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The bacterial cell envelope consists of multiple layers, including the peptidoglycan cell wall, one or two membranes, and often an external layer composed of capsular polysaccharides (CPS) or other components. How the synthesis of all these layers is precisely coordinated remains unclear. Here, we identify a mechanism that coordinates the synthesis of CPS and peptidoglycan in Streptococcus pneumoniae. We show that CPS synthesis initiates from the division septum and propagates along the long axis of the cell, organized by the tyrosine kinase system CpsCD. CpsC and the rest of the CPS synthesis complex are recruited to the septum by proteins associated with the divisome (a complex involved in septal peptidoglycan synthesis) but not the elongasome (involved in peripheral peptidoglycan synthesis). Assembly of the CPS complex starts with CpsCD, then CpsA and CpsH, the glycosyltransferases, and finally CpsJ. Remarkably, targeting CpsC to the cell pole is sufficient to reposition CPS synthesis, leading to diplococci that lack CPS at the septum. We propose that septal CPS synthesis is important for chain formation and complement evasion, thereby promoting bacterial survival inside the host. The bacterial cell envelope consists of multiple layers, the synthesis of which is coordinated through unclear mechanisms. Here, Nakamoto et al. reveal a mechanism linking the synthesis of capsular polysaccharides and cell wall peptidoglycan in pneumococci. [ABSTRACT FROM AUTHOR]

Abstract Background The pathogenicity of Plasmodium falciparum is in part due to the ability of the parasitized red blood cell (pRBC) to adhere to intra-vascular host cell receptors and serum-proteins. Binding of the pRBC is mediated by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large multi-variant molecule encoded by a family of ≈60 var genes. Methods The study of var gene transcription in the parasite clone FCR3S1.2 was performed by semi-quantitative PCR and quantitative PCR (qPCR). The expression of the major PfEMP1 in FCR3S1.2 pRBC was analysed with polyclonal sera in rosette disruption assays and immunofluorecence. Results Transcripts from var1 (FCR3S1.2var1; IT4var21) and other var genes were detected by semi-quantitative PCR but results from qPCR showed that one var gene transcript dominated over the others (FCR3S1.2var2; IT4var60). Antibodies raised in rats to the recombinant NTS-DBL1α of var2 produced in E. coli completely and dose-dependently disrupted rosettes (≈95% at a dilution of 1/5). The sera reacted with the Maurer's clefts in trophozoite stages (IFA) and to the infected erythrocyte surface (FACS) indicating that FCR3S1.2var2 encodes the dominant PfEMP1 expressed in this parasite. Conclusion The major transcript in the rosetting model parasite FCR3S1.2 is FCR3S1.2var2 (IT4var60). The results suggest that this gene encodes the PfEMP1-species responsible for the rosetting phenotype of this parasite. The activity of previously raised antibodies to the NTS-DBL1α of FCR3S1.2var1 is likely due to cross-reactivity with NTS-DBL1α of the var2 encoded PfEMP1.

Emerging resistance to current anti-malarials necessitates a more detailed understanding of the biological processes of Plasmodium falciparum proliferation, thus allowing identification of new drug targets. The well-conserved protein Receptor for Activated C-Kinase 1 (RACK1) was originally identified in mammalian cells as an anchoring protein for protein kinase C (PKC) and has since been shown to be important for cell migration, cytokinesis, transcription, epigenetics, and protein translation. The P. falciparum ortholog, PfRACK1, is expressed in blood stages of the parasite and is diffusely localized in the parasite cytoplasm. Using a destabilizing domain to allow inducible knockdown of the endogenous protein level, we evaluated the requirement for PfRACK1 during blood-stage replication. Following destabilization, the parasites demonstrate a nearly complete growth arrest at the trophozoite stage. The essential nature of PfRACK1 suggests that the protein itself or the pathways regulated by the protein are potential targets for novel anti-malarial therapeutics. [ABSTRACT FROM AUTHOR]

The human malaria parasite Plasmodium falciparum requires efficient egress out of an infected red blood cell for pathogenesis. This egress event is highly coordinated and is mediated by several signaling proteins, including the plant-like P. falciparum calcium-dependent protein kinase 5 (PfCDPK5). Knockdown of PfCDPK5 results in an egress block where parasites are trapped inside their host cells. The mechanism of this PfCDPK5-dependent block, however, remains unknown. Here, we show that PfCDPK5 colocalizes with a specialized set of parasite organelles known as micronemes and is required for their discharge, implicating failure of this step as the cause of the egress defect in PfCDPK5-deficient parasites. Furthermore, we show that PfCDPK5 cooperates with the P. falciparum cGMP-dependent kinase (PfPKG) to fully activate the protease cascade critical for parasite egress. The PfCDPK5-dependent arrest can be overcome by hyperactivation of PfPKG or by physical disruption of the arrested parasite, and we show that both treatments facilitate the release of the micronemes required for egress. Our results define the molecular mechanism of PfCDPK5 function and elucidate the complex signaling pathway of parasite egress.

Plasmodium falciparum is the most lethal of the human malaria parasites. The virulence is associated with the capacity of the infected red blood cell (iRBC) to sequester inside the deep microvasculature where it may cause obstruction of the blood-flow when binding is excessive. Rosetting, the adherence of the iRBC to uninfected erythrocytes, has been found associated with severe malaria and found to be mediated by the NTS-DBL1α-domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Here we show that the reactivity of plasma of Cameroonian children with the surface of the FCR3S1.2-iRBC correlated with the capacity to disrupt rosettes and with the antibody reactivity with a recombinant PfEMP1 (NTS-DBL1α of IT4var60) expressed by parasite FCR3S1.2. The plasma-reactivity in a microarray, consisting of 96 overlapping 15-mer long peptides covering the NTS-DBL1α domain from IT4var60 sequence, was compared with their capacity to disrupt rosettes and we identified five peptides where the reactivity were correlated. Three of the peptides were localized in subdomain-1 and 2. The other two peptide-sequences were localized in the NTS-domain and in subdomain-3. Further, principal component analysis and orthogonal partial least square analysis generated a model that supported these findings. In conclusion, human antibody reactivity with short linear-peptides of NTS-DBL1α of PfEMP1 suggests subdomains 1 and 2 to hold anti-rosetting epitopes recognized by anti-rosetting antibodies. The data suggest rosetting to be mediated by the variable areas of PfEMP1 but also to involve structurally relatively conserved areas of the molecule that may induce biologically active antibodies. [ABSTRACT FROM AUTHOR]

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1α previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1a-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1α antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface. [ABSTRACT FROM AUTHOR]

The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteineresidues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies. [ABSTRACT FROM AUTHOR]

Background: The pathogenicity of Plasmodium falciparum is in part due to the ability of the parasitized red blood cell (pRBC) to adhere to intra-vascular host cell receptors and serum-proteins. Binding of the pRBC is mediated by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large multi-variant molecule encoded by a family of ≈60 var genes. Methods: The study of var gene transcription in the parasite clone FCR3S1.2 was performed by semi-quantitative PCR and quantitative PCR (qPCR). The expression of the major PfEMP1 in FCR3S1.2 pRBC was analysed with polyclonal sera in rosette disruption assays and immunofluorecence. Results: Transcripts from var1 (FCR3S1.2var1; IT4var21) and other var genes were detected by semi-quantitative PCR but results from qPCR showed that one var gene transcript dominated over the others (FCR3S1.2var2; IT4var60). Antibodies raised in rats to the recombinant NTS-DBL1α of var2 produced in E. coli completely and dose-dependently disrupted rosettes (≈95% at a dilution of 1/5). The sera reacted with the Maurer's clefts in trophozoite stages (IFA) and to the infected erythrocyte surface (FACS) indicating that FCR3S1.2var2 encodes the dominant PfEMP1 expressed in this parasite. Conclusion: The major transcript in the rosetting model parasite FCR3S1.2 is FCR3S1.2var2 (IT4var60). The results suggest that this gene encodes the PfEMP1-species responsible for the rosetting phenotype of this parasite. The activity of previously raised antibodies to the NTS-DBL1a of FCR3S1.2var1 is likely due to cross-reactivity with NTS-DBL1a of the var2 encoded PfEMP1. [ABSTRACT FROM AUTHOR]

Abstract: A major feature of Plasmodium falciparum parasitized red blood cells (pRBC) is their capacity to sequester in the microcirculation. The binding is mediated by PfEMP1 (P. falciparum erythrocyte membrane protein 1), a variable protein encoded by the var gene family. P. falciparum avoids the host antibody response generated against previously used variants by switching the expression of PfEMP1, which may affect the disease outcome. We have here studied var gene transcription over time within the life cycle of the parasite by semi-quantitative PCR and sequencing by employing three sets of degenerate primers to the 5-prime end of the var genes (corresponding to the DBL1α-domain). To accurately determine transcript levels, subsequent in-depth analysis was made by amplifying the 10 most frequently expressed var sequences identified in each developmental stage by quantitative PCR (Q-PCR). The maximum peak in var gene transcription seems to vary in time among parasites. In five out of seven parasites, var gene transcription was found to be higher or equal at 22–26h post-invasion compared to 4–10h post-invasion. Our data indicate that the intra-isolate var gene transcription dominance order may change between different developmental stages. The transcription of var genes in field isolates is more complex than in laboratory strains and often changes after in vitro adaption of the parasite. By using semi-quantitative PCR employing degenerate primers combined with quantitative-PCR using specific primers it is possible to monitor var gene transcription in detail during the life cycle of the parasite. The work presented here suggests that trophozoite pRBC is likely to be the optimal source of RNA for predicting the translated var gene species. [Copyright &y& Elsevier]

Plasmodium falciparum, the mosquito-transmitted Apicomplexan parasite, causes the most severe form of human malaria. In the asexual blood-stage, the parasite resides within erythrocytes where it proliferates, multiplies and finally spreads to new erythrocytes. Development of drugs targeting the ribosome, the site of protein synthesis, requires specific knowledge of its structure and work cycle, and, critically, the ways they differ from those in the human host. Here, we present five cryo-electron microscopy (cryo-EM) reconstructions of ribosomes purified from P. falciparum blood-stage schizonts at sub-nanometer resolution. Atomic models were built from these density maps by flexible fitting. Significantly, our study has taken advantage of new capabilities of cryo-EM, in visualizing several structures co-existing in the sample at once, at a resolution sufficient for building atomic models. We have discovered structural and dynamic features that differentiate the ribosomes of P. falciparum from those of mammalian system. Prompted by the absence of RACK1 on the ribosome in our and an earlier study we confirmed that RACK1 does not specifically co-purify with the 80S fraction in schizonts. More extensive studies, using cryo-EM methodology, of translation in the parasite will provide structural knowledge that may lead to development of novel anti-malarials.