Multiomics analysis reveals B. MO1 as a distinct Babesia species and provides insights into its evolution and virulence.

Babesiosis, caused by protozoan parasites of the genus Babesia , is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of various Babesia species underscores the ongoing risk of new zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental shifts impacting the distribution and transmission dynamics of parasites, their vectors, and reservoir hosts. One such species, Babesia MO1, previously implicated in severe cases of human babesiosis in the midwestern United States, was initially considered closely related to B. divergens , the predominant agent of human babesiosis in Europe. Yet, uncertainties persist regarding whether these pathogens represent distinct variants of the same species or are entirely separate species. We show that although both B. MO1 and B. divergens share similar genome sizes, comprising three nuclear chromosomes, one linear mitochondrial chromosome, and one circular apicoplast chromosome, major differences exist in terms of genomic sequence divergence, gene functions, transcription profiles, replication rates and susceptibility to antiparasitic drugs. Furthermore, both pathogens have evolved distinct classes of multigene families, crucial for their pathogenicity and adaptation to specific mammalian hosts. Leveraging genomic information for B. MO1, B. divergens , and other members of the Babesiidae family within Apicomplexa provides valuable insights into the evolution, diversity, and virulence of these parasites. This knowledge serves as a critical tool in preemptively addressing the emergence and rapid transmission of more virulent strains.

Comparative proteomic analysis of kinesin-8B deficient Plasmodium berghei during gametogenesis.

Plasmodium blood stages, responsible for human to vector transmission, termed gametocytes, are the precursor cells that develop into gametes in the mosquito. Male gametogenesis works as a bottleneck for the parasite life cycle, where, during a peculiar and rapid exflagellation, a male gametocyte produces 8 intracellular axonemes that generate by budding 8 motile gametes. Understanding the molecular mechanisms of gametogenesis is key to design strategies for controlling malaria transmission. In the rodent P. berghei, the microtubule-based motor kinesin-8B (PbKIN8B) is essential for flagellum assembly during male gametogenesis and its gene disruption impacts on completion of the parasitic life cycle. In efforts to improve our knowledge about male gametogenesis, we performed an iTRAQ-based quantitative proteomic comparison of P. berghei mutants with disrupted kinesin-8B gene (ΔPbkin8B) and wild type parasites. During the 15 min of gametogenesis, ΔPbkin8B parasites exhibited important motor protein dysregulation that suggests an essential role of PbKIN8B for the correct interaction or integration of axonemal proteins within the growing axoneme. The energy metabolism of ΔPbkin8B mutants was further affected, as well as the response to stress proteins, protein synthesis, as well as chromatin organisation and DNA processes, although endomitoses seemed to occur. SIGNIFICANCE: Malaria continues to be a global scourge, mainly in subtropical and tropical areas. The disease is caused by parasites from the Plasmodium genus. Plasmodium life cycle alternates between female Anopheles mosquitoes and vertebrate hosts through bites. Gametocytes are the parasite blood forms responsible for transmission from vertebrates to vectors. Inside the mosquito midgut, after stimulation, male and female gametocytes transform into gametes resulting in fertilization. During male gametogenesis, one gametocyte generates eight intracytoplasmic axonemes that generate, by budding, flagellated motile gametes involving a process termed exflagellation. Sexual development has a central role in ensuring malaria transmission. However, molecular data on male gametogenesis and particularly on intracytoplasmic axoneme assembly are still lacking. Since rodent malaria parasites permit the combination of in vivo and in vitro experiments and reverse genetic studies, our group investigated the molecular events in rodent P. berghei gametogenesis. The P. berghei motor ATPase kinesin-8B is proposed as an important component for male gametogenesis. We generated Pbkin8B gene-disrupted gametocytes (ΔPbkin8B) that were morphologically similar to the wild- type (WT) parasites. However, in mutants, male gametogenesis is impaired, male gametocytes are disabled in their ability to assemble axonemes and to exflagellate to release gametes, reducing fertilization drastically. Using a comparative quantitative proteomic analysis, we associated the nonfunctional axoneme of the mutants with the abnormal differential expression of proteins essential to axoneme organisation and stability. We also observed a differential dysregulation of proteins involved in protein biosynthesis and degradation, chromatin organisation and DNA processes in ΔPbkin8B parasites, although DNA condensation, mitotic spindle formation and endomitoses seem to occur. This is the first functional proteomic study of a kinesin gene-disrupted Plasmodium parasite providing new insights into Plasmodium male gametogenesis.

Vital role for Plasmodium berghei Kinesin8B in axoneme assembly during male gamete formation and mosquito transmission.

Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin-8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B-gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal '9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.

Dynamic molecular events associated to Plasmodium berghei gametogenesis through proteomic approach.

Plasmodium mature sexual cycle occurs in the vector mosquitoes and ensures the transmission to a new host. Gametogenesis takes place within minutes in the vector midgut. Gametocytes have to complete a deep nuclear reorganization, quick differentiation, and in the case of male gametocytes, intracytoplasmic flagellum assembly that results in free-swimming microgametes required for macrogamete fertilization. In efforts to improve our knowledge of molecular mechanisms involved in gamete morphogenesis, we carried out a nanoLC/MSMS based quantitative proteomic analysis throughout the xanthurenic acid-induced gametogenesis of the rodent parasite P. berghei. Time-course analyses were performed 7 and 15min after gametogenesis induction. From 2617 iTRAQ-labelled peptides, 49 proteins were found differentially abundant. Proteins related to RNA translation, DNA, and protein biosynthesis were most prominent and strongly regulated. The energetic metabolic pathway, glycolysis, environmental stress response, RNA/protein biosynthesis, mitosis and axoneme formation, both related to tubulin-associated cytoskeleton dynamic, were predominant regulated cell processes at protein level during the differentiation. Our results also include 26 phosphoproteins in gametocytes/gametes. This first iTRAQ-based proteomic time course analysis of Plasmodium gamete development sheds light on the biological protein orchestration within gametogenesis. SIGNIFICANCE: Malaria is one of the most serious and widespread parasitic diseases that affected humans in medicine history. The increasing emergence of resistance of parasites from Plasmodium genus to the available antimalarial drugs and the absence of efficient vaccines require an urgent need of development of new therapeutic strategies to fight against that disease. The sexual reproduction is a key step of Plasmodium life cycle and constitutes an attractive target for the development of new therapeutic approaches since it would control malaria based on an inhibition of the parasite transmission to Anopheles, and then to humans. Male and female gamete formation (gametogenesis) is thus a biological event that is determinant for the perpetuation of the parasite in which drastic morphological and metabolic changes occur in a short time interval, resulting in the production of 8 male gametes from a male gametocyte, and fertilization of the female gamete. Development of such transmission-blocking strategies required in deep understanding of the molecular and cellular events associated to gametogenesis. Despite several studies, our understanding on gametogenesis is still incomplete and requires further investigations. This work is the first large-scale quantitative proteomic insight into the P. berghei gamete morphogenesis providing valuable time course data. Plasmodium gametogenesis clearly requires regulation of expression and phosphorylation of proteins belonging to different metabolic pathways and functions, in order to produce mature male and female gametes.

Whole genome mapping and re-organization of the nuclear and mitochondrial genomes of Babesia microti isolates.

Babesia microti is the primary causative agent of human babesiosis, an emerging pathogen that causes a malaria-like illness with possible fatal outcome in immunocompromised patients. The genome sequence of the B. microti R1 strain was reported in 2012 and revealed a distinct evolutionary path for this pathogen relative to that of other apicomplexa. Lacking from the first genome assembly and initial molecular analyses was information about the terminal ends of each chromosome, and both the exact number of chromosomes in the nuclear genome and the organization of the mitochondrial genome remained ambiguous. We have now performed various molecular analyses to characterize the nuclear and mitochondrial genomes of the B. microti R1 and Gray strains and generated high-resolution Whole Genome maps. These analyses show that the genome of B. microti consists of four nuclear chromosomes and a linear mitochondrial genome present in four different structural types. Furthermore, Whole Genome mapping allowed resolution of the chromosomal ends, identification of areas of misassembly in the R1 genome, and genomic differences between the R1 and Gray strains, which occur primarily in the telomeric regions. These studies set the stage for a better understanding of the evolution and diversity of this important human pathogen.

The traditional medicine Spilanthes acmella, and the alkylamides spilanthol and undeca-2E-ene-8,10-diynoic acid isobutylamide, demonstrate in vitro and in vivo antimalarial activity.

Spilanthes spp. are used as traditional herbal medicines in Africa and India to treat malaria. Yet, to date, there are no data on the active constituents or the most effective extraction methods for this indication. The isolated alkylamides, spilanthol and undeca-2E-ene-8,10-diynoic acid isobutylamide, found in S. acmella Murr., were shown to have IC50s of 16.5 µg/mL and 41.4 µg/mL on Plasmodium falciparum strain PFB and IC50s of 5.8 µg/mL and 16.3 µg/mL for the chloroquine resistant P. falciparum K1 strain, respectively. Further investigations revealed that at relatively low concentrations, spilanthol and the water extract of S. acmella reduced the parasitemia 59% and 53% in mice infected with P. yoelii yoelii 17XNL at 5 mg/kg and 50 mg/kg, respectively. Unexpectedly, the 95% ethanol extract of S. acmella was less effective (36% reduction in parasitemia) at 50 mg/kg. These results provide the first evidence supporting S. acmella against malaria and demonstrating active constituents in S. acmella against P. falciparum.

Blochmannia endosymbionts improve colony growth and immune defence in the ant Camponotus fellah.

BACKGROUND: Microorganisms are a large and diverse form of life. Many of them live in association with large multicellular organisms, developing symbiotic relations with the host and some have even evolved to form obligate endosymbiosis. All Carpenter ants (genus Camponotus) studied hitherto harbour primary endosymbiotic bacteria of the Blochmannia genus. The role of these bacteria in ant nutrition has been demonstrated but the omnivorous diet of these ants lead us to hypothesize that the bacteria might provide additional advantages to their host. In this study, we establish links between Blochmannia, growth of starting new colonies and the host immune response. RESULTS: We manipulated the number of bacterial endosymbionts in incipient laboratory-reared colonies of Camponotus fellah by administrating doses of an antibiotic (Rifampin) mixed in honey-solution. Efficiency of the treatment was estimated by quantitative polymerase chain reaction and Fluorescent in situ hybridization (FISH), using Blochmannia specific primers (qPCR) and two fluorescent probes (one for all Eubacterial and other specific for Blochmannia). Very few or no bacteria could be detected in treated ants. Incipient Rifampin treated colonies had significantly lower numbers of brood and adult workers than control colonies. The immune response of ants from control and treated colonies was estimated by inserting nylon filaments in the gaster and removing it after 24 h. In the control colonies, the encapsulation response was positively correlated to the bacterial amount, while no correlation was observed in treated colonies. Indeed, antibiotic treatment increased the encapsulation response of the workers, probably due to stress conditions. CONCLUSION: The increased growth rate observed in non-treated colonies confirms the importance of Blochmannia in this phase of colony development. This would provide an important selective advantage during colony founding, where the colonies are faced with severe inter and intraspecific competition. Furthermore, the bacteria improve the workers encapsulation response. Thus, these ants are likely to be less susceptible to various pathogen attacks, such as the Phoridae fly parasitoids, normally found in the vicinity of Camponotus nests. These advantages might explain the remarkable ecological success of this ant genus, comprising more than 1000 species.

Plasmodium telomeric sequences: structure, stability and quadruplex targeting by small compounds.

The increasing resistance of Plasmodium falciparum to the most commonly used antimalarial drugs makes it necessary to identify new therapeutic targets. The telomeres of the parasite could constitute an attractive target. They are composed of repetitions of a degenerate motif ((5')GGGTTYA(3'), where Y is T or C), different from the human one ((5')GGGTTA(3')). In this report we investigate the possibility of targeting Plasmodium telomeres with G-quadruplex ligands. Through solution hybridisation assays we provide evidence of the existence of a telomeric 3' G-overhang in P. falciparum genomic DNA. Through UV spectroscopy studies we demonstrate that stable G-quadruplex structures are formed at physiological temperature by sequences composed of the degenerate Plasmodium telomeric motif. Through a FRET melting assay we show stabilisation of Plasmodium telomeric G-quadruplexes by a variety of ligands. Many of the tested ligands display strong quadruplex versus duplex selectivity, but show little discrimination between human and Plasmodium telomeric quadruplexes.

Malaria and obesity: obese mice are resistant to cerebral malaria.

BACKGROUND: The relationship between malaria and obesity are largely unknown. This is partly due to the fact that malaria occurs mainly in tropical areas where, until recently, obesity was not prevalent. It now appears, however, that obesity is emerging as a problem in developing countries. To investigate the possible role of obesity on the host-parasite response to malarial infection, this study applied a murine model, which uses the existence of genetically well characterized obese mice. METHODS: The receptivity of obese homozygous ob/ob mice was compared to the receptivity of control heterozygous ob/+ lean mice after a single injection of Plasmodium berghei ANKA sporozoites. Both parasitaemia and mortality in response to infection were recorded. RESULTS: The control mice developed the expected rapid neurological syndromes associated with the ANKA strain, leading to death after six days, in absence of high parasitaemia. The obese mice, on the other hand, did not develop cerebral malaria and responded with increasing parasitaemia, which produced severe anemia leading to death 18-25 days after injection. CONCLUSION: The observed major differences in outward symptoms for malarial infection in obese versus control mice indicate a link between obesity and resistance to the infection which could be addressed by malariologists studying human malaria.

Convergent use of RhoGAP toxins by eukaryotic parasites and bacterial pathogens.

Inactivation of host Rho GTPases is a widespread strategy employed by bacterial pathogens to manipulate mammalian cellular functions and avoid immune defenses. Some bacterial toxins mimic eukaryotic Rho GTPase-activating proteins (GAPs) to inactivate mammalian GTPases, probably as a result of evolutionary convergence. An intriguing question remains whether eukaryotic pathogens or parasites may use endogenous GAPs as immune-suppressive toxins to target the same key genes as bacterial pathogens. Interestingly, a RhoGAP domain-containing protein, LbGAP, was recently characterized from the parasitoid wasp Leptopilina boulardi, and shown to protect parasitoid eggs from the immune response of Drosophila host larvae. We demonstrate here that LbGAP has structural characteristics of eukaryotic RhoGAPs but that it acts similarly to bacterial RhoGAP toxins in mammals. First, we show by immunocytochemistry that LbGAP enters Drosophila immune cells, plasmatocytes and lamellocytes, and that morphological changes in lamellocytes are correlated with the quantity of LbGAP they contain. Demonstration that LbGAP displays a GAP activity and specifically interacts with the active, GTP-bound form of the two Drosophila Rho GTPases Rac1 and Rac2, both required for successful encapsulation of Leptopilina eggs, was then achieved using biochemical tests, yeast two-hybrid analysis, and GST pull-down assays. In addition, we show that the overall structure of LbGAP is similar to that of eukaryotic RhoGAP domains, and we identify distinct residues involved in its interaction with Rac GTPases. Altogether, these results show that eukaryotic parasites can use endogenous RhoGAPs as virulence factors and that despite their differences in sequence and structure, eukaryotic and bacterial RhoGAP toxins are similarly used to target the same immune pathways in insects and mammals.

Targeted disruption of cytosolic SIR2 deacetylase discloses its essential role in Leishmania survival and proliferation.

Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity on histone and various other cellular substrates. Functional analyses of such proteins have been carried out in a number of prokaryotes and eukaryotes organisms but until now, none have described an essential function for any SIR2 genes. Here using genetic approach, we report that a cytosolic SIR2 homolog in Leishmania is determinant to parasite survival. L. infantum promastigote tolerates deletion of one wild-type LiSIR2 allele (LiSIR2+/-) but achievement of null chromosomal mutants (LiSIR2-/-) requires episomal rescue. Accordingly, plasmid cure shows that these parasites maintain episome even in absence of drug pressure. Though single LiSIR2 gene disruption (LiSIR2+/-) does not affect the growth of parasite in the promastigote form, axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Taken together these data support a stage specific requirement and/or activity of the Leishmania cytosolic SIR2 protein and reveal an unrelated essential function for the life cycle of this unicellular pathogenic organism. The lack of an effective vaccine against leishmaniasis, and the need for alternative drug treatments, makes LiSIR2 protein a new attractive therapeutic target.