A crucial role for the C‐terminal domain of exported protein 1 during the mosquito and hepatic stages of the Plasmodium bergheilife cycle

© 2019 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Intracellular Plasmodium parasites develop inside a parasitophorous vacuole (PV), a specialised compartment enclosed by a membrane (PVM) that contains proteins of both host and parasite origin. Although exported protein 1 (EXP1) is one of the earliest described parasitic PVM proteins, its function throughout the Plasmodium life cycle remains insufficiently understood. Here, we show that whereas the N-terminus of Plasmodium berghei EXP1 (PbEXP1) is essential for parasite survival in the blood, parasites lacking PbEXP1's entire C-terminal (CT) domain replicate normally in the blood but cause less severe pathology than their wild-type counterparts. Moreover, truncation of PbEXP1's CT domain not only impairs parasite development in the mosquito but also abrogates PbEXP1 localization to the PVM of intrahepatic parasites, severely limiting their replication and preventing their egress into the blood. Our findings highlight the importance of EXP1 during the Plasmodium life cycle and identify this protein as a promising target for antiplasmodial intervention.This study was sup- ported by German Research Foundation (Deutsche Forschungsgemeinschaft ‐ DFG) Grants SPP 1580 (to A.‐K. M.) and SFB1129 (to A.‐K. M.); Fundação para a Ciência e Tecnologia, Portugal (FCT‐PT) Grants UID/BIM/50005/2019 (Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado) and 02/SAICT/2017 (to M. P.). M. S.‐V. was supported by an FCT‐PT Grant PD/BD/105838/2014. D. F. was supported by FEEI and FCT‐MEC. M. P. was supported by FCT‐PT Investigador FCT 2013 and CEEC 2018 fellowship. A.‐K. M. was a recipient of a Maternity Leave Stipend by the German Center for Infection Research (DZIF, Heidelberg Site)info:eu-repo/semantics/publishedVersio

Discovery of spirooxadiazoline oxindoles with dual-stage antimalarial activity

© 2022 Published by Elsevier Masson SAS.Malaria remains a prevalent infectious disease in developing countries. The first-line therapeutic options are based on combinations of fast-acting artemisinin derivatives and longer-acting synthetic drugs. However, the emergence of resistance to these first-line treatments represents a serious risk, and the discovery of new effective drugs is urgently required. For this reason, new antimalarial chemotypes with new mechanisms of action, and ideally with activity against multiple parasite stages, are needed. We report a new scaffold with dual-stage (blood and liver) antiplasmodial activity. Twenty-six spirooxadiazoline oxindoles were synthesized and screened against the erythrocytic stage of the human malaria parasite P. falciparum. The most active compounds were also tested against the liver-stage of the murine parasite P. berghei. Seven compounds emerged as dual-stage antimalarials, with IC50 values in the low micromolar range. Due to structural similarity with cipargamin, which is thought to inhibit blood-stage P. falciparum growth via inhibition of the Na + efflux pump PfATP4, we tested one of the most active compounds for anti-PfATP4 activity. Our results suggest that this target is not the primary target of spirooxadiazoline oxindoles and further studies are ongoing to identify the main mechanism of action of this scaffold.This work was supported by FCT (Fundação para a Ciência e a Tecnologia, I.P.) through iMed.ULisboa (UID/DTP/04138/2019), project PTDC/QUI-QOR/29664/2017, and PhD fellowship SFRH/BD/137544/2018 (E. Lopes). The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). Financial support from FCT and Portugal 2020 to the Portuguese Mass Spectrometry Network (Rede Nacional de Espectrometria de Massa – RNEM; LISBOA-01-0145-FEDER-402-022125) is also acknowledged.info:eu-repo/semantics/publishedVersio

Influenza seroprotection correlates with predominant circulating viruses during 2014/15 and 2015/16 seasons in Portugal

Rede Portuguesa de Laboratórios para o Diagnóstico da GripeBACKGROUND: Population immune profile for influenza is highly affected by circulating influenza viruses, thus changing the risk of infection for influenza. This study aims to assess influenza immunity in the Portuguese population by age groups, during 2014 and 2015 and establish a relationship between seroprotection and circulating influenza viruses in 2014/15 and 2015/16 seasons. METHODS: Two cross-sectional studies were developed based on a convenience serum sample collected in June 2014 (n=626) and July 2015 (n=675) in hospitals from mainland and Azores and Madeira.Serums equally represent all age groups. Antibody titers were evaluated by HI assay for strains recommended for seasonal influenza vaccine northern hemisphere,2014/15 and 2015/2016. Seroprevalences were estimated for each strain by age group and the association with seasonal cumulative influenza-like illness (ILI) rates for influenza virus during both seasons was analised. RESULTS: In June 2014 the highest seroprotection was observed for influenza A(H3) (39.0%; 95% CI: 36.2-43.8%) and A(H1)pdm09 (29.7; 95% CI: 26.3-33.4%), with higher levels in children 5-14 years old. In 2014/2015 a dominant circulation of influenza B/Yamagata was observed with high incidence rates in individuals under 65 years old, the ones that had lower seroprotection. Although before the start of the season high protection for A(H3) was observed, the circulation of the new drift A(H3) strains had gained an immunological advantage,in accordance with A(H3) elevated incidence rates observed during 2014/15. In July 2015 the highest seroprotection was observed for influenza B/ Yamagata (55.1%; 95% CI: 51.4-58.9%), 2.4 times the estimated 2014.This increase was even more pronounced in younger (≤ 4 years old), 6.3 times increase in 2015.This fact is in agreement with the predominant influenza B virus detected and the high ILI incidence rate observed in children during 2014/2015 epidemic. Seroprotection levels for influenza A in July 2015 were not significantly different from 2014.During 2015/16 season, influenza A(H1N1)pdm09 was predominant, with high incidence rate in < 65 year old. Influenza B/Victoria lineage,although detected at low levels increased in frequency, in agreement with the lowest level of seroprotection detected in the general population before the start of 2015/2016 season (21.8%; 95% CI: 18.7-24.0%). CONCLUSIONS There was a correlation between virus circulation, incidence rates for each age group and the previous seroprotection for seasonal influenza viruses.Our study highlights the value of measuring the serological profile for influenza to establishe risk groups for infection for which an increase preventive measures, including vaccination, should be fostered.info:eu-repo/semantics/publishedVersio

Influenza severe cases in hospitals, between 2014 and 2016 in Portugal

Rede Portuguesa de Laboratórios para o Diagnóstico da GripeBackground: Since 2009, the Portuguese Laboratory Network (PLNID) for Influenza Diagnosis has integrated 15 Laboratories in mainland and Atlantic Islands of Azores and Madeira. This PLNID added an important contribute to the National Influenza Surveillance Program regarding severe and hospitalized influenza cases. The present study aims to describe influenza viruses detected in influenza like illness (ILI) cases: outpatients (Outp), hospitalized (Hosp), and intensive care units (ICU), between 2014 and 2016. Methods: The PLNID performs influenza virus diagnosis by biomolecular methodologies. Weekly reports to the National Influenza Reference Laboratory ILI cases tested for influenza. Reports include data on detecting viruses, hospital assistance, antiviral therapeutics, and information on death outcome. Were reported during two winter seasons 8059 ILI cases,being 3560 cases in 2014/15 (1024 in Outp, 1750 Hosp, and 606 in ICU) and 4499 cases in 2015/2016 (1933 in Outp, 1826 Hosp, and 740 in ICU). Results: The higher percentage of influenza positive cases were detected in Outp in both seasons, 18% during 2014/15 and 20% in 2015/16. In 2014/15,influenza cases were more frequent in individuals older than 65 years old and these required more hospitalizations,even in ICU. In 2015/16,the influenza cases were mainly detected in individuals between 15-64 years old. A higher proportion of influenza positive cases with hospitalization in ICU were observed in adults between 45-64 years old.During the study period,the predominant circulating influenza viruses were different in the two seasons: influenza B and A(H3) co-circulated in 2014/15,and influenza A(H1)pdm09 was predominant during 2015/16. Even when influenza A is notthe dominant virus, A(H3) and A(H1)pdm09 subtypes correlate with higher detection rate in hospitalized cases (Hosp and UCI), with higher frequencies in adults older than 45. Influenza B,detected in higher proportion in outpatients, was frequently relatedwith influenza cases in younger age groups: 0-4 and 5-14 years old. Conclusions: This study highlights the correlation of theinfluenza virus type/subtype that circulates in each season with the possible need for hospitalization and intensive care in special groups of the population. Circulation of influenza A subtypes can cause more frequentdisease in individuals older than 45, with need of hospitalization including intensive care. On the other hand, influenza B is more frequently associated with less severe cases and with infection in children and younger adults. Influenza B circulation might predict lower number of hospitalizations.The identification of influenza type in circulation,byPLNID ineach season, could guide action planning measures in population health care.info:eu-repo/semantics/publishedVersio

The reciprocal influence of malaria and sleeping sickness co-infections

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2013Malaria and sleeping sickness are tropical diseases that share overlapping spacial distributions in sub-Saharan Africa. Malaria is caused by several Plasmodium species, while sleeping sickness is caused by Trypanosoma brucei. Co-infections between these two eukaryotic parasites are likely to happen in humans, but this problem remains largely understudied. In this thesis, we tested whether an ongoing infection by one parasite would influence progression of a secondary infection by the other. The type of co-infection characterized most thoroughly throughout this thesis was the one in which mice were primarily infected by T. brucei and submitted to a secondary infection by P. berghei. In this case, we observed a dramatic impairment of the liver infection by the latter parasite. Immunofluorescence microscopy analysis and qRT-PCR revealed that a primary T. brucei infection is able to reduce approximately 80% P. berghei liver infection in the first 6 hours of its development, which means that elimination of Plasmodium parasites or Plasmodium-infected hepatocytes occurs very early. Several experiments were performed to address the mechanism of this T. brucei induced impairment of a P. berghei liver infection. Our results suggest that Plasmodium elimination may be immune response-dependent and indicate the involvement of pro-inflammatory cytokines, such as IFN-γ. The upregulation of this cytokine in co-infected mice leads to a production of TNF-α and nitric oxide, which may eliminate Plasmodium parasites. Interestingly, we observed that mice co-infected with T. brucei and P. berghei did not develop experimental cerebral malaria, neither when Plasmodium infection went through a liver stage or directly from infected blood. This work revealed hitherto unknown features of a Trypanosoma/Plasmodium co-infection and paved the way to a potential application in the control of malaria infections.A malária e a doença do sono são infeções tropicais que partilham uma distribuição espacial na África subsaariana. A malária é causada pelo parasita Plasmodium, enquanto a doença do sono é causada pelo parasita Trypanosoma brucei. Assim, é provável que um hospedeiro seja co-infetado por estes dois microrganismos. Contudo, o estudo da dupla infeção de Trypanosoma e Plasmodium ainda não foi muito aprofundado. Nesta tese, testámos se o decorrer da infeção por um dos parasitas poderia resultar numa diferente progressão da infeção secundária pelo outro parasita. O tipo de co-infeção mais estudado foi aquele em que ratinhos foram primeiro infetados por T. brucei e subsequentemente por P. berghei. Nesta situação observámos uma drástica redução na infeção hepática deste último parasita. Verificámos através qRT-PCR e por análise de imunofluorescência por microscopia que uma infeção por T. brucei reduz a infeção secundária de P. berghei em aproximadamente 80% (fase hepática), nas primeiras 6 horas deste estádio, o que significa que os parasitas ou os hepatócitos infetados por Plasmodium serão eliminados muito cedo. Foram realizadas diversas experiências de forma a desvendar o mecanismo pelo qual T. brucei reduz a infeção hepática de P. berghei. Os nossos resultados indicam que a diminuição da infeção seja mediada pela resposta imunitária e sugerem o envolvimento de citocinas pro-inflamatórias, nomeadamente o IFN-γ, na eliminação de Plasmodium. Tipicamente a sobre-expressão desta citocina leva à produção de TNF-α e óxido nítrico, sendo que estes poderão eliminar P. berghei em ratinhos co-infetados. Surpreendentemente, observámos que os ratinhos co-infetados por T. brucei e P. berghei não desenvolveram malária cerebral experimental, quer quando os parasitas de Plasmodium passaram pela fase hepática quer quando provieram de sangue infetado. Este trabalho revelou assim características da co-infeção por Trypanosoma/Plasmodium que eram até então desconhecidas e abre caminho a uma potencial aplicação no controlo de infeções maláricas

Coupling the cell-penetrating peptides transportan and transportan 10 to primaquine enhances its activity against liver-stage malaria parasites

This journal is © The Royal Society of Chemistry 2018Novel primaquine-cell penetrating peptide conjugates were synthesised and tested in vitro against liver stage Plasmodium berghei parasites. Generally, the conjugates were more active than the parent peptides and, in some cases, than the parent drug. These are unprecedented findings that may open a new route towards antimalarial drug rescuing.The authors are indebted to Fundação para a Ciência e Tecnologia (FCT, Portugal) for funding LAQV-REQUIMTE re- search unit through project UID/QUI/50006/2013. NV thanks FCT and FEDER (European Union) for funding project IF/00092/2014/CP1255/CT0004 and IF position. LA thanks FCT and the Medicinal Biochemistry and Biochemistry International Doctoral Programme (M2B-PhD) for PhD grant PD/BD/106035/2015.info:eu-repo/semantics/publishedVersio

Inhibition of Plasmodium Hepatic Infection by Antiretroviral Compounds

Recent WHO guidelines on control of human immunodeficiency virus (HIV) call for the widespread use of antiretroviral (AR) therapy (ART) for people living with HIV. Given the considerable overlap between infections by HIV and Plasmodium, the causative agent of malaria, it is important to understand the impact of AR compounds and ART regimens on infections by malaria parasites. We undertook a systematic approach to identify AR drugs and ART drug combinations with inhibitory activity against the obligatory hepatic stage of Plasmodium infection. Our in vitro screen of a wide array of AR drugs identified the non-nucleoside reverse transcriptase inhibitors efavirenz and etravirine (ETV), and the protease inhibitor nelfinavir, as compounds that significantly impair the development of the rodent malaria parasite P. berghei in an hepatoma cell line. Furthermore, we show that WHO-recommended ART drug combinations currently employed in the field strongly inhibit Plasmodium liver infection in mice, an effect that may be significantly enhanced by the inclusion of ETV in the treatment. Our observations are the first report of ETV as an anti-Plasmodial drug, paving the way for further evaluation and potential use of ETV-containing ARTs in regions of geographical overlap between HIV and Plasmodium infections

Excreted Trypanosoma brucei proteins inhibit Plasmodium hepatic infection

Funding: This work was supported by the Howard Hughes Medical Institute (ref. 55007419) awarded to LMF, by Fundac¸ão para a Ciência e Tecnologia (PD/ BD/138891/2018) awarded to AT, (PD/BD/ 105838/2018) awarded to MSV, (CEECIND/03322/ 2018) awarded to LMF, (CEECIND/03539/2017) awarded to MP, and by the Wellcome Trust (094476/Z/10/Z) awarded to TKS.Malaria, a disease caused by Plasmodium parasites, remains a major threat to public health globally. It is the most common disease in patients with sleeping sickness, another parasitic illness, caused by Trypanosoma brucei. We have previously shown that a T. brucei infection impairs a secondary P. berghei liver infection and decreases malaria severity in mice. However, whether this effect requires an active trypanosome infection remained unknown. Here, we show that Plasmodium liver infection can also be inhibited by the serum of a mouse previously infected by T. brucei and by total protein lysates of this kinetoplastid. Biochemical characterisation showed that the anti-Plasmodium activity of the total T. brucei lysates depends on its protein fraction, but is independent of the abundant variant surface glycoprotein. Finally, we found that the protein(s) responsible for the inhibition of Plasmodium infection is/are present within a fraction of ~350 proteins that are excreted to the bloodstream of the host. We conclude that the defence mechanism developed by trypanosomes against Plasmodium relies on protein excretion. This study opens the door to the identification of novel antiplasmodial intervention strategies.Publisher PDFPeer reviewe

Excreted Trypanosoma brucei proteins inhibit Plasmodium hepatic infection

© 2021 Temporão et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Malaria, a disease caused by Plasmodium parasites, remains a major threat to public health globally. It is the most common disease in patients with sleeping sickness, another parasitic illness, caused by Trypanosoma brucei. We have previously shown that a T. brucei infection impairs a secondary P. berghei liver infection and decreases malaria severity in mice. However, whether this effect requires an active trypanosome infection remained unknown. Here, we show that Plasmodium liver infection can also be inhibited by the serum of a mouse previously infected by T. brucei and by total protein lysates of this kinetoplastid. Biochemical characterisation showed that the anti-Plasmodium activity of the total T. brucei lysates depends on its protein fraction, but is independent of the abundant variant surface glycoprotein. Finally, we found that the protein(s) responsible for the inhibition of Plasmodium infection is/are present within a fraction of ~350 proteins that are excreted to the bloodstream of the host. We conclude that the defence mechanism developed by trypanosomes against Plasmodium relies on protein excretion. This study opens the door to the identification of novel antiplasmodial intervention strategies.This work was supported by the Howard Hughes Medical Institute (ref. 55007419) awarded to LMF, by Fundação para a Ciência e Tecnologia (PD/BD/138891/2018) awarded to AT, (PD/BD/105838/2018) awarded to MSV, (CEECIND/03322/2018) awarded to LMF, (CEECIND/03539/2017) awarded to MP, and by the Wellcome Trust (094476/Z/10/Z) awarded to TKS.info:eu-repo/semantics/publishedVersio