Comparison of the cellular and biochemical properties of Plasmodium falciparum choline and ethanolamine kinases.

International audienceThe proliferation of the malaria-causing parasite Plasmodium falciparum within the erythrocyte is concomitant with massive phosphatidylcholine and phosphatidylethanolamine biosynthesis. Based on pharmacological and genetic data, de novo biosynthesis pathways of both phospholipids appear to be essential for parasite survival. The present study characterizes PfCK (P. falciparum choline kinase) and PfEK (P. falciparum ethanolamine kinase), which catalyse the first enzymatic steps of these essential metabolic pathways. Recombinant PfCK and PfEK were expressed as His6-tagged fusion proteins from overexpressing Escherichia coli strains, then purified to homogeneity and characterized. Using murine polyclonal antibodies against recombinant kinases, PfCK and PfEK were shown to be localized within the parasite cytoplasm. Protein expression levels increased during erythrocytic development. PfCK and PfEK appeared to be specific to their respective substrates and followed Michaelis-Menten kinetics. The Km value of PfCK for choline was 135.3+/-15.5 microM. PfCK was also able to phosphorylate ethanolamine with a very low affinity. PfEK was found to be an ethanolamine-specific kinase (Km=475.7+/-80.2 microM for ethanolamine). The quaternary ammonium compound hemicholinium-3 and an ethanolamine analogue, 2-amino-1-butanol, selectively inhibited PfCK or PfEK. In contrast, the bis-thiazolium compound T3, which was designed as a choline analogue and is currently in clinical trials for antimalarial treatment, affected PfCK and PfEK activities similarly. Inhibition exerted by T3 was competitive for both PfCK and PfEK and correlated with the impairment of cellular phosphatidylcholine biosynthesis. Comparative analyses of sequences and structures for both kinase types gave insights into their specific inhibition profiles and into the dual capacity of T3 to inhibit both PfCK and PfEK

The Phosphoarginine Phosphatase PtpB from Staphylococcus aureus Is Involved in Bacterial Stress Adaptation during Infection

Staphylococcus aureus continues to be a public health threat, especially in hospital settings. Studies aimed at deciphering the molecular and cellular mechanisms that underlie pathogenesis, host adaptation, and virulence are required to develop effective treatment strategies. Numerous host-pathogen interactions were found to be dependent on phosphatases-mediated regulation. This study focused on the analysis of the role of the low-molecular weight phosphatase PtpB, in particular, during infection. Deletion of ptpB in S. aureus strain SA564 significantly reduced the capacity of the mutant to withstand intracellular killing by THP-1 macrophages. When injected into normoglycemic C57BL/6 mice, the SA564 ∆ptpB mutant displayed markedly reduced bacterial loads in liver and kidney tissues in a murine S. aureus abscess model when compared to the wild type. We also observed that PtpB phosphatase-activity was sensitive to oxidative stress. Our quantitative transcript analyses revealed that PtpB affects the transcription of various genes involved in oxidative stress adaptation and infectivity. Thus, this study disclosed first insights into the physiological role of PtpB during host interaction allowing us to link phosphatase-dependent regulation to oxidative bacterial stress adaptation during infection

Characterization of the Secreted Acid Phosphatase SapS Reveals a Novel Virulence Factor of Staphylococcus aureus That Contributes to Survival and Virulence in Mice

Staphylococcus aureus possesses a large arsenal of immune-modulating factors, enabling it to bypass the immune system’s response. Here, we demonstrate that the acid phosphatase SapS is secreted during macrophage infection and promotes its intracellular survival in this type of immune cell. In animal models, the SA564 sapS mutant demonstrated a significantly lower bacterial burden in liver and renal tissues of mice at four days post infection in comparison to the wild type, along with lower pathogenicity in a zebrafish infection model. The SA564 sapS mutant elicits a lower inflammatory response in mice than the wild-type strain, while S. aureus cells harbouring a functional sapS induce a chemokine response that favours the recruitment of neutrophils to the infection site. Our in vitro and quantitative transcript analysis show that SapS has an effect on S. aureus capacity to adapt to oxidative stress during growth. SapS is also involved in S. aureus biofilm formation. Thus, this study shows for the first time that SapS plays a significant role during infection, most likely through inhibiting a variety of the host’s defence mechanisms

Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues

Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the bhydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors

Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter

Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes

Interactions of FGFs with target cells

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Point Mutations within the Fatty Acid Synthase Type II Dehydratase Components HadA or HadC Contribute to Isoxyl Resistance in Mycobacterium tuberculosis

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A 24 bp cis-acting element essential for the transcriptional activity of Plasmodium falciparum CDP-diacylglycerol synthase gene promoter

International audienceCDP-diacylglycerol synthase (CDS) is a key rate-limiting enzyme in the phospholipid metabolism of Plasmodium falciparum, converting phosphatidic acid to CDP-diacylglycerol. The CDS gene is predominantly expressed in the mature intraerythrocytic stages. Consequently, we physically and functionally characterized the CDS gene promoter. The mRNA transcription initiation site was mapped 121 bp upstream of the CDS gene translation start site. A 1909 bp 5' upstream sequence was isolated and found to be transcriptionally active thus constituting a functional CDS promoter. Mapping of this promoter identified a 44 bp cis-acting sequence, located between -1640 and -1596 bp upstream of the ATG codon, essential for efficient transcriptional activity. This 44 bp sequence binds specifically to nuclear factors from trophozoite stage parasites. We further showed that a 24 bp element, lying within the 44 bp sequence, mediates the specific binding to nuclear proteins and shows no significant homology to known eukaryotic DNA consensus sequence elements that bind transcription factors. The deletion of the 24 bp element abrogated promoter activity, indicating that this cis-acting sequence element is essential for efficient transcription of the CDS gene

Study of the diversity and cruciality of synthesis pathways for structural phospholipids in Plasmodium species

Le paludisme, une maladie qui affecte non seulement les humains mais également divers autres vertébrés, est causé par un protozoaire du genre Plasmodium. Chez l'homme, l'espèce Plasmodium falciparum est responsable de la forme la plus grave de la maladie. Les lipides membranaires comptent parmi les éléments les plus critiques pour son développement intraérythrocytaire, et pour assurer sa prolifération. Les phospholipides (PLs), les principaux constituants membranaires, sont synthétisés de novo par la machinerie enzymatique du parasite. En préambule, nous présentons dans deux revues les voies de synthèse des glycérophospholipides chez les procaryotes et les eucaryotes en général, et faisons un examen détaillé de celles présentes dans différentes espèces de Plasmodium. Les majeurs PLs structuraux, la phosphatidylcholine (PC) et la phosphatidylethanolamine (PE), sont synthétisés par de nombreuses voies métaboliques, très similaires à celles de la levure et des plantes. Le premier travail de thèse a porté sur le large éventail de voies métaboliques chez diverses espèces de Plasmodium. Des études in silico et des expériences de marquage métabolique ont révélé des différences marquées concernant le type de voies métaboliques dans les espèces de Plasmodium infectant les rongeurs. Ces résultats ouvrent le débat sur l'origine évolutive des parasites du paludisme humain. Dans une deuxième partie, nous avons eu pour objectifs de déterminer le caractère essentiel ou non pour la survie du parasite, des voies de synthèse de novo de PC et PE (voies de Kennedy). Des études génétiques chez le parasite de rongeur P. berghei indiquent que les deux voies de Kennedy sont nécessaires au stade érythrocytaire. Ainsi, Plasmodium diffère de la levure où ces voies métaboliques sont auxiliaires et non essentielles. En parallèle, d'autres points ont été abordés concernant les enzymes clefs impliquées dans la synthèse des PLs qui sont absents chez l'hôte humain et/ou qui présentent des caractéristiques particulières par rapport à leurs homologues eucaryotes. D'autres études sont nécessaires pour caractériser plus en détail le métabolisme des PLs, incluant d'autres enzymes, leur régulation, et leur caractère essentiel, ceci tant d'un point de vue fondamental, que pour des retombées possibles en pharmacologie anti-paludique.Malaria, a disease affecting not only humans but also various other vertebrates, is caused by a protozoon of the genus Plasmodium. The Plasmodium falciparum species is responsible for the most severe form of malaria in humans. At the intraerythrocytic stage of its complex life cycle, lipids are among the most critical components to be required for parasite growth and proliferation. Phospholipids (PLs), the main membrane constituents, mostly originate from the enzymatic machinery of the parasite. Foremost, in the form of two detailed reviews, we compare the PL synthetic pathways of prokaryotes and eukaryotes in general, to those of Plasmodium species. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the major structural PLs of the parasite, are synthesized in P. falciparum through a bewildering number of routes, greatly similar to those of yeast and plants. The first asked question concerned the occurrence of this wide range of metabolic pathways among diverse Plasmodium species. In silico and metabolic labelling studies revealed marked differences in PL synthesis between rodent and non-rodents parasite species. These findings relaunch the debate on the evolutionary origin of the human malaria parasites. As a second issue, the essential nature for parasite survival of the de novo PC and PE synthetic pathways, i.e. the Kennedy pathways, was questioned. Genetic studies in the rodent Plasmodium berghei parasite indicated that both Kennedy pathways are required at the erythrocytic stage. Thus, P. berghei differs from yeast, in which these routes are auxiliary and not crucial. In parallel, other issues were addressed concerning key enzymes involved in PL synthesis, that are absent in the human host and/or that exhibit particular features compared to eukaryotic counterparts. Further studies in P. falciparum are needed to characterize in greater detail the PL metabolism i.e. individual enzymes, regulation and cruciality from a fundamental point of view and for antimalarial purposes.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF