Engineering towards catalytic use of fungal class-II peroxidases for dye-decolorizing and conversion of lignin model compounds

Background. Manganese peroxidases (MnP) and lignin peroxidases (LiP) are haem-including fungal secreted class-II peroxidases, which are interesting oxidoreductases in protein engineering aimed at design of biocatalysts for lignin and lignocellulose conversion, dye compound degradation, activation of aromatic compounds, and biofuel production. Objective. Recombinant short-type MnP (Pr-MnP3) of the white rot fungus Phlebia radiata, and its manganese-binding site (E40, E44, D186) directed variants were produced and characterized. To allow catalytic applications, enzymatic bleaching of Reactive Blue 5 and conversion of lignin-like compounds by engineered class-II peroxidases were explored. Method. Pr-MnP3 and its variants were expressed in Escherichia coli. The resultant body proteins were lysed, purified and refolded into haem-including enzymes in 6-7% protein recovery, and examined spectroscopically and kinetically. Results. Successful production of active enzymes was attained, with spectral characteristics of high-spin class-II peroxidases. Recombinant Pr-MnP3 demonstrated high affinity to Mn2+, which was noticeably affected by single (D186H/N) and double (E40H+E44H) mutations. Without addition of Mn2+, Pr-MnP3 was able to oxidize ABTS and decolorize Reactive Blue 5. Pc-LiPH8, its Trp-radical site variants, and engineered CiP-LiP demonstrated conversion of veratryl alcohol and dimeric non-phenolic lignin-model compounds (arylglycerol-β-aryl ethers) with production of veratraldehyde, which is evidence for cation radical formation with subsequent Cα-Cβ cleavage. Pc-LiPH8 and CiP variants were able to effectively oxidize and convert the phenolic dimer (guaiacylglycerol-β–guaiacyl ether). Conclusion. Our results demonstrate suitability of engineered MnP and LiP peroxidases for dye-decolorizing, and efficiency of LiP and its variants for activation and degradation of phenolic and non-phenolic lignin-like aryl ether-linked compounds.Peer reviewe

An Induced Mutation in Tomato eIF4E Leads to Immunity to Two Potyviruses

BACKGROUND: The characterization of natural recessive resistance genes and Arabidopsis virus-resistant mutants have implicated translation initiation factors of the eIF4E and eIF4G families as susceptibility factors required for virus infection and resistance function. METHODOLOGY/PRINCIPAL FINDINGS: To investigate further the role of translation initiation factors in virus resistance we set up a TILLING platform in tomato, cloned genes encoding for translation initiation factors eIF4E and eIF4G and screened for induced mutations that lead to virus resistance. A splicing mutant of the eukaryotic translation initiation factor, S.l_eIF4E1 G1485A, was identified and characterized with respect to cap binding activity and resistance spectrum. Molecular analysis of the transcript of the mutant form showed that both the second and the third exons were miss-spliced, leading to a truncated mRNA. The resulting truncated eIF4E1 protein is also impaired in cap-binding activity. The mutant line had no growth defect, likely because of functional redundancy with others eIF4E isoforms. When infected with different potyviruses, the mutant line was immune to two strains of Potato virus Y and Pepper mottle virus and susceptible to Tobacco each virus. CONCLUSIONS/SIGNIFICANCE: Mutation analysis of translation initiation factors shows that translation initiation factors of the eIF4E family are determinants of plant susceptibility to RNA viruses and viruses have adopted strategies to use different isoforms. This work also demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. We have also developed a complete tool that can be used for both forward and reverse genetics in tomato, for both basic science and crop improvement. By opening it to the community, we hope to fulfill the expectations of both crop breeders and scientists who are using tomato as their model of study

A Portrait of the Transcriptome of the Neglected Trematode, Fasciola gigantica—Biological and Biotechnological Implications

Fasciola gigantica (Digenea) is an important foodborne trematode that causes liver fluke disease (fascioliasis) in mammals, including ungulates and humans, mainly in tropical climatic zones of the world. Despite its socioeconomic impact, almost nothing is known about the molecular biology of this parasite, its interplay with its hosts, and the pathogenesis of fascioliasis. Modern genomic technologies now provide unique opportunities to rapidly tackle these exciting areas. The present study reports the first transcriptome representing the adult stage of F. gigantica (of bovid origin), defined using a massively parallel sequencing-coupled bioinformatic approach. From >20 million raw sequence reads, >30,000 contiguous sequences were assembled, of which most were novel. Relative levels of transcription were determined for individual molecules, which were also characterized (at the inferred amino acid level) based on homology, gene ontology, and/or pathway mapping. Comparisons of the transcriptome of F. gigantica with those of other trematodes, including F. hepatica, revealed similarities in transcription for molecules inferred to have key roles in parasite-host interactions. Overall, the present dataset should provide a solid foundation for future fundamental genomic, proteomic, and metabolomic explorations of F. gigantica, as well as a basis for applied outcomes such as the development of novel methods of intervention against this neglected parasite

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes

International audienceAdhesion of Plasmodium falciparum-infected red blood cells (iRBC) to human erythrocytes (i.e. rosetting) is associated with severe malaria. Rosetting results from interactions between a subset of variant PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) adhesins and specific erythrocyte receptors. Interfering with such interactions is considered a promising intervention against severe malaria. To evaluate the feasibility of a vaccine strategy targetting rosetting, we have used here the Palo Alto 89F5 VarO rosetting model. PfEMP1-VarO consists of five Duffy-Binding Like domains (DBL1-5) and one Cysteine-rich Interdomain Region (CIDR1). The binding domain has been mapped to DBL1 and the ABO blood group was identified as the erythrocyte receptor. Here, we study the immunogenicity of all six recombinant PfEMP1-VarO domains and the DBL1- CIDR1 Head domain in BALB/c and outbred OF1 mice. Five readouts of antibody responses are explored: ELISA titres on the recombinant antigen, VarO-iRBC immunoblot reactivity, VarO-iRBC surface-reactivity, capacity to disrupt VarO rosettes and the capacity to prevent VarO rosette formation. For three domains, we explore influence of the expression system on antigenicity and immunogenicity. We show that correctly folded PfEMP1 domains elicit high antibody titres and induce a homogeneous response in outbred and BALB/c mice after three injections. High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain. Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational. We also report limited cross-reactivity between some PfEMP1 VarO domains. These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting

Group A Streptococcus Vaccine Targeting the Erythrogenic Toxins SpeA and SpeB Is Safe and Immunogenic in Rabbits and Does Not Induce Antibodies Associated with Autoimmunity

Group A streptococcus (GAS) is a global pathogen associated with significant morbidity and mortality for which there is currently no licensed vaccine. Vaccine development has been slow, mostly due to safety concerns regarding streptococcal antigens associated with autoimmunity and related complications. For a GAS vaccine to be safe, it must be ensured that the antigens used in the vaccine do not elicit an antibody response that can cross-react with host tissues. In this study, we evaluated the safety of our GAS vaccine candidate called VaxiStrep in New Zealand White rabbits. VaxiStrep is a recombinant fusion protein comprised of streptococcal pyrogenic exotoxin A (SpeA) and exotoxin B (SpeB), also known as erythrogenic toxins, adsorbed to an aluminum adjuvant. The vaccine elicited a robust immune response against the two toxins in the rabbits without any adverse events or toxicity. No signs of autoimmune pathology were detected in the rabbits’ brains, hearts, and kidneys via immunohistochemistry, and serum antibodies did not cross-react with cardiac or neuronal tissue proteins associated with rheumatic heart disease or Sydenham chorea (SC). This study further confirms that VaxiStrep does not elicit autoantibodies and is safe to be tested in a first-in-human trial

Influence of the expression system on the antibody response of animals immunised with PfEMP1-VarO DBL1 recombinant constructs.

<p><b>(A and B)</b> Titration curves of individual bleed 2 sera collected from five outbred (OF1-1 to -5) (<b>A</b>) or five inbred (BALB/c-1 to -5) mice immunised with bDBL1 (<b>B</b>) on the immunising antigen (bDBL1). OF1 mice responded poorly. <b>(C)</b> Titration curves of serum from a rabbit immunised with bDBL1 compared to the pool of bleed 4 sera from OF1 mice immunised with bDBL1; antigen used in the ELISA: bDBL1. <b>(D)</b> Titration curve of rabbit IgG purified from the serum shown in (<b>C)</b>; antigen used in the ELISA: bDBL1. <b>(E to H)</b> Cross-checking on the homologous and heterologous expression product: titration curves of individual bleed 4 sera from OF1 mice immunised with bDBL1 on bDBL1 <b>(E)</b> and eDBL1 <b>(G)</b>; titration curves of individual bleed 4 sera from OF1 mice immunised with eDBL1 on eDBL1 <b>(F)</b> and bDBL1 <b>(H)</b>.</p

Immunoblot reactivity of antisera to PfEMP1-VarO-derived recombinant domains on Palo Alto VarO parasite extracts.

<p>Immunoblots were prepared from Palo Alto total extracts separated under reducing (<b>A, C</b>) or non-reducing conditions (<b>B</b>). Strips from immunoblots <b>(A)</b> and <b>(B)</b> were reacted simultaneously (2 strips in a given serum) with individual sera (dilution 1/200) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134292#sec002" target="_blank">Materials and Methods</a>) as follows: lane 1: a representative mouse pre-immune serum (all were negative), lane 2: anti-bDBL2 (bleed 4), lane 3 anti-bDBL1 (bleed 3), lane 4 anti-eDBL1 (bleed 3); lane 5: anti-bDBL1 (bleed 4), lane 6 anti-eDBL1 (bleed 4), lane 7: anti-eDBL1RA (bleed 4); lane 8: anti-eDBL0 (bleed 4). (<b>C</b>) lane 1: a representative mouse pre-immune serum (idem), lane 2: anti-eDBL1, lane 3: anti-eHead, lane 4: anti-eDBL2, lane 5: anti-pCIDR, lane 6: anti-eDBL3, lane 7: anti-eDBL4, lane 8: anti-eDBL5, lane 9: pre-immune rabbit; lane 10: rabbit anti-bDBL1.</p

Cross-reactivity of mouse sera raised to DBL1 constructs with bDBL2.

<p>ELISA assays of OF1 mouse antisera against recombinant antigens. (<b>A</b>) Reactivity of bleed 4 antisera against bDBL1, eDBL1, bDBL2 and bCIDR (dilution 1/10,000) with the cognate and heterologous antigens. Symbols for antigens are indicated on the right. (<b>B and C</b>) Titration curves of bleed 4 sera against bDBL1 <b>(B)</b> or eDBL1 <b>(C)</b> on eDBL1, bDBL1 and bDBL2 antigens. Symbols for antigens are indicated on the right.</p

PfEMP1-VarO-derived antigens and immunisation schedule.

<p><b>(A)</b> Coomassie blue staining of SDS-PAGE gels of the recombinant antigens used for immunisation and ELISA. The purity of the recombinant domains was assessed by electrophoresis of reduced proteins separated on 10% SDS PAGE gels. Antigens (1 μg per lane) were loaded as indicated. The molecular mass markers (M) are indicated. <b>(B)</b> ELISA assays of the recombinant proteins with human sera collected from 54 persons living in France and never exposed to malaria parasites (non-immune) (left panel) or individual sera from 12 Senegalese adults living in a malaria hyper-endemic setting (right panel). For details of ELISA see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134292#sec002" target="_blank">Materials and Methods</a>. Please note that reactivity of individual human sera with bDBL1 is not included as it has been reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134292#pone.0134292.ref039" target="_blank">39</a>]. <b>(C)</b> Immunisation regimen and bleeding schedule used for all antigens.</p