Staphylococcus aureus binds to the N-terminal region of corneodesmosin to adhere to the stratum corneum in atopic dermatitis

Staphylococcus aureus colonizes the skin of the majority of patients with atopic dermatitis (AD), and its presence increases disease severity. Adhesion of S. aureus to corneocytes in the stratum corneum is a key initial event in colonization, but the bacterial and host factors contributing to this process have not been defined. Here, we show that S. aureus interacts with the host protein corneodesmosin. Corneodesmosin is aberrantly displayed on the tips of villus-like projections that occur on the surface of AD corneocytes as a result of low levels of skin humectants known as natural moisturizing factor (NMF). An S. aureus mutant deficient in fibronectin binding protein B (FnBPB) and clumping factor B (ClfB) did not bind to corneodesmosin in vitro. Using surface plasmon resonance, we found that FnBPB and ClfB proteins bound with similar affinities. The S. aureus binding site was localized to the N-terminal glycine–serine-rich region of corneodesmosin. Atomic force microscopy showed that the N-terminal region was present on corneocytes containing low levels of NMF and that blocking it with an antibody inhibited binding of individual S. aureus cells to corneocytes. Finally, we found that S. aureus mutants deficient in FnBPB or ClfB have a reduced ability to adhere to low-NMF corneocytes from patients. In summary, we show that FnBPB and ClfB interact with the accessible N-terminal region of corneodesmosin on AD corneocytes, allowing S. aureus to take advantage of the aberrant display of corneodesmosin that accompanies low NMF in AD. This interaction facilitates the characteristic strong binding of S. aureus to AD corneocytes.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder, affecting 15 to 20% of children (1, 2). During disease flares, patients experience painful inflamed skin lesions accompanied by intense itch. Epidermal barrier dysfunction, increased type 2 immune responses, and recurrent skin infections are features of AD (3). The most common cause of infection is Staphylococcus aureus. This bacterium colonizes the skin of the majority of AD patients (4, 5). Isolates representing several S. aureus lineages are recovered from AD skin; however, strains from the clonal complex 1 (CC1) lineage are the most frequently isolated (6–9). The burden of S. aureus on lesional and nonlesional skin correlates with severity of the disease (10, 11). S. aureus directly influences pathogenesis, and several factors produced by the bacterium increase inflammation and exacerbate AD symptoms, including staphylococcal superantigen B and delta-toxin (12–15).Despite the clear association between S. aureus colonization and AD disease severity (11), the bacterial and host factor determinants underlying colonization are poorly understood (16). Adhesion is a critical early step in the colonization process. S. aureus adheres to corneocytes in the stratum corneum of AD skin (6, 17, 18). We previously found that clumping factor B (ClfB), a cell wall-anchored protein displayed on the surface of S. aureus, can mediate adhesion to corneocytes from AD patients (6). ClfB also binds to the alpha chain of fibrinogen and to the cornified envelope proteins loricrin and cytokeratin 10 (K10) in desquamated nasal epithelial cells (19–21). To date, ClfB is the only bacterial factor known to promote adherence to corneocytes in AD. However, a ClfB-deficient mutant retained the ability to bind to corneocytes (6), suggesting that additional bacterial factors are at play.Filaggrin deficiency is common in patients with established AD and is either genetic or caused by down-regulation of gene expression by Th-2–type cytokines (22–24). Filaggrin deficiency causes epidermal barrier defects and a loss of the hygroscopic filaggrin breakdown products that normally contribute to the natural moisturizing factor (NMF) in corneocytes (25). NMF comprises a collection of humectants, including filaggrin breakdown products urocanic acid and pyrrolidone acid, along with urea, citrate, lactate acid, and sugars, and is responsible for regulating hydration in the skin (26). Low-NMF levels are associated with a loss of hydration, increased disease severity, and abnormal corneocyte morphology (27). We showed recently that S. aureus binds more strongly to low-NMF AD corneocytes than to corneocytes with normal levels of NMF (18).Corneocytes with low NMF have very different surface topography when compared with corneocytes with normal levels of NMF (27). Aberrant “villus-like” projections (VPs) protrude from the surface of low-NMF corneocytes (18, 27). The protein corneodesmosin (CDSN) is confined to the cell–cell junctions between corneocytes in healthy skin, where homophilic interactions between the CDSN proteins on adjacent cells facilitate cell–cell cohesion (28). In AD patients, however, CDSN decorates the tips of the VPs on low-NMF corneocytes (27).This study aimed to elucidate a key component of S. aureus colonization by identifying the molecular determinants of adherence to AD corneocytes. We recognized that the occurrence of VPs on low-NMF corneocytes presents a different colonization surface to the bacterium and postulated that the accessibility of CDSN on the tips of VPs could influence pathogen adherence. We show that S. aureus can interact with CDSN and identify the S. aureus proteins promoting adherence to this host protein. We use single-cell and single-molecule atomic force microscopy (AFM), surface plasmon resonance (SPR), and ex vivo bacterial adherence studies with patient corneocytes to characterize this interaction. This study expands the repertoire of ligands for S. aureus and, crucially, links bacterial interactions with a host protein (CDSN) to binding to corneocytes taken from patients. Thus, our findings provide insights into the adhesion process and develop our understanding of the mechanisms underlying colonization of the skin of AD patients by S. aureus.     

Evaluating the potential efficacy and limitations of a phage for joint antibiotic and phage therapy of Staphylococcus aureus infections

In response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYO^Sa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYO^Sa. Because of the mode of action of PYO^Sa, S. aureus is unlikely to generate classical receptor-site mutants resistant to PYO^Sa; none were observed in the 13 clinical isolates tested. PYO^Sa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYO^Sa and antibiotics raise issues that must be addressed before PYO^Sa is employed clinically. Despite the maintenance of the phage, PYO^Sa does not clear populations of S. aureus. Due to the ascent of a phenotyically diverse array of small-colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise–resurrection dynamics of PYO^Sa and S. aureus. Critically for phage therapy, our experimental results suggest that treatment with PYO^Sa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone.

Driven by well-warranted concerns about the increasing frequencies of infections with antibiotic-resistant pathogens, there has been a resurrection of interest in, research on, and clinical trials with a therapy that predates antibiotics by more than 15 y: bacteriophage (1–4). One direction phage therapy research has taken is to engineer lytic (virulent) phages with properties that are anticipated to maximize their efficacy for treating bacterial infections in mammals (5–8). Primary among these properties are 1) a broad host range for the target bacterial species; 2) mechanisms that prevent the generation of envelope or other kinds of high-fitness resistance in the target bacteria (9); 3) the capacity to thwart the innate and adaptive immune systems of bacteria, respectively, restriction-modification and CRISPR-Cas (7, 10, 11); 4) the ability to survive, kill, and replicate on pathogenic bacteria colonizing or infecting mammalian hosts (12, 13); and 5) little or no negative effects on the treated host (9).To these five desired properties for therapeutic bacteriophages, there is a sixth that should be considered: the joint action of these phages and antibiotics (8, 14–17). Phages-only treatment may be reasonable for compassionate therapy, where the bacteria responsible for the infection are resistant to all available antibiotics (18–20). From a practical perspective, however, for phages to become widely employed for treating bacterial infections, they would have to be effective in combination with antibiotics. It would be unethical and unacceptable to clinicians and regulatory agencies to use phage-only therapy for infections that can be effectively treated with existing antibiotics.Although not specifically engineered for these properties, there is a Staphylococcal phage isolated from a therapeutic phage collection from the Eliava Institute in Tbilisi, Georgia, that we call PYO^Sa that on first consideration appears to have all six of the properties required to be an effective agent for therapy. 1) PYO^Sa is likely to have a broad host range for S. aureus. The receptor of this K-like Myoviridae is N-acetylglucosamine in the wall-teichoic acid backbone of Staphylococcus aureus and is shared among most (21), if not all, S. aureus, thereby suggesting PYO^Sa should be able to adsorb to and potentially replicate on and kill a vast number of clinical isolates of S. aureus. 2) S. aureus does not generate classical, surface modification mutants resistant to PYO^Sa. Since the structure of the receptor of PYO^Sa is critical to the viability, replication, and virulence of these bacteria, the modifications in this receptor (22) may not be consistent with the viability or pathogenicity of S. aureus (23). 3) The replication of PYO^Sa is unlikely to be prevented by restriction-modification (RM) or CRISPR-Cas. Despite a genome size of 127 KB, the PYO^Sa phage has no GATC nucleotide restriction sites for the S. aureus restriction enzyme Sau3A1 and only one restriction site, GGNCC (guanine, guanine, any nucleotide, cytosine, cytosine), for the Sau961 restriction endonuclease (24, 25). There is no evidence for a functional CRISPR-Cas system in S. aureus or, to our knowledge, other mechanisms by which S. aureus may prevent the replication of this phage (26). 4) There is evidence that PYO^Sa-like phages can replicate in mammals. Early treatment with a phage with a different name but the same properties as PYO^Sa, Statu^v, prevented mortality in otherwise lethal peritoneal infections of S. aureus in mice (27). A PYO^Sa-like phage has also been successfully used therapeutically in humans (28). 5) No deleterious effects of a PYO^Sa-like phage were observed in recent placebo-controlled trials with volunteers asymptotically colonized by S. aureus (24). 6) Finally, there is evidence to suggest synergy with antibiotics. In vitro, PYO^Sa increased the efficacy of low concentrations of antibiotics for the treatment of biofilm populations of S. aureus (14).With in vitro population and evolutionary dynamic experiments with PYO^Sa and S. aureus Newman in combination with three different bacteriostatic and six different bactericidal antibiotics, we explore just how well PYO^Sa fits the above criteria for combination antibiotic and phage therapy. Our population dynamic experiments indicate that as consequence of the ascent of potentially pathogenic PYO^Sa resistant small colony variants (SCVs), by itself, PYO^Sa does not clear S. aureus infections. After an initial decline in the density of these bacteria when confronted with PYO^Sa, despite the continued presence of these phage, the densities of the bacterial populations return to levels similar to those observed in their absence. Using mathematical models, we present a hypothesis to account for these demise resurrection population dynamics, and the continued maintenance of the phage following the ascent of PYO^Sa resistant SCVs. We test and provide evidence in support of that hypothesis with a DNA sequence analysis of the genetic basis of the SCVs. By combining PYO^Sa with antibiotics, the density of the S. aureus population can be markedly reduced. There are, however, significant differences in the effectiveness of this combination therapy depending on whether the antibiotics and phage are used simultaneously or sequentially and on whether the antibiotics are bacteriostatic or bactericidal. Treatment with PYO^Sa, followed by the administration of bactericidal antibiotics, is more effective in reducing density of these bacterial population than treatment with these antibiotics alone. The methods developed here to evaluate the clinical potential of PYO^Sa in combination with antibiotics and design protocols for treating S. aureus infections with these phages and antibiotics can be employed for these purposes for any phage and bacterium that can be cultured in vitro.     

Host fibrinogen drives antimicrobial function in Staphylococcus aureus peritonitis through bacterial-mediated prothrombin activation

The blood-clotting protein fibrinogen has been implicated in host defense following Staphylococcus aureus infection, but precise mechanisms of host protection and pathogen clearance remain undefined. Peritonitis caused by staphylococci species is a complication for patients with cirrhosis, indwelling catheters, or undergoing peritoneal dialysis. Here, we sought to characterize possible mechanisms of fibrin(ogen)-mediated antimicrobial responses. Wild-type (WT) (Fib+) mice rapidly cleared S. aureus following intraperitoneal infection with elimination of ∼99% of an initial inoculum within 15 min. In contrast, fibrinogen-deficient (Fib–) mice failed to clear the microbe. The genotype-dependent disparity in early clearance resulted in a significant difference in host mortality whereby Fib+ mice uniformly survived whereas Fib– mice exhibited high mortality rates within 24 h. Fibrin(ogen)-mediated bacterial clearance was dependent on (pro)thrombin procoagulant function, supporting a suspected role for fibrin polymerization in this mechanism. Unexpectedly, the primary host initiator of coagulation, tissue factor, was found to be dispensable for this antimicrobial activity. Rather, the bacteria-derived prothrombin activator vWbp was identified as the source of the thrombin-generating potential underlying fibrin(ogen)-dependent bacterial clearance. Mice failed to eliminate S. aureus deficient in vWbp, but clearance of these same microbes in WT mice was restored if active thrombin was administered to the peritoneal cavity. These studies establish that the thrombin/fibrinogen axis is fundamental to host antimicrobial defense, offer a possible explanation for the clinical observation that coagulase-negative staphylococci are a highly prominent infectious agent in peritonitis, and suggest caution against anticoagulants in individuals susceptible to peritoneal infections.

In addition to serving as a centerpiece of hemostasis and thrombosis, fibrin(ogen) directs local inflammation in areas of tissue damage. Fibrin(ogen) can engage an array of integrin and nonintegrin cell-surface receptors to mediate downstream effector functions of innate immune cells. Whereas fibrin(ogen)-driven inflammation is detrimental in the context of inflammatory diseases like arthritis, colitis, and musculoskeletal disease (1–4), it is an important component of host defense in the context of infection (5–8). To help counter host defense mechanisms, bacteria have evolved virulence factors that engage host hemostatic system components to manipulate the activity of coagulation and fibrinolytic factors. This is particularly true for staphylococcal species, including the common, highly virulent human pathogen Staphylococcus aureus.S. aureus is a Gram-positive pathogen that expresses numerous virulence factors that engage the hemostatic system within vertebrate hosts, including an array of products that directly bind fibrin(ogen) and control fibrin deposition (9–13). S. aureus produces two nonproteolytic prothrombin activators, collectively termed “coagulases,” that bind host prothrombin and mediate fibrin formation (14, 15). Pathogen-mediated fibrin(ogen) binding and fibrin formation have been linked to S. aureus functioning as a causative agent underlying a spectrum of diseases ranging from skin infections to life-threatening pneumonia, bacteremia/sepsis, and endocarditis. A possible exception may be peritonitis. Peritoneal infection is particularly problematic for cirrhotic patients, and in hospital-acquired infections is associated with sutures, catheters, and medical implants. Intriguingly, it is coagulase-negative staphylococci (CoNS) that account for the overwhelming majority of these infections (16–20). The basis for the high prevalence of CoNS rather than the more pathogenic coagulase-positive S. aureus, and whether there is a functional link to bacterial-driven prothrombin activation, remains undefined. Here, we explored the hypothesis that host fibrinogen and prothrombin drive a bacterial killing mechanism and defined the contribution of bacterial coagulases to infection in a murine model of acute S. aureus peritonitis.     

Staphylococcus aureus adapts to the host nutritional landscape to overcome tissue-specific branched-chain fatty acid requirement

During infection, pathogenic microbes adapt to the nutritional milieu of the host through metabolic reprogramming and nutrient scavenging. For the bacterial pathogen Staphylococcus aureus, virulence in diverse infection sites is driven by the ability to scavenge myriad host nutrients, including lipoic acid, a cofactor required for the function of several critical metabolic enzyme complexes. S. aureus shuttles lipoic acid between these enzyme complexes via the amidotransferase, LipL. Here, we find that acquisition of lipoic acid, or its attachment via LipL to enzyme complexes required for the generation of acetyl-CoA and branched-chain fatty acids, is essential for bacteremia, yet dispensable for skin infection in mice. A lipL mutant is auxotrophic for carboxylic acid precursors required for synthesis of branched-chain fatty acids, an essential component of staphylococcal membrane lipids and the agent of membrane fluidity. However, the skin is devoid of branched-chain fatty acids. We showed that S. aureus instead scavenges host-derived unsaturated fatty acids from the skin using the secreted lipase, Geh, and the unsaturated fatty acid–binding protein, FakB2. Moreover, murine infections demonstrated the relevance of host lipid assimilation to staphylococcal survival. Altogether, these studies provide insight into an adaptive trait that bypasses de novo lipid synthesis to facilitate S. aureus persistence during superficial infection. The findings also reinforce the inherent challenges associated with targeting bacterial lipogenesis as an antibacterial strategy and support simultaneous inhibition of host fatty acid salvage during treatment.

The gram-positive bacterium Staphylococcus aureus is notorious for its capacity to cause widespread pathology in nearly every organ and tissue during infection (1). Much of this success can be attributed to the ability of S. aureus to extract essential nutrients from the host milieu (2). Hence, understanding the adaptive traits that allow S. aureus to exploit in situ resources for survival is critical to devising effectual treatments for staphylococcal diseases.Lipoic acid is an organosulfur compound derived from an early-stage intermediate of fatty acid biosynthesis that is required for carbon shuttling in central metabolism via a redox-sensitive dithiolane ring at its distal end (3). In a previous study, we found that shuttling of lipoic acid to metabolic enzyme complexes by the amidotransferase, LipL, was paramount for S. aureus survival in a murine model of bacteremia but not skin infection (4). At minimum, LipL appears to be required for the transfer of lipoic acid to E2 subunits of the pyruvate dehydrogenase (PDH) and branched-chain α-ketoacid dehydrogenase (BCODH) complexes (4, 5). The absence of lipoic acid renders each complex nonfunctional (4, 5). The discrepancy in demand for lipoic acid attachment in these two infection sites suggests that the nutritional environment of the skin eases the requirement for lipoic acid–dependent metabolic processes, especially those that require PDH or BCODH complex activity. PDH is responsible for the generation of acetyl-CoA upon exit from glycolysis, while BCODH is essential for the synthesis of saturated branched-chain fatty acids (BCFAs), a major component of staphylococcal membrane phospholipids (6). Like unsaturated fatty acids (UFAs), BCFAs provide membrane fluidity to staphylococci, which neither synthesize UFAs nor encode a fatty acid (FA) desaturase that converts saturated fatty acids (SFAs) to unsaturated products (7). Since a lack of membrane fluidity triggers cell death as a result of lipid phase separation and protein segregation, bacterial survival hinges on the fine-tuning of membrane lipid composition to adapt to fluctuating environments (8).Given the importance of de novo FA synthesis to ensure bacterial membrane integrity, enzymes that are involved in this metabolic process serve as attractive targets for antibacterial drug discovery. One such drug, triclosan, binds to the enoyl-acyl carrier protein reductase, FabI, a key enzyme in the type II FA synthesis (FASII) system found in archaea and bacteria (9). FASII restriction induces FA starvation and initiates the stringent response, where high concentrations of the alarmone (p)ppGpp inhibit synthesis of the FASII substrate malonyl-CoA (10). Nevertheless, mounting evidence suggests that S. aureus can resolve triclosan interference by incorporating host-derived UFAs into its membrane (11–13). During anti-FASII–adaptive outgrowth, dissipation of (p)ppGpp levels replenishes the malonyl-CoA pool and skews distribution of the FASII substrate to favor binding with FapR, a global repressor of phospholipid synthesis genes in S. aureus (10). Ultimately, this interaction sequesters FapR to allow expression of plsX and plsC, which encode enzymes that use host UFAs to synthesize phospholipids (10, 14). UFAs are commonly found esterified as triglycerides and cholesterol esters in vertebrates (15). In humans, both compounds form the lipid core of the five major groups of lipoproteins (chylomicron, VLDL, LDL, IDL, and HDL) that transport lipids throughout the body (15). Triglycerides are also the major constituents of adipocytes and exist to a lesser extent in nonadipose cells (16, 17). It has been suggested that S. aureus secretes the glycerol ester hydrolase Geh to liberate esterified FAs from triglycerides and LDL for use as FA substrates as a nutrient acquisition strategy, although this phenomenon has not yet been tested in vivo (18, 19).Despite increasing understanding of FASII bypass by S. aureus in recent years, the overall mechanism of rescue continues to be investigated. Here, we draw upon insights derived from the study of S. aureus lipoic acid synthesis and acquisition to make connections between BCFA synthesis and bacterial survival during infection. We report that de novo BCFA synthesis by S. aureus is the primary enzymatic process that necessitates lipoic acid in vitro and during skin infection. However, the requirement for BCFA synthesis in the skin is bypassed through assimilation of UFAs from host tissue. We also provide genetic, in vivo, and chemical evidence, via treatment with the over-the-counter lipase inhibitor, orlistat, that suggests UFA acquisition depends on the secreted lipase, Geh, and the UFA binding protein, FakB2. Finally, we demonstrate that ablating both BCFA synthesis and exogenous FA uptake significantly improves staphylococcal infection outcome in mice.     

Macrophage LC3-associated phagocytosis is an immune defense against Streptococcus pneumoniae that diminishes with host aging

Streptococcus pneumoniae is a leading cause of pneumonia and invasive disease, particularly, in the elderly. S. pneumoniae lung infection of aged mice is associated with high bacterial burdens and detrimental inflammatory responses. Macrophages can clear microorganisms and modulate inflammation through two distinct lysosomal trafficking pathways that involve 1A/1B-light chain 3 (LC3)-marked organelles, canonical autophagy, and LC3-associated phagocytosis (LAP). The S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers an autophagic response in nonphagocytic cells, but the role of LAP in macrophage defense against S. pneumoniae or in age-related susceptibility to infection is unexplored. We found that infection of murine bone-marrow-derived macrophages (BMDMs) by PLY-producing S. pneumoniae triggered Atg5- and Atg7-dependent recruitment of LC3 to S. pneumoniae-containing vesicles. The association of LC3 with S. pneumoniae-containing phagosomes required components specific for LAP, such as Rubicon and the NADPH oxidase, but not factors, such as Ulk1, FIP200, or Atg14, required specifically for canonical autophagy. In addition, S. pneumoniae was sequestered within single-membrane compartments indicative of LAP. Importantly, compared to BMDMs from young (2-mo-old) mice, BMDMs from aged (20- to 22-mo-old) mice infected with S. pneumoniae were not only deficient in LAP and bacterial killing, but also produced higher levels of proinflammatory cytokines. Inhibition of LAP enhanced S. pneumoniae survival and cytokine responses in BMDMs from young but not aged mice. Thus, LAP is an important innate immune defense employed by BMDMs to control S. pneumoniae infection and concomitant inflammation, one that diminishes with age and may contribute to age-related susceptibility to this important pathogen.

Streptococcus pneumoniae (pneumococcus) commonly colonizes the nasopharynx asymptomatically but is also capable of infecting the lower respiratory tract to cause pneumonia and spreading to the bloodstream to cause septicemia and meningitis (1). Susceptibility to pneumonia and invasive disease caused by S. pneumoniae is remarkably higher in individuals aged 65 and over, leading to high rates of mortality and morbidity in the elderly population (1, 2). In countries, such as the United States and Japan, deaths due to pneumococcal pneumonia have been on the rise in parallel with the rapid growth in the elderly population (3, 4).A hallmark of pneumococcal pneumonia is a rapid and exuberant response by immune cells, such as neutrophils and macrophages. This innate immune response to S. pneumoniae lung infection is critical for pathogen clearance and the control of disease (5–7). Deficiencies in the number or function of innate phagocytic cells, such as neutropenia (8) or macrophage phagocytic receptor defects (9–12), lead to diminished pneumococcal clearance and increased risk of invasive pneumococcal disease in both mouse models and humans. Phagocytic activity in alveolar macrophages is important during early responses to subclinical infections (13–15), and during moderate S. pneumoniae lung infection, newly generated monocytes eggress from the bone marrow and migrate into the lungs, differentiating into monocyte-derived alveolar macrophages (16). In addition to directly eliminating the invading microbe, macrophages secrete key cytokines, such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), and interleukin-6 (IL-6), that regulate effector cell functions and pulmonary inflammation (17–19).Although an innate immune response is critical for pathogen clearance, poorly controlled inflammation can lead to tissue damage and mortality (20, 21). For example, in murine models, neutrophilic infiltration can enhance pulmonary damage and disrupt epithelial barrier function, leading to bacteremia and mortality (22–25). Macrophages are critical not only in regulating the early inflammatory response, but are also crucial for curtailing inflammation during the resolution phase of infection to limit tissue damage and promote healing (26, 27).Elderly individuals have higher baseline and induced levels of inflammation, a phenomenon termed inflammaging (28), that contributes to many age-associated pathological conditions, including increased susceptibility to a variety of infectious diseases, such as S. pneumoniae infection (7, 28–30). S. pneumoniae-induced inflammation, characterized by increased levels of chemokines, proinflammatory cytokines, and decreased anti-inflammatory cytokines, such as IL-10, is enhanced in the elderly (29, 31) as well as in aged mice (32, 33) and correlates with ineffective immune responses. Age-related chronic exposure to TNF-α, for instance, dampens macrophage-mediated S. pneumoniae clearance during lung infection (34), and NLR family pyrin domain containing 3 inflammasome activation in macrophages diminishes upon aging in mice (35). However, the age-related changes in macrophage effector functions leading to diminished clearance of S. pneumoniae are incompletely understood.One important means of macrophage-mediated pathogen clearance is1A/1B-light chain-3 (LC3)-associated phagocytosis (LAP), a process by which cells target phagocytosed extracellular particles for efficient degradation (36–38). LAP combines the molecular machineries of phagocytosis and autophagy, resulting in the conjugation of the autophagic marker, the microtubule-associated protein LC3, to phosphatidylethanolamine on the phagosomal membrane, generating so-called “LAPosomes” that undergo facilitated fusion with lysosomes (38, 39). Canonical autophagy targets cytoplasmic components, such as damaged subcellular organelles and intracellular microbes for sequestration into double-membrane autophagic vesicles (40, 41). In contrast, LAPosomes retain the single-membrane nature of phagosomes, and their formation requires overlapping but nonidentical genes compared to canonical autophagy (42). In addition to enabling efficient degradation of phagocytosed bacteria, LAP also plays important immune regulatory roles, such as in curtailing proinflammatory cytokine production during the subsequent innate immune response (39, 43). Indeed, the LAP-mediated microbial defense and immunomodulatory functions work together to limit tissue damage and restore homeostasis (38).S. pneumoniae triggers canonical autophagy in epithelial cells and fibroblasts, and bacteria can be found in double-membrane vacuoles whose formation is dependent on autophagic machinery (44). Many bacterial pathogens that induce autophagy produce pore-forming toxins, which can damage endosomal membranes, thus, recruiting autophagic machinery to engulf injured organelles (45). Pneumococcus-induced autophagy is dependent on the cholesterol-dependent pore-forming toxin pneumolysin (PLY), which triggers the autophagic delivery of S. pneumoniae to lysosomes and results in bacterial killing (44, 46). Recently, a kinetic examination of S. pneumoniae-targeting autophagy in fibroblasts demonstrated that canonical autophagy was preceded by early and rapid PLY-dependent LAP (47). However, the requirements for this process were somewhat different from LAP in macrophages, and the pneumococcus-containing LAPosomes did not promote bacterial clearance but required subsequent transition to canonical autophagy to reduce bacterial numbers (46, 47).In the current study, we found that S. pneumoniae infection of murine bone-marrow-derived macrophages (BMDMs) induces LAP in a PLY-dependent manners and that age-related defects in BMDM LAP contributed to diminished bactericidal activity and enhanced proinflammatory cytokine production. Our results suggest that PLY-induced LAP promotes bacterial clearance, and age-associated dysregulation of this process may contribute to enhanced bacterial survival, poorly regulated inflammation, and increased susceptibility to invasive pneumococcal disease.     

Synthesis and delivery of Streptococcus pneumoniae capsular polysaccharides by recombinant attenuated Salmonella vaccines

Streptococcus pneumoniae capsular polysaccharides (CPSs) are major determinants of bacterial pathogenicity. CPSs of different serotypes form the main components of the pneumococcal vaccines Pneumovax, Prevnar7, and Prevnar13, which substantially reduced the S. pneumoniae disease burden in developed countries. However, the laborious production processes of traditional polysaccharide-based vaccines have raised the cost of the vaccines and limited their impact in developing countries. The aim of this study is to develop a kind of low-cost live vaccine based on using the recombinant attenuated Salmonella vaccine (RASV) system to protect against pneumococcal infections. We cloned genes for seven different serotypes of CPSs to be expressed by the RASV strain. Oral immunization of mice with the RASV-CPS strains elicited robust Th1 biased adaptive immune responses. All the CPS-specific antisera mediated opsonophagocytic killing of the corresponding serotype of S. pneumoniae in vitro. The RASV-CPS2 and RASV-CPS3 strains provided efficient protection of mice against challenge infections with either S. pneumoniae strain D39 or WU2. Synthesis and delivery of S. pneumoniae CPSs using the RASV strains provide an innovative strategy for low-cost pneumococcal vaccine development, production, and use.

The bacteria Streptococcus pneumoniae (pneumococcus) continues to be a leading cause of pneumonia, meningitis, and bacteremia in young children, elderly people, and immunocompromised populations (1), and therefore is responsible for millions of cases and deaths each year worldwide (2). S. pneumoniae synthesizes immunologically distinct capsular polysaccharides (CPSs), which cover the cell surface and define serotypes. At present, there are over 90 capsular serotypes identified (3–5). The CPSs prevent S. pneumoniae from complement-mediated opsonophagocytic killing, which is believed to be an important defense mechanism during S. pneumoniae infection (6, 7). Antibodies against CPSs mediate clearance of S. pneumoniae from the lung and protect the host against pneumococcal disease (8–10). Consequently, traditional pneumococcal vaccines are mainly developed based on purified CPS or CPS conjugated to a protein carrier, in order to induce the CPS-specific immune responses (11).A 23-valent pneumococcal polysaccharide vaccine (PPV23; Pneumovax) was developed and recommended for people over 65 y or for children and adults with underlying medical conditions. The PPV23 contains 23 capsular types of purified CPSs: 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F. However, the purified CPSs are T cell-independent antigens that could not induce much IgM-to-IgG switching (12) and sustained memory immune responses (13). Therefore, the PPV23 is not effective in children younger than 2 y of age (14). The pneumococcal conjugate vaccine (PCV13; Prevnar13) developed by conjugating CPSs of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, 19A, and 23F to diphtheria toxoid (CRM197) (15) provided effective memory responses and increased immunogenicity in children under 2 y of age (16). Although glycoconjugated vaccines have played a great role in controlling S. pneumoniae infections caused by vaccine serotypes (17–23), there are also disadvantages to their use. PCV13 includes the most prevalent serotypes in the Western world and in developing countries (24, 25) and helps decrease the overall incidence of invasive pneumococcal disease. However, as the existing serotypes in PCV13 are not readily altered and the distribution of S. pneumoniae serotypes varies by geography, age, and time (26), the introduction of PCV13 resulted in variable immunogenicity among different populations (27) and the diseases caused by nonvaccine serotypes increased significantly in children younger than 5 y and in adults older than 45 y (28).The production process of the PCV involves multiple stages and rounds of purification, which greatly increases their cost and limits the vaccine use in developing countries where the disease burden is heaviest. Although the vaccine price drops significantly because of the support of the Gavi Pneumococcal Advance Market Commitment, the global average coverage of pneumonia vaccine is currently only at 47%. More efforts are still needed to reduce the vaccine price and increase immunization coverage for lower-income countries. In addition, as each polysaccharide is structurally distinct and the chemical conjugation may change its conformation and epitopes, each reaction requires optimization and the produced glycoconjugate vaccines are often poorly characterized, heterogeneous, and variably immunogenic (11, 29, 30).The protein glycan coupling technology was developed recently for biosynthesis of polysaccharide conjugate vaccine. This approach uses the oligosaccharyltransferases from Campylobacter jejuni (PglB) (31–33) or Neisseria (PglL) (34, 35). This technology still needs the complicated process of isolation and purification of protein-CPS conjugates. Another limitation for both of the PPV and PCV is that the induced predominant serum immunoglobulin G (IgG) isotype is IgG2 in responses of people older than 2 y of age (36), and neither of the two vaccines induces significant mucosal IgA responses (37). Live attenuated strains of S. pneumoniae are used for pneumococcal vaccine development as well (37, 38). However, the potential reversion to virulence of attenuated vaccines remains a major concern, as the pneumococci are naturally competent for DNA uptake from circulating wild-type strains.As an alternative approach, we focused on using recombinant attenuated Salmonella vaccine (RASV) strains as delivery platforms for pneumococcal polysaccharides. RASV strains presenting foreign antigens from unrelated pathogens are promising oral vaccine vectors, as they were developed to provide novel, needle-free, and low-cost strategies for preventing infectious diseases (39). There are gram-negative bacteria, such as Escherichia coli strains with a waaL gene deletion, which showed a capability to synthesize heterologous polysaccharides from gram-negative and gram-positive bacteria (40, 41). In our previous study (42), genes required for Salmonella O-antigen synthesis were also deleted for construction of RASV strains delivering heterologous polysaccharide antigens. Lipopolysaccharide (LPS)-associated O-antigen in gram-negative bacteria is covalently ligated to the lipid A-core in the outer membrane (OM) (43). Several lines of evidence suggest that OM proteins (OMPs), including matrix porin (OmpF) and siderophore transporter FhuA, form a tight complex with LPS (44). The OMP–LPS complexes offer high potentials for triggering T cell-dependent (TD) immune responses against O-antigen, which may help explain that antibodies to O-antigen could provide protective activity after natural infection of Salmonella Typhimurium (S. Typhimurium) (45). Attenuated Salmonella strains synthesizing Shigella O-antigens elicited strong anti-Shigella–LPS immune responses and conferred efficient protection against challenge with virulent Shigella strains in murine models (46–48). Therefore, the RASV platform may provide a novel strategy for producing bioconjugated polysaccharide vaccines against pneumococcal infections. In this study, the gene clusters for synthesis of S. pneumoniae CPSs of 2, 3, 5, 6A, 9V, 14, and 18C were cloned into our balanced lethal vector and the resultant plasmids were introduced into the RASV strain to establish a platform for vaccine development against pneumococcal infection.     

Collagen IV differentially regulates planarian stem cell potency and lineage progression

The extracellular matrix (ECM) provides a precise physical and molecular environment for cell maintenance, self-renewal, and differentiation in the stem cell niche. However, the nature and organization of the ECM niche is not well understood. The adult freshwater planarian Schmidtea mediterranea maintains a large population of multipotent stem cells (neoblasts), presenting an ideal model to study the role of the ECM niche in stem cell regulation. Here we tested the function of 165 planarian homologs of ECM and ECM-related genes in neoblast regulation. We identified the collagen gene family as one with differential effects in promoting or suppressing proliferation of neoblasts. col4-1, encoding a type IV collagen α-chain, had the strongest effect. RNA interference (RNAi) of col4-1 impaired tissue maintenance and regeneration, causing tissue regression. Finally, we provide evidence for an interaction between type IV collagen, the discoidin domain receptor, and neuregulin-7 (NRG-7), which constitutes a mechanism to regulate the balance of symmetric and asymmetric division of neoblasts via the NRG-7/EGFR pathway.

Across the animal kingdom, stem cell function is regulated by the microenvironment in the surrounding niche (1), where the concentration of molecular signals for self-renewal and differentiation can be precisely regulated (2). The niche affects stem cell biology in many processes, such as aging and tissue regeneration, as well as pathological conditions such as cancer (3). Most studies have been done in tissues with large stem cell populations, such as the intestinal crypt (4) and the hair follicle (5) in mice. Elucidation of the role of the stem cell niche in tissue regeneration requires the study of animals with high regenerative potential, such as freshwater planarians (flatworms) (6). Dugesia japonica and Schmidtea mediterranea are two well-studied species that possess the ability to regenerate any missing body part (6, 7).Adult S. mediterranea maintain a high number of stem cells (neoblasts)—∼10 to 30% of all somatic cells in the adult worm—with varying potency, including pluripotent cells (8–14). Neoblasts are the only proliferating somatic cells: they are molecularly heterogeneous, but all express piwi-1 (15–18). Lineage-committed neoblasts are “progenitors” that transiently express both piwi-1 and tissue-specific genes (15, 19). Examples include early intestinal progenitors (γ neoblast, piwi-1⁺/hnf4⁺) (8, 10, 15, 19–21) and early epidermal progenitors (ζ neoblast, piwi-1⁺/zfp-1⁺) (8, 15). Other progenitor markers include collagen for muscles (22), ChAT for neurons (23), and cavII for protonephridia (24, 25). During tissue regeneration, neoblasts are recruited to the wound site, where they proliferate then differentiate to replace the missing cell types (16, 26). Some neoblasts express the pluripotency marker tgs-1, and are designated as clonogenic neoblasts (cNeoblasts) (10, 11). cNeoblasts are located in the parenchymal space adjacent to the gut (11).Neoblasts are sensitive to γ-irradiation and can be preferentially depleted in the adult planarian (27). After sublethal γ-irradiation, remaining cNeoblasts can repopulate the stem cell pool within their niche (10, 11). The close proximity of neoblasts to the gut suggests gut may be a part of neoblast niche (28, 29). When gut integrity was impaired by silencing gata4/5/6, the egfr-1/nrg-1 ligand-receptor pair, or wwp1, maintenance of non–γ-neoblasts were also disrupted (20, 30, 31), but whether that indicates the gut directly regulates neoblast remains unclear. There is evidence indicating the dorsal-ventral (D/V) transverse muscles surrounding the gut may promote neoblast proliferation and migration, with the involvement of matrix metalloproteinase mt-mmpB (32, 33). The central nervous system has also been implicated in influencing neoblast maintenance through the expression of EGF homolog neuregulin-7 (nrg-7), a ligand for EGFR-3, affecting the balance of neoblast self-renewal (symmetric or asymmetric division) (34).In other model systems, an important component of the stem-cell niche is the extracellular matrix (ECM) (35). Germline stem cells in Drosophila are anchored to niche supporting cells with ECM on one side, while the opposite side is exposed to differentiation signals, allowing asymmetric cell fate outcomes for self-renewal or differentiation following division (36–38). Few studies have addressed the ECM in planarians, largely due to the lack of genetic tools to manipulate the genome, the absence of antibodies to specific planarian ECM homologs, or the tools required to study cell fate changes. However, the genomes of D. japonica (39–41) and S. mediterranea (41–45), and single-cell RNA-sequencing (scRNA-seq) datasets for S. mediterranea are now available (11, 46–50). A recent study of the planarian matrisome demonstrated that muscle cells are the primary source of many ECM proteins (51), which, together with those produced by neoblasts and supporting parenchymal cells, may constitute components of the neoblast niche. For example, megf6 and hemicentin restrict neoblast’s localization within the parenchyma (51, 52). Functional studies also implicate ECM-modifiers, such as matrix metalloproteases (MMPs) in neoblast migration and regeneration. For example, reducing the activity of the ECM-degrading enzymes mt-mmpA (26, 33), mt-mmpB (53), or mmp-1 (33) impaired neoblast migration, proliferation, or overall tissue growth, respectively. Neoblasts are also likely to interact with ECM components of the niche via cell surface receptors, such as β1 integrin, inactivation of which impairs brain regeneration (54, 55).Here, we identified planarian ECM homologs in silico, followed by systematic functional assessment of 165 ECM and ECM-related genes by RNA interference (RNAi), to determine the effect on neoblast repopulation in planarians challenged by a sublethal dose of γ-irradiation (10). Surprisingly, multiple classes of collagens were shown to have the strongest effects. In particular, we show that the type IV collagens (COLIV) of basement membranes (BMs), were required to regulate the repopulation of neoblasts as well as lineage progression to progenitor cells. Furthermore, our data support an interaction between COLIV and the discoidin domain receptor (DDR) in neurons that activates signaling of NRG-7 in the neoblasts to regulate neoblast self-renewal versus differentiation. Together, these data demonstrate multifaceted regulation of planarian stem cells by ECM components.     

Boosting can explain patterns of fluctuations of ratios of inapparent to symptomatic dengue virus infections

Dengue is the most prevalent arboviral disease worldwide, and the four dengue virus (DENV) serotypes circulate endemically in many tropical and subtropical regions. Numerous studies have shown that the majority of DENV infections are inapparent, and that the ratio of inapparent to symptomatic infections (I/S) fluctuates substantially year-to-year. For example, in the ongoing Pediatric Dengue Cohort Study (PDCS) in Nicaragua, which was established in 2004, the I/S ratio has varied from 16.5:1 in 2006–2007 to 1.2:1 in 2009–2010. However, the mechanisms explaining these large fluctuations are not well understood. We hypothesized that in dengue-endemic areas, frequent boosting (i.e., exposures to DENV that do not lead to extensive viremia and result in a less than fourfold rise in antibody titers) of the immune response can be protective against symptomatic disease, and this can explain fluctuating I/S ratios. We formulate mechanistic epidemiologic models to examine the epidemiologic effects of protective homologous and heterologous boosting of the antibody response in preventing subsequent symptomatic DENV infection. We show that models that include frequent boosts that protect against symptomatic disease can recover the fluctuations in the I/S ratio that we observe, whereas a classic model without boosting cannot. Furthermore, we show that a boosting model can recover the inverse relationship between the number of symptomatic cases and the I/S ratio observed in the PDCS. These results highlight the importance of robust dengue control efforts, as intermediate dengue control may have the potential to decrease the protective effects of boosting.

Dengue virus (DENV) is the most prevalent vector-borne viral disease of humans, with recent estimates of around 105 million individuals infected annually (1). It comprises four antigenically distinct serotypes, DENV-1 to -4 (2), and is transmitted to humans by Aedes aegypti and, less frequently, Aedes albopictus mosquitoes (3–5). While most studies have focused on symptomatic infections, epidemiologic studies have shown that for dengue, the majority of infections are inapparent (3, 5), that is, infections that do not cause detected disease but result in a fourfold or greater rise in antibody titers. However, large fluctuations in annual dengue inapparent:symptomatic (I/S) ratios have been documented worldwide (5). For example, cohort studies able to detect inapparent DENV infections in Nicaragua (6–9), Peru (10), and Thailand (11) have shown that the I/S ratio of DENV infections ranges widely year to year. In the Pediatric Dengue Cohort Study (PDCS) in Nicaragua, the longest running dengue cohort study, the I/S ratio has varied widely, from 16.5:1 in 2006–2007 (7) to 1.2:1 in 2009–2010 (9). We currently do not understand the drivers of these fluctuations; however, we do know that potential extrinsic drivers, such as differences in replication rates of the predominating serotype, cannot explain them (5). Gaining a mechanistic understanding of these fluctuations in the I/S ratio is likely to be critical for understanding potential drivers of epidemic potential and severe dengue disease and for enacting effective control policies.Extensive research has been conducted into the causes of DENV infection and disease, and there is now some evidence to suggest that immune interactions among viruses and strains may be responsible for fluctuating patterns (12–14). In particular, this extensive body of work has shown that severe disease occurs due to immunopathology (4, 15, 16). The most important risk factor for severe dengue disease is secondary heterologous infections (4), due in part to a phenomenon called antibody-dependent enhancement (ADE), in which antibodies from a first infection cross-react with virus from a secondary infection, leading to incomplete neutralization. The resulting partially neutralized immune complexes enhance infection into Fc receptor-bearing cells (17). Low to intermediate titers of cross-reactive anti-DENV antibodies have been shown to enhance subsequent dengue disease severity in human populations (15, 18, 19). However, neutralizing antibody titers are thought to be protective against dengue disease, and a recent study showed that higher preinfection neutralizing antibody titers correlated with lower probability of symptomatic infection in children in the PDCS (20). Importantly, individuals with inapparent heterologous secondary infections had significantly higher preinfection titers than individuals with symptomatic heterologous secondary infections (20–22), providing direct evidence that preinfection neutralizing antibody titer is an important determinant of disease outcome. Therefore, it is plausible that the variability in preinfection antibody titer could explain fluctuations in I/S ratios.Recent work has suggested that frequent exposure to DENV may boost the immune response and result in modest increases in neutralizing antibody titer (20), which in turn may protect individuals against symptomatic infection. Evidence for boosting comes from analysis of neutralizing antibodies following primary infection. Here we have defined boosting as exposures to DENV that do not lead to extensive viremia and that result in a less than fourfold rise in antibody titers. Traditionally, the temporary period of cross-protection against heterotypic serotypes following a primary infection is explained by waning cross-reactive antibodies, resulting in a decrease in neutralizing antibody titers (23). However, an analysis of neutralizing antibody titers from the PDCS showed that neutralizing antibody titers did not decrease in the time between primary and secondary DENV infection, but in fact increased marginally (20). A comparable trend was seen in Thailand (24) and in a long-term hospital-based study in Nicaragua (25, 26). The increase in neutralizing antibody titer may be due to immune boosts (20), suggesting that children may be regularly exposed to DENV without experiencing symptoms or meeting the criteria for inapparent infection. There is also evidence of a phenomenon similar to boosting in a human vaccine study (27) and in a study in nonhuman primates (28), where in both cases there was initial exposure that resulted in viremia and seroconversion and a second challenge that did not result in viremia but did result in increased antibody titers. Clearly, in years with a high incidence of dengue, we would expect boosting to occur more frequently, and thus in the years immediately following high dengue incidence, we would expect fewer symptomatic infections, as individuals would be protected against symptomatic infection due to boosts (5).Here we used mathematical models to determine which mechanisms can recover the fluctuations in the I/S ratio in DENV infections. Since our aim was to gain a conceptual qualitative understanding of the role of the impact of a range of mechanisms, we took the classic simplifying approach of not explicitly modeling the mosquito population dynamics. All models are adapted from existing dengue epidemiologic models (12, 29) and include immunity against homologous reinfection, a period of cross-protection following infection, and seasonality. For simplicity, we model the whole population but also present results from a model of the pediatric cohort from which our data are taken. With only these factors, a year-to-year variation in case number is seen, but not a variation in I/S ratio. This model was first modified to include the basic assumption that antibody titer decreases with time since infection and is predictive of infection outcome (20), to evaluate whether I/S fluctuations can be recovered by shorter periods of cross-protection between primary infections and secondary heterotypic infections for inapparent secondary infections than for symptomatic secondary infections, as previously suggested (6, 23).We then explored whether I/S ratio differences can be explained by protection against symptomatic disease due to boosting of the immune response. We define boosts as exposures to homotypic or heterotypic DENV serotypes that “boost” the immune response and result in a modest rise in antibody titers (less than fourfold rise, below the threshold of classification as an inapparent infection), possibly due to limited viremia. It is important to note that with boosting, the antibody titer that we measure might not fall. Although it was previously thought that homologous DENV infection confers lifelong immunity against the infecting serotype (30), recent work has shown that homologous DENV reinfections do occur (31). We hypothesize that a boost in antibody titer can protect an individual during subsequent infections, resulting in the development of inapparent infection instead of symptomatic infection. We show that a boosting model can recover the fluctuations in the I/S ratio, recover the inverse relationship between the number of symptomatic cases and the I/S ratio in the PDCS, and recover a positive relationship between the I/S ratio in a given year and the number of cases in the previous year, as has been previously noted (5, 11). These models suggest that boosts may be occurring frequently in endemic areas and need to be considered when constructing effective dengue control policies.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

Structure and activity of SLAC1 channels for stomatal signaling in leaves

Stomata in leaves regulate gas exchange between the plant and its atmosphere. Various environmental stimuli elicit abscisic acid (ABA); ABA leads to phosphoactivation of slow anion channel 1 (SLAC1); SLAC1 activity reduces turgor pressure in aperture-defining guard cells; and stomatal closure ensues. We used electrophysiology for functional characterizations of Arabidopsis thaliana SLAC1 (AtSLAC1) and cryoelectron microscopy (cryo-EM) for structural analysis of Brachypodium distachyon SLAC1 (BdSLAC1), at 2.97-Å resolution. We identified 14 phosphorylation sites in AtSLAC1 and showed nearly 330-fold channel-activity enhancement with 4 to 6 of these phosphorylated. Seven SLAC1-conserved arginines are poised in BdSLAC1 for regulatory interaction with the N-terminal extension. This BdSLAC1 structure has its pores closed, in a basal state, spring loaded by phenylalanyl residues in high-energy conformations. SLAC1 phosphorylation fine-tunes an equilibrium between basal and activated SLAC1 trimers, thereby controlling the degree of stomatal opening.

The stomatal pore, formed by a pair of specialized guard cells in plant leaves, serves as the gateway for water transpiration and atmospheric CO₂ influx for photosynthesis (1, 2). These pores need to be tightly controlled, as inadequate CO₂ intake is harmful and excessive water loss is devastating for plants (3). The guard cells respond to a wide range of environmental stimuli, including high CO₂ levels, O₃, low air humidity, and drought, and transduce these signals into appropriate turgor pressure changes that can adjust stomatal pore aperture (4, 5). It is well established that turgor pressure of guard cells is regulated by ion transport across the membrane, notably anions and potassium ions (6–8).The phytohormone abscisic acid (ABA) plays a central role in controlling stomatal closure via activation of a complex signaling pathway that is mediated by receptors, kinase/phosphatases, and ion channels (5, 9). It has been known that the activation of a slow anion current in guard cells is a key event leading to stomatal closure (10–13). The corresponding gene, slow anion channel 1 (SLAC1), was discovered in genetic screens for O₃ or CO₂ sensitivity in Arabidopsis; when this particular channel was mutated out, the mutants showed impaired response to stimuli (14–16).Arabidopsis thaliana SLAC1 (AtSLAC1) contains 556 amino acid (aa) residues, with a conserved transmembrane domain (TMD) between hydrophilic tails at both the N and C termini (15). Within the ABA signaling pathway, SLAC1 channel activity is controlled by a protein kinase-phosphatase pair (OST1/ABI1) (17–19). In the absence of ABA, OST1 kinase is bound and inhibited by ABI1 phosphatase (20–22). Under drought condition, ABA is accumulated into guard cells and perceived by the PYR1 receptor (23, 24). This resulting hormone–receptor complex then binds to phosphatase ABI1 to form a ternary hormone–receptor–phosphatase complex (25–28), which activates OST1 kinase, and leads to phosphorylation of SLAC1 (17, 18). Subsequently, SLAC1 releases anions (Cl⁻ and NO₃⁻) from guard cells, leading to membrane depolarization. Depolarization in turn activates potassium channels to release K⁺ via K⁺ channels and drives water out of the cell, decreasing guard cell turgor to close down the stomatal pore (8).It has been shown that SLAC1 activation is due to phosphorylation at its N terminus (17, 18, 29–31). The phosphorylation of S120 by OST1 is essential but not sufficient for SLAC1 activation monitored in Xenopus oocytes (18). S59 has been shown to be the target for both OST1 and other kinases (29, 31). In another phosphoproteomic analysis, using the fragment SLAC1¹⁻¹⁸⁶ as substrate, S86 and S113 were also shown to be phosphorylated by OST1 (32). OST1 also phosphorylates SLAC1 at the C terminus (17), with T513 identified as a potential phosphorylation site for SLAC1 activation (31). Although multiple phosphorylation sites have been proposed, the underlying mechanism for SLAC1 activation remains elusive.Our previous study on a SLAC1 bacterial homolog from Haemophilus influenzae (HiTehA) revealed a superfamily of trimeric anion channels (33) and found each protomer gated by a highly conserved phenylalanine. We observed an unusual high-energy rotameric conformation for this gating phenylalanine (F262 in HiTehA), and further mutational study on the corresponding site in AtSLAC1 (F450) suggested a unique mechanism for channel activation (33). Nevertheless, since HiTehA shares a mere 19% in sequence identity with AtSLAC1 and lacks the substantial N and C termini that are essential for channel activation, questions remain about SLAC1 structure and its regulation by kinase phosphorylation (7).Here we describe the cryoelectron microscopy (cryo-EM) structure of SLAC1 from Brachypodium distachyon (BdSLAC1) at 2.97-Å resolution, and we further characterize the channel activity on Arabidopsis SLAC1. BdSLAC1 shares 63% overall sequence identity with AtSLAC1, with 73% for the pore-forming TMD and 48% for the regulatory N and C termini. As for the bacterial homolog, each BdSLAC1 TMD comprises five tandemly repeated helical hairpins that surround a transmembrane pore in each protomer of the trimeric assembly. The SLAC1 pores are electropositive, consistent with its function as an anion channel. The regulatory N-terminal domain (190 aa) and C-terminal domain (65 aa) are flexibly associated and not discernible in the structure.We employed a SLAC1::OST1 fusion design for examining SLAC1 phosphorylation, whereby we systematically characterized critical sites responsible for channel activation. Inspired by the structure, we also conducted mutagenesis on the highly conserved phenylalanine residues related to channel gating. Altogether, our structural and functional analyses provide insights into SLAC1 gating and activity modulation. These findings allow us to propose a mechanism for finely tuned SLAC1 control of the stomatal pore in response to environmental stimuli.     

First-in-class humanized FSH blocking antibody targets bone and fat

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a K_D of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH–FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHβ subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing.

Obesity and osteoporosis affect nearly 650 million and 200 million people worldwide, respectively (1, 2). Yet the armamentarium for preventing and treating these disorders remains limited, particularly when compared with public health epidemics of a similar magnitude. It has also become increasingly clear that obesity and osteoporosis track together clinically. First, body mass does not protect against bone loss; instead, obesity can be permissive to osteoporosis and a high fracture risk (3, 4). Furthermore, the menopausal transition marks the onset not only of rapid bone loss, but also of visceral obesity and dysregulated energy balance (5–9). These physiologic aberrations have been attributed traditionally to a decline in serum estrogen, although, during the perimenopause—2 to 3 y prior to the last menstrual period—serum estrogen is within the normal range, while FSH levels rise to compensate for reduced ovarian reserve (10–12). In our view, therefore, the early skeletal and metabolic derangements cannot conceivably be explained solely by declining estrogen (13, 14).The past decade has shown that pituitary hormones can act directly on the skeleton and other tissues, a paradigm shift that is in stark contrast to previously held views on their sole regulation of endocrine targets (15–25). We and others have shown that FSH can bypass the ovary to act on G_i-coupled FSH receptors (FSHRs) on osteoclasts to stimulate bone resorption and inhibit bone formation (26, 27). This mechanism, which could underscore the bone loss during early menopause, is testified by the strong correlations between serum FSH, bone turnover, and bone mineral density (7–9, 14, 16, 26). Likewise, activating polymorphisms in the FSHR in postmenopausal women are linked to a high bone turnover and reduced bone mass (27). It therefore made biological and clinical sense to inhibit FSH action during this period to prevent bone loss.Toward this goal, we generated murine polyclonal and monoclonal antibodies to a 13-amino-acid–long binding epitope of FSHβ (28–31). The mouse and human FSHβ epitopes differ by just two amino acids; hence, blocking antibodies to the human epitope showed efficacy in mice (28). The antibodies displayed two sets of actions: they attenuated the loss of bone after ovariectomy by inhibiting bone resorption and stimulating bone formation and displayed profound effects on body composition and energy metabolism (28, 29, 31). Most notably, in a series of contemporaneously reproduced experiments, we (M.Z. and C.J.R.) found that FSH blockade reduced body fat, triggered adipocyte beiging, and increased thermogenesis in models of obesity, notably post ovariectomy and after high-fat diet (29). Our findings have been further confirmed independently by two groups who used a FSHβ–GST fusion protein or tandem repeats of the 13-amino-acid–long FSHβ epitope for studies on bone and fat, respectively (32, 33). Consistent with the mouse data, inhibiting FSH secretion using a GnRH agonist in prostate cancer patients resulted in low body fat compared with orchiectomy, wherein FSH levels are high (34). This interventional clinical trial provides evidence for a therapeutic benefit of reducing FSH levels on body fat in people. There is also new evidence that FSH blockade lowers serum cholesterol (35, 36).Thus, both emerging and validated datasets on the antiobesity, osteoprotective, and lipid-lowering actions of FSH blockade in mice and in humans prompted our current attempt to develop and characterize an array of fully humanized FSH-blocking antibodies for future testing in people. Here, we report that our lead first-in-class humanized antibody, Hu6, and two related molecules, Hu26 and Hu28, bind human FSH with a high affinity (K_Ds <10 nM), block the binding of FSH on the human FSHR, and inhibit FSH action in functional cell-based assays.     

Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus–induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

Myopia (nearsightedness) in school-age children is generally axial myopia, which is the consequence of elongation of the eyeball along the visual axis. This shape change results in blurred vision but can also lead to severe complications including cataract, retinal detachment, myopic choroidal neovascularization, glaucoma, and even blindness (1–3). Despite the current worldwide pandemic of myopia, the mechanism of myopia onset is still not understood (4–8). One hypothesis that has earned a current consensus is the suggestion that a change in the lighting environment of modern society is the cause of myopia (9, 10). Consistent with this, outdoor activity has a protective effect on myopia development (9, 11, 12), though the main reason for this effect is still under debate (7, 12, 13). One explanation is that bright outdoor light can promote the synthesis and release of dopamine in the eye, a myopia-protective neuromodulator (14–16). Another suggestion is that the distinct wavelength composition of sunlight compared with fluorescent or LED (light-emitting diode) artificial lighting may influence myopia progression (9, 10). Animal studies have shown that different wavelengths of light can affect the development of myopia independent of intensity (17, 18). The effects appear to be distinct in different species: for chicks and guinea pigs, blue light showed a protective effect on experimentally induced myopia, while red light had the opposite effect (18–22). For tree shrews and rhesus monkeys, red light is protective, and blue light causes dysregulation of eye growth (23–25).It has been shown that visible violet light (VL) has a protective effect on myopia development in mice, in chick, and in human (10, 26, 27). According to Commission Internationale de l’Eclairage (International Commission on Illumination), VL has the shortest wavelength of visible light (360 to 400 nm). These wavelengths are abundant in outside sunlight but can only rarely be detected inside buildings. This is because the ultraviolet (UV)-protective coating on windows blocks all light below 400 nm and because almost no VL is emitted by artificial light sources (10). Thus, we hypothesized that the lack of VL in modern society is one reason for the myopia boom (9, 10, 26).In this study, we combine a newly developed lens-induced myopia (LIM) model with genetic manipulations to investigate myopia pathways in mice (28, 29). Our data confirm (10, 26) that visible VL is protective but further show that delivery of VL only in the evening is sufficient for the protective effect. In addition, we show that the protective effect of VL on myopia induction requires OPN5 (neuropsin) within the retina. The absence of retinal Opn5 prevents lens-induced, VL-dependent thickening of the choroid, a response thought to play a key role in adjusting the size of the eyeball in both human and animal myopia models (30–33). This report thus identifies a cell type, the Opn5 retinal ganglion cell (RGC), as playing a key role in emmetropization. The requirement for OPN5 also explains why VL has a protective effect on myopia development.