PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion

Type IV pili (T4P) are functionally versatile filamentous nanomachines, nearly ubiquitous in prokaryotes. They are predominantly polymers of one major pilin but also contain minor pilins whose functions are often poorly defined and likely to be diverse. Here, we show that the minor pilin PilB from the T4P of Streptococcus sanguinis displays an unusual bimodular three-dimensional structure with a bulky von Willebrand factor A–like (vWA) module “grafted” onto a small pilin module via a short loop. Structural modeling suggests that PilB is only compatible with a localization at the tip of T4P. By performing a detailed functional analysis, we found that 1) the vWA module contains a canonical metal ion–dependent adhesion site, preferentially binding Mg²⁺ and Mn²⁺, 2) abolishing metal binding has no impact on the structure of PilB or piliation, 3) metal binding is important for S. sanguinis T4P–mediated twitching motility and adhesion to eukaryotic cells, and 4) the vWA module shows an intrinsic binding ability to several host proteins. These findings reveal an elegant yet simple evolutionary tinkering strategy to increase T4P functional versatility by grafting a functional module onto a pilin for presentation by the filaments. This strategy appears to have been extensively used by bacteria, in which modular pilins are widespread and exhibit an astonishing variety of architectures.

Type IV pili (T4P) are functionally versatile filaments widespread in prokaryotes, implicated in a variety of functions such as adhesion, twitching motility, DNA uptake, etc (1). T4P are helical polymers consisting of type IV pilins, usually one major pilin and several minor (low abundance) ones, assembled by conserved multiprotein machineries. These defining features are shared by a superfamily of filamentous nanomachines known as type IV filaments (T4F) (1), ubiquitous in prokaryotes (2).T4P have been intensively studied for decades in diderm bacteria because they play a central role in pathogenesis in important human pathogens (3). The following global picture of T4P biology has emerged from these studies. The pilus subunits, type IV pilins, are characterized by a short N-terminal sequence motif known as class III signal peptide, which consists of a hydrophilic leader peptide ending with a small residue (Gly or Ala), followed by a tract of 21 predominantly hydrophobic residues (4). This tract constitutes the N-terminal segment (α-1N) of an α-helix (α-1) of ∼50 residues, which is the universally conserved structural feature in type IV pilins. Usually, the α-1N helix protrudes from a globular head most often consisting of a β-sheet composed of several antiparallel β-strands, which gives pilins their characteristic “lollipop” shape (4). The hydrophilic leader peptide is then processed by a dedicated prepilin peptidase (5) after pilin translocation across the cytoplasmic membrane (CM) by the general secretory pathway (6, 7). Processed pilins remain embedded in the CM via their α-1N, generating a pool of subunits ready for polymerization. Filament assembly, which occurs from tip to base, is mediated at the CM by a complex multiprotein machinery (10 to 20 components) (1), centered on an integral membrane platform protein and a cytoplasmic extension ATPase (8). Recent cryogenic electron microscopy (cryo-EM) structures have revealed that T4P are right-handed helical polymers where pilins are held together by extensive interactions between their α-1N helices, which are partially melted and run approximately parallel to each other within the filament core (9, 10). One of the properties of T4P key for their functional versatility is their ability to retract, which has been best characterized for T4aP (where “a” denotes the subtype). In T4aP, retraction results from rapid filament depolymerization powered by the cytoplasmic retraction ATPase PilT (11), which generates important tensile forces (12, 13).Studying T4P in monoderm bacteria represents a promising alternative research avenue (14). Streptococcus sanguinis, a commensal of the oral cavity that commonly causes life-threatening infective endocarditis (IE), has emerged as a monoderm model for deciphering T4P biology (15). Our comprehensive functional analysis of S. sanguinis T4P (16) revealed that they are canonical T4aP. Indeed, filaments are 1) assembled by a multiprotein machinery similar to diderm T4aP species but simpler with only 10 components, 2) retracted by a PilT ATPase, generating tensile forces similar to diderm species, and 3) powering intense twitching motility, leading to spreading zones around bacteria growing on plates, visible by the naked eye. Subsequently, we performed a global biochemical and structural analysis of S. sanguinis T4P (17), showing that 1) they are heteropolymers composed of two major pilins, PilE1 and PilE2, rather than one as usually seen, 2) the major pilins display classical type IV pilin three-dimensional (3D) structure, and 3) the filaments contain a low abundance of three minor pilins (PilA, PilB, and PilC), which are required for piliation.The present study was prompted by a perplexing observation [i.e., the minor pilin PilB harbors a protein domain that has been extensively studied in eukaryotic proteins where it mediates adhesion to a variety of protein ligands (18)]. This suggested that PilB might be an adhesin, promoting T4P-mediated adhesion of S. sanguinis to host cells and proteins. Therefore, since both the molecular mechanisms of T4P-mediated adhesion and the exact role of minor pilins in T4P biology remain incompletely understood (1), we performed a structure/function analysis of PilB, which is reported here. This uncovered a widespread strategy for minor pilins to enhance the functional properties of T4P.     

The cyanobacterial taxis protein HmpF regulates type IV pilus activity in response to light

Motility is ubiquitous in prokaryotic organisms including the photosynthetic cyanobacteria where surface motility powered by type 4 pili (T4P) is common and facilitates phototaxis to seek out favorable light environments. In cyanobacteria, chemotaxis-like systems are known to regulate motility and phototaxis. The characterized phototaxis systems rely on methyl-accepting chemotaxis proteins containing bilin-binding GAF domains capable of directly sensing light, and the mechanism by which they regulate the T4P is largely undefined. In this study we demonstrate that cyanobacteria possess a second, GAF-independent, means of sensing light to regulate motility and provide insight into how a chemotaxis-like system regulates the T4P motors. A combination of genetic, cytological, and protein–protein interaction analyses, along with experiments using the proton ionophore carbonyl cyanide m-chlorophenyl hydrazine, indicate that the Hmp chemotaxis-like system of the model filamentous cyanobacterium Nostoc punctiforme is capable of sensing light indirectly, possibly via alterations in proton motive force, and modulates direct interaction between the cyanobacterial taxis protein HmpF, and Hfq, PilT1, and PilT2 to regulate the T4P motors. Given that the Hmp system is widely conserved in cyanobacteria, and the finding from this study that orthologs of HmpF and T4P proteins from the distantly related model unicellular cyanobacterium Synechocystis sp. strain PCC6803 interact in a similar manner to their N. punctiforme counterparts, it is likely that this represents a ubiquitous means of regulating motility in response to light in cyanobacteria.

Motility is ubiquitous in prokaryotic organisms, including both swimming motility in aqueous environments and twitching or gliding motility on solid surfaces, and enables these organisms to optimize their position in response to various environmental factors. Among the photosynthetic cyanobacteria, surface motility is widespread and facilitates phototaxis to seek out favorable light environments (1, 2), and, for multicellular filamentous cyanobacteria, plays a key role in dispersal as well as the establishment of nitrogen-fixing symbioses with eukaryotes (3) and the formation of supracellular structures (3–5).Current understanding of cyanobacterial surface motility at the molecular level has been informed primarily by studies of two model organisms, the unicellular strain Synechocystis sp. strain PCC6803 (herein Synechocystis) and the filamentous strain Nostoc punctiforme ATCC29133/{"type":"entrez-protein","attrs":{"text":"PCC73102","term_id":"1245706357"}}PCC73102, where motility is exhibited only by differentiated filaments termed “hormogonia.” Motility in both organisms is powered by a type IV pilus (T4P) system where the ATPases PilB and PilT drive the extension and subsequent retraction, respectively, of pili which adhere to the substrate and pull the cells forward (for review, see ref. 6). In Synechocystis, the T4P motors are distributed throughout the entire cell, allowing a 360 ° range of motion (7), whereas in N. punctiforme they are confined to rings at the cell poles (8), resulting in movement only along the long axis of the filament. Comparative genomics implies that this mechanism of motility is widely conserved among cyanobacteria (9).Both Synechocystis and N. punctiforme employ chemotaxis-like systems to regulate motility. One of these systems, the Hmp chemotaxis-like system of N. punctiforme (3, 10), and its orthologous counterpart, the Pil chemotaxis-like system of Synechocystis (11), includes homologs to the canonical Escherichia coli chemotaxis complex (for review, see ref. 12), including the histidine kinase CheA, the adaptor protein CheW, the response regulator CheY, and the methyl-accepting chemotaxis protein MCP. These systems are essential for motility in their respective organisms and appear to regulate the T4P motors, although there are distinct differences in the phenotypes for inactivation of the components from each. In Synechocystis, null mutations either enhance or reduce the level of surface piliation (11), whereas in N. punctiforme they disrupt the coordinated polarity, but not the overall level of piliation, and affect various other aspects of hormogonium development (3, 10). In N. punctiforme, the subcellular localization of this system has been determined and has been found arrayed in static, bipolar rings similar to the T4P motors (3). However, the signals that are perceived by the MCPs and the precise mechanism by which these systems modulate T4P activity is currently undefined.Recently, an additional component of the Hmp system, HmpF, was characterized (9). HmpF is a predicted coiled-coil protein and is ubiquitous to, but confined within, the cyanobacterial lineage (9). It is essential for accumulation of surface pili and exhibits dynamic, unipolar localization to the leading poles of most cells in hormogonium filaments (9). Based on these findings, a model has been proposed where the localization of HmpF is regulated by the other components of the Hmp system, and in turn, the unipolar accumulation of HmpF leads to the activation of the T4P motors on one side of the cell to facilitate directional movement.A second chemotaxis-like system in each organism, the Ptx system of N. punctiforme (13) and the Pix system of Synechocystis (14, 15), is essential for positive phototaxis. These systems contain MCPs with cyanobacteriochrome sensory domains capable of perceiving light (for review, see ref. 16). Disruption of the Pix system results in negative phototaxis under light conditions that normally produce a positive phototactic response (14). Several other proteins containing cyanobacteriochromes, and one containing a BLUF domain, also modulate phototaxis in Synechocystis (for review, see ref. 6). In N. punctiforme, disruption of the Ptx system abolishes the phototactic response completely, resulting in uniform movement in all directions regardless of the light conditions (13), and there are currently no other proteins reported to modulate phototaxis. More recently, a motile, wild isolate of the model unicellular cyanobacterium Synechococcus elongatus sp. PCC7942 was shown to possess a chemotaxis-like system that modulates phototaxis in a manner similar to that of the N. punctiforme Ptx system (17). How these systems influence T4P activity to facilitate phototaxis is also currently unknown.There is also a substantial body of literature on motility and phototaxis in cyanobacteria, primarily based on observational studies of various filamentous strains, that predates the development of genetically tractable model organisms (for review, see ref. 18). These reports suggested that the photosystems may serve a sensory role in modulating phototaxis and that proton motive force (PMF) powers motility (19, 20), a finding that is inconsistent with the theory that cyanobacteria possess a common T4P-based gliding motor driven by ATP hydrolysis. In this study, we help reconcile this historical data with more recent molecular studies by providing evidence that the Hmp chemotaxis-like system senses light, possibly indirectly through alterations in PMF, and in turn modulates the interaction of HmpF with the T4P base to activate the motors.     

Structure of cell–cell adhesion mediated by the Down syndrome cell adhesion molecule

The Down syndrome cell adhesion molecule (DSCAM) belongs to the immunoglobulin superfamily (IgSF) and plays important roles in neural development. It has a large ectodomain, including 10 Ig-like domains and 6 fibronectin III (FnIII) domains. Previous data have shown that DSCAM can mediate cell adhesion by forming homophilic dimers between cells and contributes to self-avoidance of neurites or neuronal tiling, which is important for neural network formation. However, the organization and assembly of DSCAM at cell adhesion interfaces has not been fully understood. Here we combine electron microscopy and other biophysical methods to characterize the structure of the DSCAM-mediated cell adhesion and generate three-dimensional views of the adhesion interfaces of DSCAM by electron tomography. The results show that mouse DSCAM forms a regular pattern at the adhesion interfaces. The Ig-like domains contribute to both trans homophilic interactions and cis assembly of the pattern, and the FnIII domains are crucial for the cis pattern formation as well as the interaction with the cell membrane. By contrast, no obvious assembly pattern is observed at the adhesion interfaces mediated by mouse DSCAML1 or Drosophila DSCAMs, suggesting the different structural roles and mechanisms of DSCAMs in mediating cell adhesion and neural network formation.

The Down syndrome cell adhesion molecule (DSCAM) was initially identified by isolating genes responsible for the phenotypes of Down syndrome (1), a genetic disease featured with cognitive and learning deficits (2). The DSCAM gene locates at the Down syndrome critical region (DSCR) on human chromosome 21 and is broadly expressed in nervous system (1, 3, 4), and its expression increases in patients with Down syndrome and in mouse models (3, 5, 6). Therefore, DSCAM has been hypothesized as a candidate gene associated with neurodevelopmental disorders and its dysregulation may lead to cognitive impairment and intellectual disability in Down syndrome (7), but the mechanism for the association between DSCAM and Down syndrome is still poorly understood.In invertebrates, Drosophila DSCAM1 (dDSCAM1) undergoes extensive alternative splicing by generating 38,016 isoforms with distinct recognition specificity (8–10), which is crucial for isoneuronal avoidance (11, 12). Loss of function or overexpression of dDSCAM1 in mutant flies causes defects or disorders in dendrite arborization (13, 14), axon guidance (15, 16), axon branching (17, 18), and synaptic targeting (11, 19, 20). Drosophila DSCAM2 (dDSCAM2) and DSCAM4 (dDSCAM4) also function in neural network formation by directing dendritic targeting but without the massive isoform diversity (21), and dDSCAM2 can mediate axonal tiling as well (22). Aplysia DSCAM (aDSCAM) is involved in transsynaptic protein localization (23).In vertebrates, two paralogous DSCAM genes, DSCAM and DSCAML1 (DSCAM-LIKE1) were identified (1, 24) and both of them could promote isoneuronal and homotypic self-avoidance (25, 26). In mouse, neurons expressing DSCAM (mDSCAM) or DSCAML1 (mDSCAML1) mutants may lose their mosaic pattern and neurite arborization (26, 27). Although the mechanism of mDSCAM-mediated self-avoidance remains unclear, it has been suggested that mDSCAM may function by masking the adhesion mediated by certain cadherin superfamily members (28). In addition, mDSCAM may also regulate neurite outgrowth (29, 30), promote cell death (31, 32), and control neuronal delamination (33). Studies have also shown that it could direct lamina-specific synaptic connections in chick (34) and be involved in cell movement in zebrafish (35). In contrast to dDSCAM1, the extensive alternative splicing has not been found for DSCAM in vertebrates, suggesting the different roles in the formation of neuronal circuits.DSCAM belongs to the immunoglobulin superfamily (IgSF) and consists of 10 immunoglobulin-like (Ig-like) domains, 6 type III fibronectin (FnIII) domains, a transmembrane domain, and a cytoplasmic domain (Fig. 1A). The domain arrangements of DSCAMs from invertebrates and vertebrates are quite similar, and the amino acid sequence identities of DSCAM among homologs are 98% between mDSCAM and hDSCAM (human), 59% between mDSCAM and mDSCAML1, and 33% between mDSCAM and dDSCAM1. The crystal structures of the N-terminal Ig-like domains of dDSCAM1 have been solved (36, 37). The eight N-terminal Ig-like domains form a dimer with a double-S–shaped conformation, which is critical for the homophilic cell adhesion (36). However, it is unclear whether the N-terminal Ig-like domains of mDSCAM and mDSCAML1 adopt a similar conformation to dDSCAM1, and the roles of other domains of DSCAM in cell adhesion remain elusive.Open in a separate windowFig. 1.Conformations of the ectodomains of mDSCAM, mDSCAML1, and dDSCAM1. (A) Diagrams of mDSCAM, mDSCAML1, and dDSCAM1 (ovals, Ig-like domains; rounded rectangles, FnIII domains; vertical rectangles, transmembrane domains; rectangles, cytoplasmic domains). (B–D) Negative staining EM images show the particles of mDSCAM-D_1–8, mDSCAM-D_9–16, and mDSCAM-D_1–16, respectively (Top, red arrows). (Scale bar, 50 nm.) The selected particles (Middle; the particles are picked from different images) and their contours (Bottom) are also listed. (Scale bar, 10 nm.) The schematic models of mDSCAM-D_1–8, mDSCAM-D_9–16, and mDSCAM-D_1–16 are shown in the Top Left Insets, respectively. (E–G) Negative staining EM images show the particles of mDSCAML1-D_1–8, mDSCAML1-D_9–16, and mDSCAML1-D_1–16, respectively (Top, red arrows). (Scale bar, 50 nm.) The selected particles (Middle) and their contours (Bottom) are also listed. (Scale bar, 10 nm.) The schematic models of mDSCAML1-D_1–8, mDSCAML1-D_9–16, and mDSCAML1-D_1–16 are shown in the Top Left Insets, respectively. (H–J) Negative staining EM images show the particles of dDSCAM1-D_1–8, dDSCAM1-D_9–16, and dDSCAM1-D_1–16, respectively (Top, red arrows). (Scale bar, 50 nm.) The selected particles (Middle) and their contours (Bottom) are also listed. (Scale bar, 10 nm.) The schematic models of dDSCAM1-D_1–8, dDSCAM1-D_9–16, and dDSCAM1-D_1–16 are shown in the Top Left Insets, respectively.Recently, electron tomography (ET) has become a powerful tool to provide three-dimensional (3D) views of biological samples (38, 39). By combining correlative light and electron microscopy (CLEM), high-pressure freezing and freeze substitution (HPF-FS), ultrathin sectioning and ET, the 3D structure of cellular or tissue samples can be reconstructed at nanometer resolution, revealing the molecular architecture of macromolecules in situ (40–43). Here we characterize the structures of mDSCAM, mDSCAML1, and dDSCAMs by electron microscopy (EM) as well as other biochemical and biophysical methods and reconstruct the 3D views of the mDSCAM-mediated adhesion interface by electron tomography, thereby unveiling the in situ structural model and the potential mechanism of cell adhesion by DSCAM.     

From the Cover: High diversity of West African bat malaria parasites and a tight link with rodent Plasmodium taxa

As the only volant mammals, bats are captivating for their high taxonomic diversity, for their vital roles in ecosystems—particularly as pollinators and insectivores—and, more recently, for their important roles in the maintenance and transmission of zoonotic viral diseases. Genome sequences have identified evidence for a striking expansion of and positive selection in gene families associated with immunity. Bats have also been known to be hosts of malaria parasites for over a century, and as hosts, they possess perhaps the most phylogenetically diverse set of hemosporidian genera and species. To provide a molecular framework for the study of these parasites, we surveyed bats in three remote areas of the Upper Guinean forest ecosystem. We detected four distinct genera of hemosporidian parasites: Plasmodium, Polychromophilus, Nycteria, and Hepatocystis. Intriguingly, the two species of Plasmodium in bats fall within the clade of rodent malaria parasites, indicative of multiple host switches across mammalian orders. We show that Nycteria species form a very distinct phylogenetic group and that Hepatocystis parasites display an unusually high diversity and prevalence in epauletted fruit bats. The diversity and high prevalence of novel lineages of chiropteran hemosporidians underscore the exceptional position of bats among all other mammalian hosts of hemosporidian parasites and support hypotheses of pathogen tolerance consistent with the exceptional immunology of bats.Malaria is a mosquito-borne epidemic human disease caused by protozoan parasites of the genus Plasmodium. Four different species known to cause human malaria have been studied intensively over several decades, and in the recent past two additional species have also been verified as human malaria parasites (1, 2). However, human-infecting Plasmodium species represent only a small fraction of over 550 species in the order Haemosporida that are classified into 17 extant genera (3). One hallmark of all hemosporidian parasites is the obligate host switch between a vertebrate intermediate host and an arthropod vector as a definitive host. However, across this family, a diverse array of intermediate hosts are used, including several orders of mammals, birds, squamate reptiles, turtles, and crocodilians (4). Based on solitary reports over the last century, it is thought that parasites belonging to at least seven hemosporidian genera can infect bats (Chiroptera), most of which are likely exclusive to this order (5).Bats have an almost worldwide distribution, feature diverse life history traits, and play important ecological roles (6). Chiroptera is the second largest order of mammals after the Rodentia, with an estimated 1,232 living species and 18 families, which represent ∼20% of all living mammalian species (7). Bats are also important reservoir hosts for numerous emerging and highly pathogenic viruses (8, 9). In marked contrast, the hemosporidian parasites of bats remain largely unstudied, despite the first records dating back to the late 19th century (10). The corresponding vectors for most bat parasites remain unknown. Similarly, the phylogenetic relationships for the majority of these parasites remain enigmatic.Here, we present a unique systematic analysis of Haemosporida in a diverse species assemblage of bats. For this study, we performed surveys in three remnants of the Upper Guinean forest ecosystem, considered one of the world’s biologically most diverse, but also one of the most endangered terrestrial ecosystems (Fig. 1). Our analysis highlights the overall diversity of chiropteran hemosporidian parasites and reveals distinct host–parasite associations.Open in a separate windowFig. 1.Bat sampling areas in West Africa. Bats were captured during the dry season between November and December 2006 in Taï National Park, Côte d’Ivoire, in December 2008 in the Forêt Classée de Pic de Fon in the Simandou range of Guinea, and between November and December 2010 in the Putu range in southeastern Liberia.     

Indirect costs of a nontarget pathogen mitigate the direct benefits of a virus-resistant transgene in wild Cucurbita

Virus-resistant transgenic squash are grown throughout the United States and much of Mexico and it is likely that the virus-resistant transgene (VRT) has been introduced to wild populations repeatedly. The evolutionary fate of any resistance gene in wild populations and its environmental impacts depend upon trade-offs between the costs and benefits of the resistance gene. In a 3-year field study using a wild gourd and transgenic and nontransgenic introgressives, we measured the effects of the transgene on fitness, on herbivory by cucumber beetles, on the incidence of mosaic viruses, and on the incidence of bacterial wilt disease (a fatal disease vectored by cucumber beetles). In each year, the first incidence of zucchini yellow mosaic virus occurred in mid-July and spread rapidly through the susceptible plants. We found that the transgenic plants had greater reproduction through both male and female function than the susceptible plants, indicating that the VRT has a direct fitness benefit for wild gourds under the conditions of our study. Moreover, the VRT had no effect on resistance to cucumber beetles or the incidence of wilt disease before the spread of the virus. However, as the virus spread through the fields, the cucumber beetles became increasingly concentrated upon the healthy (mostly transgenic) plants, which increased exposure to and the incidence of wilt disease on the transgenic plants. This indirect cost of the VRT (mediated by a nontarget herbivore and pathogen) mitigated the overall beneficial effect of the VRT on fitness.