Marine Invertebrates: A Promissory Still Unexplored Source of Inhibitors of Biomedically Relevant Metallo Aminopeptidases Belonging to the M1 and M17 Families.

Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Peptidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many biochemical and physiological processes. They are also involved in several pathophysiological processes. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids of proteins or peptide substrates. They are distributed in many phyla and play critical roles in physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development of therapeutic agents for human diseases, including cancer, hypertension, central nervous system disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as malaria. The relevance of aminopeptidases has driven the search and identification of potent and selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an important and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with foreseen biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further studies with inhibitors isolated from marine invertebrates in different biomedical models associated with the activity of these families of exopeptidases.

Marine gregarine genomes reveal the breadth of apicomplexan diversity with a partially conserved glideosome machinery.

Our current view of the evolutionary history, coding and adaptive capacities of Apicomplexa, protozoan parasites of a wide range of metazoan, is currently strongly biased toward species infecting humans, as data on early diverging apicomplexan lineages infecting invertebrates is extremely limited. Here, we characterized the genome of the marine eugregarine Porospora gigantea, intestinal parasite of Lobsters, remarkable for the macroscopic size of its vegetative feeding forms (trophozoites) and its gliding speed, the fastest so far recorded for Apicomplexa. Two highly syntenic genomes named A and B were assembled. Similar in size (~ 9 Mb) and coding capacity (~ 5300 genes), A and B genomes are 10.8% divergent at the nucleotide level, corresponding to 16-38 My in divergent time. Orthogroup analysis across 25 (proto)Apicomplexa species, including Gregarina niphandrodes, showed that A and B are highly divergent from all other known apicomplexan species, revealing an unexpected breadth of diversity. Phylogenetically these two species branch sisters to Cephaloidophoroidea, and thus expand the known crustacean gregarine superfamily. The genomes were mined for genes encoding proteins necessary for gliding, a key feature of apicomplexans parasites, currently studied through the molecular model called glideosome. Sequence analysis shows that actin-related proteins and regulatory factors are strongly conserved within apicomplexans. In contrast, the predicted protein sequences of core glideosome proteins and adhesion proteins are highly variable among apicomplexan lineages, especially in gregarines. These results confirm the importance of studying gregarines to widen our biological and evolutionary view of apicomplexan species diversity, and to deepen our understanding of the molecular bases of key functions such as gliding, well known to allow access to the intracellular parasitic lifestyle in Apicomplexa.

Rapid protein evolution, organellar reductions, and invasive intronic elements in the marine aerobic parasite dinoflagellate Amoebophrya spp.

BACKGROUND: Dinoflagellates are aquatic protists particularly widespread in the oceans worldwide. Some are responsible for toxic blooms while others live in symbiotic relationships, either as mutualistic symbionts in corals or as parasites infecting other protists and animals. Dinoflagellates harbor atypically large genomes (~ 3 to 250 Gb), with gene organization and gene expression patterns very different from closely related apicomplexan parasites. Here we sequenced and analyzed the genomes of two early-diverging and co-occurring parasitic dinoflagellate Amoebophrya strains, to shed light on the emergence of such atypical genomic features, dinoflagellate evolution, and host specialization. RESULTS: We sequenced, assembled, and annotated high-quality genomes for two Amoebophrya strains (A25 and A120), using a combination of Illumina paired-end short-read and Oxford Nanopore Technology (ONT) MinION long-read sequencing approaches. We found a small number of transposable elements, along with short introns and intergenic regions, and a limited number of gene families, together contribute to the compactness of the Amoebophrya genomes, a feature potentially linked with parasitism. While the majority of Amoebophrya proteins (63.7% of A25 and 59.3% of A120) had no functional assignment, we found many orthologs shared with Dinophyceae. Our analyses revealed a strong tendency for genes encoded by unidirectional clusters and high levels of synteny conservation between the two genomes despite low interspecific protein sequence similarity, suggesting rapid protein evolution. Most strikingly, we identified a large portion of non-canonical introns, including repeated introns, displaying a broad variability of associated splicing motifs never observed among eukaryotes. Those introner elements appear to have the capacity to spread over their respective genomes in a manner similar to transposable elements. Finally, we confirmed the reduction of organelles observed in Amoebophrya spp., i.e., loss of the plastid, potential loss of a mitochondrial genome and functions. CONCLUSION: These results expand the range of atypical genome features found in basal dinoflagellates and raise questions regarding speciation and the evolutionary mechanisms at play while parastitism was selected for in this particular unicellular lineage.

Programmed Multiple C-H Bond Functionalization of the Privileged 4-hydroxyquinoline Template.

The introduction of substituents on bare heterocyclic scaffolds can selectively be achieved by directed C-H functionalization. However, such methods have only occasionally been used, in an iterative manner, to decorate various positions of a medicinal scaffold to build chemical libraries. We herein report the multiple, site selective, metal-catalyzed C-H functionalization of a "programmed" 4-hydroxyquinoline. This medicinally privileged template indeed possesses multiple reactive sites for diversity-oriented functionalization, of which four were targeted. The C-2 and C-8 decorations were directed by an N-oxide, before taking benefit of an O-carbamoyl protection at C-4 to perform a Fries rearrangement and install a carboxamide at C-3. This also released the carbonyl group of 4-quinolones, the ultimate directing group to functionalize position 5. Our study highlights the power of multiple C-H functionalization to generate diversity in a biologically relevant library, after showing its strong antimalarial potential.

Structure-activity relationship and molecular modelling studies of quinazolinedione derivatives MMV665916 as potential antimalarial agent.

A series of new quinazolinedione derivatives have been readily synthesized and evaluated for their in vitro antiplasmodial growth inhibition activity. Most of the compounds inhibited P. falciparum FcB1 strain in the low to medium micromolar concentration. The 2-ethoxy 8ag', 2-trifluoromethoxy 8ai' and 4-fluoro-2-methoxy 8ak' showed the best inhibitory activity with EC50 values around 5 µM and were non-toxic to the primary human fibroblast cell line AB943. However, these compounds were less potent than the original hit MMV665916, which showed remarkable growth inhibition with EC50 value of 0.4 µM and presented the highest selectivity index (SI > 250). In addition, a novel approach for determining the docking poses of these quinazolinedione derivatives with their potential protein target, the P. falciparum farnesyltransferase PfFT, was investigated.

Why the -omic future of Apicomplexa should include gregarines.

Gregarines, a polyphyletic group of apicomplexan parasites infecting mostly non-vertebrates hosts, remains poorly known at taxonomic, phylogenetic and genomic levels. However, it represents an essential group for understanding evolutionary history and adaptive capacities of apicomplexan parasites to the remarkable diversity of their hosts. Because they have a mostly extracellular lifestyle, gregarines have developed other cellular developmental forms and host-parasite interactions, compared with their much better studied apicomplexan cousins, intracellular parasites of vertebrates (Hemosporidia, Coccidia, Cryptosporidia). This review highlights the promises offered by the molecular exploration of gregarines, that have been until now left on the side of the road of the comparative -omic exploration of apicomplexan parasites. Elucidating molecular bases for both their ultrastructural, functional and behavioural similarities and differences, compared with those of the typical apicomplexan models, is expected to provide entirely novel clues on the adaptive capacities developed by Apicomplexa over evolution. A challenge remains to identify which gregarines should be explored in priority, as recent metadata from open and host-associated environments have confirmed how underestimated is our current view on true gregarine biodiversity. It is now time to turn to gregarines to widen the currently highly skewed view we have of adaptive mechanisms developed by Apicomplexa.

Aminobenzosuberone derivatives as PfA-M1 inhibitors: Molecular recognition and antiplasmodial evaluation.

Aminobenzosuberone-based PfA-M1 inhibitors were explored as novel antimalarial agents against two different Plasmodium falciparum strains. The 4-phenyl derivative 7c exhibited the most encouraging growth inhibitory activity with IC50 values of 6.5-11.2 µM. X-ray crystal structures and early assessment of DMPK/ADME-Tox parameters allowed us to initiate structure-based drug design approach and understand the liabilities (such as potential metabolic and aqueous solubility issues) as well as identify the opportunities for improvement of this aminobenzosuberone series. It also suggested that compound 7c should be regarded as an attractive chemical tool to investigate the different biological roles of this multifunctional PfA-M1 protein.

Aminobenzosuberone Scaffold as a Modular Chemical Tool for the Inhibition of Therapeutically Relevant M1 Aminopeptidases.

The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to investigate their specificity, series of enzyme inhibition assays were carried out against a diversity of proteases, covering representative members of aspartic, cysteine, metallo and serine endopeptidases and including eight members of the monometallic M1 family of aminopeptidases as well as two members of the bimetallic M17 and M28 aminopeptidase families. This aminobenzosuberone scaffold indeed demonstrated selective inhibition of M1 aminopeptidases to the exclusion of other tested protease families; it was particularly potent against mammalian APN and its bacterial/parasitic orthologues EcPepN and PfAM1.

Selective inhibition of PfA-M1, over PfA-M17, by an amino-benzosuberone derivative blocks malaria parasites development in vitro and in vivo.

BACKGROUND: Plasmodium falciparum M1 family aminopeptidase is currently considered as a promising target for anti-malarial chemotherapy. Several series of inhibitors developed by various research groups display IC50/Ki values down to nM range on native PfA-M1 or recombinant forms and block the parasite development in culture at µM to sub-µM concentrations. A handful of these inhibitors has been tested on murine models of malaria and has shown anti plasmodial in vivo activity. However, most of these inhibitors do also target the other neutral malarial aminopeptidase, PfA-M17, often with lower Ki values, which questions the relative involvement and importance of each enzyme in the parasite biology. RESULTS: An amino-benzosuberone derivative from a previously published collection of chemicals targeting specifically the M1-aminopeptidases has been identified; it is highly potent on PfA-M1 (Ki = 50 nM) and devoid of inhibitory activity on PfA-M17 (no inhibition up to 100 µM). This amino-benzosuberone derivative (T5) inhibits, in the µM range, the in vitro growth of two P. falciparum strains, 3D7 and FcB1, respectively chloroquino-sensitive and resistant. Evaluated in vivo, on the murine non-lethal model of malaria Plasmodium chabaudi chabaudi, this amino-benzosuberone derivative was able to reduce the parasite burden by 44 and 40% in a typical 4-day Peters assay at a daily dose of 12 and 24 mg/kg by intraperitoneal route of administration. CONCLUSIONS: The evaluation of a highly selective inhibitor of PfA-M1, over PfA-M17, active on Plasmodium parasites in vitro and in vivo, highlights the relevance of PfA-M1 in the biological development of the parasite as well as in the list of promising anti-malarial targets to be considered in combination with current or future anti-malarial drugs.

KBE009: An antimalarial bestatin-like inhibitor of the Plasmodium falciparum M1 aminopeptidase discovered in an Ugi multicomponent reaction-derived peptidomimetic library.

Malaria is a global human parasitic disease mainly caused by the protozoon Plasmodium falciparum. Increased parasite resistance to current drugs determines the relevance of finding new treatments against new targets. A novel target is the M1 alanyl-aminopeptidase from P. falciparum (PfA-M1), which is essential for parasite development in human erythrocytes and is inhibited by the pseudo-peptide bestatin. In this work, we used a combinatorial multicomponent approach to produce a library of peptidomimetics and screened it for the inhibition of recombinant PfA-M1 (rPfA-M1) and the in vitro growth of P. falciparum erythrocytic stages (3D7 and FcB1 strains). Dose-response studies with selected compounds allowed identifying the bestatin-based peptidomimetic KBE009 as a submicromolar rPfA-M1 inhibitor (Ki=0.4µM) and an in vitro antimalarial compound as potent as bestatin (IC50=18µM; without promoting erythrocyte lysis). At therapeutic-relevant concentrations, KBE009 is selective for rPfA-M1 over porcine APN (a model of these enzymes from mammals), and is not cytotoxic against HUVEC cells. Docking simulations indicate that this compound binds PfA-M1 without Zn2+ coordination, establishing mainly hydrophobic interactions and showing a remarkable shape complementarity with the active site of the enzyme. Moreover, KBE009 inhibits the M1-type aminopeptidase activity (Ala-7-amido-4-methylcoumarin substrate) in isolated live parasites with a potency similar to that of the antimalarial activity (IC50=82µM), strongly suggesting that the antimalarial effect is directly related to the inhibition of the endogenous PfA-M1. These results support the value of this multicomponent strategy to identify PfA-M1 inhibitors, and make KBE009 a promising hit for drug development against malaria.

Genetic impairment of parasite myosin motors uncovers the contribution of host cell membrane dynamics to Toxoplasma invasion forces.

BACKGROUND: The several-micrometer-sized Toxoplasma gondii protozoan parasite invades virtually any type of nucleated cell from a warm-blooded animal within seconds. Toxoplasma initiates the formation of a tight ring-like junction bridging its apical pole with the host cell membrane. The parasite then actively moves through the junction into a host cell plasma membrane invagination that delineates a nascent vacuole. Recent high resolution imaging and kinematics analysis showed that the host cell cortical actin dynamics occurs at the site of entry while gene silencing approaches allowed motor-deficient parasites to be generated, and suggested that the host cell could contribute energetically to invasion. In this study we further investigate this possibility by analyzing the behavior of parasites genetically impaired in different motor components, and discuss how the uncovered mechanisms illuminate our current understanding of the invasion process by motor-competent parasites. RESULTS: By simultaneously tracking host cell membrane and cortex dynamics at the site of interaction with myosin A-deficient Toxoplasma, the junction assembly step could be decoupled from the engagement of the Toxoplasma invasive force. Kinematics combined with functional analysis revealed that myosin A-deficient Toxoplasma had a distinct host cell-dependent mode of entry when compared to wild-type or myosin B/C-deficient Toxoplasma. Following the junction assembly step, the host cell formed actin-driven membrane protrusions that surrounded the myosin A-deficient mutant and drove it through the junction into a typical vacuole. However, this parasite-entry mode appeared suboptimal, with about 40 % abortive events for which the host cell membrane expansions failed to cover the parasite body and instead could apply deleterious compressive forces on the apical pole of the zoite. CONCLUSIONS: This study not only clarifies the key contribution of T. gondii tachyzoite myosin A to the invasive force, but it also highlights a new mode of entry for intracellular microbes that shares early features of macropinocytosis. Given the harmful potential of the host cell compressive forces, we propose to consider host cell invasion by zoites as a balanced combination between host cell membrane dynamics and the Toxoplasma motor function. In this light, evolutionary shaping of myosin A with fast motor activity could have contributed to optimize the invasive potential of Toxoplasma tachyzoites and thereby their fitness.

Redescription of Dicyemennea eledones (Wagener, 1857) (Phylum Dicyemida) from Eledone cirrhosa (Lamarck, 1798) (Mollusca: Cephalopoda: Octopoda).

Dicyemids are common parasites found in the kidneys of many cephalopods. Species identification previously relied on old species descriptions containing considerable confusions, casting doubt on taxonomy and identification. Detailed morphological description and genotyping of all developmental stages are required for an exact taxonomy. To this end, we undertook the redescription of the dicyemid Dicyemennea eledones (Wagener, 1857), infecting the cephalopod Eledone cirrhosa (Lamarck). Samples were collected off Concarneau in the Bay of Biscay, France, and off La Goulette in the Gulf of Tunis, Tunisia. Dicyemennea eledones is a large species, with adults reaching c.7,000 µm in length. The vermiform stages are characterised by having 23 peripheral cells, a conical calotte and an axial cell that extends to the base of the propolar cells. An anterior abortive axial cell is present in vermiform embryos. Infusoriform embryos consist of 37 cells; a single nucleus is present in each urn cell and the refringent bodies, which were not always seen, are possibly liquid. For the first time, an 18S rDNA sequence is generated for D. eledones, illustrating genetic differences with the other dicyemid 18S rDNA sequences available in databases. This sequence can now be used for D. eledones barcoding, making the identification of the species easier and more reliable.

Access to new endoperoxide derivatives by electrochemical oxidation of substituted 3-azabicyclo[4.1.0]hept-4-enes.

A series of substituted 3-azabicyclo[4.1.0]hept-4-ene derivatives were prepared and analysed by cyclic voltammetry. Preparative aerobic electrochemical oxidation reactions were then carried out. Three original endoperoxides were isolated, characterised and subjected to antimalarial and cytotoxicity activity assays.

Parasitic Protists: Diversity of Adaptations to a Parasitic Lifestyle.

Parasitic protists cause some of the most well-known human and animal diseases such as malaria, toxoplasmosis, amoebic meningitis, sleeping sickness, leishmaniosis, and diarrheal illness of protozoan origin (e [...].

Genome Sequence and Assessment of Safety and Potential Probiotic Traits of Lactobacillus johnsonii CNCM I-4884.

The probiotic strain Lactobacillus johnsonii CNCM I-4884 exhibits anti-Giardia activity in vitro and in vivo in a murine model of giardiasis. The aim of this study was the identification and characterization of the probiotic potential of L. johnsonii CNCM I-4884, as well as its safety assessment. This strain was originally classified as Lactobacillus gasseri based on 16S gene sequence analysis. Whole genome sequencing led to a reclassification as L. johnsonii. A genome-wide search for biosynthetic pathways revealed a high degree of auxotrophy, balanced by large transport and catabolic systems. The strain also exhibits tolerance to low pH and bile salts and shows strong bile salt hydrolase (BSH) activity. Sequencing results revealed the absence of antimicrobial resistance genes and other virulence factors. Phenotypic tests confirm that the strain is susceptible to a panel of 8 antibiotics of both human and animal relevance. Altogether, the in silico and in vitro results confirm that L. johnsonii CNCM I-4884 is well adapted to the gastrointestinal environment and could be safely used in probiotic formulations.

Nutrient Acquisition and Attachment Strategies in Basal Lineages: A Tough Nut to Crack in the Evolutionary Puzzle of Apicomplexa.

Apicomplexa are unicellular eukaryotes that parasitise a wide spectrum of invertebrates and vertebrates, including humans. In their hosts, they occupy a variety of niches, from extracellular cavities (intestine, coelom) to epicellular and intracellular locations, depending on the species and/or developmental stages. During their evolution, Apicomplexa thus developed an exceptionally wide range of unique features to reach these diversified parasitic niches and to survive there, at least long enough to ensure their own transmission or that of their progeny. This review summarises the current state of knowledge on the attachment/invasive and nutrient uptake strategies displayed by apicomplexan parasites, focusing on trophozoite stages of their so far poorly studied basal representatives, which mostly parasitise invertebrate hosts. We describe their most important morphofunctional features, and where applicable, discuss existing major similarities and/or differences in the corresponding mechanisms, incomparably better described at the molecular level in the more advanced Apicomplexa species, of medical and veterinary significance, which mainly occupy intracellular niches in vertebrate hosts.

Age and Giardia intestinalis Infection Impact Canine Gut Microbiota.

Giardia intestinalis is a flagellated protozoan responsible for giardiosis (also called giardiasis in humans), the most prevalent and widespread parasitic infection in humans and mammals worldwide. The intestinal microbiota is highly diverse and any alteration in its composition may impact on the health of the host. While studies on the mouse model of giardiosis described the role of the gut microbiota in host susceptibility to infection by the parasite, little is known about the gut microbiota during natural infections in dogs and particularly in puppies. In this study, we monitored naturally G. intestinalis-infected puppies for 3 months and quantified cyst excretion every 2 weeks. All puppies remained subclinically infected during the sampling period as confirmed by fecal examination. In parallel, we performed 16S Illumina sequencing of fecal samples from the different time points to assess the impact of G. intestinalis infection on gut microbiota development of the puppies, as well as gut health markers of immunity such as fecal IgA and calprotectin. Sequencing results revealed that the canine fecal microbiota of Giardia-infected puppies becomes more complex and less diverse with increasing age. In addition, significant differences in the structure of the microbiota were observed between puppies with high and low Giardia cyst excretion. Chronic subclinical G. intestinalis infection appears to be associated with some detrimental structural changes in the gut microbiota. G. intestinalis-associated dysbiosis is characterized by an enrichment of facultative anaerobic, mucus-degrading, pro-inflammatory species and opportunistic pathogens, as well as a reduction of Lactobacillus johnsonii at specific time points. Calprotectin levels increased with age, suggesting the establishment of chronic low-grade inflammation in puppies. Further work is needed to demonstrate whether these alterations in the canine gut microbiota could lead to a dysbiosis-related disease, such as irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD).

Integrative taxonomy confirms that Gregarina garnhami and G. acridiorum (Apicomplexa, Gregarinidae), parasites of Schistocerca gregaria and Locusta migratoria (Insecta, Orthoptera), are distinct species.

Orthoptera are infected by about 60 species of gregarines assigned to the genus Gregarina Dufour, 1828. Among these species, Gregarina garnhami Canning, 1956 from Schistocerca gregaria (Forsskål, 1775) was considered by Lipa et al. in 1996 to be synonymous with Gregarina acridiorum (Léger 1893), a parasite of several orthopteran species including Locusta migratoria (Linné, 1758). Here, a morphological study and molecular analyses of the SSU rDNA marker demonstrate that specimens of S. gregaria and specimens of L. migratoria are infected by two distinct Gregarina species, G. garnhami and G. acridiorum, respectively. Validation of the species confirms that molecular analyses provide useful taxonomical information. Phenotypic plasticity was clearly observed in the case of G. garnhami: the morphology of its trophozoites, gamonts and syzygies varied according to the geographical location of S. gregaria and the subspecies infected.

TITLE: La taxonomie intégrative confirme que Gregarina garnhami et G. acridiorum (Apicomplexa, Gregarinidae), parasites de Schistocerca gregaria et Locusta migratoria (Insecta, Orthoptera), sont des espèces distinctes. ABSTRACT: Les orthoptères sont parasités par environ soixante espèces de grégarines affiliées au genre Gregarina Dufour, 1828. Parmi ces espèces Gregarina garnhami Canning, 1956 décrite chez Schistocerca gregaria (Forskål, 1775), a été mise en synonymie par Lipa et al. en 1996 avec Gregarina acridiorum (Léger 1893), parasite de plusieurs espèces d'orthoptères dont Locusta migratoria (Linné, 1758). Ici, une étude morphologique et des analyses moléculaires du marqueur SSU rDNA démontrent que les spécimens de S. gregaria et ceux de L. migratoria sont infectés par 2 espèces distinctes de grégarines, Gregarina garnhami et Gregarina acridiorum, respectivement. La validation de ces espèces confirme l'importance des informations fournies par les analyses moléculaires dans les études taxonomiques. Une plasticité phénotypique a été clairement observée dans le cas de G. garnhami : la morphologie de ses trophozoïtes, gamontes et syzygies varie selon la localisation géographique et la sous-espèce de S. gregaria infectée.

Assessing functional annotation transfers with inter-species conserved coexpression: application to Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum is the main causative agent of malaria. Of the 5 484 predicted genes of P. falciparum, about 57% do not have sufficient sequence similarity to characterized genes in other species to warrant functional assignments. Non-homology methods are thus needed to obtain functional clues for these uncharacterized genes. Gene expression data have been widely used in the recent years to help functional annotation in an intra-species way via the so-called Guilt By Association (GBA) principle. RESULTS: We propose a new method that uses gene expression data to assess inter-species annotation transfers. Our approach starts from a set of likely orthologs between a reference species (here S. cerevisiae and D. melanogaster) and a query species (P. falciparum). It aims at identifying clusters of coexpressed genes in the query species whose coexpression has been conserved in the reference species. These conserved clusters of coexpressed genes are then used to assess annotation transfers between genes with low sequence similarity, enabling reliable transfers of annotations from the reference to the query species. The approach was used with transcriptomic data sets of P. falciparum, S. cerevisiae and D. melanogaster, and enabled us to propose with high confidence new/refined annotations for several dozens hypothetical/putative P. falciparum genes. Notably, we revised the annotation of genes involved in ribosomal proteins and ribosome biogenesis and assembly, thus highlighting several potential drug targets. CONCLUSIONS: Our approach uses both sequence similarity and gene expression data to help inter-species gene annotation transfers. Experiments show that this strategy improves the accuracy achieved when using solely sequence similarity and outperforms the accuracy of the GBA approach. In addition, our experiments with P. falciparum show that it can infer a function for numerous hypothetical genes.