i>. <i>hirsuta</i> PODs, characteristic PODs amino acid residues and genes’ intron positions.

<p>Numbers at nodes are bootstrap percentages from 100 replicates. The outgroup containing the three peroxidase genes of <i>Auricularia subglabra</i> (XM_007341671.1, XM_007341680.1 and XM_007347393.1) is highlighted in pink. Characteristic LiP residues are highlighted in violet; characteristic MnP residues–in green and red. Non-standard aa residues are highlighted in blue. The numeration of aa residues is corresponding to <i>P</i>. <i>chrysosporium</i> PODs (MnP1 and LiP -H8) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173813#pone.0173813.ref037" target="_blank">37</a>].</p

DataSheet_1_Estimation of anti-orthopoxvirus immunity in Moscow residents and potential risks of spreading Monkeypox virus.docx

WHO has declared the outbreak of monkeypox as a public health emergency of international concern. In less than three months, monkeypox was detected in more than 30 000 people and spread to more than 80 countries around the world. It is believed that the immunity formed to smallpox vaccine can protect from monkeypox infection with high efficiency. The widespread use of Vaccinia virus has not been carried out since the 1980s, which raises the question of the level of residual immunity among the population and the identification of groups requiring priority vaccination. We conducted a cross-sectional serological study of remaining immunity among Moscow residents. To do this, a collection of blood serum samples of age group over 30 years old was formed, an in-house ELISA test system was developed, and a virus neutralization protocol was set up. Serum samples were examined for the presence of IgG antibodies against Vaccinia virus (n=2908), as well as for the ability to neutralize plaque formation with a Vaccinia virus MNIIVP-10 strain (n=299). The results indicate the presence of neutralizing antibody titer of 1/20 or more in 33.3 to 53.2% of people older than 45 years. Among people 30-45 years old who probably have not been vaccinated, the proportion with virus neutralizing antibodies ranged from 3.2 to 6.7%. Despite the higher level of antibodies in age group older than 66 years, the proportion of positive samples in this group was slightly lower than in people aged 46-65 years. The results indicate the priority of vaccination in groups younger than 45, and possibly older than 66 years to ensure the protection of the population in case of spread of monkeypox among Moscow residents. The herd immunity level needed to stop the circulation of the virus should be at least 50.25 – 65.28%.</p