Convergent evolution toward an improved growth rate and a reduced resistance range in Prochlorococcus strains resistant to phage

Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.Large bacterial populations are present in the oceans, playing important roles in primary production and the biogeochemical cycling of matter. These bacterial communities are highly diverse (1–4) yet form stable and reproducible bacterial assemblages under similar environmental conditions (5–7).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (8–11). Although studied only rarely in marine organisms (12–16), this coexistence is likely to be the result of millions of years of coevolution between these antagonistic interacting partners, as has been well documented for other systems (17–20). From the perspective of the bacteria, survival entails the selection of cells that are resistant to infection, preventing viral production and enabling the continuation of the cell lineage. Resistance mechanisms include passively acquired spontaneous mutations in cell surface molecules that prevent phage entry into the cell and other mechanisms that actively terminate phage infection intracellularly, such as restriction–modification systems and acquired resistance by CRISPR-Cas systems (21, 22). Mutations in the phage can also occur that circumvent these host defenses and enable the phage to infect the recently emerged resistant bacterium (23).Acquisition of resistance by bacteria is often associated with a fitness cost. This cost is frequently, but not always, manifested as a reduction in growth rate (24–27). Recently, an additional type of cost of resistance was identified, that of enhanced infection whereby resistance to one phage leads to greater susceptibility to other phages (14, 15, 28).Over the years, a number of models have been developed to explain coexistence in terms of the above coevolutionary processes and their costs (16, 29–32). In the arms race model, repeated cycles of host mutation and virus countermutation occur, leading to increasing breadths of host resistance and viral infectivity. However, experimental evidence generally indicates that such directional arms race dynamics do not continue indefinitely (25, 33, 34). Therefore, models of negative density-dependent fluctuations due to selective trade-offs, such as kill-the-winner, are often invoked (20, 33, 35, 36). In these models, fluctuations are generally considered to occur between rapidly growing competition specialists that are susceptible to infection and more slowly growing resistant strains that are considered defense specialists. Such negative density-dependent fluctuations are also likely to occur between strains that have differences in viral susceptibility ranges, such as those that would result from enhanced infection (30).The above coevolutionary processes are considered to be among the major mechanisms that have led to and maintain diversity within bacterial communities (32, 35, 37–39). These processes also influence genetic microdiversity within populations of closely related bacteria. This is especially the case for cell surface-related genes that are often localized to genomic islands (14, 40, 41), regions of high gene content, and gene sequence variability among members of a population. As such, populations in nature display an enormous degree of microdiversity in phage susceptibility regions, potentially leading to an assortment of subpopulations with different ranges of susceptibility to coexisting phages (4, 14, 30, 40).Prochlorococcus is a unicellular cyanobacterium that is the numerically dominant photosynthetic organism in vast oligotrophic expanses of the open oceans, where it contributes significantly to primary production (42, 43). Prochlorococcus consists of a number of distinct ecotypes (44–46) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (47–49).Previously, we found that resistance to phage infection occurs frequently in two high-light–adapted Prochlorococcus ecotypes through spontaneous mutations in cell surface-related genes (14). These genes are primarily localized to genomic island 4 (ISL4) that displays a high degree of genetic diversity in environmental populations (14, 40). Although about a third of Prochlorococcus-resistant strains had no detectable associated cost, the others came with a cost manifested as either a slower growth rate or enhanced infection by other phages (14). In nature, Prochlorococcus seems to be growing close to its intrinsic maximal growth rate (50–52). This raises the question as to the fate of emergent resistant Prochlorococcus lineages in the environment, especially when resistance is accompanied with a high growth rate fitness cost.To begin addressing this question, we investigated the phenotype of Prochlorococcus strains with time after the acquisition of resistance. We found that resistant strains evolved toward an improved growth rate and a reduced resistance range. Whole-genome sequencing and PCR screening of many of these strains revealed that these phenotypic changes were largely due to additional, compensatory mutations, leading to increased genetic diversity. These findings suggest that the oceans are populated with rapidly growing Prochlorococcus cells with varying degrees of resistance and provide an explanation for how a multitude of presumably resistant Prochlorococcus cells are growing close to their maximal known growth rate in nature.     

Lemon-shaped halo archaeal virus His1 with uniform tail but variable capsid structure

Lemon-shaped viruses are common in nature but so far have been observed to infect only archaea. Due to their unusual shape, the structures of these viruses are challenging to study and therefore poorly characterized. Here, we have studied haloarchaeal virus His1 using cryo-electron tomography as well as biochemical dissociation. The virions have different sizes, but prove to be extremely stable under various biochemical treatments. Subtomogram averaging of the computationally extracted virions resolved a tail-like structure with a central tail hub density and six tail spikes. Inside the tail there are two cavities and a plug density that separates the tail hub from the interior genome. His1 most likely uses the tail spikes to anchor to host cells and the tail hub to eject the genome, analogous to classic tailed bacteriophages. Upon biochemical treatment that releases the genome, the lemon-shaped virion transforms into an empty tube. Such a dramatic transformation demonstrates that the capsid proteins are capable of undergoing substantial quaternary structural changes, which may occur at different stages of the virus life cycle.There seems to be only a limited number of different virus particle architectures (virion-based structural lineages) due to the limited protein-fold space (1–3). Examples of the uncommon architectures are spindle-, bottle-, and droplet-shaped virions, so far found only in archaeal viruses (4). Archaea, organisms forming the third domain of cellular life, are known to thrive in both moderate and extreme environments (5–7). Interestingly, archaeal viruses are morphologically diverse, resembling eukaryotic viruses in this respect (4). Of these morphotypes, the spindle-shaped (also known as lemon-shaped) viruses are the most common ones in archaea-dominated habitats (8–15). It appears that this architecture is unique. However, deeper structural and biochemical analyses are needed to confirm this claim.Lemon-shaped virions are wider in the middle and narrow toward the ends. Three types of such viruses have been described based on the virion appearance: (i) those with one very short tail, (ii) those with one long tail, and (iii) those having two long tails (16–18). However, based on the comparison of the structural proteins, it was recently proposed that all known lemon-shaped viruses could be classified into two evolutionary lineages or viral families: Fuselloviridae, containing type i viruses, and Bicaudaviridae, containing type ii and iii viruses (19). Most of the isolated lemon-shaped viruses belong to the family Fuselloviridae, whose type species is the Sulfolobus spindle-shaped virus 1 (SSV1). Fuselloviruses infect hyperthermophilic crenarchaea and have one short tail with tail fibers (4, 20). The physical properties of the studied spindle-shaped viruses have recently been summarized (21). However, 3D structural understanding of lemon-shaped viruses remains limited.His1 is the only high-salinity lemon-shaped virus isolate, and it infects an extremely halophilic euryarchaeon, Haloarcula hispanica, and morphologically resembles fuselloviruses (22). However, unlike fuselloviruses, which have a circular double-stranded DNA (dsDNA) genome, His1 has a linear dsDNA genome encoding a putative type-B DNA polymerase; consequently, His1 has been classified in the floating genus Salterprovirus (20, 23). The 14,462-bp genome of His1 is predicted to have 35 ORFs, and 4 of these have been shown to encode structural proteins of the virion (21, 23). His1 virion contains one major capsid protein (MCP), VP21. In addition, a few minor ones have been detected: VP11, VP26, and VP27. Interestingly, VP21 exists in two forms. One form is lipid modified, although there is no detectable lipid bilayer in the His1 virion (21). The His1 MCP is 47% similar to the MCP of SSV1, indicating that His1 and fuselloviruses may share a common ancestor, and it has been proposed that His1 could be classified into the family Fuselloviridae (19, 21). In addition to the DNA polymerase, the His1 genome is predicted to encode an ATPase and a glycosyltransferase (21, 23). His1, like the other lemon-shaped viruses, is nonlytic, and many of them also encode an integrase (16, 17, 21, 24–26). Despite its typically hypersaline environment, His1 tolerates a variety of salinities, from 50 mM up to 4 M NaCl (21).Few attempts have been made to determine the structures of nonicosahedral viruses, mainly due to the fact that their pleomorphic nature introduces great challenges in particle structure classification and averaging. Recent advances in image-processing methods in cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) have been demonstrated to be a useful tool in the structural studies of nonicosahedral viruses, such as Tula hantavirus (27) and the immature capsid in HIV-1 virus (28), and some archaeal viruses, such as pleomorphic, two-tailed spindle-shaped, and linear viruses (17, 21, 29) and bottle-shaped and filamentous archaeal viruses (30–32). Recent structural studies of the spindle-shaped virus SSV1 revealed its 3D structure with a spindle body and a short tail at one end (33).Here, we used cryo-ET and symmetry-free and model-free subtomogram averaging to reveal the previously unrecognized tail organization of the lemon-shaped virus His1, with a central tail hub and six surrounding tail spikes. Further analysis of a larger population of subtomograms showed variable dimensions of the lemon particles but a constant structure of the tail. Biochemical analysis of the virion under different chemical conditions revealed unexpected biochemical properties of the His1 virion, which may be relevant to the life cycle of this virus.     

Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens

The rapid reorganization and polarization of actin filaments (AFs) toward the pathogen penetration site is one of the earliest cellular responses, yet the regulatory mechanism of AF dynamics is poorly understood. Using live-cell imaging in Arabidopsis, we show that polarization coupled with AF bundling involves precise spatiotemporal control at the site of attempted penetration by the nonadapted barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We further show that the Bgh-triggered AF mobility and organelle aggregation are predominately driven by the myosin motor proteins. Inactivation of myosins by pharmacological inhibitors prevents bulk aggregation of organelles and blocks recruitment of lignin-like compounds to the penetration site and deposition of callose and defensive protein, PENETRATION 1 (PEN1) into the apoplastic papillae, resulting in attenuation of penetration resistance. Using gene knockout analysis, we demonstrate that highly expressed myosins XI, especially myosin XI-K, are the primary contributors to cell wall-mediated penetration resistance. Moreover, the quadruple myosin knockout mutant xi-1 xi-2 xi-i xi-k displays impaired trafficking pathway responsible for the accumulation of PEN1 at the cell periphery. Strikingly, this mutant shows not only increased penetration rate but also enhanced overall disease susceptibility to both adapted and nonadapted fungal pathogens. Our findings establish myosins XI as key regulators of plant antifungal immunity.In nature, plants are constantly exposed to a large number of pathogens including fungi, bacteria, and viruses. In response, plants have evolved multiple layers of defense mechanisms to resist the pathogen attack (1). The first line of plant defense against fungi is penetration resistance that is achieved by localized cell wall appositions (CWAs), also called papillae, on an inner surface of cell walls at the site of fungal penetration (2). CWAs consist primarily of callose (β-1,3-glucan), lignin, cell wall proteins, and reactive oxygen species (2–4). The focal deposition of these elements in papillae appears to be an early and essential factor in plant penetration resistance (5).Studies on the genetic basis of penetration resistance have revealed that entry control of A. thaliana against nonadapted powdery mildews largely depends on several PENETRATION (PEN) genes (PEN1, PEN2, and PEN3). All three PEN proteins are also recruited to attempted fungal penetration sites (6–11). Intriguing findings show that the focal accumulation of PEN1 and PEN3 occurs outside the plasma membrane and within papillae or haustorial encasements (3, 11, 12). Disruption of actin cytoskeleton by pharmacological inhibitors blocks PEN3–GFP accumulation at most penetration sites, but has a lesser effect on the recruitment of GFP–PEN1 to these sites (11), suggesting that transport pathways mediating PEN1 and PEN3 recruitment and export to the apoplastic papillae are distinct.Accumulation of dynamically moving cytoplasm near the pathogen penetration site is the most striking and microscopically visible early response in epidermal cells (2). The secretory vesicles and organelles, including peroxisomes, Golgi, mitochondria, and the nucleus, also move toward penetration sites (7, 13). In addition to the deposition of cell wall reinforcements and focal accumulation of penetration-related proteins such as PEN3, the accretion of cytoplasm and organelles at sites of attempted fungal penetration involves reorganization of actin cytoskeleton, which forms a radial array focused on penetration site (10, 11, 14–18). Consistent with this finding, disruption of AFs hampers penetration resistance, leading to increased penetration frequency by various fungal and oomycete pathogens (15–17, 19). However, the mechanisms that drive AF dynamics and active transport of cellular components toward sites of attempted pathogen penetration remain elusive. Myosins are molecular motors responsible for AF-based motility (20). Recently, plant class XI myosins were implicated in the organization of actin cytoskeleton, organelle and vesicle transport, cell expansion, and plant growth (21–27). Although none of the individual myosin gene knockouts produces plant growth defects (22), progressive elimination of two to four highly expressed myosins results in concomitant reduction in cell and plant size (23, 24). However, relatively little is known about the functions of myosins in plant–pathogen interactions (28, 29).Using pharmacological and genetic approaches to disrupt myosin function in Arabidopsis, we show that transient assembly and polarization of actin filament (AF) bundles toward the fungal penetration site are regulated by myosin motors. Furthermore, we demonstrate that plant myosins contribute to focal aggregation of a battery of cellular defense activities at the infection site and papillary deposition of cell wall appositions of lignin-like compounds, callose and PEN1, and are required for plant penetration resistance.     

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania

Innovative strategies are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. Here we develop a sensitive method, which we term Cosmid Sequencing (or “Cos-Seq”), based on functional cloning coupled to next-generation sequencing. Cos-Seq identified >60 loci in the Leishmania genome that were enriched via drug selection with methotrexate and five major antileishmanials (antimony, miltefosine, paromomycin, amphotericin B, and pentamidine). Functional validation highlighted both known and previously unidentified drug targets and resistance genes, including novel roles for phosphatases in resistance to methotrexate and antimony, for ergosterol and phospholipid metabolism genes in resistance to miltefosine, and for hypothetical proteins in resistance to paromomycin, amphothericin B, and pentamidine. Several genes/loci were also found to confer resistance to two or more antileishmanials. This screening method will expedite the discovery of drug targets and resistance mechanisms and is easily adaptable to other microorganisms.Leishmaniasis is a neglected tropical disease causing significant morbidity and mortality worldwide (1). It is caused by parasites of the genus Leishmania that cycle between the flagellar promastigote form in the gut of the insect vector and the nonmotile amastigote stage in the vertebrate host. Given the lack of an effective antileishmanial vaccine, control of leishmaniasis relies mainly on chemotherapy. Only a few antileishmanial drugs are available, and their efficacy is severely limited by toxicity, cost, and drug resistance (2, 3). New methods for expediting the discovery of drug targets and resistance mechanisms in Leishmania would aid the reassessment of current therapies and the development of new, effective drugs.Next-generation sequencing (NGS) technologies have enabled the high-throughput and genome-scale screening of eukaryotic pathogens, and have been useful in identifying drug targets and elucidating drug resistance mechanisms (4, 5). The development of RNA interference (RNAi) target sequencing (RIT-Seq) in kinetoplastid parasites, such as Trypanosoma brucei (6), has revealed numerous genes associated with drug action (7); however, RNAi-based screening is not applicable to Leishmania, because most species lack functional RNAi machinery (8). In Leishmania spp., copy number variation and single-nucleotide polymorphism were detected in drug-resistant parasites using NGS (9, 10; reviewed in ref 11). Gain-of-function screening using genomic cosmid libraries is also a proven approach to studying drug resistance in Leishmania (12). Cosmid-based functional cloning was first implemented for studying lipophosphoglycan biosynthesis (13) and later successfully applied to study nucleoside transport (14, 15) and drug resistance (16–21). The technique has proven powerful, albeit with limitations; for instance, it is not easily amenable to high-throughput screening, because clones require individual characterization, and is biased toward the selection of cosmids conferring dominant phenotypes, leaving out less conspicuous candidates.In this study, such limitations were alleviated by combining genome-wide cosmid-based functional screening with NGS, a strategy that we term Cosmid Sequencing (or “Cos-Seq”). This method allows us to study the dynamics of cosmid enrichment under selective drug pressure and to isolate an unprecedented number of both known and previously unidentified antileishmanial targets and resistance genes.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies

Broadly neutralizing HIV antibodies (bNAbs) can recognize carbohydrate-dependent epitopes on gp120. In contrast to previously characterized glycan-dependent bNAbs that recognize high-mannose N-glycans, PGT121 binds complex-type N-glycans in glycan microarrays. We isolated the B-cell clone encoding PGT121, which segregates into PGT121-like and 10-1074–like groups distinguished by sequence, binding affinity, carbohydrate recognition, and neutralizing activity. Group 10-1074 exhibits remarkable potency and breadth but no detectable binding to protein-free glycans. Crystal structures of unliganded PGT121, 10-1074, and their likely germ-line precursor reveal that differential carbohydrate recognition maps to a cleft between complementarity determining region (CDR)H2 and CDRH3. This cleft was occupied by a complex-type N-glycan in a “liganded” PGT121 structure. Swapping glycan contact residues between PGT121 and 10-1074 confirmed their importance for neutralization. Although PGT121 binds complex-type N-glycans, PGT121 recognized high-mannose-only HIV envelopes in isolation and on virions. As HIV envelopes exhibit varying proportions of high-mannose- and complex-type N-glycans, these results suggest promiscuous carbohydrate interactions, an advantageous adaptation ensuring neutralization of all viruses within a given strain.Antibodies are essential for the success of most vaccines (1), and antibodies against HIV appear to be the only correlate of protection in the recent RV144 anti-HIV vaccine trial (2). Some HIV-1–infected patients develop broadly neutralizing serologic activity against the gp160 viral spike 2–4 y after infection (3–10), but these antibodies do not generally protect infected humans because autologous viruses escape through mutation (11–13). Nevertheless, broadly neutralizing activity puts selective pressure on the virus (13) and passive transfer of broadly neutralizing antibodies (bNAbs) to macaques protects against simian/human immunodeficiency virus (SHIV) infection (14–24). It has therefore been proposed that vaccines that elicit such antibodies may be protective against HIV infection in humans (10, 25–28).The development of single-cell antibody cloning techniques revealed that bNAbs target several different epitopes on the HIV-1 gp160 spike (29–35). The most potent HIV-1 bNAbs recognize the CD4 binding site (CD4bs) (31, 34, 36) and carbohydrate-dependent epitopes associated with the variable loops (32, 33, 37, 38), including the V1/V2 (antibodies PG9/PG16) (33) and V3 loops (PGTs) (32). Less is known about carbohydrate-dependent epitopes because the antibodies studied to date are either unique examples or members of small clonal families.To better understand the neutralizing antibody response to HIV-1 and the epitope targeted by PGT antibodies, we isolated members of a large clonal family dominating the gp160-specific IgG memory response from the clade A-infected patient who produced PGT121. We report that PGT121 antibodies segregate into two groups, a PGT121-like and a 10-1074–like group, according to sequence, binding affinity, neutralizing activity, and recognition of carbohydrates and the V3 loop. The 10-1074 antibody and related family members exhibit unusual potent neutralization, including broad reactivity against newly transmitted viruses. Unlike previously characterized carbohydrate-dependent bNAbs, PGT121 binds to complex-type, rather than high-mannose, N-glycans in glycan microarray experiments. Crystal structures of PGT121 and 10-1074 compared with structures of their germ-line precursor and a structure of PGT121 bound to a complex-type N-glycan rationalize their distinct properties.