Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis

Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane-binding properties of this protein. Recombinant anthrolysin O (rALO35-512) and two N-terminally truncated versions of ALO (rALO390-512 and rALO403-512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35-512, but not rALO390-512 or rALO403-512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35-512 and rALO403-512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35-512 and rALO403-512, whereas other sterols tested did not support binding. The rALO403-512--membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35-512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides.     

Cellular Functions and X-ray Structure of Anthrolysin O, a Cholesterol-dependent Cytolysin Secreted by Bacillus anthracis

Anthrolysin O (ALO) is a pore-forming, cholesterol-dependent cytolysin (CDC) secreted by Bacillus anthracis, the etiologic agent for anthrax. Growing evidence suggests the involvement of ALO in anthrax pathogenesis. Here, we show that the apical application of ALO decreases the barrier function of human polarized epithelial cells as well as increases intracellular calcium and the internalization of the tight junction protein occludin. Using pharmacological agents, we also found that barrier function disruption requires increased intracellular calcium and protein degradation. We also report a crystal structure of the soluble state of ALO. Based on our analytical ultracentrifugation and light scattering studies, ALO exists as a monomer. Our ALO structure provides the molecular basis as to how ALO is locked in a monomeric state, in contrast to other CDCs that undergo antiparallel dimerization or higher order oligomerization in solution. ALO has four domains and is globally similar to perfringolysin O (PFO) and intermedilysin (ILY), yet the highly conserved undecapeptide region in domain 4 (D4) adopts a completely different conformation in all three CDCs. Consistent with the differences within D4 and at the D2-D4 interface, we found that ALO D4 plays a key role in affecting the barrier function of C2BBE cells, whereas PFO domain 4 cannot substitute for this role. Novel structural elements and unique cellular functions of ALO revealed by our studies provide new insight into the molecular basis for the diverse nature of the CDC family.Cholesterol-dependent cytolysins (CDCs)⁴ are a family of pore-forming toxins from many organisms, including but not limited to the genera Archanobacterium, Bacillus, Clostridium, Listeria, and Streptococcus. Recently, work in vertebrates has revealed that CDCs and membrane attack complex/perforin superfamily domain-containing proteins share a similar fold, suggesting that vertebrates use a similar mechanism for defense against infection (1, 2). A common feature of the CDC family is the requirement of cholesterol in the membrane to form pores (3). In addition to cholesterol, certain members of the family also require a cellular receptor, such as CD59 for the toxin ILY from Streptococcus intermedius (4). The specific mechanism by which CDCs form pores is not completely resolved; however, what is generally known is that ring-shaped oligomerization at the cellular membrane is followed by large conformational changes in each unit of the oligomer, resulting in the insertion of a β-barrel into the cellular membrane (5). Pore formation results in a variety of downstream signaling effects, including but not limited to the influx of Ca²⁺ into the cell (6).A good deal is known about structures of the prepore conformation of CDCs. The crystal structures of prepore PFO, from Clostridium perfringens, and ILY have previously been elucidated (7, 8). Each structure shows a characteristic four-domain architecture, in which domain 4 (D4) is involved in membrane recognition, domain 3 (D3) is involved in β-sheet insertion, and domain 2 (D2) is the hinge region that undergoes a large conformational change (9-11). Nevertheless, despite the similarities, structural differences in D4 orientation and the conformation of a highly conserved segment named the undecapeptide region confer functional differences to PFO and ILY (8). Noting these differences, we decided to explore the structure and function of another member of the CDC family, anthrolysin O (ALO).ALO is secreted by Bacillus anthracis, the etiologic agent for anthrax. ALO is chromosomally encoded by a gene whose regulation is poorly understood, and it is highly homologous to other members of the CDC family (12). ALO has been shown to have hemolytic and cytolytic activity (13, 14). Although clinical studies have shown that B. anthracis is weakly hemolytic (15), anthrax bacteria do produce biologically relevant amounts of hemolytic ALO, although the levels of expression are under complex regulation and are dependent on the culture media and growth conditions (12, 13, 16). At lower concentrations, ALO can disrupt cell signaling (13, 14). Search for a cellular receptor of ALO has lead to the conclusion that it is a TLR4 agonist (17). However, it is not known that ALO binds to TLR4 directly and, if so, whether ALO also binds other cellular receptors.In addition to ALO, B. anthracis secrete ∼400 proteins, termed the anthrax secretome (18). Of those, two exotoxins, edema toxin (ET) and lethal toxin (LT) have been characterized in greatest detail. ET raises intracellular cAMP to pathologic levels, whereas LT impairs mitogenic and stress responses by inactivating mitogen-activating protein kinase kinase (19, 20). The complex interplay between these two toxins on various aspects of host cellular functions have been demonstrated (20-25). ALO could also work in conjunction with other anthrax virulence factors to modulate their cellular toxicity. For example, ALO and LF together induce macrophage apoptosis, whereas ALO and PLC play a redundant role in a murine inhalation anthrax model (17, 26). Interplay among anthrax secreted factors on cells relevant to anthrax infection is just beginning to be understood. This network of interactions is vital to the molecular basis of how anthrax bacteria interact with the hosts during anthrax infection.Anthrax infection initiates when B. anthracis spores enter the host through one of three routes: cutaneous, inhalational, or gastrointestinal (GI) (27, 28). All three routes of infection can lead to systemic infection and are ultimately lethal. Different from inhalational anthrax, spores are ingested and germinate on or within the epithelium of the GI tract in GI anthrax (29). This is primarily based on pathological observations that primary lesions of the GI tract are found in GI anthrax, whereas no primary lesions of the lung are found in inhalational anthrax (29). Inhalational anthrax is a disease of choice for biological weapons because of its high infectivity and mortality (30). The initiation of GI anthrax requires much higher doses of spores than inhalational anthrax, and the molecular basis for the initiation of GI anthrax remains elusive (31).Since the primary function of GI epithelia is to control the flux of material into the body, disruption of this barrier can lead to movement of bacteria into the surrounding tissue (32). The barrier is produced by a matrix of transmembrane and membrane-associated proteins. These cell to cell contacts, or tight junctions, are sometimes altered during bacterial infection to specifically disrupt the barrier function of epithelial cells. Using a functional model for the gut epithelium, human gut epithelial Caco-2 brush border expressor (C2BBE) cells, we report that ALO decreases the barrier function of C2BBE cells through disruption of tight junctions. We also show that ALO disruption of barrier function is dependent on epithelial cell polarity. We also present the crystal structure of the soluble state of ALO and compare it with the known structures of other CDCs. In addition, we show that ALO exists primarily as a monomer, in contrast to its closely related homologue PFO, which exists as a dimer. Finally, we used domain swapping to examine the structural components that confer specificity of ALO to gut epithelial cells.     

Analysis of the role of the COL1 domain and its adjacent cysteine-containing sequence in the chain assembly of type IX collagen

The mechanisms of chain selection and assembly of type IX collagen, a heterotrimer 1(IX)2(IX)3(IX), must differ from that of fibrillar collagens since it lacks the characteristic C-propeptide of these latter molecules. We have tested the hypothesis that the information required for this process is contained within the C-terminal triple helical disulfide-bonded region (LMW). The reassociations of the purified LMW fragments of pepsinized bovine type IX collagen were followed by the formation of disulfide-bonded multimers. Our data demonstrate that only three triple helical assemblies form readily, (1)₃, (2)₃, and 123. The information required for chain selection and assembly is thus, at least in part, contained in the studied fragments. Molecular stoichiometries different from the classical heterotrimer may thus also form under certain conditions.     

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d- galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 &mgr;m recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.      

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.     

Nucleotide sequence and functional properties of a sodium-dependent citrate transport system from Klebsiella pneumoniae.

The gene of the sodium-dependent citrate transport system from Klebsiella pneumoniae (citS) is located on plasmid pES3 (Schwarz, E., and Oesterhelt, D. (1985) EMBO J. 4, 1599-1603) and encodes a 446-amino acid protein. Transport of citrate via this citrate transport protein (CitS) is dependent on the presence of sodium ions and is inhibited by magnesium ions. The delta pH (pH gradient across the membrane) is the major driving force for uptake. It is postulated that, in analogy with the proton-dependent citrate carrier (CitH) of K. pneumoniae (van der Rest, M. E., Abee, T., Molenaar, D., and Konings, W. N. (1990) Eur. J. Biochem. 195, 71-77), only one of the protonated species of citrate is recognized by CitS and that citrate is translocated across the membrane in symport with protons and sodium ions. The hydrophobicity profile of CitS suggests that the protein is very hydrophobic and contains 12 membrane-spanning segments. These segments are not centered around a hydrophilic core as has been suggested for other transport proteins, but the protein is asymmetrical with seven transmembrane segments in front of a large hydrophilic loop and five after this loop. The amino acid sequence is highly similar to a citrate transport system of Lactococcus lactis subsp. lactis var. diacetylactis (CitP) (David, S., van der Rest, M. E., Driessen, A. J. M., Simons, G., and de Vos, W. M. (1990) J. Bacteriol. 172, 5789-5794) and less similar to CitH of K. pneumoniae. We conclude that the citS gene of K. pneumoniae encodes a sodium-dependent citrate transport system that belongs to a novel subclass of transport proteins.     

Rapid damage to membranes of Neisseria gonorrhoeae caused by human neutrophil granule extracts

Neisseria gonorrhoeae were exposed to extracts of human neutrophil granules and effects on gonococcal growth and membranes were determined. Enumeration of gonococci by phase-contrast microscopy at 0 and 60 min revealed that they underwent very limited cell division after exposure to granule extract. At 60 min, treated gonococci tended to clump, and some lost their refractivity under phase-contrast optics, indicating membrane damage. Treated and untreated gonococci utilized oxygen at similar rates at time 0; treated gonococci utilized oxygen at a relatively constant rate for 60 min, even though colony-forming ability (i.e. viability) decreased by 90%, whereas untreated gonococci showed a steadily increasing rate of oxygen consumption over the same period, which essentially paralleled increase in colony-forming ability. Membrane ultrastructure of untreated and treated gonococci was compared in thin section by transmission electron microscopy. Extract treatment resulted in a time-related increase in disruption of the bacterial outer membrane, which became apparent almost immediately after treatment. This was accompanied by increasingly aberrant septum structure. Extract treatment also increased the resolution of peptidoglycan by electron microscopy, as early as 10 min after treatment. These data suggest that extract treatment of gonococci caused a rapid loss of the ability to form colonies on agar concomitant with alteration of gonococcal peptidoglycan and outer-membrane structure, but with little alteration of inner-membrane function.     

Efficiency clustering for low-density microarrays and its application to QPCR

Background  

Pathway-targeted or low-density arrays are used more and more frequently in biomedical research, particularly those arrays that are based on quantitative real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or probes, and they bring with it the promise of being able to reliably measure differences in target levels without the need to establish absolute standard curves for each and every target. To achieve reliable quantification all primer pairs or array probes must perform with the same efficiency.     

Molecular systematics of primary reptilian lineages and the tuatara mitochondrial genome

We provide phylogenetic analyses for primary Reptilia lineages including, for the first time, Sphenodon punctatus (tuatara) using data from whole mitochondrial genomes. Our analyses firmly support a sister relationship between Sphenodon and Squamata, which includes lizards and snakes. Using Sphenodon as an outgroup for select squamates, we found evidence indicating a sister relationship, among our study taxa, between Serpentes (represented by Dinodon) and Varanidae. Our analyses support monophyly of Archosauria, and a sister relationship between turtles and archosaurs. This latter relationship is congruent with a growing set of morphological and molecular analyses placing turtles within crown Diapsida and recognizing them as secondarily anapsid (lacking a skull fenestration). Inclusion of Sphenodon, as the only surviving member of Sphenodontia (with fossils from the mid-Triassic), helps to fill a sampling gap within previous analyses of reptilian phylogeny. We also report a unique configuration for the mitochondrial genome of Sphenodon, including two tRNA(Lys) copies and an absence of ND5, tRNA(His), and tRNA(Thr) genes.