Coordinated Regulation of the Size and Number of Polyhydroxybutyrate Granules by Core and Accessory Phasins in the Facultative Microsymbiont Sinorhizobium fredii NGR234

ABSTRACT The exact roles of various granule-associated proteins (GAPs) of polyhydroxybutyrate (PHB) are poorly investigated, particularly for bacteria associated with plants. In this study, four structural GAPs, named phasins PhaP1 to PhaP4, were identified and demonstrated as true phasins colocalized with PHB granules in Sinorhizobium fredii NGR234, a facultative microsymbiont of Vigna unguiculata and many other legumes. The conserved PhaP2 dominated in regulation of granule size under both free-living and symbiotic conditions. PhaP1, another conserved phasin, made a higher contribution than accessory phasins PhaP4 and PhaP3 to PHB biosynthesis at stationary phase. PhaP3, with limited phyletic distribution on the symbiosis plasmid of Sinorhizobium, was more important than PhaP1 in regulating PHB biosynthesis in V. unguiculata nodules. Under the test conditions, no significant symbiotic defects were observed for mutants lacking individual or multiple phaP genes. The mutant lacking two PHB synthases showed impaired symbiotic performance, while mutations in individual PHB synthases or a PHB depolymerase yielded no symbiotic defects. This phenomenon is not related to either the number or size of PHB granules in test mutants within nodules. Distinct metabolic profiles and cocktail pools of GAPs of different phaP mutants imply that core and accessory phasins can be differentially involved in regulating other cellular processes in the facultative microsymbiont S. fredii NGR234. IMPORTANCE Polyhydroxybutyrate (PHB) granules are a store of carbon and energy in bacteria and archaea and play an important role in stress adaptation. Recent studies have highlighted distinct roles of several granule-associated proteins (GAPs) in regulating the size, number, and localization of PHB granules in free-living bacteria, though our knowledge of the role of GAPs in bacteria associated with plants is still limited. Here we report distinct roles of core and accessory phasins associated with PHB granules of Sinorhizobium fredii NGR234, a broad-host-range microsymbiont of diverse legumes. Core phasins PhaP2 and PhaP1 are conserved major phasins in free-living cells. PhaP2 and accessory phasin PhaP3, encoded by an auxiliary gene on the symbiosis plasmid, are major phasins in nitrogen-fixing bacteroids in cowpea nodules. GAPs and metabolic profiles can vary in different phaP mutants. Contrasting symbiotic performances between mutants lacking PHB synthases, depolymerase, or phasins were revealed.

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The Sinorhizobium fredii HH103 MucR1 Global Regulator Is Connected With the nod Regulon and Is Required for Efficient Symbiosis With Lotus burttii and Glycine max cv. Williams

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-06-16-0116-r ◽

2016 ◽

Vol 29 (9) ◽

pp. 700-712 ◽

Cited By ~ 11

Author(s):

Sebastián Acosta-Jurado ◽

Cynthia Alias-Villegas ◽

Pilar Navarro-Gómez ◽

Susanne Zehner ◽

Piedad del Socorro Murdoch ◽

...

Keyword(s):

Glycine Max ◽

Cell Aggregation ◽

Broad Host Range ◽

Rna Seq ◽

Free Living ◽

Sinorhizobium Fredii ◽

Global Regulators ◽

Enhanced Production ◽

Free Living Conditions

Sinorhizobium fredii HH103 is a rhizobial strain showing a broad host range of nodulation. In addition to the induction of bacterial nodulation genes, transition from a free-living to a symbiotic state requires complex genetic expression changes with the participation of global regulators. We have analyzed the role of the zinc-finger transcriptional regulator MucR1 from S. fredii HH103 under both free-living conditions and symbiosis with two HH103 host plants, Glycine max and Lotus burttii. Inactivation of HH103 mucR1 led to a severe decrease in exopolysaccharide (EPS) biosynthesis but enhanced production of external cyclic glucans (CG). This mutant also showed increased cell aggregation capacity as well as a drastic reduction in nitrogen-fixation capacity with G. max and L. burttii. However, in these two legumes, the number of nodules induced by the mucR1 mutant was significantly increased and decreased, respectively, with respect to the wild-type strain, indicating that MucR1 can differently affect nodulation depending on the host plant. RNA-Seq analysis carried out in the absence and the presence of flavonoids showed that MucR1 controls the expression of hundreds of genes (including some related to EPS production and CG transport), some of them being related to the nod regulon.

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The Sinorhizobium (Ensifer) fredii HH103 Nodulation Outer Protein NopI Is a Determinant for Efficient Nodulation of Soybean and Cowpea Plants

Applied and Environmental Microbiology ◽

10.1128/aem.02770-16 ◽

2016 ◽

Vol 83 (5) ◽

Cited By ~ 14

Author(s):

Irene Jiménez-Guerrero ◽

Francisco Pérez-Montaño ◽

Carlos Medina ◽

Francisco Javier Ollero ◽

Francisco Javier López-Baena

Keyword(s):

Adenylate Cyclase ◽

Host Cell ◽

Host Range ◽

Pathogenic Bacteria ◽

Defense Responses ◽

Host Cells ◽

Broad Host Range ◽

Sinorhizobium Fredii ◽

Symbiotic Efficiency ◽

Content Type

ABSTRACT The type III secretion system (T3SS) is a specialized secretion apparatus that is commonly used by many plant and animal pathogenic bacteria to deliver proteins, termed effectors, to the interior of the host cells. These effectors suppress host defenses and interfere with signal transduction pathways to promote infection. Some rhizobial strains possess a functional T3SS, which is involved in the suppression of host defense responses, host range determination, and symbiotic efficiency. The analysis of the genome of the broad-host-range rhizobial strain Sinorhizobium fredii HH103 identified eight genes that code for putative T3SS effectors. Three of these effectors, NopL, NopP, and NopI, are Rhizobium specific. In this work, we demonstrate that NopI, whose amino acid sequence shows a certain similarity with NopP, is secreted through the S. fredii HH103 T3SS in response to flavonoids. We also determined that NopL can be considered an effector since it is directly secreted to the interior of the host cell as demonstrated by adenylate cyclase assays. Finally, the symbiotic phenotype of single, double, and triple nopI, nopL, and nopP mutants in soybean and cowpea was assayed, showing that NopI plays an important role in determining the number of nodules formed in both legumes and that the absence of both NopL and NopP is highly detrimental for symbiosis. IMPORTANCE The paper is focused on three Rhizobium-specific T3SS effectors of Sinorhizobium fredii HH103, NopL, NopP, and NopI. We demonstrate that S. fredii HH103 is able to secrete through the T3SS in response to flavonoids the nodulation outer protein NopI. Additionally, we determined that NopL can be considered an effector since it is secreted to the interior of the host cell as demonstrated by adenylate cyclase assays. Finally, nodulation assays of soybean and cowpea indicated that NopI is important for the determination of the number of nodules formed and that the absence of both NopL and NopP negatively affected nodulation.

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Targeting Toxoplasma Tubules: Tubulin, Microtubules, and Associated Proteins in a Human Pathogen

Eukaryotic Cell ◽

10.1128/ec.00225-14 ◽

2014 ◽

Vol 14 (1) ◽

pp. 2-12 ◽

Cited By ~ 26

Author(s):

Naomi Morrissette

Keyword(s):

Opportunistic Infections ◽

Genetic Recombination ◽

Flagellar Apparatus ◽

Content Type ◽

Obligate Intracellular ◽

Spiral Trajectory ◽

Parasite Survival ◽

Associated Proteins ◽

Flagellar Axoneme

ABSTRACTToxoplasma gondiiis an obligate intracellular parasite that causes serious opportunistic infections, birth defects, and blindness in humans. Microtubules are critically important components of diverse structures that are used throughout theToxoplasmalife cycle. As in other eukaryotes, spindle microtubules are required for chromosome segregation during replication. Additionally, a set of membrane-associated microtubules is essential for the elongated shape of invasive “zoites,” and motility follows a spiral trajectory that reflects the path of these microtubules.Toxoplasmazoites also construct an intricate, tubulin-based apical structure, termed the conoid, which is important for host cell invasion and associates with proteins typically found in the flagellar apparatus. Last, microgametes specifically construct a microtubule-containing flagellar axoneme in order to fertilize macrogametes, permitting genetic recombination. The specialized roles of these microtubule populations are mediated by distinct sets of associated proteins. This review summarizes our current understanding of the role of tubulin, microtubule populations, and associated proteins inToxoplasma; these components are used for both novel and broadly conserved processes that are essential for parasite survival.

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Identification of Obscure yet Conserved Actin-Associated Proteins in Giardia lamblia

Eukaryotic Cell ◽

10.1128/ec.00041-14 ◽

2014 ◽

Vol 13 (6) ◽

pp. 776-784 ◽

Cited By ~ 20

Author(s):

Alexander R. Paredez ◽

Arash Nayeri ◽

Jennifer W. Xu ◽

Jana Krtková ◽

W. Zacheus Cande

Keyword(s):

Protein Identification ◽

Binding Proteins ◽

Affinity Purification ◽

Actin Binding ◽

Interacting Proteins ◽

Actin Binding Proteins ◽

Content Type ◽

Cellular Processes ◽

Multidimensional Protein Identification Technology ◽

Associated Proteins

ABSTRACTConsistent with its proposed status as an early branching eukaryote,Giardiahas the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing fromGiardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins inGiardiausing affinity purification coupled with mass spectroscopy (multidimensional protein identification technology [MudPIT]) and have identified >80 putative actin-binding proteins. Several of these have homology to conserved proteins known to complex with actin for functions in the nucleus and flagella. We validated localization and interaction for seven of these proteins, including 14-3-3, a known cytoskeletal regulator with a controversial relationship to actin. Our results indicate that althoughGiardialacks canonical actin-binding proteins, there is a conserved set of actin-interacting proteins that are evolutionarily indispensable and perhaps represent some of the earliest functions of the actin cytoskeleton.

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An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease

Journal of Bacteriology ◽

10.1128/jb.00453-16 ◽

2016 ◽

Vol 199 (1) ◽

Cited By ~ 16

Author(s):

Richard E. Wiemels ◽

Stephanie M. Cech ◽

Nikki M. Meyer ◽

Caleb A. Burke ◽

Andy Weiss ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Virulence Factors ◽

Virulence Factor ◽

Active Form ◽

Ppiase Activity ◽

Content Type ◽

Associated Proteins

ABSTRACT Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus. IMPORTANCE Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.

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Evolution of the Arsenal ofLegionella pneumophilaEffectors To Modulate Protist Hosts

mBio ◽

10.1128/mbio.01313-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 13

Author(s):

Ashley Best ◽

Yousef Abu Kwaik

Keyword(s):

Legionella Pneumophila ◽

Effector Proteins ◽

Host Cells ◽

Human Pathogen ◽

Intracellular Growth ◽

Content Type ◽

Human Macrophages ◽

Cellular Processes ◽

Host Interaction

ABSTRACTWithin the human host,Legionella pneumophilareplicates within alveolar macrophages, leading to pneumonia. However,L. pneumophilais an aquatic generalist pathogen that replicates within a wide variety of protist hosts, including amoebozoa, percolozoa, and ciliophora. The intracellular lifestyles ofL. pneumophilawithin the two evolutionarily distant hosts macrophages and protists are remarkably similar. Coevolution with numerous protist hosts has shaped plasticity of the genome ofL. pneumophila, which harbors numerous proteins encoded by genes acquired from primitive eukaryotic hosts through interkingdom horizontal gene transfer. The Dot/Icm type IVb translocation system translocates ∼6,000 effectors amongLegionellaspecies and >320 effector proteins inL. pneumophilainto host cells to modulate a plethora of cellular processes to create proliferative niches. Since many of the effectors have likely evolved to modulate cellular processes of primitive eukaryotic hosts, it is not surprising that most of the effectors do not contribute to intracellular growth within human macrophages. Some of the effectors may modulate highly conserved eukaryotic processes, while others may target protist-specific processes that are absent in mammals. The lack of studies to determine the role of the effectors in adaptation ofL. pneumophilato various protists has hampered the progress to determine the function of most of these effectors, which are routinely studied in mouse or human macrophages. Since many protists restrictL. pneumophila, utilization of such hosts can also be instrumental in deciphering the mechanisms of failure ofL. pneumophilato overcome restriction of certain protist hosts. Here, we review the interaction ofL. pneumophilawith its permissive and restrictive protist environmental hosts and outline the accomplishments as well as gaps in our knowledge ofL. pneumophila-protist host interaction andL. pneumophila’s evolution to become a human pathogen.

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Comprehensive analysis of yeast ESCRT-III composition in single ESCRT-III deletion mutants

Biochemical Journal ◽

10.1042/bcj20190141 ◽

2019 ◽

Vol 476 (14) ◽

pp. 2031-2046 ◽

Cited By ~ 2

Author(s):

Christian Heinzle ◽

Lara Mücke ◽

Thomas Brune ◽

Ralf Kölling

Keyword(s):

Family Members ◽

Cell Fractionation ◽

Endosomal Sorting ◽

Cellular Processes ◽

Associated Proteins ◽

Bona Fide ◽

Main Component ◽

The Impact

Abstract The endosomal sorting complex required for transport (ESCRT)-III is associated with a multitude of cellular processes involving membrane remodeling and abscission. The exact composition of ESCRT-III and the contribution of individual ESCRT-III family members to these diverse functions is unclear. Most of the currently available information about ESCRT-III was obtained with tagged, largely non-functional proteins, which may not correctly reflect the in vivo situation. Here, we performed a comprehensive biochemical analysis of ESCRT-III localization and composition in yeast under purely native conditions. Most of our findings are in line with the current concepts about ESCRT-III, but some findings are unexpected and call for adjustments to the model. In particular, our data suggest that the distinction between bona fide ESCRT-III components and ESCRT-III associated proteins is not justified. We detected a single complex containing all ESCRT-III members (except of Chm7) with Did2 as its main component. The classical core components were present in equimolar amounts. Our analysis of the impact of single deletions on the composition of ESCRT-III confirmed the central role of Snf7 for ESCRT-III assembly. For the other ESCRT-III family members predictions could be made about their role in ESCRT-III assembly. Furthermore, our cell fractionation points to a role of Vps20 at the endoplasmic reticulum.

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Life at Home and on the Roam: Genomic Adaptions Reflect the Dual Lifestyle of an Intracellular, Facultative Symbiont

mSystems ◽

10.1128/msystems.00057-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 8

Author(s):

Ilia Burgsdorf ◽

Kim M. Handley ◽

Rinat Bar-Shalom ◽

Patrick M. Erwin ◽

Laura Steindler

Keyword(s):

Defense Mechanisms ◽

Genetic Regulation ◽

Free Living ◽

Symbiotic Interaction ◽

Genomic Features ◽

Content Type ◽

Intracellular Symbiont ◽

Stem Lineage ◽

Associated Proteins ◽

Sponge Symbionts

ABSTRACT “Candidatus Synechococcus feldmannii” is a facultative intracellular symbiont of the Atlanto-Mediterranean sponge Petrosia ficiformis. Genomic information of sponge-associated cyanobacteria derives thus far from the obligate and extracellular symbiont “Candidatus Synechococcus spongiarum.” Here we utilized a differential methylation-based approach for bacterial DNA enrichment combined with metagenomics to obtain the first draft genomes of “Ca. Synechococcus feldmannii.” By comparative genomics, we revealed that some genomic features (e.g., iron transport mediated by siderophores, eukaryotic-like proteins, and defense mechanisms, like CRISPR-Cas [clustered regularly interspaced short palindromic repeats-associated proteins]) are unique to both symbiont types and absent or rare in the genomes of taxonomically related free-living cyanobacteria. These genomic features likely enable life under the conditions found inside the sponge host. Interestingly, there are many genomic features that are shared by “Ca. Synechococcus feldmannii” and free-living cyanobacteria, while they are absent in the obligate symbiont “Ca. Synechococcus spongiarum.” These include genes related to cell surface structures, genetic regulation, and responses to environmental stress, as well as the composition of photosynthetic genes and DNA metabolism. We speculate that the presence of these genes confers on “Ca. Synechococcus feldmannii” its facultative nature (i.e., the ability to respond to a less stable environment when free-living). Our comparative analysis revealed that distinct genomic features depend on the nature of the symbiotic interaction: facultative and intracellular versus obligate and extracellular. IMPORTANCE Given the evolutionary position of sponges as one of the earliest phyla to depart from the metazoan stem lineage, studies on their distinct and exceptionally diverse microbial communities should yield a better understanding of the origin of animal-bacterium interactions. While genomes of several extracellular sponge symbionts have been published, the intracellular symbionts have, so far, been elusive. Here we compare the genomes of two unicellular cyanobacterial sponge symbionts that share an ancestor but followed different evolutionary paths—one became intracellular and the other extracellular. Counterintuitively, the intracellular cyanobacteria are facultative, while the extracellular ones are obligate. By sequencing the genomes of the intracellular cyanobacteria and comparing them to the genomes of the extracellular symbionts and related free-living cyanobacteria, we show how three different cyanobacterial lifestyles are reflected by adaptive genomic features.

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Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants

Applied and Environmental Microbiology ◽

10.1128/aem.03454-15 ◽

2015 ◽

Vol 82 (4) ◽

pp. 1305-1315 ◽

Cited By ~ 14

Author(s):

Yue Zhen Li ◽

Dan Wang ◽

Xue Ying Feng ◽

Jian Jiao ◽

Wen Xin Chen ◽

...

Keyword(s):

Critical Role ◽

Nodule Occupancy ◽

Phosphotransferase System ◽

Carbon Utilization ◽

Sinorhizobium Fredii ◽

Content Type ◽

Symbiotic Interactions ◽

Mutant Strains ◽

Negative Role

ABSTRACTThe nitrogen phosphotransferase system (PTSNtr) consists of EINtr, NPr, and EIIANtr. The active phosphate moiety derived from phosphoenolpyruvate is transferred through EINtrand NPr to EIIANtr.Sinorhizobium frediican establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study,S. frediistrains with mutations inptsPandptsO(encoding EINtrand NPr, respectively) formed ineffective nodules on soybeans, while a strain with aptsNmutation (encoding EIIANtr) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-β-hydroxybutyrate granules in bacteroids were observed in nodules infected by theptsPorptsOmutant strains but not in those infected with theptsNmutant strain. However, these defects of theptsPandptsOmutant strains were recovered inptsP ptsNandptsO ptsNdouble-mutant strains, implying a negative role of unphosphorylated EIIANtrin symbiosis. Moreover, the symbiotic defect of theptsPmutant was also recovered by expressing EINtrwith or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTSNtrin symbiosis was also observed when related PTSNtrmutant strains ofS. frediiwere inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTSNtrin addition to the negative role of unphosphorylated EIIANtr.

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Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti

Infection and Immunity ◽

10.1128/iai.00107-16 ◽

2016 ◽

Vol 84 (5) ◽

pp. 1574-1584 ◽

Cited By ~ 10

Author(s):

Rosalynn L. Ord ◽

Marilis Rodriguez ◽

Jeny R. Cursino-Santos ◽

Hyunryung Hong ◽

Manpreet Singh ◽

...

Keyword(s):

Direct Role ◽

Parasite Invasion ◽

Content Type ◽

Apicomplexan Parasites ◽

Babesia Divergens ◽

Associated Proteins ◽

Identification And Characterization

Apicomplexan parasites include those of the generaPlasmodium,Cryptosporidium, andToxoplasmaand those of the relatively understudied zoonotic genusBabesia. In humans, babesiosis, particularly transfusion-transmitted babesiosis, has been emerging as a major threat to public health. Like malaria, the disease pathology is a consequence of the parasitemia which develops through cyclical replication ofBabesiaparasites in host erythrocytes. However, there are no exoerythrocytic stages inBabesia, so targeting of the blood stage and associated proteins to directly prevent parasite invasion is the most desirable option for effective disease control. Especially promising among such molecules are the rhoptry neck proteins (RONs), whose homologs have been identified in many apicomplexan parasites. RONs are involved in the formation of the moving junction, along with AMA1, but no RON has been identified and characterized in anyBabesiaspp. Here we identify the RON2 proteins ofBabesia divergens(BdRON2) andB. microti(BmRON2) and show that they are localized apically and that anti-BdRON2 antibodies are significant inhibitors of parasite invasionin vitro. Neither protein is immunodominant, as both proteins react only marginally with sera from infected animals. Further characterization of the direct role of both BdRON2 and BmRON2 in parasite invasion is required, but knowledge of the level of conformity of RON2 proteins within the apicomplexan phylum, particularly that of the AMA1-RON2 complex at the moving junction, along with the availability of an animal model forB. microtistudies, provides a key to target this complex with a goal of preventing the erythrocytic invasion of these parasites and to further our understanding of the role of these conserved ligands in invasion.

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