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Red blood cell enzymopathies – effects on malaria in-fection: the importance of host cell membrane in host-parasite interactions revealed by atomic force micros-copy and quantitative proteomics
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A malária é das doenças infecciosas mais letais do mundo, provocando milhões de casos clínicos e centenas de milhares de mortes todos os anos, principalmente na África subsaariana em crianças com menos de cinco anos. Homem e Plasmodium falciparum têm interagido durante milhares de anos, moldando o genoma um do outro. Esta interac-ção fez com que a malária fosse das mais importantes forças selectivas a actuar no genoma humano. Diversas variantes genéticas do eritrócito têm sido mantidas no genoma humano devido à protecção que conferem contra infecção, ou pelo menos contra doença grave. A deficiência em glucose-6-fosfato desidrogenase (G6PD) e em piruvato cinase (PK) são duas dessas variantes, ambas relacionadas com o metabolismo da célula hospedeira.
Apesar de vários estudos terem analisado a protecção contra malária conferida por estas variantes, ainda não se conhece totalmente o mecanismo de protecção e a dinâmica da interacção célula hospedeira-parasita durante a infecção. Como tal, este estudo teve como objectivo compreender melhor: 1) como é que o desenvolvimento do parasita é afectado por estas deficiências; 2) como é que célula hospedeira e parasita interagem du-rante a infecção de eritrócitos com estas deficiências; e 3) qual o mecanismo envolvido na protecção conferida contra doença grave.
Através de microscopia de força atómica (AFM) e de proteómica quantitativa baseada em espectrometria de massa, compararam-se eritrócitos não infectados e infectados, e analisaram-se extractos de proteínas, tanto da célula hospedeira, como do parasita. Foi possível simular as deficiências em G6PD e em PK na célula hospedeira através de su-plementação com cloreto de cálcio (no caso da deficiência em G6PD) ou com fluoreto de sódio (no caso da deficiência em PK). A deficiência em PK também foi simulada através da adição directa de bifosfoglicerato, um produto intermédio de metabolismo. As mem-branas de eritrócitos com deficiência (replicada) em PK, tanto infectados como não in-fectados, foram analisadas com AFM. As metodologias de proteómica quantitativa per-mitiram analisar extractos de proteínas de eritrócitos controlo ou com deficiência em G6PD, infectados e não infectados.
No seguimento de estudos anteriores (apesar das diferenças não serem significativas), observou-se que a invasão foi inferior em culturas de parasita em eritrócitos com defici-ência em G6PD. A análise feita com AFM revelou diferenças significativas entre membranas de eritrócitos controlo ou com deficiência (replicada) em PK, enquanto a aná-lise quantitativa dos proteomas de parasita e célula hospedeira demonstrou expressão di-ferencial de proteínas associadas ao citoesqueleto do eritrócito, nomeadamente a beta-espectrina. A análise de interacções proteína-proteína revelou grupos de proteínas associ-ados a actividade do proteassoma, resposta ao stress, glicólise e membrana/citoesqueleto da célula hospedeira.
Estes resultados apontam claramente para a membrana do eritrócito como sendo fun-damental na interacção parasita-hospedeiro. Os resultados sugerem ainda que alterações na membrana do eritrócito devido a deficiências em G6PD e em PK são responsáveis por uma menor invasão parasitária e, consequentemente, por menores densidades parasitárias. Estes resultados permitem pensar na possibilidade de terapias direccionadas ao hospe-deiro que podem ser desenvolvidas para melhorar a protecção contra malária, mesmo para pessoas sem qualquer tipo de mecanismo inato de protecção.
Malaria is one of the deadliest infectious diseases worldwide, causing millions of clinical cases and hundreds of thousands of deaths every year, especially in children under five in sub-Saharan African countries. Plasmodium falciparum and human host have in-teracted for thousands of years, shaping each other’s genomes. This long interaction has made malaria one of the most important selective forces acting on the human genome. Many human genetic variants related to the red blood cell (RBC) have been maintained in the human genome due to their protection against malaria infection, at least against severity of disease. Glucose-6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase (PK) deficiency are two of such variants, both specifically linked to host cell me-tabolism. Although several studies have focused on protection against malaria caused by RBC variants, the exact mechanism of protection and the dynamics between host cell and par-asite during infection are not fully understood yet for G6PD and PK deficiencies. There-fore, this study aimed at better understanding: 1) how parasite development is affected by these deficiencies; 2) how host cell and parasite interact during infection of RBCs with these enzymopathies; and 3) what is the mechanism involved in protection against sever-ity of disease. Atomic force microscopy (AFM) and mass spectrometry-based quantitative prote-omics were used to analyse and compare uninfected and infected RBCs and both host and parasite protein extracts. Mimicked G6PD and PK deficiencies in the host cell were achieved by calcium chloride (for G6PD deficiency) or sodium fluoride (for PK defi-ciency) supplementation. PK deficiency was also mimicked by direct addition of the gly-colysis intermediate metabolite 2,3-diphpsphoglycerate. RBC membrane of (mimicked) PK deficient uninfected and infected host cells were analysed using AFM. Quantitative proteomics methodologies were used for the analysis of G6PD deficient and control RBCs proteins extracts, both for uninfected and infected host cells. In line with previous studies (and although not significantly different), invasion was shown to be consistently lower in parasite cultures grown in deficient RBCs. More im-portantly, AFM analysis revealed significant differences between membranes of (mim-icked) PK deficient and control RBCs, whereas quantitative analyses of the proteomes of both parasite and host cell indicated differential expression of important RBC membrane-associated cytoskeleton proteins, such as beta-spectrin. Protein-protein interaction analy-sis also revealed some clusters associated with proteasomal activity, response to stress, glycolysis and host cell membrane/cytoskeleton. Results clearly point to the RBC membrane playing a pivotal role in the interaction between host cell and parasite and suggest that RBC membrane alterations due to RBC enzymopathies such as G6PD-def and PK-def are responsible for reduced parasite inva-sion and, therefore, for lower parasite densities. This opens the possibility for host-di-rected therapies to be developed and used to improve host protection against malaria, even for people without any kind of innate protective mechanism.
Malaria is one of the deadliest infectious diseases worldwide, causing millions of clinical cases and hundreds of thousands of deaths every year, especially in children under five in sub-Saharan African countries. Plasmodium falciparum and human host have in-teracted for thousands of years, shaping each other’s genomes. This long interaction has made malaria one of the most important selective forces acting on the human genome. Many human genetic variants related to the red blood cell (RBC) have been maintained in the human genome due to their protection against malaria infection, at least against severity of disease. Glucose-6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase (PK) deficiency are two of such variants, both specifically linked to host cell me-tabolism. Although several studies have focused on protection against malaria caused by RBC variants, the exact mechanism of protection and the dynamics between host cell and par-asite during infection are not fully understood yet for G6PD and PK deficiencies. There-fore, this study aimed at better understanding: 1) how parasite development is affected by these deficiencies; 2) how host cell and parasite interact during infection of RBCs with these enzymopathies; and 3) what is the mechanism involved in protection against sever-ity of disease. Atomic force microscopy (AFM) and mass spectrometry-based quantitative prote-omics were used to analyse and compare uninfected and infected RBCs and both host and parasite protein extracts. Mimicked G6PD and PK deficiencies in the host cell were achieved by calcium chloride (for G6PD deficiency) or sodium fluoride (for PK defi-ciency) supplementation. PK deficiency was also mimicked by direct addition of the gly-colysis intermediate metabolite 2,3-diphpsphoglycerate. RBC membrane of (mimicked) PK deficient uninfected and infected host cells were analysed using AFM. Quantitative proteomics methodologies were used for the analysis of G6PD deficient and control RBCs proteins extracts, both for uninfected and infected host cells. In line with previous studies (and although not significantly different), invasion was shown to be consistently lower in parasite cultures grown in deficient RBCs. More im-portantly, AFM analysis revealed significant differences between membranes of (mim-icked) PK deficient and control RBCs, whereas quantitative analyses of the proteomes of both parasite and host cell indicated differential expression of important RBC membrane-associated cytoskeleton proteins, such as beta-spectrin. Protein-protein interaction analy-sis also revealed some clusters associated with proteasomal activity, response to stress, glycolysis and host cell membrane/cytoskeleton. Results clearly point to the RBC membrane playing a pivotal role in the interaction between host cell and parasite and suggest that RBC membrane alterations due to RBC enzymopathies such as G6PD-def and PK-def are responsible for reduced parasite inva-sion and, therefore, for lower parasite densities. This opens the possibility for host-di-rected therapies to be developed and used to improve host protection against malaria, even for people without any kind of innate protective mechanism.
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Palavras-chave
Parasitologia Genética Plasmodium falciparum Deficiências enzimáticas do eritrócito Microscopia da força atómica Proteómica quantitativa baseada em espectrometria de massa Interações parasita-hospedeiro