Isolation of viable Babesia bovis merozoites to study parasite invasion

AbstractBabesia parasite invades exclusively red blood cell (RBC) in mammalian host and induces alterations to host cell for survival. Despite the importance of Babesia in livestock industry and emerging cases in humans, their basic biology is hampered by lack of suitable biological tools. In this study, we aimed to develop a synchronization method for Babesia bovis which causes the most pathogenic form of bovine babesiosis. Initially, we used compound 2 (C2), a specific inhibitor of cyclic GMP-dependent protein kinase (PKG), and a derivative of C2, ML10. While both inhibitors were able to prevent B. bovis egress from RBC and increased percentage of binary forms, removal of inhibitors from culture did not result in a synchronized egress of parasites. Because using PKG inhibitors alone was not efficient to induce a synchronized culture, we isolated viable and invasive B. bovis merozoites and showed dynamics of merozoite invasion and development in RBCs. Using isolated merozoites we showed that BbVEAP, VESA1-export associated protein, is essential for parasite development in the RBC while has no significant role in invasion. Given the importance of invasion for the establishment of infection, this study paves the way for finding novel antigens to be used in control strategies against bovine babesiosis.

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Inhibition of Plasmodium Liver Infection by Ivermectin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02005-16 ◽

2016 ◽

Vol 61 (2) ◽

Cited By ~ 13

Author(s):

António M. Mendes ◽

Inês S. Albuquerque ◽

Marta Machado ◽

Joana Pissarra ◽

Patrícia Meireles ◽

...

Keyword(s):

Control Strategies ◽

Parasite Development ◽

Mammalian Host ◽

Mosquito Vector ◽

Content Type ◽

Vector Borne Diseases ◽

Host Infection ◽

Liver Infection

ABSTRACT Avermectins are powerful endectocides with an established potential to reduce the incidence of vector-borne diseases. Here, we show that several avermectins inhibit the hepatic stage of Plasmodium infection in vitro. Notably, ivermectin potently inhibits liver infection in vivo by impairing parasite development inside hepatocytes. This impairment has a clear impact on the ensuing blood stage parasitemia, reducing disease severity and enhancing host survival. Ivermectin has been proposed as a tool to control malaria transmission because of its effects on the mosquito vector. Our study extends the effect of ivermectin to the early stages of mammalian host infection and supports the inclusion of this multipurpose drug in malaria control strategies.

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Evaluating the Babesia bovis infection of cattle in China with enzyme-linked immunosorbent assay (ELISA)

Acta Parasitologica ◽

10.1515/ap-2015-0103 ◽

2015 ◽

Vol 60 (4) ◽

Cited By ~ 1

Author(s):

Congshan Yang ◽

Junlong Liu ◽

Anyan Li ◽

Youquan Li ◽

Aihong Liu ◽

...

Keyword(s):

Immunosorbent Assay ◽

Control Strategies ◽

Babesia Bovis ◽

Enzyme Linked Immunosorbent Assay ◽

Bovine Babesiosis ◽

Serum Samples ◽

The World ◽

Positive Rate ◽

Cattle Serum ◽

The Mean

AbstractBabesia bovis is an important pathogen of bovine babesiosis and causes serious constraints on the health and productivity of domestic cattle in the tropical and subtropical areas of the world. Aiming to clarify the prevalence of B. bovis in China, a total of 2,364 cattle serum samples were randomly collected from 45 different areas of 17 provinces in China. Antibodies against B. bovis were tested by enzyme-linked immunosorbent assay (ELISA) using a recombinant C-terminal antigen of B. bovis rhoptry-associated protein-1 (RAP-1) to evaluate the prevalence of B. bovis. The results showed that the parasite was present in all of the investigated 17 provinces. The positive rate was from 6.40 to 47.27%, and the mean rate was 24.92%. These survey data will provide important information for designing control strategies for bovine babesiosis in China.

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Differential expression of calcium-dependent protein kinase 4, tubulin tyrosine ligase, and methyltransferase by xanthurenic acid-induced Babesia bovis sexual stages

Parasites & Vectors ◽

10.1186/s13071-021-04902-3 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Hala E. Hussein ◽

Wendell C. Johnson ◽

Naomi S. Taus ◽

Janaina Capelli-Peixoto ◽

Carlos E. Suarez ◽

...

Keyword(s):

Babesia Bovis ◽

Xanthurenic Acid ◽

Bovine Babesiosis ◽

Dependent Protein Kinase ◽

Tick Vector ◽

Calcium Dependent ◽

Dependent Protein ◽

Sexual Stages ◽

Calcium Dependent Protein Kinase

Abstract Background Babesia bovis is one of the most significant tick-transmitted pathogens of cattle worldwide. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. Each life stage has developmental forms that differ in morphology and metabolism. Differentiation between these forms is highly regulated in response to changes in the parasite’s environment. Understanding the mechanisms by which Babesia parasites respond to environmental changes and the transmission cycle through the biological vector is critically important for developing bovine babesiosis control strategies. Results In this study, we induced B. bovis sexual stages in vitro using xanthurenic acid and documented changes in morphology and gene expression. In vitro induced B. bovis sexual stages displayed distinctive protrusive structures and surface ruffles. We also demonstrated the upregulation of B. bovis calcium-dependent protein kinase 4 (cdpk4), tubulin-tyrosine ligase (ttl), and methyltransferase (mt) genes by in vitro induced sexual stages and during parasite development within tick midguts. Conclusions Similar to other apicomplexan parasites, it is likely that B. bovis upregulated genes play a vital role in sexual reproduction and parasite transmission. Herein, we document the upregulation of cdpk4, ttl, and mt genes by both B. bovis in vitro induced sexual stages and parasites developing in the tick vector. Understanding the parasite's biology and identifying target genes essential for sexual reproduction will enable the production of non-transmissible live vaccines to control bovine babesiosis. Graphical abstract

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The fibrinolytic system enables the onset of Plasmodium infection in the mosquito vector and the mammalian host

Science Advances ◽

10.1126/sciadv.abe3362 ◽

2021 ◽

Vol 7 (6) ◽

pp. eabe3362 ◽

Cited By ~ 1

Author(s):

Thiago Luiz Alves e Silva ◽

Andrea Radtke ◽

Amanda Balaban ◽

Tales Vicari Pascini ◽

Zarna Rajeshkumar Pala ◽

...

Keyword(s):

Plasminogen Activators ◽

Fibrinolytic System ◽

Matrix Proteins ◽

Parasite Development ◽

Mammalian Host ◽

Mosquito Vector ◽

Plasminogen Activation ◽

Plasmodium Infection ◽

Human Plasminogen ◽

Vertebrate Hosts

Plasmodium parasites must migrate across proteinaceous matrices to infect the mosquito and vertebrate hosts. Plasmin, a mammalian serine protease, degrades extracellular matrix proteins allowing cell migration through tissues. We report that Plasmodium gametes recruit human plasminogen to their surface where it is processed into plasmin by corecruited plasminogen activators. Inhibition of plasminogen activation arrests parasite development early during sexual reproduction, before ookinete formation. We show that increased fibrinogen and fibrin in the blood bolus, which are natural substrates of plasmin, inversely correlate with parasite infectivity of the mosquito. Furthermore, we show that sporozoites, the parasite form transmitted by the mosquito to humans, also bind plasminogen and plasminogen activators on their surface, where plasminogen is activated into plasmin. Surface-bound plasmin promotes sporozoite transmission by facilitating parasite migration across the extracellular matrices of the dermis and of the liver. The fibrinolytic system is a potential target to hamper Plasmodium transmission.

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STO-609, a specific inhibitor of the Ca2+/calmodulin-dependent protein kinase kinase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)30856-7 ◽

2003 ◽

Vol 278 (6) ◽

pp. 4368

Author(s):

Hiroshi Tokumitsu ◽

Hiroyuki Inuzuka ◽

Yumi Ishikawa ◽

Masahiko Ikeda ◽

Ikutaro Saji ◽

...

Keyword(s):

Protein Kinase ◽

Specific Inhibitor ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Protein Kinase Kinase

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ANP and BNP Exert Anti-Inflammatory Action via NPR-1/cGMP Axis by Interfering with Canonical, Non-Canonical, and Alternative Routes of Inflammasome Activation in Human THP1 Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22010024 ◽

2020 ◽

Vol 22 (1) ◽

pp. 24

Author(s):

Letizia Mezzasoma ◽

Vincenzo Nicola Talesa ◽

Rita Romani ◽

Ilaria Bellezza

Keyword(s):

Natriuretic Peptide ◽

Specific Inhibitor ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Caspase 8 ◽

Inflammasome Activation ◽

Intracellular Cgmp ◽

Dependent Protein ◽

Anti Inflammatory ◽

Alternative Routes

Dysregulated inflammasome activation and interleukin (IL)-1β production are associated with several inflammatory disorders. Three different routes can lead to inflammasome activation: a canonical two-step, a non-canonical Caspase-4/5- and Gasdermin D-dependent, and an alternative Caspase-8-mediated pathway. Natriuretic Peptides (NPs), Atrial Natriuretic Peptide (ANP) and B-type Natriuretic Peptide (BNP), binding to Natriuretic Peptide Receptor-1 (NPR-1), signal by increasing cGMP (cyclic guanosine monophosphate) levels that, in turn, stimulate cGMP-dependent protein kinase-I (PKG-I). We previously demonstrated that, by counteracting inflammasome activation, NPs inhibit IL-1β secretion. Here we aimed to decipher the molecular mechanism underlying NPs effects on THP-1 cells stimulated with lipopolysaccharide (LPS) + ATP. Involvement of cGMP and PKG-I were assessed pre-treating THP-1 cells with the membrane-permeable analogue, 8-Br-cGMP, and the specific inhibitor KT-5823, respectively. We found that NPs, by activating NPR-1/cGMP/PKG-I axis, lead to phosphorylation of NLRP3 at Ser295 and to inflammasome platform disassembly. Moreover, by increasing intracellular cGMP levels and activating phosphodiesterases, NPs interfere with both Gasdermin D and Caspase-8 cleavage, indicating that they disturb non-canonical and alternative routes of inflammasome activation. These results showed that ANP and BNP anti-inflammatory and immunomodulatory actions may involve the inhibition of all the known routes of inflammasome activation. Thus, NPs might be proposed for the treatment of the plethora of diseases caused by a dysregulated inflammasome activation.

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Applying Machine Learning to Predict the Exportome of Bovine and Canine Babesia Species That Cause Babesiosis

Pathogens ◽

10.3390/pathogens10060660 ◽

2021 ◽

Vol 10 (6) ◽

pp. 660

Author(s):

Stephen J. Goodswen ◽

Paul J. Kennedy ◽

John T. Ellis

Keyword(s):

Machine Learning ◽

Severe Disease ◽

Economic Loss ◽

Babesia Bovis ◽

Training Data ◽

Babesia Bigemina ◽

Bovine Babesiosis ◽

Canine Babesiosis ◽

Clinically Significant ◽

Significant Disease

Babesia infection of red blood cells can cause a severe disease called babesiosis in susceptible hosts. Bovine babesiosis causes global economic loss to the beef and dairy cattle industries, and canine babesiosis is considered a clinically significant disease. Potential therapeutic targets against bovine and canine babesiosis include members of the exportome, i.e., those proteins exported from the parasite into the host red blood cell. We developed three machine learning-derived methods (two novel and one adapted) to predict for every known Babesia bovis, Babesia bigemina, and Babesia canis protein the probability of being an exportome member. Two well-studied apicomplexan-related species, Plasmodium falciparum and Toxoplasma gondii, with extensive experimental evidence on their exportome or excreted/secreted proteins were used as important benchmarks for the three methods. Based on 10-fold cross validation and multiple train–validation–test splits of training data, we expect that over 90% of the predicted probabilities accurately provide a secretory or non-secretory indicator. Only laboratory testing can verify that predicted high exportome membership probabilities are creditable exportome indicators. However, the presented methods at least provide those proteins most worthy of laboratory validation and will ultimately save time and money.

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Developmental competence and antigen switch frequency can be uncoupled in Trypanosoma brucei

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1912711116 ◽

2019 ◽

Vol 116 (45) ◽

pp. 22774-22782 ◽

Cited By ~ 1

Author(s):

Kirsty R. McWilliam ◽

Alasdair Ivens ◽

Liam J. Morrison ◽

Monica R. Mugnier ◽

Keith R. Matthews

Keyword(s):

Trypanosoma Brucei ◽

Antigenic Variation ◽

Experimental Studies ◽

Developmental Competence ◽

Parasite Development ◽

Mammalian Host ◽

Mechanical Transmission ◽

African Trypanosomes ◽

Infection Dynamic ◽

Developmental Capacity

African trypanosomes use an extreme form of antigenic variation to evade host immunity, involving the switching of expressed variant surface glycoproteins by a stochastic and parasite-intrinsic process. Parasite development in the mammalian host is another feature of the infection dynamic, with trypanosomes undergoing quorum sensing (QS)-dependent differentiation between proliferative slender forms and arrested, transmissible, stumpy forms. Longstanding experimental studies have suggested that the frequency of antigenic variation and transmissibility may be linked, antigen switching being higher in developmentally competent, fly-transmissible, parasites (“pleomorphs”) than in serially passaged “monomorphic” lines that cannot transmit through flies. Here, we have directly tested this tenet of the infection dynamic by using 2 experimental systems to reduce pleomorphism. Firstly, lines were generated that inducibly lose developmental capacity through RNAi-mediated silencing of the QS signaling machinery (“inducible monomorphs”). Secondly, de novo lines were derived that have lost the capacity for stumpy formation by serial passage (“selected monomorphs”) and analyzed for their antigenic variation in comparison to isogenic preselected populations. Analysis of both inducible and selected monomorphs has established that antigen switch frequency and developmental capacity are independently selected traits. This generates the potential for diverse infection dynamics in different parasite populations where the rate of antigenic switching and transmission competence are uncoupled. Further, this may support the evolution, maintenance, and spread of important trypanosome variants such as Trypanosoma brucei evansi that exploit mechanical transmission.

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Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes

Zygote ◽

10.1017/s0967199401001356 ◽

2001 ◽

Vol 9 (4) ◽

pp. 309-316 ◽

Cited By ~ 21

Author(s):

Carsten Krischek ◽

Burkhard Meinecke

Keyword(s):

Map Kinase ◽

Protein Kinases ◽

Chromatin Condensation ◽

Specific Inhibitor ◽

Histone H1 ◽

Dependent Manner ◽

Free Medium ◽

Concentration Dependent Manner ◽

Dependent Protein

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 μM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 μM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to ≥22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.

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CaMKII Potentiates Store-Operated Ca2+ Entry Through Enhancing STIM1 Aggregation and Interaction with Orai1

Cellular Physiology and Biochemistry ◽

10.1159/000488835 ◽

2018 ◽

Vol 46 (3) ◽

pp. 1042-1054 ◽

Cited By ~ 2

Author(s):

Shu Li ◽

Jingyi Xue ◽

Zhipeng Sun ◽

Tiantian Liu ◽

Lane Zhang ◽

...

Keyword(s):

Protein Kinase ◽

Confocal Microscopy ◽

Specific Inhibitor ◽

Potential Mechanism ◽

Additional Insight ◽

Dependent Protein Kinase ◽

Store Depletion ◽

Selective Channel ◽

Dependent Protein ◽

Insight Into

Background/Aims: Upon Ca2+ store depletion, stromal interaction molecule 1 (STIM1) oligomerizes, redistributes near plasmalemma to interact with Ca2+ selective channel-forming subunit (Orai1) and initiates store-operated Ca2+ entry (SOCE). Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a regulator of SOCE, but how CaMKII regulates SOCE remains obscure. Methods: Using Fura2, confocal microscopy, co-immunoprecipitation, specific blocker and overexpression/knockdown approaches, we evaluated STIM1 aggregation and its interaction with Orai1, and SOCE upon Ca2+ store depletion in thapsigargin (TG) treated HEK293 and HeLa cells. Results: Overexpression of CaMKIIδ enhanced TG-induced STIM1 co-localization and interaction with Orai1 as well as SOCE. In contrast, CaMKIIδ knockdown and a specific inhibitor of CaMKII suppressed them. In addition, overexpression or knockdown of CaMKIIδ in TG treated cells exhibited increased or reduced STIM1 clustering and plasmalemma redistribution, respectively. Conclusion: CaMKII up-regulates SOCE by increasing STIM1 aggregation and interaction with Orai1. This study provides an additional insight into SOCE regulation and a potential mechanism for CaMKII involvement in some pathological situations through crosstalk with SOCE.

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