Population dynamics in colonizing vancomycin-resistant Enterococcus faecium isolated from immunosuppressed patients.

OBJECTIVES: Vancomycin-resistant Enterococcus faecium and Enterococcus faecalis (VRE) are a common cause of healthcare-associated infections. Whole genome sequencing-based typing methods yield the highest discriminatory power for outbreak surveillance in the hospital. We analysed the clonal composition of enteric VRE populations of at-risk patients over several weeks to characterise VRE population diversity and dynamics. METHODS: Five bone marrow transplant recipients (three colonised with vanA-positive isolates, two colonised with vanB-positive isolates) contributed three rectal swabs over a course of several weeks. Fourteen VRE colonies per swab were analysed by core genome multi locus sequence typing (cgMLST) and typing of the van-element. RESULTS: VRE populations were clonally diverse in three of five patients, and population composition changed dynamically over the time of observation. Besides new acquisition of VRE isolates, shared van-elements localised on nearly identical plasmids between clonally different isolates indicate horizontal gene transfer as a mechanism behind VRE population diversity within single patients. CONCLUSION: Outbreak detection relies on typing of isolates, usually by analysing one isolate per patient. We here show that this approach is insufficient for outbreak surveillance of VRE in highly vulnerable patients, as it does not take into account VRE population heterogeneity and horizontal gene transfer of the resistance element.

Prebiotic Nucleoside Synthesis: The Selectivity of Simplicity.

Ever since the discovery of nucleic acids 150 years ago,[1] major achievements have been made in understanding and decrypting the fascinating scientific questions of the genetic code.[2] However, the most fundamental question about the origin and the evolution of the genetic code remains a mystery. How did nature manage to build up such intriguingly complex molecules able to encode structure and function from simple building blocks? What conditions were required? How could the precursors survive the unhostile environment of early Earth? Over the past decades, promising synthetic concepts were proposed providing clarity in the field of prebiotic nucleic acid research. In this Minireview, we show the current status and various approaches to answer these fascinating questions.

Direct Prebiotic Pathway to DNA Nucleosides.

It is assumed that RNA played a key role in the origin of life, and the transition to more complex but more stable DNA for continuous information storage and replication requires the development of a ribonucleotide reductase to obtain the deoxyribonucleotides from ribonucleotides. This step, as well as an alternative path from abiotic molecules to DNA-based life is completely unknown. Shown here is the formation of deoxyribonucleosides under relevant prebiotic conditions in water in high regio- and stereoselectivity, from all canonical purine and pyrimidine bases, by condensation with acetaldehyde and sugar-forming precursors. Thus, a continuous path to deoxyribonucleosides, starting from simple, prebiotically available molecules has been discovered. Furthermore, the deoxyapionucleosides (DApiNA) were identified as a potential DNA progenitor. The results suggest that the DNA world evolved much earlier than previously assumed.

Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export.

Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite Plasmodium falciparum. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.

Uncovering common principles in protein export of malaria parasites.

For proliferation, the malaria parasite Plasmodium falciparum needs to modify the infected host cell extensively. To achieve this, the parasite exports proteins containing a Plasmodium export element (PEXEL) into the host cell. Phosphatidylinositol-3-phosphate binding and cleavage of the PEXEL are thought to mediate protein export. We show that these requirements can be bypassed, exposing a second level of export control in the N terminus generated after PEXEL cleavage that is sufficient to distinguish exported from nonexported proteins. Furthermore, this region also corresponds to the export domain of a second group of exported proteins lacking PEXELs (PNEPs), indicating shared export properties among different exported parasite proteins. Concordantly, export of both PNEPs and PEXEL proteins depends on unfolding, revealing translocation as a common step in export. However, translocation of transmembrane proteins occurs at the parasite plasma membrane, one step before translocation of soluble proteins, indicating unexpectedly complex translocation events at the parasite periphery.

Development and host cell modifications of Plasmodium falciparum blood stages in four dimensions.

Blood stages of Plasmodium falciparum cause the pathology of malaria; however, the progression of the parasite through this complex part of the life cycle has never been visualized. In this study, we use four-dimensional imaging to show for the first time the development of individual parasites in erythrocytes and the concomitant host cell modifications. Our data visualize an unexpectedly dynamic parasite, provide a reference for this life cycle stage and challenge the model that protein export in P. falciparum is linked to the biogenesis of host cell modifications termed Maurer's clefts. Our results provide a novel view of the blood-stage development, Maurer's cleft development and protein export in malaria parasites, and open the door to study dynamic processes, drug effects and the phenotype of mutants.