Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex

Pathogen recognition by nucleotide-binding oligomerization domain-like receptor (NLR) results in the formation of a macromolecular protein complex (inflammasome) that drives protective inflammatory responses in the host. It is thought that the number of inflammasome complexes forming in a cell is determined by the number of NLRs being activated, with each NLR initiating its own inflammasome assembly independent of one another; however, we show here that the important foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) simultaneously activates at least two NLRs, whereas only a single inflammasome complex is formed in a macrophage. Both nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 and nucleotide-binding domain and leucine-rich repeat pyrin domain 3 are simultaneously present in the same inflammasome, where both NLRs are required to drive IL-1β processing within the Salmonella-infected cell and to regulate the bacterial burden in mice. Superresolution imaging of Salmonella-infected macrophages revealed a macromolecular complex with an outer ring of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain and an inner ring of NLRs, with active caspase effectors containing the pro–IL-1β substrate localized internal to the ring structure. Our data reveal the spatial localization of different components of the inflammasome and how different members of the NLR family cooperate to drive robust IL-1β processing during Salmonella infection.Inflammasomes are cytosolic multimeric protein complexes formed in the host cell in response to the detection of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Formation of the inflammasome in response to PAMPs is critical for host defense because it facilitates processing of the proinflammatory cytokines pro–IL-1β and pro–IL-18 into their mature forms (1). The inflammasome also initiates host cell death in the form of pyroptosis, releasing macrophage-resident microbes to be killed by other immune mechanisms (2). The current paradigm is that there are individual, receptor-specific inflammasomes consisting of one nucleotide-binding oligomerization domain-like receptor (NLR; leucine-rich repeat–containing) or PYHIN [pyrin domain and hematopoietic expression, interferon-inducible nature, and nuclear localization (HIN) domain-containing] receptor, the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD; ASC), and caspase-1 (3). How the protein constituents of the inflammasome are spatially orientated is unclear.Nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 (NLRC4) and nucleotide-binding domain and leucine-rich repeat pyrin domain 3 (NLRP3) are the best-characterized inflammasomes, especially with respect to their responses to pathogenic bacteria. The NLRC4 inflammasome is activated primarily by bacteria, including Aeromonas veronii (4), Escherichia coli (5), Listeria monocytogenes (6, 7), Pseudomonas aeruginosa (5), Salmonella enterica serovar Typhimurium (S. Typhimurium) (5, 8–10), and Yersinia species (11). In mouse macrophages, the NLRC4 inflammasome responds to flagellin and type III secretion system-associated needle or rod proteins (5, 8, 9) after their detection by NLR family, apoptosis inhibitory protein (NAIP) 5 or NAIP6 and NAIP1 or NAIP2, respectively (12–15). Phosphorylation of NLRC4 at a single, evolutionarily conserved residue, Ser 533, by PKCδ kinase is required for NLRC4 inflammasome assembly (16). The NLRP3 inflammasome is activated by a large repertoire of DAMPs, including ATP, nigericin, maitotoxin, uric acid crystals, silica, aluminum hydroxide, and muramyl dipeptide (17–20). NLRP3 is also activated by bacterial PAMPs from many species, including Aeromonas species (4, 21), L. monocytogenes (6, 7, 22), Neisseria gonorrhoeae (23), S. Typhimurium (10), Streptococcus pneumoniae (24), and Yersinia species (11). The mechanisms by which NLRC4 and NLRP3 inflammasomes contribute to host defense against bacterial pathogens are emerging; however, little is known about the dynamics governing inflammasome assembly in infections caused by bacteria that activate multiple NLRs, such as S. Typhimurium (10), A. veronii (4), and Yersinia (11).NLRP3 does not have a CARD and requires ASC to interact with the CARD of procaspase-1. This interaction requires a charged interface around Asp27 of the procaspase-1 CARD (25). Whether ASC is also required for the assembly of the NLRC4 inflammasome is less clear. NLRC4 contains a CARD that can interact directly with the CARD of procaspase-1 (26); however, ASC is required for some of the responses driven by NLRC4 (27). Macrophages infected with S. Typhimurium or other pathogens exhibit formation of a distinct cytoplasmic ASC focus or speck, which can be visualized under the microscope and is indicative of inflammasome activation (10, 28, 29). Our laboratory and others have shown that only one ASC speck is formed per cell irrespective of the stimulus used (29–32). However, many bacteria activate two or more NLRs, and it is unclear whether a singular inflammasome is formed at a time or if multiple inflammasomes are formed independent of each other, with each inflammasome containing one member of the NLR family.In this study, we describe the endogenous molecular constituents of the Salmonella-induced inflammasome and their spatial orientation. In cross-section, ASC forms a large external ring with the NLRs and caspases located internally. Critically, NLRC4, NLRP3, caspase-1, and caspase-8 coexist in the same ASC speck to coordinate pro–IL-1β processing. All ASC specks observed contained both NLRC4 and NLRP3. These results suggest that Salmonella infection induces a single inflammasome protein complex containing different NLRs and recruiting multiple caspases to coordinate a multifaceted inflammatory response to infection.     

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.     

Cytosolic flagellin-induced lysosomal pathway regulates inflammasome-dependent and -independent macrophage responses

NAIP5/NLRC4 (neuronal apoptosis inhibitory protein 5/nucleotide oligomerization domain-like receptor family, caspase activation recruitment domain domain-containing 4) inflammasome activation by cytosolic flagellin results in caspase-1–mediated processing and secretion of IL-1β/IL-18 and pyroptosis, an inflammatory cell death pathway. Here, we found that although NLRC4, ASC, and caspase-1 are required for IL-1β secretion in response to cytosolic flagellin, cell death, nevertheless, occurs in the absence of these molecules. Cytosolic flagellin-induced inflammasome-independent cell death is accompanied by IL-1α secretion and is temporally correlated with the restriction of Salmonella Typhimurium infection. Despite displaying some apoptotic features, this peculiar form of cell death do not require caspase activation but is regulated by a lysosomal pathway, in which cathepsin B and cathepsin D play redundant roles. Moreover, cathepsin B contributes to NAIP5/NLRC4 inflammasome-induced pyroptosis and IL-1α and IL-1β production in response to cytosolic flagellin. Together, our data describe a pathway induced by cytosolic flagellin that induces a peculiar form of cell death and regulates inflammasome-mediated effector mechanisms of macrophages.Flagellin, the monomeric subunit of flagella present in Gram-negative and Gram-positive bacteria, is one of the few protein structures that can activate both transmembrane and cytosolic pattern recognition receptors of the innate immune system. Extracellular flagellin is recognized by the transmembrane Toll-like receptor (TLR)5 (1). On the other hand, flagellin can be directly delivered into the cytosol by transport systems, such as the type III secretion system (T3SS) of Salmonella (2) and the type IV secretion system (T4SS) of Legionella (3). Once in the cytosol, flagellin is sensed by the inflammasome complex comprised of the NOD-like receptor (NLR) proteins neuronal apoptosis inhibitory protein (NAIP)5 and NLRC4 [NLR family, caspase activation recruitment domain (CARD) domain-containing 4] (2–5).Both TLR5 and NAIP5/NLRC4 receptors recognize conserved regions of flagellin. TLR5 is thought to detect a region of flagellin located in the D1 domain (6), whereas a sequence of three leucine residues that is present in the C-terminal D0 domain of flagellin is required to activate the NAIP5/NLRC4 inflammasome (7). Despite some redundant roles that are attributed to NLRC4 and NAIP5 in flagellin-mediated macrophage activation (7), a new model for NAIP5/NLRC4 inflammasome activation in response to flagellin was recently proposed (8, 9). In this model, NAIP5 acts as an immune sensor protein that specifically binds to flagellin (9). The interaction between NAIP5 and flagellin promotes the recruitment of NLRC4 through the NOD domain. The formation of this protein complex leads to the association of NLRC4 with procaspase-1 via CARD-CARD interactions. Additionally, NLRC4 can recruit the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), which also contains a CARD domain and is able to recruit and process procaspase-1.Caspase-1 activation results in the cleavage and secretion of biologically active forms of the inflammatory cytokines interleukin (IL)-1β and IL-18 (10) and the induction of a form of cell death named pyroptosis (11). The activation of caspase-1 in response to cytosolic flagellin by the NAIP5/NLRC4 inflammasome complex can also induce other effector mechanisms to restrict infections, such as caspase-7–dependent phagosome maturation (4, 12) and the activation of inducible nitric oxide synthase (iNOS) by macrophages (13). Both of these effector mechanisms lead to the inhibition of Legionella pneumophila replication. Importantly, caspase-1–induced IL-1β and IL-18 are not involved in phagosome maturation (4, 12), induction of pyroptosis (14), or iNOS activation (13), suggesting that caspase-1 mediates independent effects that cooperate to clear infections.Although the NAIP5/NLRC4 inflammasome complex is involved in the control of many bacterial infections, such as infection with Salmonella Typhimurium (2, 5), Shigella flexneri (15), Pseudomonas aeruginosa (16, 17), L. pneumophila (3, 4), and Listeria monocytogenes (18), the precise effector mechanism mediated by these receptors is not completely understood. Among the NAIP5/NLRC4 inflammasome-mediated effector mechanisms that have been implicated with intracellular bacterial replication control, pyroptosis has received great attention.Pyroptosis has been described as a programmed cell death pathway that uniquely depends on caspase-1 (19). Recently, it was demonstrated that the enteric pathogenic bacteria Escherichia coli, Citrobacter rodentium, and Vibrio cholerae and the cholera toxin B subunit can trigger the activation of a noncanonical inflammasome that targets caspase-11 (also known as caspase-4 in humans and related to caspase-1) (20). These stimuli induce cell death in a caspase-11–dependent fashion, but the process is not dependent on ASC, NLRC4, or caspase-1. Interestingly, this process of cell death (also named pyroptosis) is accompanied by the secretion of IL-1α but not by the secretion of IL-1β (which requires caspase-1). Importantly, the 129 mouse strain that was used to generate the first caspase-1^−/− mutants (21, 22) harbors a mutation in the caspase-11 locus that impairs caspase-11 function. Because of the close proximity in the genome between the caspase-1 and caspase-11 genes, the two proteins cannot be segregated by recombination. Therefore, these caspase-1^−/− mice are also defective for caspase-11 (20).Importantly, although pyroptosis is regulated by caspase activation, similarly to apoptosis, inhibition of or genetic deficiency in apoptotic caspase does not rescue cells from pyroptosis (11, 23). In addition, pyroptosis and apoptosis provide distinct outcomes for the immune response, which may be explained by the different morphological and biochemical changes that are observed in cells undergoing these forms of cell death (24, 25). Activation of caspase-1/11 results in the rapid formation of pores in the plasma membrane that dissipate cellular ionic gradients. This process allows the influx of water into the cells, resulting in cell swelling, osmotic lysis, and the release of intracellular contents (25, 26). The loss of plasma membrane integrity and the secretion of inflammatory mediators during pyroptosis, including IL-1β and IL-18, results in the induction of a strong inflammatory response (27). The inflammatory milieu produced by pyroptosis could result in the recruitment of effector cells to the site of infection as a mechanism of pathogen clearance. Recently, it was demonstrated that the ectopic expression of the Salmonella flagellin protein FliC during the intracellular phase of infection triggers pyroptosis of infected cells in vivo (14). The bacteria released by the pyroptotic macrophages were controlled by infiltrating neutrophils through a reactive oxygen species-dependent mechanism.Despite the evidence implicating pyroptosis as an important host defense mechanism to clear intracellular pathogens, the molecular regulation of pyroptosis is poorly understood. Here, we analyzed the regulation of macrophage death using purified flagellin as a single, death-inducing stimulus. Our data demonstrate that cytosolic flagellin is able to induce cell death in the absence of caspase-1/11. Although displaying some apoptotic features, such as cell shrinkage and the formation of membrane blebs, cytosolic flagellin-induced caspase-1/11–independent cell death does not require apoptotic caspases but depends on lysosomal events. Similar to pyroptosis, cytosolic flagellin-induced caspase-1/11–independent cell death results in the release of intracellular inflammatory contents. Caspase-1/11–independent cell death also contributes to the control of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) infection by macrophages, supporting the existence of an effector mechanism important to restrict bacterial infection. Finally, our data provide evidences that lysosomal cathepsins also regulate IL-1β secretion and pyroptosis in response to cytosolic flagellin. Taken together, our results suggests lysosome events as a central regulator of both inflammasome-dependent and inflammasome-independent macrophage responses induced by cytosolic flagellin.     

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.     

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).     

Hyperpolarization-activated,cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K⁺ and Na⁺ ions, and is selectively blocked by Cs⁺ and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway.Hyperpolarization-activated, cyclic nucleotide-gated (HCN), cation nonselective ion channels generate hyperpolarization-activated inward currents (I_h) and thus tend to stabilize membrane potential (1–3). In addition, binding of cyclic nucleotides (cAMP and cGMP) to the C-terminal cyclic nucleotide binding domain (CNBD) enhances I_h and thus couples membrane excitability with intracellular signaling pathways (2, 4). HCN channels are widely important for numerous systemic functions such as hormonal regulation, heart contractility, epilepsy, pain, central pattern generation, sensory perception (4–15), and learning and memory (16–24).However, in previous studies it has been difficult to relate the cellular effects of HCN channels directly to their behavioral effects, because of the immense complexity of the mammalian brain. We have therefore investigated the role of HCN channels in Aplysia, which has a numerically simpler nervous system (25). We first identified and characterized an HCN gene in Aplysia, and showed that it codes for a channel that has many similarities to the mammalian HCN channel. We found that the Aplysia HCN channel is predominantly expressed in motor neurons including LFS neurons in the siphon withdrawal reflex circuit (26, 27). We therefore investigated simple forms of learning of that reflex in a semiintact preparation (28–30) and found that HCN current is involved in classical conditioning and enhances the NMDA-like current in the motor neurons. These results provide a direct connection between HCN channels and behavioral learning and suggest a postsynaptic mechanism of that effect. HCN current in turn is enhanced by nitric oxide (NO), a transmitter of facilitatory interneurons, and thus may contribute to the postsynaptic role of NO during conditioning.     

Novel serologic biomarkers provide accurate estimates of recent Plasmodium falciparum exposure for individuals and communities

Tools to reliably measure Plasmodium falciparum (Pf) exposure in individuals and communities are needed to guide and evaluate malaria control interventions. Serologic assays can potentially produce precise exposure estimates at low cost; however, current approaches based on responses to a few characterized antigens are not designed to estimate exposure in individuals. Pf-specific antibody responses differ by antigen, suggesting that selection of antigens with defined kinetic profiles will improve estimates of Pf exposure. To identify novel serologic biomarkers of malaria exposure, we evaluated responses to 856 Pf antigens by protein microarray in 186 Ugandan children, for whom detailed Pf exposure data were available. Using data-adaptive statistical methods, we identified combinations of antibody responses that maximized information on an individual’s recent exposure. Responses to three novel Pf antigens accurately classified whether an individual had been infected within the last 30, 90, or 365 d (cross-validated area under the curve = 0.86–0.93), whereas responses to six antigens accurately estimated an individual’s malaria incidence in the prior year. Cross-validated incidence predictions for individuals in different communities provided accurate stratification of exposure between populations and suggest that precise estimates of community exposure can be obtained from sampling a small subset of that community. In addition, serologic incidence predictions from cross-sectional samples characterized heterogeneity within a community similarly to 1 y of continuous passive surveillance. Development of simple ELISA-based assays derived from the successful selection strategy outlined here offers the potential to generate rich epidemiologic surveillance data that will be widely accessible to malaria control programs.Many countries have extensive programs to reduce the burden of Plasmodium falciparum (Pf), the parasite responsible for most malaria morbidity and mortality (1). Effectively using limited resources for malaria control or elimination and evaluating interventions require accurate measurements of the risk of being infected with Pf (2–15). To reflect the rate at which individuals are infected with Pf in a useful way, metrics used to estimate exposure in a community need to account for dynamic changes over space and time, especially in response to control interventions (16–18).A variety of metrics can be used to estimate Pf exposure, but tools that are more precise and low cost are needed for population surveillance. Existing metrics have varying intrinsic levels of precision and accuracy and are subject to a variety of extrinsic factors, such as cost, time, and availability of trained personnel (19). For example, entomological measurements provide information on mosquito to human transmission for a community but are expensive, require specially trained staff, and lack standardized procedures, all of which reduce precision and/or make interpretation difficult (19–22). Parasite prevalence can be measured by detecting parasites in the blood of individuals from a cross-sectional sample of a community and is, therefore, relatively simple and inexpensive to perform, but results may be imprecise, especially in areas of low transmission (19, 23), and biased by a number of factors, including immunity and access to antimalarial treatment (5, 6, 19, 23–25). The burden of symptomatic disease in a community can be estimated from routine health systems data; however, such data are frequently unreliable (5, 26–28) and generally underestimate the prevalence of Pf infection in areas of intense transmission. Precise and quantitative information about exposure at an individual level can be reliably obtained from cohort studies by measuring the incidence of asymptomatic and/or symptomatic Pf infection (i.e., by measuring the molecular force of infection) (29–35). Unfortunately, the expense of cohort studies limits their use to research settings. The end result is that most malaria-endemic regions lack reliable, timely data on Pf exposure, limiting the capabilities of malaria control programs to guide and evaluate interventions.Serologic assays offer the potential to provide incidence estimates for symptomatic and asymptomatic Pf infection, which are currently obtained from cohort studies, at the cost of cross-sectional studies (36–38). Although Pf infections are transient, a record of infection remains detectable in an individual’s antibody profile. Thus, appropriately chosen antibody measurements integrated with age can provide information about an individual’s exposure history. Antibodies can be measured by simple ELISAs and obtained from dried blood spots, which are easy to collect, transport, and store (39–41). Serologic responses to Pf antigens have been explored as potential epidemiological tools (42–45), and estimated rates of seroconversion to well-characterized Pf antigens accurately reflect stable rates of exposure in a community, whereas distinct changes in these rates are obtained from successful interventions (22, 39, 41, 46–53). However, current serologic assays are not designed to detect short-term or gradual changes in Pf exposure or measure exposure to infection at an individual level. The ability to calibrate antibody responses to estimates of exposure in individuals could allow for more flexible sampling of a population (e.g., not requiring age stratification), improve accuracy of exposure estimates from small sample sizes, and better characterize heterogeneity in exposure within a community.Different Pf antigens elicit antibody responses with different magnitudes and kinetics, providing a large and diverse set of potential biomarkers for exposure (38, 54–58). We hypothesized that new and more highly informative serologic biomarkers better able to characterize an individual’s recent exposure history could be identified by analyzing antibody responses to a large number of candidate Pf antigens in participants with well-characterized exposure histories. To test this hypothesis, we probed plasma from participants in two cohort studies in Uganda against a protein microarray containing 856 Pf antigens. The primary aim of this analysis was to identify responses to select antigens that were most informative of recent exposure using robust, data-adaptive statistical methods. Each participant’s responses to these selected antigens were used as predictors for two primary outcomes of their recent exposure to Pf: (i) days since last Pf infection and (ii) the incidence of symptomatic malaria in the last year. These individual-level estimates were then aggregated across a population to assess community-level malaria exposure. The selection strategy presented here identified accurate biomarkers of exposure for children living in areas of moderate to high Pf exposure and illustrates the utility of this flexible and broadly applicable approach.     

UNC93B1 is essential for the plasma membrane localization and signaling of Toll-like receptor 5

The proper trafficking and localization of Toll-like receptors (TLRs) are important for specific ligand recognition and efficient signal transduction. The TLRs sensing bacterial membrane components are expressed on the cell surface and recruit signaling adaptors to the plasma membrane upon stimulation. On the contrary, the nucleotide-sensing TLRs are mostly found inside cells and signal from the endolysosomes in an acidic pH-dependent manner. Trafficking of the nucleotide-sensing TLRs from the endoplasmic reticulum to the endolysosomes strictly depends on UNC93B1, and their signaling is completely abolished in the 3d mutant mice bearing the H412R mutation of UNC93B1. In contrast, UNC93B1 was considered to have no role for the cell surface-localized TLRs and signaling via TLR1, TLR2, TLR4, and TLR6 is normal in the 3d mice. Unexpectedly, we discovered that TLR5, a cell surface receptor for bacterial protein flagellin, also requires UNC93B1 for plasma membrane localization and signaling. TLR5 physically interacts with UNC93B1, and the cells from the 3d or UNC93B1-deficient mice not only lack TLR5 at the plasma membrane but also fail to secret cytokines and to up-regulate costimulatory molecules upon flagellin stimulation, demonstrating the essential role of UNC93B1 in TLR5 signaling. Our study reveals that the role of UNC93B1 is not limited to the TLRs signaling from the endolysosomes and compels the further probing of the mechanisms underlying the UNC93B1-assisted differential targeting of TLRs.Toll-like receptors (TLRs) sense unique microbial structures or host-derived molecules released from stressed or dying cells to initiate the innate immune responses (1). TLRs are composed of three domains: the leucine-rich repeat (LRR) domain responsible for ligand binding, a single transmembrane domain, and the cytoplasmic Toll/IL-1 receptor homology domain by which TLRs recruit adaptor molecules for downstream signal transduction. Activated TLRs stimulate the NF-κB, MAPK, and IFN regulatory factor pathways, leading to the expression of diverse inflammatory cytokines, chemokines, and type I interferons. TLRs also activate antigen presenting cells to induce costimulatory molecules and coordinate various aspects of adaptive immune responses (2).The members of the TLR family can be classified into two groups based on their subcellular localization patterns (3–5). TLR1, TLR2, TLR4, and TLR6, which mainly recognize the components of bacterial cell membrane, are located on the cell surface and initiate signaling thereat. In contrast, the nucleotide-sensing TLRs such as TLR3, TLR7, TLR8, TLR9, and TLR13 are largely found in endolysosomes and require an acidic environment for their efficient signaling. Additionally, TLR11 and TLR12, the sensors for Toxoplasma protein profilin, are also expressed inside cells and transmit signals in an acidic pH-dependent manner (6–8). All the intracellular TLRs commonly bind to a multispanning membrane protein UNC93B1, which is required for their proper localization and signaling (6–13). One missense mutation (H412R) of UNC93B1, found in a chemically mutagenized mouse strain called 3d, hinders binding of UNC93B1 with TLRs and prevents their exit from the endoplasmic reticulum (ER) (9–11). Consequently, signaling by all endosomal TLRs is abolished in the cells from 3d mice. In contrast, trafficking and signaling of the cell surface-localized TLRs such as TLR2 and TLR4 are not affected by the UNC93B1 mutation (9, 11).The proper localization of TLRs is critical not only for efficient signaling but also for preventing undesirable receptor hyperactivation (14, 15). Especially, sequestration of the nucleotide-sensing TLRs in endolysosomes significantly contributes to attenuating the immune stimulation by host-derived nucleotides abundant in the extracellular spaces (14). Structural discrimination of microbial vs. mammalian nucleotides is not straightforward, and a mutant TLR9 protein, engineered to artificially localize at the plasma membrane, responds to mammalian DNA as well as the CpG oligonucleotides mimicking bacterial DNA. As a result, mice expressing such mutant TLR9 succumb to systemic autoinflammation and die prematurely (15). Therefore, regulatory mechanisms for localization and trafficking of TLRs need to be tightly controlled.TLR5 recognizes flagellin, the major protein subunit of bacterial flagellum, and functions as a critical innate sensor for flagellated bacteria in all mucous organs (16–18). TLR5 plays an important role in intestinal homeostasis mediating the immune adaptation to symbiotic microflora as well as defense against pathogenic bacterial infection (19–21). In addition, systemic injection of flagellin confers protection against ionizing radiation in a TLR5-dependent manner, implying that TLR5 agonism might be clinically used for radioprotection (22). TLR5 overexpressed in the intestinal epithelial cells was exclusively found on the basolateral surface, accounting for the selective induction of proinflammatory cytokine by basolateral but not by apical flagellin (17). Also, we recently demonstrated that endogenous TLR5 is expressed at the cell surface of mouse neutrophils, monocytes, and dendritic cells (DCs) in a TLR-specific chaperone PRAT4A-dependnet manner (23). However, other regulatory mechanisms for the localization of TLR5 at the plasma membrane are unknown. Here, we show that UNC93B1 binds to TLR5, travels to the plasma membrane with the receptor, and is required for flagellin-induced signaling at the cell surface.     

Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory

Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-β′1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.Although forgetting commonly has a negative connotation, it is a functional process that shapes memory and cognition (1–4). Recent studies, including work in relatively simple invertebrate models, have started to reveal basic biological mechanisms underlying forgetting (5–15). In Drosophila, single-session Pavlovian conditioning by pairing an odor (conditioned stimulus, CS) with electric shock (unconditioned stimulus, US) induces aversive memories that are short-lasting (16). The memory performance of fruit flies is observed to drop to a negligible level within 24 h, decaying rapidly early after training and slowing down thereafter (17). Memory decay or forgetting requires the activation of the small G protein Rac, a signaling protein involved in actin remodeling, in the mushroom body (MB) intrinsic neurons (6). These so-called Kenyon cells (KCs) are the neurons that integrate CS–US information (18, 19) and support aversive memory formation and retrieval (20–22). In addition to Rac, forgetting also requires the DAMB dopamine receptor (7), which has highly enriched expression in the MB (23). Evidence suggests that the dopamine-mediated forgetting signal is conveyed to the MB by dopamine neurons (DANs) in the protocerebral posterior lateral 1 (PPL1) cluster (7, 24). Therefore, forgetting of olfactory aversive memory in Drosophila depends on a particular set of intracellular molecular pathways within KCs, involving Rac, DAMB, and possibly others (25), and also receives modulation from extrinsic neurons. Although important cellular evidence supporting the hypothesis that memory traces are erased under these circumstances is still lacking, these findings lend support to the notion that forgetting is an active, biologically regulated process (17, 26).Although existing studies point to the MB circuit as essential for forgetting, several questions remain to be answered. First, whereas the molecular pathways for learning and forgetting of olfactory aversive memory are distinct and separable (6, 7), the neural circuits seem to overlap. Rac-mediated forgetting has been localized to a large population of KCs (6), including the γ-subset, which is also critical for initial memory formation (21, 27). The site of action of DAMB for forgetting has yet to be established; however, the subgroups of PPL1-DANs implicated in forgetting are the same as those that signal aversive reinforcement and are required for learning (28–30). It leaves open the question of whether the brain circuitry underlying forgetting and learning is dissociable, or whether forgetting and learning share the same circuit but are driven by distinct activity patterns and molecular machinery (26). Second, shock reinforcement elicits multiple memory traces through at least three dopamine pathways to different subdomains in the MB lobes (28, 29). Functional imaging studies have also revealed Ca²⁺-based memory traces in different KC populations (31). It is poorly understood how forgetting of these memory traces differs, and it remains unknown whether there are multiple regulatory neural pathways. Notably, when PPL1-DANs are inactivated, forgetting still occurs, albeit at a lower rate (7). This incomplete block suggests the existence of an additional pathway(s) that conveys forgetting signals to the MB. Third, other than memory decay over time, forgetting is also observed through interference (32, 33), when new learning or reversal learning is introduced after training (6, 34, 35). Time-based and interference-based forgetting shares a similar dependence on Rac and DAMB (6, 7). However, it is not known whether distinct circuits underlie forgetting in these different contexts.In the current study, we focus on the diverse set of MB extrinsic neurons (MBENs) that interconnect the MB lobes with other brain regions, which include 34 MB output neurons (MBONs) of 21 types and ∼130 dopaminergic neurons of 20 types in the PPL1 and protocerebral anterior medial (PAM) clusters (36, 37). These neurons have been intensively studied in olfactory memory formation, consolidation, and retrieval in recent years (e.g., 24, 28–30, 38–48); however, their roles in forgetting have not been characterized except for the aforementioned PPL1-DANs. In a functional screen, we unexpectedly found that several Gal4 driver lines of MBENs showed significantly better 3-h memory retention when the Gal4-expressing cells were inactivated. The screen has thus led us to identify two types of MBENs that are not involved in initial learning but play important and additive roles in mediating memory decay. Furthermore, neither of these MBEN types is required for reversal learning, supporting the notion that there is a diversity of neural circuits that drive different forms of forgetting.