RBC Invasion Research Articles

Immunization with PfGBP130 generates antibodies that inhibit RBC invasion by P. falciparum parasites.

Despite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions. To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies. We are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates.

Transcriptome analysis reveals molecular targets of erythrocyte invasion phenotype diversity in natural Plasmodium falciparum isolates from Cameroon

Further understanding of the molecular mediators of alternative RBC invasion phenotypes in endemic malaria parasites will support malaria blood-stage vaccine or drug development. This study investigated the prevalence of sialic acid (SA)-dependent and SA-independent RBC invasion pathways in endemic Plasmodium falciparum parasites from Cameroon and compared the schizont stage transcriptomes in these two groups to uncover the wider repertoire of transcriptional variation associated with the use of alternative RBC invasion pathway phenotypes. A two-color flow cytometry-based invasion-inhibition assay against RBCs treated with neuraminidase, trypsin, and chymotrypsin and deep RNA sequencing of schizont stages harvested in the first ex vivo replication cycle in culture were employed in this investigation. RBC invasion phenotypes were determined for 63 isolates from asymptomatic children with uncomplicated malaria. Approximately 80% of the isolates invaded neuraminidase-treated but not chymotrypsin-treated RBCs, representing SA-independent pathways of RBC invasion. The schizont transcriptome profiles of 16 isolates with invasion phenotypes revealed a total of 5,136 gene transcripts, with 85% of isolates predicted at schizont stages. Two distinct transcriptome profile clusters belonging to SA-dependent and SA-independent parasites were obtained by data reduction with principal component analysis. Differential analysis of gene expression between the two clusters implicated, in addition to the well-characterized adhesins, the upregulation of genes encoding proteins mediating merozoite organelle discharges as well as several conserved, virulent, merozoite-associated, and exported proteins. The latter majority have been shown to have structural and physiological relevance to RBC surface remodeling and immune evasion in malaria and thus have potential as anti-invasion targets.

Invasion-inhibitory peptides chosen by natural selection analysis as an antimalarial strategy

Plasmodium vivax’s biological complexity has restricted in vitro culture development for characterising antigens involved in erythrocyte invasion and their immunological relevance. The murine model is proposed as a suitable alternative in the search for therapeutic candidates since Plasmodium yoelii uses homologous proteins for its invasion. The AMA-1 protein is essential for parasite invasion of erythrocytes as it is considered an important target for infection control. This study has focused on functional PyAMA-1 peptides involved in host-pathogen interaction; the protein is located in regions under negative selection as determined by bioinformatics analysis. It was found that pyama1 has two highly conserved regions amongst species (>70%) under negative selection. Fourteen synthetic peptides spanning both conserved regions were evaluated; 5 PyAMA-1 peptides having high specific binding (HABP) to murine erythrocytes were identified. The parasite’s invasion inhibition capability was analysed through in vitro assays, suggesting that peptides 42681 (43-ENTERSIKLINPWDKYMEKY-62), 42903 (206-RYSSNDANNENQPFSFTPEK-225) and 42904 (221-FTPEKIENYKDLSYLTKNLR-240) had greater than 50% inhibition profile and restricted P. yoelii intra-erythrocyte development. This work proposes that the screening of conserved HABPs under negative selective pressure might be good candidates for developing a synthetic anti-malarial vaccine since they share functionally-relevant characteristics, such as interspecies conservation, specific RBC binding profile, invasion and parasite development inhibition capability, and the predicted B-epitopes within were recognised by sera obtained from experimentally-infected mice.

Genetic Evidence That Dimerization of Glycophorin a Is Critical for Red Cell Invasion By Plasmodium Falciparum but Not for the Binding of EBA-175

Introduction: Genome-wide association studies showed that resistance to malaria may be linked to the GYPA/B/E locus (Leffler et al, Science, 2017) and it is assumed that GYPA is the receptor for P. falciparum EBA-175 ligand (Sim et al, Science, 1994). The crystal structure of the erythrocyte binding domain of EBA-175 (RII) suggests that its binding to GYPA induces dimerization of RII by assembling GYPA extracellular domain (Tolia et al, Cell, 2005), a model consistent with studies pointing out the importance of the dimer status of GYPA in this interaction (Durainsingh et al, PNAS, 2003). Thanks to the description of a new GYPA variant, we investigated the role of GYPA dimerization in RBC invasion by P. falciparum. Results: Whole exome sequencing in patient with an alloantibody to a high-prevalence antigen of unknown specificity led to the finding of eight candidate variants including a homozygous 284T>C GYPA mutation resulting in ILE95THR substitution and previously reported only at heterozygous state (frequency 0.0038 % in the Genome Aggregation Database). Others RBCs with rare blood type including Dantu and En(a-) were as reactive as the standard panel cells and the antibody was claimed to be of unknown specificity. Regarding the importance of Ile95 for GYPA dimerization (Trenker et al, JACS, 2015), we analyzed GYPA organization in RBC membranes from the proband and his homozygous brother. Indeed, immunoblotting with a specific anti-GYPA antibody showed the absence of the dimeric form of GYPA in the Ile95Thr and Dantu RBCs. Flow cytometry showed a slight decrease of GYPA but normal expression of Band3 and Wrb antigen in both variants. However, a second anti-GYPA antibody recognizing another epitope of this protein showed a strong decrease of GYPA in these variants. These results indicate a structural alteration of the GYPA in both Ile95Thr and Dantu RBCs. Interestingly, flow cytometry revealed that recombinant PfEBA-175 is able to bind the monomeric form of GYPA in Ile95Thr but not in Dantu RBCs. Finally, ex vivo invasion assays showed a drastic decreased invasion for all Ile95Thr, Dantu, En(a-) and Mk/Mk RBCs, suggesting a crucial role of GYPA dimerization for RBC invasion by P.falciparum. Conclusion: We reported a new Ile95Thrh variant of GYPA organized in a monomeric form at the RBC membrane. Although EBA-175 binding is normal, this variant seems to be resistant to P. falciparum invasion, as it is the case for Dantu and En(a-) variants. Our findings provide genetic evidence that dimerization of GYPA is critical for RBC invasion by Plasmodium falciparum but not for the binding of EBA-175. This outcome could explain the poorly elucidated mechanism of Dantu RBC resistance to P. falciparum invasion.

The RNA structurome in the asexual blood stages of malaria pathogen plasmodium falciparum

ABSTRACT Plasmodium falciparum is a deadly human pathogen responsible for the devastating disease called malaria. In this study, we measured the differential accumulation of RNA secondary structures in coding and non-coding transcripts from the asexual developmental cycle in P. falciparum in human red blood cells. Our comprehensive analysis that combined high-throughput nuclease mapping of RNA structures by duplex RNA-seq, SHAPE-directed RNA structure validation, immunoaffinity purification and characterization of antisense RNAs collectively measured differentially base-paired RNA regions throughout the parasite’s asexual RBC cycle. Our mapping data not only aligned to a diverse pool of RNAs with known structures but also enabled us to identify new structural RNA regions in the malaria genome. On average, approximately 71% of the genes with secondary structures are found to be protein coding mRNAs. The mapping pattern of these base-paired RNAs corresponded to all regions of mRNAs, including the 5ʹ UTR, CDS and 3ʹ UTR as well as the start and stop codons. Histone family genes which are known to form secondary structures in their mRNAs and transcripts from genes which are important for transcriptional and post-transcriptional control, such as the unique plant-like transcription factor family, ApiAP2, DNA-/RNA-binding protein, Alba3 and proteins important for RBC invasion and malaria cytoadherence also showed strong accumulation of duplex RNA reads in various asexual stages in P. falciparum. Intriguingly, our study determined stage-specific, dynamic relationships between mRNA structural contents and translation efficiency in P. falciparum asexual blood stages, suggesting an essential role of RNA structural changes in malaria gene expression programs.

Genome-Wide Scans for Ghanaian Plasmodium falciparum Genes Under Selection From Local and Chinese Host Populations.

Initial malarial infection mostly causes symptomatic illness in humans. Infection that is not fatal induces complete protection from severe illness and death, and thus complete protection from severe illness or death is granted with sufficient exposure. However, malaria parasite immunity necessitates constant exposure. Therefore, it is important to evaluate lowered immunity and recurrent susceptibility to symptomatic disease in lower transmission areas. We aimed to investigate selection pressure based on transmission levels, antimalarial drug use, and environmental factors. We whole genome sequenced (WGS) P. falciparum clinical samples from Chinese hosts working in Ghana and compared the results with the WGS data of isolates from native Ghanaians downloaded from pf3k. The P. falciparum samples were generally clustered according to their geographic origin, and Chinese imported samples showed a clear African origin with a slightly different distribution from the native Ghanaian samples. Moreover, samples collected from two host populations showed evidence of differences in the intensity of selection. Compared with native Ghanaian samples, the China-imported isolates exhibited a higher proportion of monoclonal infections, and many genes associated with RBC invasion and immune evasion were found to be under less selection pressure. There was no significant difference in the selection of drug-resistance genes due to a similar artemisinin-based combination therapy medication profile. Local selection of malarial parasites is considered to be a result of differences in the host immunity or disparity in the transmission opportunities of the host. In China, most P. falciparum infections were imported from Africa, and under these circumstances, distinct local selective pressures may be caused by varying acquired immunity and transmission intensity. This study revealed the impact of host switching on the immune system, and it may provide a better understanding of the mechanisms that enable clinical immunity to malaria.

Global distribution of single amino acid polymorphisms in Plasmodium vivax Duffy-binding-like domain and implications for vaccine development efforts.

Plasmodium vivax (Pv) malaria continues to be geographically widespread with approximately 15 million worldwide cases annually. Along with other proteins, Duffy-binding proteins (DBPs) are used by plasmodium for RBC invasion and the parasite-encoded receptor binding regions lie in their Duffy-binding-like (DBL) domains—thus making it a prime vaccine candidate. This study explores the sequence diversity in PvDBL globally, with an emphasis on India as it remains a major contributor to the global Pv malaria burden. Based on 1358 PvDBL protein sequences available in NCBI, we identified 140 polymorphic sites within 315 residues of PvDBL. Alarmingly, country-wise mapping of SAAPs from field isolates revealed varied and distinct polymorphic profiles for different nations. We report here 31 polymorphic residue positions in the global SAAP profile, most of which map to the PvDBL subdomain 2 (α1–α6). A distinct clustering of SAAPs distal to the DARC-binding sites is indicative of immune evasive strategies by the parasite. Analyses of PvDBL-neutralizing antibody complexes revealed that between 24% and 54% of interface residues are polymorphic. This work provides a framework to recce and expand the polymorphic space coverage in PvDBLs as this has direct implications for vaccine development studies. It also emphasizes the significance of surveying global SAAP distributions before or alongside the identification of vaccine candidates.

Babesia bovis BOV57, a Theileria parva P67 homolog, is an invasion-related, neutralization-sensitive antigen

Babesia bovis BOV57, which is a homolog of the Theileria parva vaccine candidate antigen P67, is expressed in both the tick and blood stages of the life cycle of this parasite. However, the vaccine potential of BOV57 remained to be investigated. In the present study, we generated recombinant BOV57 (rBOV57) and prepared polyclonal antibodies against rBOV57 in mice and rabbits. Indirect immunofluorescence assays conducted with the mouse anti-rBOV57 antibody demonstrated that BOV57 localized at the apical end of paired merozoites in infected bovine red blood cells, whereas the antigen was found in the parasite membrane around the apical end of intraerythrocytic single and extracellular merozoites. In an invasion-inhibition assay, the rabbit anti-rBOV57 antibody potentially inhibited RBC invasion of B. bovis merozoites in vitro. In addition, the invasion inhibition mediated by rabbit anti-rBOV57 antibody resulted in a reduced growth rate of B. bovis in the in vitro culture. These findings indicated that B. bovis BOV57 plays a critical role in the invasion of merozoites into red blood cells, suggesting its potential as a subunit vaccine candidate against B. bovis infection in cattle. Furthermore, we analyzed the genetic diversity of bov57 gene sequences isolated from Sri Lanka, Mongolia, the Philippines, and Vietnam. The bov57 gene sequences derived from Mongolia, the Philippines, and Vietnam were conserved, whereas insertion and/or deletion mutations resulted in sequence diversity among the Sri Lankan samples. In summary, BOV57 is an invasion-related, neutralization-sensitive antigen encoded by the bov57 gene, which displays higher sequence diversity than previously reported.

Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (Pv RON1)

BackgroundPlasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1) and examine its antigenicity in natural P. vivax infections.MethodsThe PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA.ResultsIn the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax.ConclusionsThis study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that PvRON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended.

A reliable ex vivo invasion assay of human reticulocytes by Plasmodium vivax

AbstractCurrently, there are no reliable RBC invasion assays to guide the discovery of vaccines against Plasmodium vivax, the most prevalent malaria parasite in Asia and South America. Here we describe a protocol for an ex vivo P vivax invasion assay that can be easily deployed in laboratories located in endemic countries. The assay is based on mixing enriched cord blood reticulocytes with matured, trypsin-treated P vivax schizonts concentrated from clinical isolates. The reliability of this assay was demonstrated using a large panel of P vivax isolates freshly collected from patients in Thailand.

Protein Targeting to the Parasitophorous Vacuole Membrane of Plasmodium falciparum

Red blood cell (RBC) invasion and parasitophorous vacuole (PV) formation by Plasmodium falciparum are critical for the development and pathogenesis of malaria, a continuing global health problem. Expansion of the PV membrane (PVM) during growth is orchestrated by the parasite. This is particularly important in mature RBCs, which lack internal organelles and no longer actively synthesize membranes. Pfs16, a 16-kDa integral PVM protein expressed by gametocytes, was chosen as a model for studying the trafficking of material from the parasite across the PV space to the PVM. The locations of Pfs16-green fluorescent protein (GFP) reporter proteins containing distinct regions of Pfs16 were tracked from RBC invasion to emergence. Inclusion of the 53 C-terminal amino acids (aa) of Pfs16 to a GFP reporter construct already containing the N-terminal secretory signal sequence was sufficient for targeting to and retention on the PVM. An amino acid motif identified in this region was also found in seven other known PVM proteins. Removal of the 11 C-terminal aa did not affect PVM targeting, but membrane retention was decreased. Additionally, during emergence from the PVM and RBC, native Pfs16 and the full-length Pfs16-GFP reporter protein were found to concentrate on the ends of the gametocyte. Capping was not observed in constructs lacking the amino acids between the N-terminal secretory signal sequence and the transmembrane domain, suggesting that this region, which is not required for PVM targeting, is involved in capping. This is the first report to define the amino acid domains required for targeting to the P. falciparum PVM.

Population genetics of GYPB and association study between GYPB*S/s polymorphism and susceptibility to P. falciparum infection in the Brazilian Amazon.

BackgroundMerozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil.MethodsTwo groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection.Results GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity.ConclusionEpidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is associated with the GPB S+ variant in this population.

Purinergic signalling is involved in the malaria parasite Plasmodium falciparum invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca(2+) levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca(2+) increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca(2+) levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca(2+) in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca(2+)](c). Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca(2+)](c) in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells. The online version of this article (doi:10.1007/s11302-010-9202-y) contains supplementary material, which is available to authorized users.

Isolation of viable Plasmodium falciparum merozoites to define erythrocyte invasion events and advance vaccine and drug development

During blood-stage infection by Plasmodium falciparum, merozoites invade RBCs. Currently there is limited knowledge of cellular and molecular invasion events, and no established assays are available to readily measure and quantify invasion-inhibitory antibodies or compounds for vaccine and drug studies. We report the isolation of viable merozoites that retain their invasive capacity, at high purity and yield, purified by filtration of highly synchronous populations of schizonts. We show that the half-life of merozoite invasive capacity after rupture is 5 min at 37 degrees C, and 15 min at room temperature. Studying the kinetics of invasion revealed that 80% of invasion events occur within 10 min of mixing merozoites and RBCs. Invasion efficiency was maximum at low merozoite-to-RBC ratios and occurred efficiently in the absence of serum and with high concentrations of dialyzed nonimmune serum. We developed and optimized an invasion assay by using purified merozoites that enabled invasion-inhibitory activity of antibodies and compounds to be measured separately from other mechanisms of growth inhibition; the assay was more sensitive for detecting inhibitory activity than established growth-inhibition assays. Furthermore, with the use of purified merozoites it was possible to capture and fix merozoites at different stages of invasion for visualization by immunofluorescence microscopy and EM. We thereby demonstrate that processing of the major merozoite antigen merozoite surface protein-1 occurs at the time of RBC invasion. These findings have important implications for defining invasion events and molecular interactions, understanding immune interactions, and identifying and evaluating inhibitors to advance vaccine and drug development.

A Presenilin-like protease associated with Plasmodium falciparum micronemes is involved in erythrocyte invasion

We describe identification of a Plasmodium falciparum microneme protease involved in RBC invasion. From the yeast two-hybrid screening of a P. falciparum cDNA library, we have identified a 47 kDa membrane protein that interacted with the 5ABC domain of human RBC band 3. This protein shared homology with a Presenilin-type aspartyl protease, the signal peptide peptidase (SPP). An antibody raised against a predicted exposed region of this protein reacted specifically to a single band of ∼47 kDa in the P. falciparum protein extract. Immunofluorescence microscopy suggested that this protein co-localized with the microneme protein EBA-175 in schizonts, and immunoelectron microscopy established that it is primarily localized to micronemes in merozoites. Functional characterization of Plasmodium falciparum signal peptide peptidase (PfSPP), demonstrates that an antibody to PfSPP blocks RBC invasion by P. falciparum in vitro. Native and recombinant PfSPP bound directly to the 5ABC domain of band 3 in solution and the binding of PfSPP to RBCs was chymotrypsin-sensitive, but trypsin and neuraminidase-resistant. Together, these results suggest that host band 3 interacts with PfSPP during RBC invasion presumably following parasite microneme discharge. PfSPP is the first microneme-associated intramembrane aspartyl protease identified in the apicomplexan parasites that interacts with a major transmembrane receptor on host erythrocytes.

Involvement of a Host Erythrocyte Sialic Acid Content in Babesia bovis Infection

Host sialic acid (SA) has recently been suggested to play an important role in erythrocyte (RBC) infection by Babesia spp. The present study attempted to further determine the specific type of SAs important in the RBC invasion. Bovine RBC was found to bear abundant alpha2-3-linked SA residues but not alpha2-6-linked SA in nature, confirmed by flow cytometric analysis of the neuraminidase (Nm)-treated RBCs. Lectin-blot analyses revealed the removal of alpha2-3-linked SAs from the 97-, 33-, and 31-kDa bands by the Nm treatment. Addition of the Nm-treated RBCs into an in vitro culture of B. bovis resulted in a decreased population of the parasitized RBCs. The thin smear samples from the cultures were then observed under a confocal laser scanning microscope after staining with the alpha2-3-linked SA-specific lectin: a selective invasion of B. bovis was found only in the intact RBCs bearing the SAs, but not in the desialylated RBCs. Furthermore, a significant reduction of the parasitized RBCs was also observed in the culture supplemented with exogenous 3'-sialyllactose containing the alpha2-3-linked SAs. However, the complete inhibition of parasite proliferation was not achieved in the culture. These findings indicate that while the alpha2-3-linked SA-dependent pathway is needed for highly efficient invasion of host RBCs by B. bovis, there might also be other potential alternative pathways.

A Conserved Subtilisin Protease Identified in Babesia divergens Merozoites

Invasion of erythrocytes is an integral part of the Babesia divergens life cycle. Serine proteases have been shown to play an important role in invasion by related Apicomplexan parasites such as the malaria parasite Plasmodium falciparum. Here we demonstrate the presence of two dominant serine proteases in asexual B. divergens using a biotinylated fluorophosphonate probe. One of these active serine proteases (p48) and its precursors were recognized by anti-PfSUB1 antibodies. These antibodies were used to clone the gene encoding a serine protease using a B. divergens cDNA library. BdSub-1 is a single copy gene with no introns. The deduced gene product (BdSUB-1) clearly belongs to the subtilisin superfamily and shows significant homology to Plasmodium subtilisins, with the highest degree of sequence identity around the four catalytic residues. Like subtilisin proteases in other Apicomplexan parasites, BdSUB-1 undergoes two steps of processing during activation in the secretory pathway being finally converted to an active form (p48). The mature protease is concentrated in merozoite dense granules, apical secretory organelles involved in erythrocyte invasion. Anti-PfSUB1 antibodies have a potent inhibitory effect on erythrocyte invasion by B. divergens merozoites in vitro. This report demonstrates conservation of the molecular machinery involved in erythrocyte invasion by these two Apicomplexan parasites and paves the way for a comparative analysis of other molecules that participate in this process in the two parasites.

Enhanced invasion of blood group A1 erythrocytes by Plasmodium falciparum

Invasion of the malaria parasite Plasmodium falciparum into human erythrocytes is a highly complex multi-step procedure. It involves parasite ligands on the merozoite interacting with receptors on the erythrocyte surface. P. falciparum is able to exploit multiple receptor ligand interactions to achieve invasion and to date, several erythrocyte surface receptors have already been identified [1]. However, several as yet unidentified receptors on the erythrocyte surface have also been postulated to play a role in invasion and the specific molecular interactions between merozoite surface ligands and erythrocyte receptors remain elusive. Inhibition of RBC invasion would prevent infection and consequently disease, therefore considerable effort has gone into identification and characterization of molecules involved in this process.

Bartonella henselae invasion of feline erythrocytes in vitro.

Bartonella henselae, the causative agent of cat scratch disease, establishes long-term bacteremia in cats, in which it attaches to and invades feline erythrocytes (RBC). Feline RBC invasion was assessed in vitro, based on gentamicin selection for intracellular bacteria or by laser confocal microscopy and digital sectioning. Invasion rates ranged from 2 to 20% of the inoculum, corresponding to infection of less than 1% of the RBC. Invasion was a slow process, requiring >8 h before significant numbers of intracellular bacteria were detected. Pretreatment of the bacteria with trypsin, or of the RBC with trypsin or neuraminidase, had no effect, but pronase pretreatment of RBC resulted in a slight increase in invasion frequency. The ability to model B. henselae invasion of feline RBC in vitro should permit identification of bacterial surface components involved in this process and elucidate the significance of RBC invasion to transmission and infection in cats.

A cell biologist's view of host cell recognition and invasion by malarial parasites

The migration of various Apicomplexan parasites through host cells and tissues is examined. Two approaches are taken. First a comparative examination of the invasive stages from an ultrastructural and biochemical viewpoint. Second a critical review of the mechanism of recognition and invasion of host cells. The ultrastructural organization of the invasive stages is highly conserved and invariably includes three classes of organelle whose probable functions can be deduced. These organelle classes are: (i) The rhoptry/microneme/osmiophilic body/microsphere complex. These electron-dense, membrane-limited organelles are situated close to the parasite plasmalemma, and are often concentrated or specifically localized within the apical complex. Their contents are probably secreted by the parasite and induce various modifications in the host cell plasmalemma. However, it is concluded that the exact role of these organelles has not yet been determined nor their mechanism of action adequately evaluated. (ii) The microtubule cytoskeleton and associated structures (e.g., apical rings and conoid, etc.). These are structural organelles, and the microtubules probably direct the locomotion of the parasites. Locomotion is possibly achieved by a directed capping reaction, (iii) The plasmalemma and inner membrane vacuoles. The plasmalemma has a glycocalyx of varying complexity which includes molecules capable of host cell recognition and binding. It is speculated that host cell invasion is achieved by the directed capping of these molecules. Whilst appreciating the very specific nature of host cell recognition and invasion in some parasite stages (e.g., RBC invasion by the malarial merozoite), a marked contrast is observed in the ability of other stages of the malarial life-cycle (sporozoite and ookinete) to invade, migrate through, and escape from varied types of host cell. The latter breadth of ability is even more evident in other members of the phylum. Questions are therefore raised on the relevance of specific recognition of host cell membranes in all cases of trans-membrane migration. It is speculated that receptors are required for the induction of a parasitophorous vacuole (and thus host cell survival), whereas receptors are of little relevance when the membrane and host cell are directly ruptured e.g., during escape from the previously parasitized host cell (in which case survival of the host cell is unimportant).