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The human malaria parasite Plasmodium falciparum invades red blood cells (RBCs) and exports parasite proteins to transform the host cell for its survival. These exported proteins facilitate cytoadherence of the infected RBC (iRBC) to endothelial cells of small blood vessels, protecting iRBCs from splenic clearance. The parasite protein PfEMP1 and the host protein CD36 play a major role in P. falciparum iRBC cytoadherence. The murine parasite Plasmodium berghei is a widely used experimental model that combines high genetic tractability with access to in vivo studies. The P. berghei iRBC also sequesters by CD36-binding via an unknown parasite ligand and few parasite proteins, including EMAP1 and EMAP2, have been localised to the iRBC membrane. We have identified a new protein named EMAP3 and demonstrated its export to the iRBC membrane where it likely interacts with EMAP1, with only EMAP3 exposed on the outer surface of the iRBC. Parasites lacking EMAP3 display no significant reduction in growth or sequestration, indicating that EMAP3 is not a major CD36-binding protein. The outer-surface location of EMAP3 offers a new scaffold for displaying P. falciparum proteins on the surface of the P. berghei iRBC, providing a platform to screen in vivo for putative inhibitors of P. falciparum cytoadherence.

FEBS+ meeting Exploring molecular frontiers, held in Pula, Croatia, in autumn 2024 was jointly organized by the Croatian, Finnish, and Swedish Member Societies of the Federation of European Biochemical Societies (FEBS). The congress covered a wide variety of different biochemical themes, some of the highlights of which are presented in this ‘In the Limelight’ issue of FEBS Open Bio. We hope that, in addition to being scientifically useful and interesting, these articles will give a glimpse of the excellent science presented in the FEBS3+ meeting Exploring molecular frontiers and encourage attendance to future FEBS3+ meetings.

Type IV secretion systems (T4SS) are versatile nanomachines responsible for the transfer of DNA and proteins across cell envelopes. From their ancestral role in conjugation, these systems have diversified into a superfamily with functions ranging from horizontal gene transfer to the delivery of toxins to eukaryotic and prokaryotic hosts. Recent structural and functional studies have uncovered unexpected architectural variations not only among Gram-negative systems but also between Gram-negative and Gram-positive systems. Despite this diversity, a conserved set of core proteins is maintained across the superfamily. To facilitate cross-system comparisons, we propose in this review a unified nomenclature for conserved T4SS subunits found in both Gram-negative and Gram-positive systems. We further highlight conserved and divergent mechanistic and architectural principles across bacterial lineages, and we discuss the diversity of emerging T4SSs whose unique structures and functions expand our understanding of this highly adaptable secretion superfamily.

Despite recent advances in our understanding of the structure and function of conjugative Type 4 Secretion Systems (T4SSs), there is still only very scarce data available for the ones from Gram-positive (G+) bacteria. This is a problem, as conjugative T4SSs are main drivers for the spread of antibiotic resistance genes and virulence factors. Here, we aim to increase our understanding of G+ systems, by using bioinformatic approaches to identify proteins that are conserved in all conjugative T4SS machineries and reviewing the current knowledge available for these components. We then combine this information with the most recent advances in structure prediction technologies to propose a structural model for a G+ T4SS from the model system encoded on pCF10. By doing so, we show that conjugative G+ T4SSs likely have more in common with their Gram-negative counterparts than previously expected, and we highlight the potential of predicted structural models to serve as a starting point for experimental design.

Yersinia pathogenicity is dependent on polarized translocation of effectorproteins via the type III secretion system (T3SS). The tip complex situatedon the needle structure of the T3SS is required for contact with the eukaryotichost membrane and is to an extent composed of pentameric LcrV. LcrVis a multifunctional protein that also acts as a regulator of the T3SS by virtueof forming a high-affinity complex in the cytoplasm with its chaperone, LcrG.By employing a structure-based approach centered on mass spectrometry,FRET and NMR spectroscopy, we demonstrated that the LcrV-LcrG complexis best described as a multivalent complex, and that the N-terminaldomain of LcrV contributes by negatively affecting the LcrG binding affinity.The N-terminal domain of LcrV is dynamic and undergoes a conformationalchange to accommodate LcrG binding. 19F NMR spectroscopy analysissuggests that the conformational change is an intrinsic property of the protein,which agrees with a conformational selection model. An analysis ofeffector secretion into a culture supernatant demonstrated that the low synthesisand low secretion phenotypes of a LcrV mutant where the N-terminaldomain has been removed are linked to the structure, interactions and stabilityof the LcrV N-terminal domain. In summary, our results add insightsinto the dynamics of LcrV and its complex with LcrG.

The bacterial cell-wall peptidoglycan is made of glycan strands crosslinked by short peptide stems. Crosslinks are catalyzed by DD-transpeptidases (4,3-crosslinks) and LD-transpeptidases (3,3-crosslinks). However, recent research on non-model species has revealed novel crosslink types, suggesting the existence of uncharacterized enzymes. Here, we identify an LD-transpeptidase, LDT_Go, that generates 1,3-crosslinks in the acetic-acid bacterium Gluconobacter oxydans. LDT_Go-like proteins are found in Alpha- and Betaproteobacteria lacking LD3,3-transpeptidases. In contrast with the strict specificity of typical LD- and DD-transpeptidases, LDT_Go can use non-terminal amino acid moieties for crosslinking. A high-resolution crystal structure of LDT_Go reveals unique features when compared to LD3,3-transpeptidases, including a proline-rich region that appears to limit substrate access, and a cavity accommodating both glycan chain and peptide stem from donor muropeptides. Finally, we show that DD-crosslink turnover is involved in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans.

Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has three predicted extracellular hydrolase domains: metallo-peptidase (LytM), soluble lytic transglycosylase (SLT), and cysteine, histidine-dependent amidohydrolases/peptidases (CHAP). Here, we report the structure of the LytM domain and show that its active site is degenerate and lacks the active site metal. Furthermore, we show that only the predicted SLT domain is functional in vitro and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Furthermore, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information for understanding the function of Gram-positive T4SSs.

IMPORTANCE: Antibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from Enterococcus faecalis. This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in E. faecalis as well as other medically relevant Gram-positive bacteria. Antibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from Enterococcus faecalis. This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in E. faecalis as well as other medically relevant Gram-positive bacteria.

Conjugative dissemination of mobile genetic elements (MGEs) among bacteria is initiated by assembly of the relaxosome at the MGE's origin-of-transfer (oriT) sequence. A critical but poorly defined step of relaxosome assembly involves recruitment of the catalytic relaxase to its DNA strand-specific nicking site within oriT. Here, we present evidence by AlphaFold modeling, affinity pulldowns, and in vivo site-directed photocrosslinking that the TraK Ribbon–Helix–Helix DNA-binding protein recruits TraI to oriT through a dynamic interaction in which TraI's C-terminal unstructured domain (TraICTD) wraps around TraK's C-proximal tetramerization domain. Upon relaxosome assembly, conformational changes disrupt this contact, and TraICTD instead self-associates as a prerequisite for relaxase catalytic functions or substrate engagement with the transfer channel. These findings delineate key early-stage processing reactions required for conjugative dissemination of a model MGE.

Mechanosensitive ion channels play an essential role in reacting to environmental signals and sustaining cell integrity by facilitating ion flux across membranes. For obligate intracellular pathogens like microsporidia, adapting to changes in the host environment is crucial for survival and propagation. Despite representing a eukaryote of extreme genome reduction, microsporidia have expanded the gene family of mechanosensitive ion channels of small conductance (mscS) through repeated gene duplication and horizontal gene transfer. All microsporidian genomes characterized to date contain mscS genes of both eukaryotic and bacterial origin. Here, we investigated the cryo-electron microscopy structure of the bacterially derived mechanosensitive ion channel of small conductance 2 (MscS2) from Nematocida displodere, an intracellular pathogen of Caenorhabditis elegans. MscS2 is the most compact MscS-like channel known and assembles into a unique superstructure in vitro with six heptameric MscS2 channels. Individual MscS2 channels are oriented in a heterogeneous manner to one another, resembling an asymmetric, flexible six-way cross joint. Finally, we show that microsporidian MscS2 still forms a heptameric membrane channel, however the extreme compaction suggests a potential new function of this MscS-like protein.

A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded type IV secretion systems (T4SSs). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many, their function remains unclear. Here, we characterize PrgE, a proposed SSB from the Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very unusual DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string supported by its N-terminal tail. In vitro studies highlight the plasticity of PrgE oligomerization and confirm the importance of the N-terminus. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from eukaryotic SSBs.