Nathan W. Schmidt

Arginine-rich cell-penetrating peptides.

Arginine‐rich cell‐penetrating peptides are short cationic peptides capable of traversing the plasma membranes of eukaryotic cells. While successful intracellular delivery of many biologically active macromolecules has been accomplished using these peptides, their mechanisms of cell entry are still under investigation. Recent dialogue has centered on a debate over the roles that direct translocation and endocytotic pathways play in internalization of cell‐penetrating peptides. In this paper, we review the evidence for the broad range of proposed mechanisms, and show that each distinct process requires negative Gaussian membrane curvature as a necessary condition. Generation of negative Gaussian curvature by cell‐penetrating peptides is directly related to their arginine content. We illustrate these concepts using HIV TAT as an example.

Memory CD8 T cell responses exceeding a large but definable threshold provide long-term immunity to malaria

Infection of mice with sporozoites of Plasmodium berghei or Plasmodium yoelii has been used extensively to evaluate liver-stage protection by candidate preerythrocytic malaria vaccines. Unfortunately, repeated success of such vaccines in mice has not translated readily to effective malaria vaccines in humans. Thus, mice may be used better as models to dissect basic parameters required for immunity to Plasmodium-infection than as preclinical vaccine models. In turn, this basic information may aid in the rational design of malaria vaccines. Here, we describe a model of circumsporozoite-specific memory CD8 T cell generation that protects mice against multiple P. berghei sporozoite challenges for at least 19 months. Using this model we defined a threshold frequency of memory CD8 T cells in the blood that predicts long-term sterilizing immunity against liver-stage infection. Importantly, the number of Plasmodium-specific memory CD8 T cells required for immunity greatly exceeds the number required for resistance to other pathogens. In addition, this model allowed us to identify readily individual immunized mice that exceed or fall below the protective threshold before infection, information that should greatly facilitate studies to dissect basic mechanisms of protective CD8 T cell memory against liver-stage Plasmodium infection. Furthermore, the extremely large threshold in memory CD8 T cell frequencies required for long-term protection in mice may have important implications for development of effective malaria vaccines.

Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions

Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using “nunchuck” CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay (“negative Gaussian”) membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.

Extreme CD8 t cell requirements for anti-malarial liver-stage immunity following immunization with radiation attenuated sporozoites

Radiation-attenuated Plasmodium sporozoites (RAS) are the only vaccine shown to induce sterilizing protection against malaria in both humans and rodents. Importantly, these “whole-parasite” vaccines are currently under evaluation in human clinical trials. Studies with inbred mice reveal that RAS-induced CD8 T cells targeting liver-stage parasites are critical for protection. However, the paucity of defined T cell epitopes for these parasites has precluded precise understanding of the specific characteristics of RAS-induced protective CD8 T cell responses. Thus, it is not known whether quantitative or qualitative differences in RAS-induced CD8 T cell responses underlie the relative resistance or susceptibility of immune inbred mice to sporozoite challenge. Moreover, whether extraordinarily large CD8 T cell responses are generated and required for protection following RAS immunization, as has been described for CD8 T cell responses following single-antigen subunit vaccination, remains unknown. Here, we used surrogate T cell activation markers to identify and track whole-parasite, RAS-vaccine-induced effector and memory CD8 T cell responses. Our data show that the differential susceptibility of RAS-immune inbred mouse strains to Plasmodium berghei or P. yoelii sporozoite challenge does not result from host- or parasite-specific decreases in the CD8 T cell response. Moreover, the surrogate activation marker approach allowed us for the first time to evaluate CD8 T cell responses and protective immunity following RAS-immunization in outbred hosts. Importantly, we show that compared to a protective subunit vaccine that elicits a CD8 T cell response to a single epitope, diversifying the targeted antigens through whole-parasite RAS immunization only minimally, if at all, reduced the numerical requirements for memory CD8 T cell-mediated protection. Thus, our studies reveal that extremely high frequencies of RAS-induced memory CD8 T cells are required, but may not suffice, for sterilizing anti-Plasmodial immunity. These data provide new insights into protective CD8 T cell responses elicited by RAS-immunization in genetically diverse hosts, information with relevance to developing attenuated whole-parasite vaccines.

Mechanism of a Prototypical Synthetic Membrane-Active Antimicrobial: Efficient Hole-Punching Via Interaction With Negative Intrinsic Curvature Lipids

Phenylene ethynylenes comprise a prototypical class of synthetic antimicrobial compounds that mimic antimicrobial peptides produced by eukaryotes and have broad-spectrum antimicrobial activity. We show unambiguously that bacterial membrane permeation by these antimicrobials depends on the presence of negative intrinsic curvature lipids, such as phosphatidylethanolamine (PE) lipids, found in high concentrations within bacterial membranes. Plate-killing assays indicate that a PE-knockout mutant strain of Escherichia coli drastically out-survives the wild type against the membrane-active phenylene ethynylene antimicrobials, whereas the opposite is true when challenged with traditional metabolic antibiotics. That the PE deletion is a lethal mutation in normative environments suggests that resistant bacterial strains do not evolve because a lethal mutation is required to gain immunity. PE lipids allow efficient generation of negative curvature required for the circumferential barrel of an induced membrane pore; an inverted hexagonal HII phase, which consists of arrays of water channels, is induced by a small number of antimicrobial molecules. The estimated antimicrobial occupation in these water channels is nonlinear and jumps from ≈1 to 3 per 4 nm of induced water channel length as the global antimicrobial concentration is increased. By comparing to exactly solvable 1D spin models for magnetic systems, we quantify the cooperativity of these antimicrobials.

Differential Effector Pathways Regulate Memory CD8 T Cell Immunity against Plasmodium berghei versus P. yoelii Sporozoites

Malaria results in >1,000,000 deaths per year worldwide. Although no licensed vaccine exists, much effort is currently focused on subunit vaccines that elicit CD8 T cell responses directed against Plasmodium parasite liver stage Ags. Multiple immune-effector molecules play a role in antimicrobial immunity mediated by memory CD8 T cells, including IFN-γ, perforin, TRAIL, Fas ligand, and TNF-α. However, it is not known which pathways are required for memory CD8 T cell-mediated immunity against liver stage Plasmodium infection. In this study, we used a novel immunization strategy to generate memory CD8 T cells in the BALB/c mouse model of P. berghei or P. yoelii sporozoite infection to examine the role of immune-effector molecules in resistance to the liver stage infection. Our studies reveal that endogenous memory CD8 T cell-mediated protection against both parasite species is, in part, dependent on IFN-γ, whereas perforin was only critical in protection against P. yoelii. We further show that neutralization of TNF-α in immunized mice markedly reduces memory CD8 T cell-mediated protection against both parasite species. Thus, our studies identify IFN-γ and TNF-α as important components of the noncytolytic pathways that underlie memory CD8 T cell-mediated immunity against liver stage Plasmodium infection. Our studies also show that the effector pathways that memory CD8 T cells use to eliminate liver stage infection are, in part, Plasmodium species specific.

Platelet-derived CD154 enables T-cell priming and protection against Listeria monocytogenes challenge

Collagen exposure in tissue activates platelets, initiates wound healing, and modulates adaptive immunity. In this report, data are presented to demonstrate a requirement for platelet-derived CD154 for both collagen-induced augmentation of T-cell immunity and induction of pro-tective immunity to Listeria challenge. Specifically, we demonstrate that Ad5 encoding the membrane-bound form of ovalbumin (Ad5-mOVA) delivered in collagen induces higher ovalbumin-specific cytotoxic T lymphocyte (CTL) activity in a dose-dependent manner compared with Ad5-mOVA delivered in PBS. Increased CTL activity was dependent on the ability of platelets to respond to collagen and to express CD154. Furthermore, mice immunized with low-dose Ad5-mOVA in collagen were able to control a challenge of Listeria monocytogenes recombinant for ovalbumin expression (Lm-OVA), whereas mice immunized with low-dose Ad5-mOVA in PBS were not. These data indicate that in a physiologic setting that mimics wounding, platelets perform a sentinel function when antigen dose is too low to provoke an efficient immune response, and can enhance the generation of antigen-specific CD8 T cells that are functionally relevant to the host.

Influence of salts and natural organic matter on the stability of bacteriophage MS2.

The stability of functionalized nanoparticles generally results from both steric and electrostatic interactions. Viruses like bacteriophage MS2 have adopted similar strategies for stability against aggregation, including a net negative charge under natural water conditions and using polypeptides that form loops extending from the surface of the protein capsid for stabilization. In natural systems, dissolved organic matter can adsorb to and effectively functionalize nanoparticle surfaces, affecting the fate and transport of these nanoparticles. We used time-resolved dynamic light scattering to measure the aggregation kinetics of a model virus, bacteriophage MS2, across a range of solution chemistries to determine what factors might destabilize viruses in aquatic systems. In monovalent electrolytes (LiCl, NaCl, and KCl), aggregation of MS2 could not be induced within a reasonable kinetic time frame, and MS2 was stable even at salt concentrations greater than 1.0 M. Aggregation of MS2 could be induced in divalent electrolytes when we employed Ca(2+). This trend was also observed in solutions containing 10 mg/L Suwannee River organic matter (SROM) reference material. Even at Ca(2+) concentrations as high 200 mM, diffusion-controlled aggregation was never achieved, demonstrating an additional barrier to aggregation. These results were confirmed by small-angle X-ray scattering experiments, which indicate a transition from repulsive to attractive interactions between MS2 virus particles as monovalent salts are replaced by divalent salts.

Composition of the gut microbiota modulates the severity of malaria.

Significance Plasmodium infections cause >200 million cases of malaria and ∼1 million deaths annually. Although these infections result in disease states that range from asymptomatic to life-threatening, factors that contribute to disease severity remain poorly defined. This report demonstrates that the assemblage of microbes in the gut can modulate the severity of malaria. Mice from different vendors with differences in their gut microbiome showed significant differences in pathology after infection with Plasmodium. Among the bacterial populations that were different between “resistant” and “susceptible” mice were Lactobacillus and Bifidobacterium, and treatment of mice with Lactobacillus and Bifidobacterium resulted in decreased Plasmodium burden. These results identify both a previously unidentified risk factor for severe malaria and a potential new avenue of treatment. Plasmodium infections result in clinical presentations that range from asymptomatic to severe malaria, resulting in ∼1 million deaths annually. Despite this toll on humanity, the factors that determine disease severity remain poorly understood. Here, we show that the gut microbiota of mice influences the pathogenesis of malaria. Genetically similar mice from different commercial vendors, which exhibited differences in their gut bacterial community, had significant differences in parasite burden and mortality after infection with multiple Plasmodium species. Germfree mice that received cecal content transplants from “resistant” or “susceptible” mice had low and high parasite burdens, respectively, demonstrating the gut microbiota shaped the severity of malaria. Among differences in the gut flora were increased abundances of Lactobacillus and Bifidobacterium in resistant mice. Susceptible mice treated with antibiotics followed by yogurt made from these bacterial genera displayed a decreased parasite burden. Consistent with differences in parasite burden, resistant mice exhibited an elevated humoral immune response compared with susceptible mice. Collectively, these results identify the composition of the gut microbiota as a previously unidentified risk factor for severe malaria and modulation of the gut microbiota (e.g., probiotics) as a potential treatment to decrease parasite burden.

Engineering persister-specific antibiotics with synergistic antimicrobial functions

Most antibiotics target growth processes and are ineffective against persister bacterial cells, which tolerate antibiotics due to their reduced metabolic activity. These persisters act as a genetic reservoir for resistant mutants and constitute a root cause of antibiotic resistance, a worldwide problem in human health. We re-engineer antibiotics specifically for persisters using tobramycin, an aminoglycoside antibiotic that targets bacterial ribosomes but is ineffective against persisters with low metabolic and cellular transport activity. By giving tobramycin the ability to induce nanoscopic negative Gaussian membrane curvature via addition of 12 amino acids, we transform tobramycin itself into a transporter sequence. The resulting molecule spontaneously permeates membranes, retains the high antibiotic activity of aminoglycosides, kills E. coli and S. aureus persisters 4–6 logs better than tobramycin, but remains noncytotoxic to eukaryotes. These results suggest a promising paradigm to renovate traditional antibiotics.