A ribosome-inactivating protein in a Drosophila defensive symbiont

Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense.Symbiosis is now recognized to be a key driver of evolutionary novelty and complexity (1, 2), and symbioses between microbes and multicellular hosts are understood as essential to the health and success of diverse lineages, from plants to humans (3). Insects, in particular, have widespread associations with symbiotic bacteria, with most insect species infected by maternally transmitted endosymbionts (4, 5). Although many insect symbionts perform roles essential for host survival, such as supplementing nutrition, others are facultative and not strictly required by their hosts. These facultative symbionts have evolved diverse and intriguing strategies to maintain themselves in host populations despite loss from imperfect maternal transmission and metabolic costs to the host. These range from manipulating host reproduction to increase their own transmission (6, 7), such as by killing male hosts, to providing context-dependent fitness benefits (8). Recently, it has become clear that different insect endosymbionts have independently evolved to protect their hosts against diverse natural enemies that so far include pathogenic fungi (9), RNA viruses (10, 11), parasitoid wasps (12), parasitic nematodes (13), and predatory spiders (14, 15). This suggests that defense might be a common aspect of many insect symbioses and demonstrates that symbionts can serve as dynamic and heritable sources of protection against natural enemies (8).Despite a growing appreciation of the importance of symbiont-mediated defense in insects, key questions remain. Most demonstrations of defense have been under laboratory conditions, and the importance of symbiont-mediated protection in natural systems is unclear in most cases (16). At the same time, the proximate causes of defense are largely unknown, although recent studies have provided some intriguing early insights: A Pseudomonas symbiont of rove beetles produces a polyketide toxin thought to deter predation by spiders (14), Streptomyces symbionts of beewolves produce antibiotics to protect the host from fungal infection (17), and bacteriophages encoding putative toxins are required for Hamiltonella defensa to protect its aphid host from parasitic wasps (18), whereas the causes of other naturally occurring defensive symbioses are unresolved. From an applied perspective, the ongoing goal of exploiting insect symbioses to arrest disease transmission to humans from insect vectors (19) makes a deeper understanding of the factors contributing to ecologically relevant and evolutionarily durable defensive symbioses urgently needed.Here, we investigate the mechanism underlying one of the most striking examples of an ecologically important defensive symbiosis. Drosophila neotestacea is a woodland fly that is widespread across North America and is commonly parasitized by the nematode Howardula aoronymphium. Infection normally sterilizes flies (20); however, when flies harbor a strain of the inherited symbiont Spiroplasma—a Gram-positive bacterium in the class Mollicutes—they remarkably tolerate Howardula infection without loss of fecundity, and infection intensity is substantially reduced (13). The benefit conferred by this protection lends a substantial selective advantage to Spiroplasma-infected flies and has led to Spiroplasma’s recent spread across North America, with symbiont-infected flies rapidly replacing uninfected ones (21). Spiroplasma is a diverse and widespread lineage of arthropod-associated bacteria that can be commensal, pathogenic, or mutualistic (22). Maternal transmission has arisen numerous times in Spiroplasma, including strains that are well known as male-killers (22). In addition to defense against nematodes in D. neotestacea, other strains of Spiroplasma have recently been shown to protect flies and aphids against parasitic wasps and pathogenic fungi, respectively (23–25), but in no case is the mechanism of defense understood.In theory, there are multiple avenues by which a symbiont may protect its host that include competing with parasites for limiting resources, priming host immunity, or producing factors to directly attack parasites (26). We previously assessed these possibilities in the defensive Spiroplasma from D. neotestacea (27); our findings best supported a role for toxins in defense, with Spiroplasma encoding a highly expressed putative ribosome-inactivating protein (RIP). RIPs are widespread across plants and some bacteria and include well-known plant toxins of particular human concern such as ricin, as well as important virulence factors in human toxigenic strains of Escherichia coli and Shigella (28, 29). RIPs characteristically exert their cytotoxic effects through depurination of eukaryotic 28S ribosomal RNAs (rRNAs) at a highly conserved adenine in the α-sarcin/ricin loop (SRL) of the rRNA by cleaving the N-glycosidic bond between the rRNA backbone and adenine (30, 31). The proliferation of RIPs across different lineages implies functional significance, but their ecological roles are unclear, although they often appear to have antiviral or other defensive roles (29, 32). Here, we find that Spiroplasma expresses a functional RIP distinct from previously characterized toxins that appears to specifically affect Howardula rRNA in flies coinfected with Spiroplasma and Howardula. This work suggests the mechanisms used in defensive associations to protect the host from disease as well as intriguing ecological roles for RIPs in a tripartite defensive symbiosis.     

Host adaptation to viruses relies on few genes with different cross-resistance properties

Host adaptation to one parasite may affect its response to others. However, the genetics of these direct and correlated responses remains poorly studied. The overlap between these responses is instrumental for the understanding of host evolution in multiparasite environments. We determined the genetic and phenotypic changes underlying adaptation of Drosophila melanogaster to Drosophila C virus (DCV). Within 20 generations, flies selected with DCV showed increased survival after DCV infection, but also after cricket paralysis virus (CrPV) and flock house virus (FHV) infection. Whole-genome sequencing identified two regions of significant differentiation among treatments, from which candidate genes were functionally tested with RNAi. Three genes were validated—pastrel, a known DCV-response gene, and two other loci, Ubc-E2H and CG8492. Knockdown of Ubc-E2H and pastrel also led to increased sensitivity to CrPV, whereas knockdown of CG8492 increased susceptibility to FHV infection. Therefore, Drosophila adaptation to DCV relies on few major genes, each with different cross-resistance properties, conferring host resistance to several parasites.Parasites impose a strong fitness cost on their hosts as they develop and reproduce at the expenses of host resources. Therefore, it is expected that host strategies will be selected to cope with parasite burden. There is an ample variety of such strategies, from behavioral to intracellular responses (1). Because the range of possibilities is very broad, it is difficult to predict which strategy, if any, will evolve in host populations upon parasite attack. Moreover, in natural populations, hosts are exposed simultaneously to several parasite species and many other selection pressures. If these selection pressures do not vary independently of each other, a clear establishment of causality between changes in host traits and the selection pressure posed by a given parasite species may be hampered.Experimental evolution enables the establishment of a direct link between the selection imposed by a given environment and the genetic and phenotypic changes observed in a population. The explanatory power of this methodology relies on three major characteristics: (i) knowledge of the ancestral state; (ii) control of the selection forces driving different sets of replicated populations; and (iii) the ability to follow the dynamics of a process, instead of measuring only its end-product (2). In addition, this methodology allows addressing the consequences of the adaptation process for the performance in other environments (3–5).Experimental evolution coupled with whole-genome approaches can provide a nearly unbiased view of the actual targets of selection, a long-standing aim of evolutionary biology (2). To this day few examples exist in which these combined methodologies have been used in multicellular sexual organisms in which most adaptation comes from standing genetic variation (SGV) instead of novel mutations (6–10). However, despite the centrality of host–parasite interactions in evolutionary biology and several experimental evolution studies in host–parasite systems (11–16), to our knowledge, no study of host–parasite interactions has combined experimental evolution with genomics.Another important aspect of experimental evolution is that it allows the measurement of the consequences of evolving in one environment for the performance in other environments (3). Indeed, adaptation to one environment may entail a fitness decrease in other environments, possibly hampering future evolution in such settings (17, 18). Despite being common, these costs are not universal (4) even within experiments (17). Moreover, adapting to one environment may even lead to increased performance in other environments (e.g., 5, 19). In host–parasite interactions, this question is particularly important because of the epidemiological consequences of infecting or resisting multiple hosts or parasites, respectively.Despite ample knowledge of the genes triggered by parasite attacks against Drosophila, only a few key studies have analyzed how an outbred fly population may adapt to a given parasite (11–13, 15). However, the genetic basis and the consequences of such adaptation for host susceptibility to other parasites have not been determined.It has been shown that natural Drosophila melanogaster populations contain SGV for resistance against natural viruses. Whereas some studies show that most of this variation can be attributed to a limited number of genes with major effect (20–23), others indicate that a significant fraction of the genetic variation for resistance is polygenic (24, 25). Interestingly, the alleles that contribute to the variation in resistance to a given virus are of genes unrelated to the canonical insect antiviral defense pathways (26). Moreover, this variation may be rather specific in mediating responses to distinct natural pathogens (21).Here, we addressed the genetics of host adaptation to parasites and the effects in cross-resistance in a D. melanogaster–virus system. To this aim, we performed experimental evolution of an outbred D. melanogaster population exposed to a natural viral parasite (Drosophila C virus or DCV), analyzed the basis for the response using a genome-wide approach, and functionally tested candidate genes for their role in the response against DCV and other parasites.     

Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins

Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival. The destruction of PVs by IFNγ-inducible guanylate binding protein (GBP) and immunity-related GTPase (IRG) host proteins is central to a successful immune response directed against numerous PV-resident pathogens. However, the mechanism by which IRGs and GBPs cooperatively detect and destroy PVs is unclear. We find that host cell priming with IFNγ prompts IRG-dependent association of Toxoplasma- and Chlamydia-containing vacuoles with ubiquitin through regulated translocation of the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). This initial ubiquitin labeling elicits p62-mediated escort and deposition of GBPs to PVs, thereby conferring cell-autonomous immunity. Hypervirulent strains of Toxoplasma gondii evade this process via specific rhoptry protein kinases that inhibit IRG function, resulting in blockage of downstream PV ubiquitination and GBP delivery. Our results define a ubiquitin-centered mechanism by which host cells deliver GBPs to PVs and explain how hypervirulent parasites evade GBP-mediated immunity.Pathogen-containing vacuoles (PVs) provide a safe haven to many intracellular bacterial and protozoan pathogens (1). Within the vacuolar enclosure of PVs, these pathogens can accumulate nutrients required for microbial growth. Moreover, life within the vacuolar niche shields microbes from cytoplasmic immune sensors that, once activated, can trigger proinflammatory and cell-autonomous immune responses (1). Accordingly, many intracellular pathogens such as the bacterium Chlamydia trachomatis and the protozoan Toxoplasma gondii have successfully adapted to a vacuolar lifestyle.For the host to successfully combat infections with PV-resident microbes, the innate immune system must target PVs and its inhabitants for destruction. Critical mediators of host-directed attacks on PVs are two families of IFNγ-inducible GTPases: immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) (2). Members of both GTPase families play roles in host-mediated lysis of PVs, a process resulting in the release of microbes into the host cell cytoplasm, subsequent killing of PV-expelled microbes, and host cell death (3–8). Additionally, GBPs help deliver cytosolic subunits of the antimicrobial NADPH oxidase NOX2 for assembly on phagosomal membranes, orchestrate the capture of PV-resident microbes inside degradative autophagolysosomes, and promote the activation of canonical and noncanonical inflammasome pathways (5, 8–12). As a critical first step underlying most if not all of these known GBP-controlled cell-autonomous immune responses, GBPs must locate to their intracellular microbial targets.GBPs belong to the dynamin superfamily of large GTPases (13). Similar to other members of the dynamin superfamiliy, GBPs can assemble as oligomers in a nucleotide-dependent fashion (13). Binding of GTP results in dimer formation; subsequent GTP hydrolysis prompts conformational changes that enable GBPs to assemble as tetramers (14, 15). Mutations in the G domain that reduce nucleotide binding affinities and hydrolytic activity block GBP oligomerization, constrain the localization of GBPs to the cytoplasm, and prevent GBPs from binding to PV membranes (9, 15–18). These observations support a model in which GBP monomers are diffusely distributed in the cytoplasm and GBP oligomers associate with membranes. However, these observations fail to account for the specificity with which oligomeric GBPs agglomerate on PV membranes.PVs formed by C. trachomatis and T. gondii recruit not only GBPs but also members of the IRG family of IFNγ-inducible GTPase (4, 19). The IRG protein family can be divided into two subgroups: IRGM and GKS proteins (20). Whereas GKS proteins feature the canonical glycine–lysine–serine (GKS) P-loop sequence, IRGM proteins have a substitution of a lysine for a methionine in their P-loop sequence (20). IRGM and GKS proteins also differ in their subcellular localization: IRGM proteins associate with endomembranes, whereas monomeric GDP-bound GKS proteins predominantly reside within the host cell cytoplasm (4, 17, 21, 22). Once GKS proteins transition into a GTP-bound active state, they can bind to PV membranes (21). IRGM proteins inhibit this activation step and thereby guard IRGM-decorated membranes against GKS protein targeting (17, 21). Because PV membranes surrounding either C. trachomatis or T. gondii are largely devoid of IRGM proteins, they are the preferred GKS binding substrate following a “missing-self” principle of immune targeting (17, 23). In IRGM-deficient cells, however, GKS proteins enter the active state prematurely, form protein aggregates, mislocalize, and thus fail to bind to PVs (17, 21). Although these previous observations help explain how IRGM proteins promote the delivery of GKS proteins to PVs, IRGM proteins also control the subcellular localization of GBPs through an uncharacterized mechanism (6, 17, 24–26).Here, we report a previously unidentified host-directed ubiquitination pathway involved in innate immunity. We demonstrate that Chlamydia- and Toxoplasma-containing vacuoles become ubiquitin-decorated upon IFNγ priming of their host cells. IFNγ-dependent association of ubiquitin with PVs requires IFNγ-inducible IRG proteins and the E3 ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). Experimental removal of the IFNγ-inducible ubiquitination pathway dramatically diminishes the p62-dependent delivery of GBPs to PVs and thereby renders host cells more susceptible to infections. Thus, our observations imply that ubiquitin serves as a host-induced pattern that marks intracellular structures as immune targets for members of the GBP family of host defense proteins.     

Local host specialization,host-switching,and dispersal shape the regional distributions of avian haemosporidian parasites

The drivers of regional parasite distributions are poorly understood, especially in comparison with those of free-living species. For vector-transmitted parasites, in particular, distributions might be influenced by host-switching and by parasite dispersal with primary hosts and vectors. We surveyed haemosporidian blood parasites (Plasmodium and Haemoproteus) of small land birds in eastern North America to characterize a regional parasite community. Distributions of parasite populations generally reflected distributions of their hosts across the region. However, when the interdependence between hosts and parasites was controlled statistically, local host assemblages were related to regional climatic gradients, but parasite assemblages were not. Moreover, because parasite assemblage similarity does not decrease with distance when controlling for host assemblages and climate, parasites evidently disperse readily within the distributions of their hosts. The degree of specialization on hosts varied in some parasite lineages over short periods and small geographic distances independently of the diversity of available hosts and potentially competing parasite lineages. Nonrandom spatial turnover was apparent in parasite lineages infecting one host species that was well-sampled within a single year across its range, plausibly reflecting localized adaptations of hosts and parasites. Overall, populations of avian hosts generally determine the geographic distributions of haemosporidian parasites. However, parasites are not dispersal-limited within their host distributions, and they may switch hosts readily.A regional community can be thought of as a set of species whose distributions partially overlap within a large geographic area (1, 2). The structure of the regional community (i.e., the relative abundances of species across space and the degree to which populations cooccur) is governed by local (e.g., interspecific competition) and regional (e.g., species diversification and dispersal) processes (3). Although regional communities include all species, parasites and pathogens are rarely considered integral community members (4). Indeed, impacts of parasites on community structure are frequently associated with epidemics—often following introductions to nonnative regions—that have driven naïve hosts to extinction or near extinction (5–7). However, parasites likely play a critical role in shaping regional community structure. Parasites can comprise a large proportion of the community biomass (8), form the majority of links in a community food web (9), and influence regional diversity by variously accelerating (10) or slowing (11) host diversification.Nevertheless, few studies have investigated the processes influencing the regional community structure of both parasites and their hosts. Parasite populations are integrated into community studies with difficulty, partly because these populations are distributed across multiple dimensions—space, host species, and host individuals (12)—and also because parasites are difficult to sample. Moreover, although parasites tend to specialize on one or a few host species, host-breadth may vary across a parasite’s range (13).Regional studies of birds and their dipteran-vectored haemosporidian (“malaria”) blood parasites (14–19) have shown that many parasites are heterogeneously distributed across space despite the availability of suitable hosts. Specialized associations between specific parasites and vectors (20–22) may drive such heterogeneity, although a recent analysis suggests that parasite–host compatibility is also important (23), and local coevolutionary relationships between parasites and their hosts likely influence geographic distributions of both host and parasite populations (11, 14, 15). However, most regional studies of these parasites have focused on individual host species (24–30).Here, we investigate the regional community structure of avian hosts and their haemosporidian parasites with respect to abiotic and biotic drivers of both host and parasite distributions. We surveyed local assemblages of avian haemosporidian parasites across eastern North America and related the distributions of individual parasite lineages to regional climate variation and to the distributions and abundances of their avian hosts. Community dissimilarities between sampling locations based on host assemblage structure (i.e., the relative abundances of potential host species) were positively correlated with those based on parasite assemblage structure, suggesting interdependence of host and parasite population distributions. However, when controlling statistically for that interdependence, local host assemblages responded strongly to environmental gradients and differed more with increasing geographic separation, whereas parasite assemblages did not. This finding suggests that haemosporidian parasites disperse readily across the distributions of their host populations in eastern North America, independently of difference in climate and geographic distance. The degree to which some parasite lineages specialized on particular hosts varied across years and locations, and the nonrandom parasite lineage turnover across the distribution of one well-sampled host species suggested that adaptations of hosts and parasites may also shape regional community structure. Despite evidence of pathogenicity of haemosporidian parasites in birds (31), correlations between host abundances and parasite relative abundances across the region were statistically indistinguishable from random. Taken together, these results suggest that the distributions of parasite populations largely follow the distributions of their hosts but that parasites readily switch hosts and may replace each other across the ranges of individual hosts, resulting in a complex and dynamic regional community.     

De novo production of the plant-derived alkaloid strictosidine in yeast

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.     

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.     

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid–bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.Plasmids serve as extrachromosomal DNA platforms for the reassortment, mobilization, and maintenance of antibiotic resistance genes in bacteria, enabling host cells to colonize environments flooded with antimicrobials and to take advantage of resources freed by the extinction of nonresistant competitors. Fueled by these selective forces and aided by their itinerant nature, plasmids disseminate resistance genes worldwide shortly after new antibiotics are developed, which is a major clinical concern (1–3). However, in antibiotic-free environments, such genes are dispensable. There, the cost that plasmid carriage imposes on cells constitutes a disadvantage in the face of competition from other cells and, because plasmids depend on their hosts to survive, also a threat to their own existence.Many plasmids keep low copy numbers (CNs) to minimize the problem above, because it reduces burdens to host cells. However, this also decreases their chances to fix in descendant cells, a new survival challenge (4). To counteract this, plasmids have evolved stability functions. Partition systems pull replicated plasmid copies to opposite poles in host cells, facilitating their inheritance by daughter cells (5). Plasmids also bear postsegregational killing (PSK) systems, which encode a stable toxin and a labile antitoxin (TA) pair that eliminates plasmid-free cells produced by occasional replication or partition failures. Regular production of the labile antitoxin protects plasmid-containing cells from the toxin. However, antitoxin replenishment is not possible in cells losing the plasmid, and this triggers their elimination (5).TA pairs are common in plasmids disseminating antibiotic resistance in bacterial pathogens worldwide (2, 6–10). The link of these systems to PSK and the exiguous list of alternatives in the pipeline have led some to propose that chemicals activating these TA pairs may constitute a powerful antibiotic approach against these organisms (5, 11–13). However, the appropriateness of these TA pairs as therapeutic targets requires unequivocal understanding of their function in plasmids. Although PSK systems encode TA pairs, not all TA pairs might function as PSK systems, as suggested by their abundance in bacterial chromosomes, where PSK seems unnecessary (14–16). Moreover, the observation that many plasmids bear several TA pairs (6–10) raises the intriguing question of why they would need more than one PSK system, particularly when they increase the metabolic burden that plasmids impose on host cells (17). Because PSK functions are not infallible, their gathering may provide a mechanism for reciprocal failure compensation, minimizing the number of cells that escape killing upon plasmid loss (5). Alternatively, some TA pairs may stabilize plasmids by mechanisms different from PSK, and their grouping might not necessarily reflect functional redundancy (18).This may be the case in plasmid R1, which encodes TA pairs hok-sok (host killing-suppressor of killing) and kis(pemI)-kid(pemK) (19–23). Inconsistent with PSK, we had noticed that activation of toxin Kid occurred in cells that still contained R1, and that this happened when CNs were insufficient to ensure plasmid transmission to descendant cells. We also found that Kid cleaved mRNA at UUACU sites, which appeared well suited to trigger a response that prevented plasmid loss and increased R1 CNs without killing cells, as suggested by our results. In view of all this, we argued that Kid and Kis functioned as a rescue system for plasmid R1, and not as a PSK system (24). This proposal cannot be supported by results elsewhere, suggesting that Kid may cleave mRNA at simpler UAH sites (with H being A, C, or U) (25, 26), a view that has prevailed in the literature (14, 16, 27–29). Moreover, other observations indicate that our past experiments may have been inappropriate to conclude that Kid does not kill Escherichia coli cells (30, 31). Importantly, Kid, Kis, and other elements that we found essential for R1 rescue are conserved in plasmids conferring resistance to extended-spectrum β-lactamases, a worrying threat to human health (1, 6–10, 32). Therapeutic options to fight pathogens carrying these plasmids are limited, and activation of Kid may be perceived as a good antibiotic alternative. Because the potential involvement of this toxin in plasmid rescue advises against such approach, we aimed to ascertain here the mode of action; the effects on cells; and, ultimately, the function of Kid (and Kis) in R1.     

The pathogen Batrachochytrium dendrobatidis disturbs the frog skin microbiome during a natural epidemic and experimental infection

Symbiotic microbial communities may interact with infectious pathogens sharing a common host. The microbiome may limit pathogen infection or, conversely, an invading pathogen can disturb the microbiome. Documentation of such relationships during naturally occurring disease outbreaks is rare, and identifying causal links from field observations is difficult. This study documented the effects of an amphibian skin pathogen of global conservation concern [the chytrid fungus Batrachochytrium dendrobatidis (Bd)] on the skin-associated bacterial microbiome of the endangered frog, Rana sierrae, using a combination of population surveys and laboratory experiments. We examined covariation of pathogen infection and bacterial microbiome composition in wild frogs, demonstrating a strong and consistent correlation between Bd infection load and bacterial community composition in multiple R. sierrae populations. Despite the correlation between Bd infection load and bacterial community composition, we observed 100% mortality of postmetamorphic frogs during a Bd epizootic, suggesting that the relationship between Bd and bacterial communities was not linked to variation in resistance to mortal disease and that Bd infection altered bacterial communities. In a controlled experiment, Bd infection significantly altered the R. sierrae microbiome, demonstrating a causal relationship. The response of microbial communities to Bd infection was remarkably consistent: Several bacterial taxa showed the same response to Bd infection across multiple field populations and the laboratory experiment, indicating a somewhat predictable interaction between Bd and the microbiome. The laboratory experiment demonstrates that Bd infection causes changes to amphibian skin bacterial communities, whereas the laboratory and field results together strongly support Bd disturbance as a driver of bacterial community change during natural disease dynamics.Symbiotic interactions between microbes and multicellular organisms are ubiquitous. In recent years, research to understand the complex microbial communities living in or on multicellular organisms (termed the microbiome) has sparked fundamental changes in our understanding of the biology of metazoans (1–5). The microbiome can affect host health directly by influencing metabolism (6), development (7), inflammation (8), or behavior (9), but it may also influence host health indirectly through interactions with infectious pathogens. The microbiome may interact with pathogens through competition for resources, release of antimicrobial compounds, contact-dependent antagonism, or modulation of the host immune response (10), and an “imbalanced” microbiome may leave the host more susceptible to pathogen infection (11, 12). At the same time, an invading pathogen may disrupt the microbiome (10, 13–15). Thus, the microbiome may play a role in disease resistance, or may itself be disturbed or altered by invading pathogens. Although a wealth of recent research has described associations between microbiome composition and a variety of syndromes in both humans and animals (16–25), documentation of microbiome responses to natural epidemics of known infectious pathogens is rare.Chytridiomycosis is an emerging infectious disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Bd is an aquatic fungus that infects the skin of amphibians and disrupts osmoregulation, a critical function of amphibian skin (26). Chytridiomycosis can be fatal, and the severity of disease symptoms has been linked to Bd load, which is a measure of the density of Bd cells infecting the host (27, 28). Bd has a broad host range spanning hundreds of amphibian species, and has been implicated in population extinctions and species declines worldwide (29–34). Efforts to understand and mitigate the effects of Bd have led to research examining the potential for symbiotic bacteria to increase resistance to infection by the pathogen (35, 36). Bacterial species isolated from the skin of amphibians have been shown to inhibit the growth of Bd and other fungal pathogens in culture (37–39), possibly by producing antifungal metabolites (40, 41). In a controlled laboratory experiment, inundation of Rana muscosa with the bacterium Janthinobacterium lividium protected frogs from subsequent Bd infection (42). These and other studies highlight the possible role of bacteria in resistance to chytridiomycosis, but critical questions remain. First, most research has focused on the ability of cultured bacteria to prevent Bd infection, whereas very little is known about whether Bd infection alters the diverse skin microbiome. Examining this latter concept is critical both to a basic understanding of how the microbiome interacts with pathogens and to conservation efforts because Bd-induced perturbations of the microbiome could undermine attempts to mitigate effects of Bd infection through augmentation with particular bacteria. A second knowledge gap is the paucity of comprehensive culture-independent assessments of the amphibian microbiome, which are important because the vast majority of environmental and symbiotic microbes are not readily cultured, and culture-based methods can lead to severe underestimates of diversity and biased assessment of community composition (43). Few studies have applied next-generation sequencing methods to characterize the microbial communities on amphibian skin (44–47), and, to our knowledge, none have done so in the context of Bd infection. A final challenge to understanding interactions between Bd and bacteria stems from the difficulties of drawing direct connections between laboratory and field studies. Laboratory studies are essential for definitive identification of cause and effect. However, complex natural microbiomes can be impossible to recreate in the laboratory, and field studies are needed to show whether processes identified in the laboratory are relevant in nature.We present paired laboratory and field studies using high-throughput 16S amplicon pyrosequencing both to document associations between Bd infection and the amphibian skin bacterial microbiome in nature and to deduce causal relationships in an experiment. Our work centers on the Sierra Nevada yellow-legged frog, Rana sierrae, which is severely threatened by, and has already suffered drastic declines due to, Bd (28, 48). We surveyed frogs from four distinct R. sierrae populations to test if differences in skin bacterial communities are associated with the intensity of pathogen infection. We then conducted a laboratory experiment to establish causal relationships underlying Bd-bacterial community associations. The data establish a strong effect of Bd infection on the composition of the amphibian skin bacterial microbiome that is consistent between the laboratory experiment and naturally occurring Bd dynamics in wild frog populations.     

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.     

Global divergence of the human follicle mite Demodex folliculorum: Persistent associations between host ancestry and mite lineages

Microscopic mites of the genus Demodex live within the hair follicles of mammals and are ubiquitous symbionts of humans, but little molecular work has been done to understand their genetic diversity or transmission. Here we sampled mite DNA from 70 human hosts of diverse geographic ancestries and analyzed 241 sequences from the mitochondrial genome of the species Demodex folliculorum. Phylogenetic analyses recovered multiple deep lineages including a globally distributed lineage common among hosts of European ancestry and three lineages that primarily include hosts of Asian, African, and Latin American ancestry. To a great extent, the ancestral geography of hosts predicted the lineages of mites found on them; 27% of the total molecular variance segregated according to the regional ancestries of hosts. We found that D. folliculorum populations are stable on an individual over the course of years and that some Asian and African American hosts maintain specific mite lineages over the course of years or generations outside their geographic region of birth or ancestry. D. folliculorum haplotypes were much more likely to be shared within families and between spouses than between unrelated individuals, indicating that transmission requires close contact. Dating analyses indicated that D. folliculorum origins may predate modern humans. Overall, D. folliculorum evolution reflects ancient human population divergences, is consistent with an out-of-Africa dispersal hypothesis, and presents an excellent model system for further understanding the history of human movement.Human evolution did not take place in isolation but instead occurred alongside that of many closely associated species. Phylogeographic studies of human-associated species—such as lice and rodents, as well as certain bacteria and viruses—have suggested, eliminated, and confirmed hypotheses about human history (1–10). For example, these studies have provided details about the timing and nature of the original human migration out of Africa, the spread of humans within and among continents, and the domestication of large vertebrates.Mites of the genus Demodex live in the hair follicles and sebaceous glands of humans and provide a promising system with which to explore further the details of human evolution. The association between Demodex and Homo sapiens is likely to be an ancient one: The broad distribution of these mites across mammal species (11), coupled with the ancient date of divergence estimated between the two species known to be found on humans (12), suggests that Demodex originated and diversified with early mammals. Furthermore, Demodex seem likely to have been carried along whenever their hosts migrated, because they are ubiquitous inhabitants of human skin (13, 14). Finally, in comparison with the other human associates that have been studied to date, Demodex mites are more tightly associated with human bodies than are lice, while their generation times are slower than those of bacteria and viruses but are faster than those of rodents, making them a complementary system with which to understand the evolution of both humans and human associates.Two species of Demodex are known to inhabit the skin of humans. Histological studies suggest that each occupies a different niche: Demodex folliculorum resides in the hair follicle and is often found near the skin surface, whereas Demodex brevis is generally found deep in the sebaceous glands (15). As a result, the frequency of D. folliculorum movement from one host to another may be greater than that of D. brevis. A recent phylogenetic analysis of Demodex, including the two human associates, shows geographically structured genetic variation in D. brevis in which individuals of European descent and those of temperate Asian (Chinese) descent exhibit up to 6% divergence in nuclear ribosomal 18S sequence (14). In contrast, studies based on 18S rDNA and 16S mtDNA suggest that D. folliculorum exhibits no clear geographic structure among hosts from China, Spain, Brazil, and the United States (14, 16, 17). However, without additional sampling it is impossible to know whether the absence of apparent geographic structure in D. folliculorum truly reflects high rates of global gene flow or instead is an artifact of limited global sampling and the particular genetic loci studied.Key to understanding the global phylogeography of these mites is an understanding of how they move among hosts. The transfer of mites from mother to progeny and between mating partners has been demonstrated in nonhuman mammals (18–21). However, the movement of Demodex among human hosts has not been characterized. If human mites are transferred between hosts at high rates, the resulting high rates of migration could account for the limited geographic structure observed in D. folliculorum to date.Here we used a 930-bp fragment of the mitochondrial genome to evaluate the genetic diversity and phylogeography of D. folliculorum among 70 human hosts of diverse geographic origins and ancestries. Our samples included people of European, Asian, African, and Latin American descent, the majority of whom currently live in the United States, providing the most broadly sampled evolutionary tree to date for any Demodex species.Additionally, we investigated Demodex transmission among humans in two ways. First, we sampled multiple mites from a single host individual over the course of 3 y to characterize the diversity and stability of the mite population. Second, we examined the relationships among mites on three sets of parents and their adult progeny; because of the close association among family members, we hypothesized that mite lineages are more likely to be shared within families than between unrelated hosts.The study of Demodex mites speaks to the story of human evolution as well as the coevolution between symbiont and host. Moreover, understanding these mites and their microbes will have applied value, because they have been linked to skin disorders such as rosacea and blepharitis (22, 23). Whatever the influence of mites on these disorders may be, it may depend on the mite lineages inhabiting a particular host. Ultimately, elucidating the evolution and transmission of Demodex mites not only will be a useful step toward understanding the evolutionary history of humans but also will be critical to contextualizing their role in human health.     

The autophagic machinery ensures nonlytic transmission of mycobacteria

In contrast to mechanisms mediating uptake of intracellular bacterial pathogens, bacterial egress and cell-to-cell transmission are poorly understood. Previously, we showed that the transmission of pathogenic mycobacteria between phagocytic cells also depends on nonlytic ejection through an F-actin based structure, called the ejectosome. How the host cell maintains integrity of its plasma membrane during the ejection process was unknown. Here, we reveal an unexpected function for the autophagic machinery in nonlytic spreading of bacteria. We show that ejecting mycobacteria are escorted by a distinct polar autophagocytic vacuole. If autophagy is impaired, cell-to-cell transmission is inhibited, the host plasma membrane becomes compromised and the host cells die. These findings highlight a previously unidentified, highly ordered interaction between bacteria and the autophagic pathway and might represent the ancient way to ensure nonlytic egress of bacteria.In recent years, our understanding of the interactions between the host autophagic machinery and intracellular pathogens has rapidly expanded. These interactions are complex; although, in many cases, the engagement of autophagy protects the host by capturing and destroying the pathogen, some bacteria actively subvert this pathway to promote their own survival (reviewed in ref. 1). Autophagy has also been suggested to promote cell-to-cell transmission of Brucella (2, 3), although the molecular mechanisms are unknown.Both Mycobacterium tuberculosis, which causes tuberculosis in humans, and the closely related species M. marinum have been shown to interact with the autophagy machinery of their host cell (4–7). After uptake by immune phagocytes, the bacteria arrest phagosomal maturation and convert their vacuole into a replication-permissive compartment. Both bacteria can translocate into the host cell cytosol dependent on an intact Region-of-Difference-1-locus (RD1) (8–11). The genomic RD1-locus encodes a secretion system, ESX-1 (Type-VII secretion system), which has been associated with mycobacterial virulence (ref. 12, reviewed in refs. 13 and 14). Once in the cytosol, M. marinum becomes ubiquitinated (4) likely recruiting adaptor proteins, such as members of the sequestosome-1 family (SQSTM1), which also bind LC3 (microtubule-associated proteins 1A/1B light chains 3A/LC3A and 3B/LC3B), here referred to as Atg8, on autophagosomal membranes. In this way, bacteria are normally targeted to autophagosomes and killed, but M. marinum efficiently escapes this fate, most probably by shedding the ubiquitinated material as a decoy (4). However, infection by M. tuberculosis can be overcome by stimulating the classic autophagic pathway (15) and autophagy can reduce the bacterial burden in vivo (7).It was previously thought that M. marinum and M. tuberculosis leave their host cell by inducing necrotic or apoptotic cell death (16). However, we recently showed that these bacteria also exit their host cell and spread via an F-actin structure, termed the ejectosome (17). This form of egress, which is common to M. tuberculosis and M. marinum in the amoeba Dictyostelium, is nonlytic for the host cell, even though its plasma membrane is perforated at the site of ejection. Previously, we showed that ejectosome formation is dependent on ESAT-6 (Early secretory antigenic target 6), a secreted virulence factor encoded in the RD1-locus, and the Dictyostelium small GTPase RacH. However, both the structure and mechanistic details of ejectosome function remain unknown.Using the Dictyostelium–M. marinum system (9, 17, 18) to further dissect the mechanism of ejectosome formation and function, we demonstrate an unexpected role for autophagic membranes in both mycobacteria egress and concomitant cell-to-cell transmission.     

Distinct dopamine neurons mediate reward signals for short- and long-term memories

Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.Memory of a momentous event persists for a long time. Whereas some forms of long-term memory (LTM) require repetitive training (1–3), a highly relevant stimulus such as food or poison is sufficient to induce LTM in a single training session (4–7). Recent studies have revealed aspects of the molecular and cellular mechanisms of LTM formation induced by repetitive training (8–11), but how a single training induces a stable LTM is poorly understood (12).Appetitive olfactory learning in fruit flies is suited to address the question, as a presentation of a sugar reward paired with odor induces robust short-term memory (STM) and LTM (6, 7). Odor is represented by a sparse ensemble of the 2,000 intrinsic neurons, the Kenyon cells (13). A current working model suggests that concomitant reward signals from sugar ingestion cause associative plasticity in Kenyon cells that might underlie memory formation (14–20). A single activation session of a specific cluster of dopamine neurons (PAM neurons) by sugar ingestion can induce appetitive memory that is stable over 24 h (19), underscoring the importance of sugar reward to the fly.The mushroom body (MB) is composed of the three different cell types, α/β, α′/β′, and γ, which have distinct roles in different phases of appetitive memories (11, 21–25). Similar to midbrain dopamine neurons in mammals (26, 27), the structure and function of PAM cluster neurons are heterogeneous, and distinct dopamine neurons intersect unique segments of the MB lobes (19, 28–34). Further circuit dissection is thus crucial to identify candidate synapses that undergo associative modulation.By activating distinct subsets of PAM neurons for reward signaling, we found that short- and long-term memories are independently formed by two complementary subsets of PAM cluster dopamine neurons. Conditioning flies with nutritious and nonnutritious sugars revealed that the two subsets could represent different reinforcing properties: sweet taste and nutritional value of sugar. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct reward signals.     

Mechanisms of NDV-3 vaccine efficacy in MRSA skin versus invasive infection

Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge development of an effective vaccine targeting Staphylococcus aureus. This study evaluated the efficacy and immunologic mechanisms of a vaccine containing a recombinant glycoprotein antigen (NDV-3) in mouse skin and skin structure infection (SSSI) due to methicillin-resistant S. aureus (MRSA). Compared with adjuvant alone, NDV-3 reduced abscess progression, severity, and MRSA density in skin, as well as hematogenous dissemination to kidney. NDV-3 induced increases in CD3+ T-cell and neutrophil infiltration and IL-17A, IL-22, and host defense peptide expression in local settings of SSSI abscesses. Vaccine induction of IL-22 was necessary for protective mitigation of cutaneous infection. By comparison, protection against hematogenous dissemination required the induction of IL-17A and IL-22 by NDV-3. These findings demonstrate that NDV-3 protective efficacy against MRSA in SSSI involves a robust and complementary response integrating innate and adaptive immune mechanisms. These results support further evaluation of the NDV-3 vaccine to address disease due to S. aureus in humans.The bacterium Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSIs), including cellulitis, furunculosis, and folliculitis (1–4), and a common etiologic agent of impetigo (5), erysipelas (6), and superinfection in atopic dermatitis (7). This bacterium is a significant cause of surgical or traumatic wound infections (8, 9), as well as decuibitus and diabetic skin lesions (10). Moreover, SSSI is an important risk factor for systemic infection. The skin is a key portal of entry for hematogenous dissemination, particularly in association with i.v. catheters. S. aureus is now the second most common bloodstream isolate in healthcare settings (11), and SSSI is a frequent source of invasive infections such as pneumonia or endocarditis (12, 13). Despite a recent modest decline in rates of methicillin-resistant S. aureus (MRSA) infection in some cohorts (13), infections due to S. aureus remain a significant problem (14, 15). Even with appropriate therapy, up to one-third of patients diagnosed with S. aureus bacteremia succumb—accounting for more attributable annual deaths than HIV, tuberculosis, and viral hepatitis combined (16).The empiric use of antibiotics in healthcare-associated and community-acquired settings has increased S. aureus exposure to these agents, accelerating selection of resistant strains. As a result, resistance to even the most recently developed agents is emerging at an alarming pace (17, 18). The impact of this trend is of special concern in light of high rates of mortality associated with invasive MRSA infection (e.g., 15–40% in bacteremia or endocarditis), even with the most recently developed antistaphylococcal therapeutics (19, 20). Moreover, patients who experience SSSI due to MRSA exhibit high 1-y recurrence rates, often prompting surgical debridement (21) and protracted antibiotic treatment.Infections due to MRSA are a special concern in immune-vulnerable populations, including hemodialysis (22), neutropenic (23, 24), transplantation (25), and otherwise immunosuppressed patients (26, 27), and in patients with inherited immune dysfunctions (28–31) or cystic fibrosis (32). Patients having deficient interleukin 17 (IL-17) or IL-22 responses (e.g., signal transduction mediators STAT3, DOCK8, or CARD9 deficiencies) exhibit chronic or “cold” abscesses, despite high densities of pathogens such as S. aureus (33, 34). For example, patients with Chronic Granulomatous Disease (CGD; deficient Th1 and oxidative burst response) have increased risk of disseminated S. aureus infection. In contrast, patients with Job’s Syndrome (deficient Th17 response) typically have increased risk to SSSI and lung infections, but less so for systemic S. aureus bacteremia (35, 36). This pattern contrasts that observed in neutropenic or CGD patients (37). These themes suggest efficacious host defenses against MRSA skin and invasive infections involve complementary but distinct molecular and cellular immune responses.From these perspectives, vaccines or immunotherapeutics that prevent or lessen severity of MRSA infections, or that enhance antibiotic efficacy, would be significant advances in patient care and public health. However, to date, there are no licensed prophylactic or therapeutic vaccine immunotherapies for S. aureus or MRSA infection. Unfortunately, efforts to develop vaccines targeting S. aureus capsular polysaccharide type 5 or 8 conjugates, or the iron-regulated surface determinant B protein, have not been successful thus far (38, 39). Likewise, passive immunization using monoclonal antibodies targeting the S. aureus adhesin clumping factor A (ClfA, tefibazumab) (40) or lipoteichoic acid (pagibaximab) (41) have not shown efficacy against invasive infections in human clinical studies to date. Moreover, the striking recurrence rates of SSSI due to MRSA imply that natural exposure does not induce optimal preventive immunity or durable anamnestic response to infection or reinfection. Thus, significant challenges exist in the development of an efficacious vaccine targeting diseases caused by S. aureus (42) that are perhaps not optimally addressed by conventional approaches.The NDV-3 vaccine reflects a new strategy to induce durable immunity targeting S. aureus. Its immunogen is engineered from the agglutinin-like sequence 3 (Als3) adhesin/invasin of Candida albicans, which we discovered to be a structural homolog of S. aureus adhesins (43). NDV-3 is believed to cross-protect against S. aureus and C. albicans due to sequence (T-cell) and conformational (B-cell) epitopes paralleled in both organisms (44). Our prior data have shown that NDV-3 is efficacious in murine models of hematogenous and mucosal candidiasis (45), as well as S. aureus bacteremia (46–48). Recently completed phase I clinical trials demonstrate the safety, tolerability, and immunogenicity of NDV-3 in humans (49).