Elizabeth S. Egan

Zebrafish organizer development and germ-layer formation require nodal-related signals

The vertebrate body plan is established during gastrulation, when cells move inwards to form the mesodermal and endodermal germ layers. Signals from a region of dorsal mesoderm, which is termed the organizer, pattern the body axis by specifying the fates of neighbouring cells,. The organizer is itself induced by earlier signals. Although members of the transforming growth factor-β (TGF-β) and Wnt families have been implicated in the formation of the organizer, no endogenous signalling molecule is known to be required for this process. Here we report that the zebrafish squint (sqt) and cyclops (cyc) genes have essential, although partly redundant, functions in organizer development and also in the formation of mesoderm and endoderm. We show that the sqt gene encodes a member of the TGF-β superfamily that is related to mouse nodal. cyc encodes another nodal-related protein,, which is consistent with our genetic evidence that sqt and cyc have overlapping functions. The sqt gene is expressed in a dorsal region of the blastula that includes the extraembryonic yolk syncytial layer (YSL). The YSL has been implicated as a source of signals that induce organizer development and mesendoderm formation,. Misexpression of sqt RNA within the embryo or specifically in the YSL induces expanded or ectopic dorsal mesoderm. These results establish an essential role for nodal-related signals in organizer development and mesendoderm formation.

Distinct Replication Requirements for the Two Vibrio cholerae Chromosomes

Studies of prokaryotic chromosome replication have focused almost exclusively on organisms with one chromosome. We defined and characterized the origins of replication of the two Vibrio cholerae chromosomes, oriCI(vc) and oriCII(vc). OriCII(vc) differs from the origin assigned by bioinformatic analysis and is unrelated to oriCI(vc). OriCII(vc)-based replication requires an internal 12 base pair repeat and two hypothetical genes that flank oriCII(vc). One of these genes is conserved among diverse genera of the family Vibrionaceae and encodes an origin binding protein. The other gene codes for an RNA and not a protein. OriCII(vc)- but not oriCI(vc)-based replication is negatively regulated by a DNA sequence adjacent to oriCII(vc). There is an unprecedented requirement for DNA adenine methyltransferase in both oriCI(vc)- and oriCII(vc)-based replication. Our studies of replication in V. cholerae indicate that microorganisms having multiple chromosomes may utilize unique mechanisms for the control of replication.

Divided genomes: negotiating the cell cycle in prokaryotes with multiple chromosomes.

Historically, the prokaryotic genome was assumed to consist of a single circular replicon. However, as more microbial genome sequencing projects are completed, it is becoming clear that multipartite genomes comprised of more than one chromosome are not unusual among prokaryotes. Chromosomes are distinguished from plasmids by the presence of essential genes as well as characteristic cell cycle‐linked replication kinetics; unlike plasmids, chromosomes initiate replication once per cell cycle. The existence of multipartite prokaryotic genomes raises several questions regarding how multiple chromosomes are replicated and segregated during the cell cycle. These divided genomes also introduce questions regarding chromosome evolution and genome stability. In this review, we discuss these and other issues, with particular emphasis on the cholera pathogen Vibrio cholerae.

Plasmodium falciparum transmission stages accumulate in the human bone marrow

Sexual stages of the human malaria parasite Plasmodium falciparum use the hematopoietic system of the bone marrow as a developmental niche. Digging Deep for Malaria Parasites Malaria remains a major public health problem in developing countries. The pathogenesis of the most deadly of human malaria parasites, Plasmodium falciparum, is related to the ability of infected red blood cells to sequester in the microvasculature of deep tissues. Using an existing tissue collection from malaria autopsy cases, Joice et al. now reveal that P. falciparum transmission stages sequester in the hematopoietic system of the human bone marrow. This finding suggests that new mechanisms may be involved in the sequestration of these transmission stages and that the hematopoietic system may be a major site of formation, development, and maturation of malaria transmission stages. Transmission of Plasmodium falciparum malaria parasites requires formation and development of gametocytes, yet all but the most mature of these sexual parasite forms are absent from the blood circulation. We performed a systematic organ survey in pediatric cases of fatal malaria to characterize the spatial dynamics of gametocyte development in the human host. Histological studies revealed a niche in the extravascular space of the human bone marrow where gametocytes formed in erythroid precursor cells and underwent development before reentering the circulation. Accumulation of gametocytes in the hematopoietic system of human bone marrow did not rely on cytoadherence to the vasculature as does sequestration of asexual-stage parasites. This suggests a different mechanism for the sequestration of gametocytes that could potentially be exploited to block malaria transmission.

MicroReview: Divided genomes: negotiating the cell cycle in prokaryotes with multiple chromosomes

Historically, the prokaryotic genome was assumed to consist of a single circular replicon. However, as more microbial genome sequencing projects are completed, it is becoming clear that multipartite genomes comprised of more than one chromosome are not unusual among prokaryotes. Chromosomes are distinguished from plasmids by the presence of essential genes as well as characteristic cell cycle‐linked replication kinetics; unlike plasmids, chromosomes initiate replication once per cell cycle. The existence of multipartite prokaryotic genomes raises several questions regarding how multiple chromosomes are replicated and segregated during the cell cycle. These divided genomes also introduce questions regarding chromosome evolution and genome stability. In this review, we discuss these and other issues, with particular emphasis on the cholera pathogen Vibrio cholerae.

Independent Control of Replication Initiation of the Two Vibrio cholerae Chromosomes by DnaA and RctB

Although the two Vibrio cholerae chromosomes initiate replication in a coordinated fashion, we show here that each chromosome appears to have a specific replication initiator. DnaA overproduction promoted overinitiation of chromosome I and not chromosome II. In contrast, overproduction of RctB, a protein that binds to the origin of replication of chromosome II, promoted overinitiation of chromosome II and not chromosome I.

A forward genetic screen identifies erythrocyte CD55 as essential for Plasmodium falciparum invasion

A way to dissect malarias secrets Malaria has exerted a strong selective force on the human genome. However, efforts to identify host susceptibility factors have been hindered by the absence of a nucleus in red blood cells. Egan et al. developed an approach involving blood stem cells to discover host factors critical for Plasmodium falciparum infection of red blood cells. The authors identified an essential host receptor for parasite invasion that could provide a target for malaria therapeutics. Science, this issue p. 711 A screening approach reveals host factors critical for human malaria parasite invasion of red blood cells. Efforts to identify host determinants for malaria have been hindered by the absence of a nucleus in erythrocytes, which precludes genetic manipulation in the cell in which the parasite replicates. We used cultured red blood cells derived from hematopoietic stem cells to carry out a forward genetic screen for Plasmodium falciparum host determinants. We found that CD55 is an essential host factor for P. falciparum invasion. CD55-null erythrocytes were refractory to invasion by all isolates of P. falciparum because parasites failed to attach properly to the erythrocyte surface. Thus, CD55 is an attractive target for the development of malaria therapeutics. Hematopoietic stem cell–based forward genetic screens may be valuable for the identification of additional host determinants of malaria pathogenesis.

Synchronous replication initiation of the two Vibrio cholerae chromosomes.

The genome of Vibrio cholerae, the causative agent of cholera, is divided between two circular replicons. Genomic analysis has revealed that several other bacterial species have more than one chromosome. However, to date, the dynamics of chromosome replication in bacteria with more than one chromosome have not been investigated. As in V. cholerae chromosome II (1.07 Mb) is smaller than chromosome I (2.96 Mb) and contains fewer essential genes, it was proposed that it originated as a plasmid [1]. The replication of chromosome II shows both plasmid and chromosome-like features [2]. Whereas prokaryotic and eukaryotic chromosomes replicate once per cell cycle [3], plasmids replicate autonomously and independently of the host chromosome [4]. Here we report that replication of both V. cholerae chromosomes is initiated synchronously, once per cell cycle. To decipher the replication kinetics of the two V. cholerae chromosomes, we used MeselsonStahl density shift experiments [5]. With this method, nonsynchronized cells are initially grown in medium containing heavy isotopes and then switched to medium with light isotopes. As the cells undergo semi-conservative DNA replication, heavy (HH) DNA gives rise to heavy-light (HL) DNA. Light-light (LL) DNA appears only after the initiation of HL DNA replication. Therefore, the time until appearance of LL DNA defines the minimal inter-replication period: the time interval between successive initiation events from the same origin [6]. There has been some debate in the literature regarding the degree of autonomy of the replication of certain low-copy plasmids, particularly the F-plasmid [7–9]. However, in density shift experiments, the inter-replication periods of the Escherichia coli chromosome and those of resident low copy plasmids clearly differ, which reflects the disparate factors controlling replication of the chromosome and plasmids [10,11]. We grew V. cholerae in ‘heavy’ medium until early exponential phase, and then shifted the cells to ‘light’ medium. At various times after this switch, DNA was extracted, sheared, and separated according to density by centrifugation in CsCl gradients. Gradient fractions were hybridized with radioactive DNA probes specific for the ori region of chromosome I (chrI) or chromosome II (chrII) to determine the relative amount of DNA in each fraction. As expected, with increasing time after the switch from heavy to light medium, the DNA density became lower, transitioning from HH to HL to LL (Figure 1). The persistence of some HH DNA until 60 min after the switch, which is longer than the doubling time of the culture (see Supplemental data), suggests that some cells were slow to recover from the density shift. Strikingly, the time at which LL DNA was first apparent (∼40 min) was the same for both chromosomes, indicating that they have the same inter-replication period. Thus, although the replicons differ in size, the time between successive initiation events is the same for both origins. Furthermore, this time was equal to the doubling time of the cells (see Supplemental data, Figure S1), indicating that each

Autorepression of RctB, an Initiator of Vibrio cholerae Chromosome II Replication

The RctB protein binds to the origin of replication of Vibrio cholerae chromosome II (chrII) and is required for oriCIIVc-based replication. Here, we found that RctB acts as an autorepressor, inhibiting rctB transcription. Integration host factor promotes rctB transcription, while Dam and DnaA, factors required for replication of both V. cholerae chromosomes, influence RctB autorepression. Thus, RctB appears to regulate chrII replication as both an initiator and a transcription repressor, and its synthesis is modulated by factors that govern replication of both chromosomes.

Host-parasite interactions that guide red blood cell invasion by malaria parasites

Purpose of reviewMalaria is caused by the infection and proliferation of parasites from the genus Plasmodium in red blood cells (RBCs). A free Plasmodium parasite, or merozoite, released from an infected RBC must invade another RBC host cell to sustain a blood-stage infection. Here, we review recent advances on RBC invasion by Plasmodium merozoites, focusing on specific molecular interactions between host and parasite. Recent findingsRecent work highlights the central role of host–parasite interactions at virtually every stage of RBC invasion by merozoites. Biophysical experiments have for the first time measured the strength of merozoite–RBC attachment during invasion. For P. falciparum, there have been many key insights regarding the invasion ligand PfRh5 in particular, including its influence on host species tropism, a co-crystal structure with its RBC receptor basigin, and its suitability as a vaccine target. For P. vivax, researchers identified the origin and emergence of the parasite from Africa, demonstrating a natural link to the Duffy-negative RBC variant in African populations. For the simian parasite P. knowlesi, zoonotic invasion into human cells is linked to RBC age, which has implications for parasitemia during an infection and thus malaria. SummaryNew studies of the molecular and cellular mechanisms governing RBC invasion by Plasmodium parasites have shed light on various aspects of parasite biology and host cell tropism, and indicate opportunities for malaria control.