Antibiotics induce redox-related physiological alterations as part of their lethality

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H₂O₂ sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H₂O₂. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.The increasing incidence of antibiotic-resistant infections coupled with a declining antibiotic pipeline has created a global public health threat (1–6). Therefore there is a pressing need to expand our conceptual understanding of how antibiotics act and to use insights gained from such efforts to enhance our antibiotic arsenal. It has been proposed that different classes of bactericidal antibiotics, regardless of their drug–target interactions, generate varying levels of deleterious reactive oxygen species (ROS) that contribute to cell killing (7, 8). This unanticipated notion, built upon important prior work (9–11), has been extended and supported by multiple laboratories investigating wide-ranging drug classes (e.g., β-lactams, aminoglycosides, and fluoroquinolones) and bacterial species (e.g., Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baumannii, Burkholderia cepecia, Streptococcus pneumonia, Enterococcus faecalis) using independent lines of evidence (12–39). Importantly, these ongoing efforts have served to refine aspects of the initial model and show that antibiotic-induced ROS generation is a more complex process than originally suggested, likely involving additional means of production.The redox stress component of antibiotic lethality is hypothesized to derive from alterations to multiple core aspects of cellular physiology and stress response activation. Specifically, this component includes alterations to central metabolism, cellular respiration, and iron metabolism initiated by drug-mediated disruptions of target-specific processes and resulting cellular damage (Fig. 1A). Important support for this hypothesis can be found in pathogenic clinical isolates whose drug tolerance involves mutations in oxidative stress response and defense genes and not exclusively in drug target mutagenesis (40–48).Open in a separate windowFig. 1.Bactericidal antibiotics promote the generation of toxic reactive species. (A) Bactericidal antibiotics of different classes are capable of inducing cell death by interfering with their primary targets and corrupting target-specific processes, resulting in lethal cellular damage. Target-specific interactions trigger stress responses that induce redox-related physiological alterations resulting in the formation of toxic reactive species, including ROS, which further contribute to cellular damage and death. (B) Treatment of wild-type E. coli with ampicillin (Amp, 5 μg/mL), gentamicin (Gent, 5 μg/mL), or norfloxacin (Nor, 250 ng/mL) induces ROS, detectable by several chemically diverse fluorescent dyes with ranging specificity. One-way ANOVA was performed to determine statistical significance against the no-dye autofluorescence control. The dyes used were 5/6-carboxy-2'',7''-dichlorodihydrofluorescein diacetate (Carboxy-H₂DCFDA); 5/6-chloromethyl-2'',7''-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA); 4-amino-5-methylamino-2´,7´-diflurorescein diacetate (DAF-FM); 2'',7''-dichlorodihydrofluorescein diacetate (H₂DCFDA); 3′-(p-hydroxyphenyl) fluorescein (HPF); OxyBURST Green (Oxyburst); and Peroxy-Fluor 2 (PF2). (C) Treatment of a quinolone-resistant strain (gyrA17) with norfloxacin does not produce detectable ROS. Data shown reflect mean ± SEM of three or more technical replicates. Where appropriate, statistical significance is shown and computed against the no-treatment control or no-dye control (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).Recent critiques of this evolving model have misinterpreted an essential aspect of the hypothesis. Specifically, these recent studies (49–51) are predicated on the notion that ROS are the sole arbiters of antibiotic lethality, thereby implying that the model suggests that antibiotics do not kill by disrupting their well-established, target-specific processes. However, the evolving model is completely consistent with the literature indicating that bactericidal antibiotics are capable of inducing lethal cellular damage via interference with target-specific processes, ultimately resulting in cell death. Rather than refute this traditional view of antibiotic action, the hypothesis extends it by suggesting that an additional component of toxicity results from ROS, which are generated as a downstream physiological consequence of antibiotics interacting with their traditional targets. In this respect, reactive species are thought to contribute causatively to drug lethality.However, an important gap exists in our general understanding of how bacteria respond physiologically to antibiotic–target interactions on a system-wide level, how these responses contribute to antibiotic killing, and how the extracellular environment protects or exacerbates the intracellular contributions to cell death. Here, we use a multidisciplinary set of biochemical, enzymatic, biophysical, and genetic assays to address these issues and expand our understanding of antibiotic-induced physiological responses and factors contributing to antibiotic lethality. Data from the present work indicate that antibiotic lethality is accompanied by ROS generation and that such reactive species causatively contribute to antibiotic lethality.     

Hemolysis-induced lethality involves inflammasome activation by heme

The increase of extracellular heme is a hallmark of hemolysis or extensive cell damage. Heme has prooxidant, cytotoxic, and inflammatory effects, playing a central role in the pathogenesis of malaria, sepsis, and sickle cell disease. However, the mechanisms by which heme is sensed by innate immune cells contributing to these diseases are not fully characterized. We found that heme, but not porphyrins without iron, activated LPS-primed macrophages promoting the processing of IL-1β dependent on nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). The activation of NLRP3 by heme required spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K⁺ efflux, whereas it was independent of heme internalization, lysosomal damage, ATP release, the purinergic receptor P2X7, and cell death. Importantly, our results indicated the participation of macrophages, NLRP3 inflammasome components, and IL-1R in the lethality caused by sterile hemolysis. Thus, understanding the molecular pathways affected by heme in innate immune cells might prove useful to identify new therapeutic targets for diseases that have heme release.Hemolysis, hemorrhage, and rhabdomyolysis cause the release of large amounts of hemoproteins to the extracellular space, which, once oxidized, release the heme moiety, a potentially harmful molecule due to its prooxidant, cytotoxic, and inflammatory effects (1, 2). Scavenging proteins such as haptoglobin and hemopexin bind hemoglobin and heme, respectively, promoting their clearance from the circulation and delivery to cells involved with heme catabolism. Heme oxygenase cleaves heme and generates equimolar amounts of biliverdin, carbon monoxide (CO) and iron (2). Studies using mice deficient for haptoglobin (Hp), hemopexin (Hx), and heme oxygenase 1 (HO-1) demonstrate the importance of these proteins in controlling the deleterious effects of heme. Both Hp^−/− and Hx^−/− mice have increased renal damage after acute hemolysis induced by phenyhydrazine (Phz) compared with wild-type mice (3, 4). Mice lacking both proteins present splenomegaly and liver inflammation composed of several foci with leukocyte infiltration after intravascular hemolysis (5). Hx protect mice against heme-induced endothelial damage improving liver and cardiovascular function (6–8). Lack of heme oxygenase 1 (Hmox1^−/−) causes iron overload, increased cell death, and tissue inflammation under basal conditions and upon inflammatory stimuli (9–15). This salutary effect of HO-1 has been attributed to its effect of reducing heme amounts as well as generating the cytoprotective molecules, biliverdin and CO.Heme induces neutrophil migration in vivo and in vitro (16, 17), inhibits neutrophil apoptosis (18), triggers cytokine and lipid mediator production by macrophages (19, 20), and increases the expression of adhesion molecules and tissue factor on endothelial cells (21–23). Heme cooperates with TNF, causing hepatocyte apoptosis in a mechanism dependent on reactive oxygen species (ROS) generation (12). Whereas heme-induced TNF production depends on functional toll-like receptor 4 (TLR4), ROS generation in response to heme is TLR4 independent (19). We recently observed that heme triggers receptor-interacting protein (RIP)1/3-dependent macrophage-programmed necrosis through the induction of TNF and ROS (15). The highly unstable nature of iron is considered critical for the ability of heme to generate ROS and to cause inflammation. ROS generated by heme has been mainly attributed to the Fenton reaction. However, recent studies suggest that heme can generate ROS through multiple sources, including NADPH oxidase and mitochondria (22, 24–27).Heme causes inflammation in sterile and infectious conditions, contributing to the pathogenesis of hemolytic diseases, subarachnoid hemorrhage, malaria, and sepsis (11, 13, 24, 28), but the mechanisms by which heme operates in different conditions are not completely understood. Blocking the prooxidant effects of heme protects cells from death and prevents tissue damage and lethality in models of malaria and sepsis (12, 13, 15). Importantly, two recent studies demonstrated the pathogenic role of heme-induced TLR4 activation in a mouse model of sickle cell disease (29, 30). These results highlight the great potential of understanding the molecular mechanisms of heme-induced inflammation and cell death as a way to identify new therapeutic targets.Hemolysis and heme synergize with microbial molecules for the induction of inflammatory cytokine production and inflammation in a mechanism dependent on ROS and Syk (24). Processing of pro–IL-1β is dependent on caspase-1 activity, requiring assembly of the inflammasome, a cytosolic multiprotein complex composed of a NOD-like receptor, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 (31–33). The most extensively studied inflammasome is the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). NLRP3 and pro–IL-1β expression are increased in innate immune cells primed with NF-κB inducers such as TLR agonists and TNF (34, 35). NLRP3 inflammasome is activated by several structurally nonrelated stimuli, such as endogenous and microbial molecules, pore-forming toxins, and particulate matter (34, 35). The activation of NLRP3 involves K⁺ efflux, increase of ROS and Syk phosphorylation. Importantly, critical roles of NLRP3 have been demonstrated in a vast number of diseases (34, 36). We hypothesize that heme causes the activation of the inflammasome and secretion of IL-1β. Here we found that heme triggered the processing and secretion of IL-1β dependently on NLRP3 inflammasome in vitro and in vivo. The activation of NLRP3 by heme was dependent on Syk, ROS, and K⁺ efflux, but independent of lysosomal leakage, ATP release, or cell death. Finally, our results indicated the critical role of macrophages, the NLRP3 inflammasome, and IL-1R to the lethality caused by sterile hemolysis.     

From the Cover: Socially mediated induction and suppression of antibiosis during bacterial coexistence

Despite their importance for humans, there is little consensus on the function of antibiotics in nature for the bacteria that produce them. Classical explanations suggest that bacteria use antibiotics as weapons to kill or inhibit competitors, whereas a recent alternative hypothesis states that antibiotics are signals that coordinate cooperative social interactions between coexisting bacteria. Here we distinguish these hypotheses in the prolific antibiotic-producing genus Streptomyces and provide strong evidence that antibiotics are weapons whose expression is significantly influenced by social and competitive interactions between competing strains. We show that cells induce facultative responses to cues produced by competitors by (i) increasing their own antibiotic production, thereby decreasing costs associated with constitutive synthesis of these expensive products, and (ii) by suppressing antibiotic production in competitors, thereby reducing direct threats to themselves. These results thus show that although antibiotic production is profoundly social, it is emphatically not cooperative. Using computer simulations, we next show that these facultative strategies can facilitate the maintenance of biodiversity in a community context by converting lethal interactions between neighboring colonies to neutral interactions where neither strain excludes the other. Thus, just as bacteriocins can lead to increased diversity via rock–paper–scissors dynamics, so too can antibiotics via elicitation and suppression. Our results reveal that social interactions are crucial for understanding antibiosis and bacterial community dynamics, and highlight the potential of interbacterial interactions for novel drug discovery by eliciting pathways that mediate interference competition.The discovery and development of antibiotics to fight bacterial diseases is one of the great triumphs in modern medicine (1). However, increasing rates of antimicrobial resistance require innovative strategies to replenish antimicrobial drug pipelines (2, 3). Several novel antibiotics have been discovered in previously unexplored habitats (4) or uncultured microbes (5). By contrast, a second potential source of novel agents, silent antibiotic gene clusters in well-characterized organisms, remains unexploited because the factors that elicit their production are unknown (1). Identifying these factors requires understanding the ecological and evolutionary roles of antibiotics in the competitive and social context in which they are used in nature (6, 7). Here we test the role of social and competitive dynamics on antibiosis in the prolific antibiotic-producing bacterial genus Streptomyces. Simultaneously, we distinguish competing hypotheses for the role of antibiotics in nature.Streptomycetes are a diverse group of filamentous bacteria that produce some two-thirds of all known antibiotics (8). Although the antibiotics they produce have classically been viewed as intermicrobial weapons (6, 9), this perspective is increasingly questioned on two grounds (10–13). First, antibiotic concentrations in soil are believed to be too low to kill or inhibit competing bacteria (9). Second, subinhibitory (sub-MIC) concentrations of antibiotics induce responses in exposed organisms, such as increased biofilm formation (14) or expression of virulence genes (11, 15) that may benefit these target cells (10). Thus, rather than weapons, these arguments have led to the idea that antibiotics are cooperative signals (16) used for intercellular communication, that they are “collective regulators of the homeostasis of microbial communities” (12).However, evidence of response to sub-MIC antibiotic concentrations does not imply that antibiotics are signals or a form of communication. Communication can be partitioned according to the costs and benefits associated with production and response (17). A signal is a form of mutually beneficial communication between the sender of a signal and its recipient. A cue, by contrast, elicits a response that benefits only the recipient, sometimes to the detriment of the sender. Finally, suppression or attenuation (18) elicits a response that harms the recipient and benefits the producer (19, 20). Whereas signals are a form of cooperation, the unidirectional benefits associated with cues and suppression imply that these are forms of competition.Distinguishing whether antibiotics are cooperative signals or competitive weapons requires partitioning communication into these contrasting modes (6, 19, 20) and examining the role of antibiotics in the competitive and social context in which they are used.     

Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton

Meiosis generates haploid cells or spores for sexual reproduction. As a prelude to haploidization, homologous chromosomes pair and recombine to undergo segregation during the first meiotic division. During the entire meiotic prophase of the yeast Saccharomyces cerevisiae, chromosomes perform rapid movements that are suspected to contribute to the regulation of recombination. Here, we investigated the impact of ionizing radiation (IR) on movements of GFP–tagged bivalents in live pachytene cells. We find that exposure of sporulating cultures with >40 Gy (4-krad) X-rays stalls pachytene chromosome movements. This identifies a previously undescribed acute radiation response in yeast meiosis, which contrasts with its reported radioresistance of up to 1,000 Gy in survival assays. A modified 3′-end labeling assay disclosed IR-induced dsDNA breaks (DSBs) in pachytene cells at a linear dose relationship of one IR-induced DSB per cell per 5 Gy. Dihydroethidium staining revealed formation of reactive oxygen species (ROS) in irradiated cells. Immobility of fuzzy-appearing irradiated bivalents was rescued by addition of radical scavengers. Hydrogen peroxide-induced ROS did reduce bivalent mobility similar to 40 Gy X IR, while they failed to induce DSBs. IR- and H₂O₂-induced ROS were found to decompose actin cables that are driving meiotic chromosome mobility, an effect that could be rescued by antioxidant treatment. Hence, it appears that the meiotic actin cytoskeleton is a radical-sensitive system that inhibits bivalent movements in response to IR- and oxidant-induced ROS. This may be important to prevent motility-driven unfavorable chromosome interactions when meiotic recombination has to proceed in genotoxic environments.Exposure to ionizing irradiation (IR) has dire consequences for the cell, because it causes the formation of radicals and reactive oxygen species (ROS) that can oxidize and damage cellular components including proteins and DNA (1), whereas protection from IR-induced radical-mediated protein oxidation can lead to significant radio resistance (2). At the DNA level, IR leads to single-stranded and double-stranded DNA breaks (DSBs), with the latter being a severe threat to cellular survival (3). To cope with DSBs that may arise physiologically and/or by genotoxic environmental impacts such as IR, the cell repairs DSBs by two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), which predominate in the G1 and G2 phase of the cell cycle, respectively, and underlie cell-cycle-dependent sensitivities to IR exposure (4, 5). In the G2 phase and in the first meiotic prophase, DSB repair is mediated by HR, in yeast meiosis addressing ∼150 DSBs (6, 7) that are formed by the Topo2-related endonuclease/transesterase enzyme SPO11 (8). Absence of DSBs and the resulting compromised spore viability (9) can be partially rescued by ionizing irradiation (10). In all, yeast cells exhibit a high resistance to IR (11–13) which is also true for cells in prophase I (10, 14). In the meiosis of numerous species, programmed Spo11-induced DSBs are instrumental for homologous chromosome search and pairing and provide the substrate for HR, generating two outcomes: noncrossovers (NCO) and crossovers (CO) that allow for homolog segregation in the meiosis I division (reviewed by refs. 15 and 16). For CO to occur, homologous chromosomes need to encounter and pair lengthwise (synapse) during first meiotic prophase (see ref. 17). Homolog pairing occurs after completion of premeiotic DNA replication. Live cell studies in the synaptic meiosis of the yeast Saccharomyces cerevisiae (2n = 32) have shown that meiotic telomeres (18), chromosomes, and bivalents undergo a striking mobility throughout the entire prophase I (19–21), which contrasts with the relative immobility of pachytene bivalents in mammalian prophase I (22). It has been found that rapid and continuous telomere and chromosome movements in budding yeast meiocytes depend on actin polymerization (18–20) and an intact meiotic telomere complex (21, 23, 24). Besides a general mobility of chromosomes throughout prophase I, single bivalents are capable to rapidly move away and return to the motile chromosome mass, a behavior termed “maverick” formation (19) or rapid chromosome movements (20, 21).Exposure to ionizing radiation induces a plethora of physicochemical effects in the irradiated cells including DNA damage (1, 3). Extensive research addressing the adverse effects of IR exposure using yeast as a model system had largely been directed toward mutation induction, DSB repair, and cell cycle effects (e.g., 11–13, 25, 26). Meiotic yeast cells exposed to 50–80 krad (500–800 Gy) X or γ irradiation have been shown to exhibit a profound reduction in cell survival, particularly when exposed in the G1 cell cycle phase that lacks a sister chromatid for repair (4). Irradiated meiotic yeast cells exhibit mutations and chromosome missegregation at meiosis I, leading to reduced sporulation (5, 10, 27). While previous studies addressed late deterministic effects in irradiated yeast cells such as DNA repair, mutations, and cell survival, we were interested in the immediate consequences of IR exposure on motile meiotic chromosomes. Bivalent mobility can be expected to promote chromosomal rearrangements, if it continues after the formation of ectopic unregulated DSBs. Chromosomal translocations have, for instance, been observed after irradiation of mitotic budding yeast cells (28) and of meiotic prophase cells of mice (29). Furthermore, meiotic chromosome mobility has been proposed to be involved in regulating (adverse) chromosomal interactions (30). To study the consequences of IR exposure on meiotic chromosome mobility we followed live bivalent movements in X-irradiated and nonirradiated yeast cells expressing the GFP-tagged version of the synaptonemal complex protein ZIP1 (19) undergoing sporulation.     

Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria

The current epidemic of infections caused by antibiotic-resistant Gram-positive bacteria requires the discovery of new drug targets and the development of new therapeutics. Lipoteichoic acid (LTA), a cell wall polymer of Gram-positive bacteria, consists of 1,3-polyglycerol-phosphate linked to glycolipid. LTA synthase (LtaS) polymerizes polyglycerol-phosphate from phosphatidylglycerol, a reaction that is essential for the growth of Gram-positive bacteria. We screened small molecule libraries for compounds inhibiting growth of Staphylococcus aureus but not of Gram-negative bacteria. Compound 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] blocked phosphatidylglycerol binding to LtaS and inhibited LTA synthesis in S. aureus and in Escherichia coli expressing ltaS. Compound 1771 inhibited the growth of antibiotic-resistant Gram-positive bacteria and prolonged the survival of mice with lethal S. aureus challenge, validating LtaS as a target for the development of antibiotics.Methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide-resistant enterococci (GRE), and antibiotic-resistant Clostridium difficile are examples for newly emerging or reemerging drug-resistant Gram-positive pathogens with significant human morbidity (1). As commensals of the human skin or intestine, these microbes are continuously exposed to antibiotics and have evolved resistance traits against commonly used therapeutics (2). To develop novel antibiotics, a suitable molecular target must be identified (3). Ideal targets are found only in bacteria, not in humans, and are positioned in the bacterial envelope, accessible for small molecule inhibitors yet out of reach of multi–drug-resistance transporters protecting cytoplasmic targets (3, 4). Such attributes are found in penicillin-binding proteins and their inhibitors, β-lactam or glycopeptide antibiotics; however, efforts to identify other extracellular targets in Gram-positive bacteria have met with little success (5).Gram-positive bacteria incorporate lipoteichoic acids (LTAs) into their envelope to scavenge magnesium ions, direct autolysins to subcellular cell wall locations, and enable bacterial cell division (6, 7). LTA from S. aureus and other bacteria is composed of 1,3-polyglycerol-phosphate linked to glycolipid, which provides for LTA anchoring in membranes (8). The glycolipid moiety is composed of β-gentiobiosyldiacylglycerol [glucosyl-(1→6)-glucosyl-(1→3)-diacylglycerol (Glc₂-DAG)] (8, 9). PgcA (α-phosphoglucomutase), GtaB (UTP:α-glucose-1-phosphate uridyl transferase), and YpfP (glycosyl-transferase) catalyze the three steps of glycolipid synthesis (10–12), whereas LtaA transports Glc₂-DAG across the lipid bilayer (10). S. aureus ltaA, ypfP, pgcA, or gtaB mutants cannot assemble Glc₂-DAG but continue to synthesize polyglycerol-phosphate (10, 12). Glycolipid synthesis mutants are viable; however, the variants display an increase in size and aberrant cell shapes (10, 12).LTA synthesis involves the polymerization of polyglycerol-phosphate and its transfer to Glc₂-DAG (13). This reaction is catalyzed by LTA synthase (LtaS), a protein with five transmembrane domains in its N-terminal domain and an extracellular sulfatase-like domain (pfam00884) at the C-terminal end (14). Genetic depletion or loss of the ltaS gene in S. aureus results in severe cell division defects, a phenotype that is exacerbated when staphylococci are grown at >30 °C (14, 15). Characterization of LtaS in Bacillus anthracis, Bacillus subtilis, and Listeria monocytogenes confirmed that membrane proteins with pfam00884 domains are indeed responsible for the synthesis of polyglycerol-phosphate LTA (7). Where examined, ltaS mutants exhibited diminished viability, increased cell size, and altered morphology (16–19). These results suggested that LtaS of Gram-positive bacteria may represent an extracellular target for the development of antibiotics against drug-resistant Gram-positive bacteria. We here provide proof for this hypothesis by isolating a small molecule inhibitor of LTA synthesis.     

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

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

Contribution of reactive oxygen species to cerebral amyloid angiopathy,vasomotor dysfunction,and microhemorrhage in aged Tg2576 mice

Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid β peptide (Aβ) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer’s Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aβ-induced vessel dysfunction, but the mechanisms by which insoluble Aβ in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE—a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.Cerebral amyloid angiopathy (CAA) is characterized by amyloid deposition within walls of leptomeningeal and cortical arterioles. Among the several types of amyloid proteins causing CAA, fibrillar amyloid β (Aβ) is by far the most common (1). This pathological form of Aβ is also the major constituent of neuritic plaques in patients with Alzheimer’s disease (AD) (2). Aβ is a 39- to 43-amino acid peptide that is produced from the amyloid precursor protein (APP) via sequential proteolytic cleavage processed by β- and γ-secretases (3, 4). Aβ₄₀ is the predominant Aβ species present in CAA whereas Aβ₄₂ is the major Aβ species present in neuritic plaques. CAA is a very common disorder, pathologically affecting about one-third of all elderly patients (>60 y of age) and about 90% of patients with AD (5, 6). CAA is a well-recognized cause of intracerebral hemorrhage (7, 8). It is also a major contributor to ischemic stroke and dementia (2, 9–12)—two conditions in which CAA-induced impairment in cerebral arteriole function is likely to play a fundamental role (13).Multiple lines of evidence indicate that soluble Aβ monomers and insoluble Aβ fibrils in the form of CAA cause significant cerebrovascular (CV) impairment. Ex vivo studies with isolated cerebral arterioles show that synthetic Aβ₄₀ (and to a lesser degree Aβ₄₂) induces direct vessel constriction, enhanced response to vasoconstrictors, and reduced response to vasodilators (14–22). Similar results have been demonstrated with synthetic Aβ₄₀ topically applied to the cerebral cortex (23, 24), results that are generally supported by in vivo studies (20, 23, 25). For example, Iadecola and coworkers have shown that young APP transgenic mice (Tg2576) exposed to elevated levels of Aβ₄₀ and Aβ₄₂ (but no CAA) have reduced baseline cerebral blood flow (CBF) and decreased CBF responses to topical vasodilators (23, 24, 26). We have shown similar CV deficits in young Tg2576 mice (13). Moreover, we provided the most direct evidence to date that endogenous soluble Aβ plays a causal role in these CV deficits when we found that depletion of soluble Aβ via γ-secretase inhibition restores CV function in young Tg2576 mice (13).Fibrillar Aβ in the form of CAA produces even greater degrees of CV impairment. Evidence for this notion comes from several experimental studies from different laboratories that show reduced pial arteriole responses (27) and diminished CBF responses (27, 28) to a variety of vasodilatory stimuli in aged APP mice with CAA vs. young APP mice without CAA. Our past work examining pial arteriole function in young vs. aged Tg2576 mice shows similar age-dependent CV deficits (13). Moreover, multiple additional observations from our study show that CAA (and not prolonged exposure to soluble Aβ and/or mutant APP) is the principle cause of the severe CV dysfunction noted in aged Tg2576 mice: (i) The severity of the vasomotor deficits noted in these mice is dependent on the presence and extent of CAA; (ii) even small amounts of CAA are associated with profound vasomotor impairment; and (iii) the CV dysfunction noted in CAA-ladened arteries is poorly responsive to depletion of soluble Aβ via γ-secretase inhibition (13).Regarding the mechanism of soluble Aβ-induced CV deficits, increased reactive oxygen species (ROS) are strongly implicated. Cerebral arterioles exposed to exogenous Aβ₄₀ develop significant oxidative stress (29), and various anti-ROS strategies have been shown to improve Aβ₄₀-induced vessel dysfunction (16, 23). Similarly, cerebral arterioles of young APP mice producing elevated levels of endogenous Aβ₄₀ and Aβ₄₂ (but no CAA) display oxidative stress (19), and the CV deficits found in these mice can be attenuated by both genetic and pharmacologic anti-ROS interventions (15, 19, 20, 30). In particular, ROS derived from NADPH oxidase—one of two major sources of ROS in the cerebrovasculature (31–33)—have been implicated (28–30, 34, 35).Regarding the mechanism of CAA-induced CV deficits, far less is known; however, three recent findings suggest that ROS may play a role. First, CAA-affected vessels were shown to have significantly greater oxidative stress than CAA-free vessels of aged Tg2576 mice (36). Second, genetic knockdown of mitochondrial superoxide dismutase 2 (SOD2)—which increases mitochondria-derived ROS—was shown to exacerbate CAA pathology in aged APP mice (37). Third, genetic depletion of the catalytic subunit Nox2 of NADPH oxidase was shown to reduce oxidative stress and improve CV function in aged Tg2576 mice (28, 35). Importantly, the latter studies did not examine for the presence of CAA, nor did they assess for the effect of CAA on cerebral arteriole function (28, 35). To address this critical knowledge gap, we examined the effect of the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, on CAA-induced CV dysfunction in aged Tg2576 mice. The effect of these agents on CAA formation and CAA-related microhemorrhage was also examined.     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

CLE-CLAVATA1 peptide-receptor signaling module regulates the expansion of plant root systems in a nitrogen-dependent manner

Morphological plasticity of root systems is critically important for plant survival because it allows plants to optimize their capacity to take up water and nutrients from the soil environment. Here we show that a signaling module composed of nitrogen (N)-responsive CLE (CLAVATA3/ESR-related) peptides and the CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase is expressed in the root vasculature in Arabidopsis thaliana and plays a crucial role in regulating the expansion of the root system under N-deficient conditions. CLE1, -3, -4, and -7 were induced by N deficiency in roots, predominantly expressed in root pericycle cells, and their overexpression repressed the growth of lateral root primordia and their emergence from the primary root. In contrast, clv1 mutants showed progressive outgrowth of lateral root primordia into lateral roots under N-deficient conditions. The clv1 phenotype was reverted by introducing a CLV1 promoter-driven CLV1:GFP construct producing CLV1:GFP fusion proteins in phloem companion cells of roots. The overaccumulation of CLE2, -3, -4, and -7 in clv1 mutants suggested the amplitude of the CLE peptide signals being feedback-regulated by CLV1. When CLE3 was overexpressed under its own promoter in wild-type plants, the length of lateral roots was negatively correlated with increasing CLE3 mRNA levels; however, this inhibitory action of CLE3 was abrogated in the clv1 mutant background. Our findings identify the N-responsive CLE-CLV1 signaling module as an essential mechanism restrictively controlling the expansion of the lateral root system in N-deficient environments.Living organisms have developed dynamic strategies to explore nutrients in the environment. Morphological plasticity of plant roots and microorganisms is often compared with foraging behavior of animals. Plant roots are highly dynamic systems because they can modify their structure to reach nutrient resources in soil and optimize their nutrient uptake capacities. This strategy appears to be associated with morphological adaptation, because plants are sessile in nature and nutrient availabilities in soil are often altered by surrounding biotic and abiotic factors and climate changes. Morphological modifications of plant root systems are particularly prominent when they grow in soil environments with unbalanced nutrient availabilities (1–4). Among the essential elements required for plant growth, nitrogen (N) has a particularly strong effect on root development (1–6). Lateral roots can be developed in N-rich soil patches where adequate amounts of nitrate (NO₃⁻) or ammonium (NH₄⁺) are available, whereas this local outgrowth of lateral roots is restricted in N-deficient patches (7–9). In addition to these local N responses, lateral root growth is stimulated in response to mild N deficiency and suppressed under excess N supply by systemic plant signals carrying information on the nutritional status of distant plant organs (4, 10–13). These morphological responses are important for plant fitness and N acquisition, despite the cost for structuring the root system architecture (2, 6). However, lateral root growth is not sustained when plants are deprived of N for an extended period (4). Under such severe circumstances, the development of new lateral roots should rather be restricted to prevent the risk of extending roots into N-poor environments. Economizing the cost for root development appears to be an important morphological strategy for plant survival.To modify root traits in response to changing N availabilities, plants use various types of signaling molecules including hormones and small RNAs (10, 13–17). In particular, auxin signaling proteins and auxin transporters have been proven essential for lateral root development in response to local nitrate supplies (10, 14–17). These proteins are involved in increasing auxin sensitivity or auxin accumulation at lateral root initials or lateral root tips exposed to NO₃⁻, and the NRT1.1 nitrate transporter has been suggested to play a key role in NO₃⁻ sensing (8, 17, 18). In addition, mutations of the nitrate transporter NRT2.1 have been shown to repress or stimulate lateral root initiation depending on N conditions and sucrose supply (12, 19). Thus, N-dependent root development is apparently under control of complex mechanisms, although its signaling components have remained largely unidentified. In this study, we have identified several homologs of the CLE (CLAVATA3/ESR-related) gene family (20–24) to be up-regulated by N deficiency and involved in this yet unresolved regulatory mechanism. CLAVATA3 (CLV3) is known as a signaling peptide that binds to the CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase (LRR-RLK) and controls stem cell differentiation in the shoot apical meristem (25–32). CLE-receptor signaling modules are also known to control meristem function in the primary and lateral roots (33–35). The N-responsive CLE peptides described in the present study belong to the group of CLE peptides with the highest sequence similarity to CLAVATA3 (CLV3) (21–23) and may partly substitute for CLV3 in the shoot apical meristem (31, 36, 37). Our present findings indicate that the N-responsive CLE peptides and CLV1 are signaling components required for translating an N-deficient nutritional status into a morphological response inhibiting the outgrowth of lateral root primordia in Arabidopsis. The present study demonstrates a unique function of the CLE-CLV1 signaling module in roots and provides new insights into signaling mechanisms regulating the expansion of the plant root system in N-deficient environments.     

Transition paths,diffusive processes,and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.Protein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1, 2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7–15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16–25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the free energy minimum of the unfolded state. The development of ultrafast laser spectroscopy (29, 30) and single-molecule (27, 28, 31) techniques have made it possible to establish upper bounds on the transition path time (t_TP) ranging from <200 and <10 μs by earlier (27) and more recent (28), respectively, direct single-molecule FRET to <2 μs (30) by bulk relaxation measurements. Consistent with these observations, recent extensive atomic simulations have also provided estimated t_TP values of the order of ∼1 μs (32, 33). These advances offer exciting prospects of characterizing the productive events along folding TPs.It is timely, therefore, to further the theoretical investigation of TP-related questions (19). To this end, we used coarse-grained C_α models (14) to perform extensive simulations of the folding trajectories of small proteins with 56- to 86-aa residues. These tractable models are useful, because despite significant progress, current atomic models cannot provide the same degree of sampling coverage for proteins of comparable sizes (32, 33). In addition to structural insights, this study provides previously unexplored vantage points to compare the diffusion and TST pictures of folding. Deviations of folding behaviors from TST predictions are not unexpected, because TST is mostly applicable to simple gas reactions; however, the nature and extent of the deviations have not been much explored. Our explicit-chain simulation data conform well to the diffusion picture but not as well to TST. In particular, the preexponential factors of the simulated folding rates exhibit a small but appreciable variation that depends on native topology. These findings and others reported below underscore the importance of single-molecule measurements (13, 27, 28, 31, 34, 35) in assessing the merits of proposed scenarios and organizing principles of folding (7–25, 36, 37).