Proline transporters ProT and PutP are required for Staphylococcus aureus infection.

Proline acquired via specific transporters can serve as a proteinogenic substrate, carbon and nitrogen source, or osmolyte. Previous reports have documented that Staphylococcus aureus, a major community and nosocomial pathogen, encodes at least four proline transporters, PutP, OpuC, OpuD, and ProP. A combination of genetic approaches and 3H-proline transport assays reveal that a previously unrecognized transporter, ProT, in addition to PutP, are the major proline transporters in S. aureus. Complementation experiments using constitutively expressed non-cognate promoters found that proline transport via OpuD, OpuC, and ProP is minimal. Both proline biosynthesis from arginine and proline transport via ProT are critical for growth when S. aureus is grown under conditions of high salinity. Further, proline transport mediated by ProT or PutP are required for growth in media with and without glucose, indicating both transporters function to acquire proline for proteinogenic purposes in addition to acquisition of proline as a carbon/nitrogen source. Lastly, inactivation of proT and putP resulted in a significant reduction (5 log10) of bacterial burden in murine skin-and-soft tissue infection and bacteremia models, suggesting that proline transport is required to establish a S. aureus infection.

Albumin Enhances the Rate at Which Coxsackievirus B3 Strain 28 Converts to A-Particles.

Three strains of coxsackievirus B3 (CVB3) differ by single mutations in capsid protein VP1 or VP3 and also differ in stability at 37°C in tissue culture medium. Among these strains, the CVB3/28 parent strain has been found to be uniquely sensitive to a component in fetal bovine serum (FBS) identified as serum albumin. In cell culture medium, serum increased the rate of CVB3/28 conversion to noninfectious particles at least 2-fold. The effect showed a saturable dose response. Rates of conversion to noninfectious virus with high concentrations of soluble coxsackievirus and adenovirus receptor (sCAR) were similar with and without FBS, but FBS amplified the catalytic effect of 100 nM sCAR nearly 3-fold. Such effects in other systems are due to nonessential activating cofactors.IMPORTANCE A factor other than the virus receptor expressed by target cells has been found to accelerate the loss of an enterovirus (CVB3/28) infectious titer, with little effect on nearly identical mutant strains. The destabilizing factor in fetal bovine serum, identified as albumin, does not interfere with the catalytic activity of soluble receptor at saturating receptor concentrations and amplifies the catalytic activity of the soluble receptor at a concentration that otherwise produces about one-third the saturated receptor-catalyzed rate of virus decay. This finding evidences the possibility that other virus-"priming" ligands may also be nonessential activating cofactors that serve to accelerate receptor-catalyzed viral eclipse.

Protease-Mediated Growth of Staphylococcus aureus on Host Proteins Is opp3 Dependent.

Staphylococcus aureus has the ability to cause infections in multiple organ systems, suggesting an ability to rapidly adapt to changing carbon and nitrogen sources. Although there is little information about the nutrients available at specific sites of infection, a mature skin abscess has been characterized as glucose depleted, indicating that peptides and free amino acids are an important source of nutrients for the bacteria. Our studies have found that mutations in enzymes necessary for growth on amino acids, including pyruvate carboxykinase (ΔpckA) and glutamate dehydrogenase (ΔgudB), reduced the ability of the bacteria to proliferate within a skin abscess, suggesting that peptides and free amino acids are important for S. aureus growth. Furthermore, we found that collagen, an abundant host protein that is present throughout a skin abscess, serves as a reservoir of peptides. To liberate peptides from the collagen, we identified that the host protease, MMP-9, as well as the staphylococcal proteases aureolysin and staphopain B function to cleave collagen into peptide fragments that can support S. aureus growth under nutrient-limited conditions. Moreover, the oligopeptide transporter Opp3 is the primary staphylococcal transporter responsible for peptide acquisition. Lastly, we observed that the presence of peptides (3-mer to 7-mer) induces the expression of aureolysin, suggesting that S. aureus has the ability to detect peptides in the environment.IMPORTANCEStaphylococcus aureus has the ability to cause infections in a variety of niches, suggesting a robust metabolic capacity facilitating proliferation under various nutrient conditions. The mature skin abscess is glucose depleted, indicating that peptides and free amino acids are important sources of nutrients for S. aureus Our studies have found that mutations in both pyruvate carboxykinase and glutamate dehydrogenase, enzymes that function in essential gluconeogenesis reactions when amino acids serve as the major carbon source, reduce bacterial burden in a murine skin abscess model. Moreover, peptides liberated from collagen by host protease MMP-9 as well as the staphylococcal protease aureolysin support S. aureus growth in an Opp3-dependent manner under nutrient-limited conditions. Additionally, the presence of peptides induces aureolysin expression. Overall, these studies define one pathway by which S. aureus senses a nutrient-limiting environment and induces factors that function to acquire and utilize carbon from host-derived sources.

MOPS and coxsackievirus B3 stability.

Study of coxsackievirus B3 strain 28 (CVB3/28) stability using MOPS to improve buffering in the experimental medium revealed that MOPS (3-morpholinopropane-1-sulfonic acid) increased CVB3 stability and the effect was concentration dependent. Over the pH range 7.0-7.5, virus stability was affected by both pH and MOPS concentration. Computer-simulated molecular docking showed that MOPS can occupy the hydrophobic pocket in capsid protein VP1 where the sulfonic acid head group can form ionic and hydrogen bonds with Arg95 and Asn211 near the pocket opening. The effects of MOPS and hydrogen ion concentrations on the rate of virus decay were modeled by including corresponding parameters in a recent kinetic model. These results indicate that MOPS can directly associate with CVB3 and stabilize the virus, possibly by altering capsid conformational dynamics.

Three capsid amino acids notably influence coxsackie B3 virus stability.

Coxsackievirus B3 strain 28 (CVB3/28) is less stable at 37 °C than eight other CVB3 strains with which it has been compared, including four in this study. In a variant CVB3/28 population selected for increased stability at 37 °C, the capsid proteins of the stable variant differed from the parental CVB3/28 by two mutations in Vp1 and one mutation in Vp3, each of which resulted in altered protein sequences. Each of the amino acid changes was individually associated with a more stable virus. Competition between CVB3/28 and a more stable derivative of the strain showed that propagation of the less stable virus was favoured in receptor-rich HeLa cells.

Kinetic models for receptor-catalyzed conversion of coxsackievirus B3 to A-particles.

UNLABELLED: The immunoglobulin superfamily protein receptors for poliovirus, human rhinovirus, and coxsackievirus B (CVB) serve to bind the viruses to target cells and to facilitate the release of the virus genome by catalyzing the transition from the mature infectious virus to the A-particle uncoating intermediate. Receptor binding sites characterized by two equilibrium dissociation constants have been identified. The site with higher affinity is best observed at warmer temperatures and appears to correlate with the reversible conformational state in which the capsid is permeable to small molecules and peptides that are buried in the crystal structures are exposed. Measurements of CVB conversion to inactive particles over time in the presence of varied concentrations of soluble coxsackievirus and adenovirus receptor showed that the observed first-order rate constant varies with receptor concentration. The dose-response data, previously modeled as the sum of first-order reactions, have been used to evaluate models for the receptor-catalyzed conversion of CVB that include the high- and low-affinity binding sites associated with capsid breathing. Allosteric models wherein receptor binding shifts the equilibrium toward the open capsid conformation, in which the high-affinity binding site is available, best fit the data. IMPORTANCE: This paper compares models that relate the structural, mechanistic, and kinetic details of receptor-virus interactions known from previous work with human enteroviruses. New models are derived using recent results from receptor-catalyzed conversion of coxsackievirus B3 to non-infectious A-particles. Of those considered, the acceptable models include the capsid breathing cycle and two conformation-dependent receptor binding sites. The results indicate that the receptor enhancement of virus conversion to A-particles involves allostery through conformation selection.

Kinetic and structural analysis of coxsackievirus B3 receptor interactions and formation of the A-particle.

UNLABELLED: The coxsackievirus and adenovirus receptor (CAR) has been identified as the cellular receptor for group B coxsackieviruses, including serotype 3 (CVB3). CAR mediates infection by binding to CVB3 and catalyzing conformational changes in the virus that result in formation of the altered, noninfectious A-particle. Kinetic analyses show that the apparent first-order rate constant for the inactivation of CVB3 by soluble CAR (sCAR) at physiological temperatures varies nonlinearly with sCAR concentration. Cryo-electron microscopy (cryo-EM) reconstruction of the CVB3-CAR complex resulted in a 9.0-Šresolution map that was interpreted with the four available crystal structures of CAR, providing a consensus footprint for the receptor binding site. The analysis of the cryo-EM structure identifies important virus-receptor interactions that are conserved across picornavirus species. These conserved interactions map to variable antigenic sites or structurally conserved regions, suggesting a combination of evolutionary mechanisms for receptor site preservation. The CAR-catalyzed A-particle structure was solved to a 6.6-Šresolution and shows significant rearrangement of internal features and symmetric interactions with the RNA genome. IMPORTANCE: This report presents new information about receptor use by picornaviruses and highlights the importance of attaining at least an ∼9-Šresolution for the interpretation of cryo-EM complex maps. The analysis of receptor binding elucidates two complementary mechanisms for preservation of the low-affinity (initial) interaction of the receptor and defines the kinetics of receptor-catalyzed conformational change to the A-particle.

HeLa cell heterogeneity and coxsackievirus B3 cytopathic effect: implications for inter-laboratory reproducibility of results.

Concerns over cell line identities and contamination have led investigators to acquire fresh stocks of HeLa CCL-2 cells, but results with the HeLa CCL-2 cells do not always reproduce results with HeLa cells that have long history in the laboratory. When used for TCID(50) assays of Coxsackievirus B3/28 (CVB3/28), HeLa CCL-2 cells returned titers for CVB3/28 that were more than ten-fold lower than titers obtained using laboratory HeLa cells. The viral cytopathic effect was less distinct in the HeLa CCL-2 cultures, suggestive of a mixed population of cells with varied susceptibility to viral cytopathic effect. Analysis of short tandem repeat markers confirmed the identities of the cell lines as HeLa. Subpopulations in the HeLa CCL-2 culture, separated easily based on the speed with which they were released by trypsin-EDTA, differed in their susceptibilities to CVB3/28 cytopathic effect, and in their expression of the Coxsackievirus and adenovirus receptor (CAR). The distinctions between Lab HeLa and HeLa CCL-2 cells were less obvious when infected with CVB3/RD, a strain selected for growth in RD cells. Results that differ among laboratories may be due to the use of HeLa cell strains with different histories, and experiments using HeLa CCL-2 available from the American Type Culture Collection are probably incapable of reproducing many of the published studies of Coxsackievirus that have used HeLa cells with laboratory-dependent histories.

Variations of coxsackievirus B3 capsid primary structure, ligands, and stability are selected for in a coxsackievirus and adenovirus receptor-limited environment.

While group B coxsackieviruses (CVB) use the coxsackievirus and adenovirus receptor (CAR) as the receptor through which they infect susceptible cells, some CVB strains are known for their acquired capacity to bind other molecules. The CVB3/RD strain that emerged from a CVB3/Nancy population sequentially passaged in the CAR-poor RD cell line binds decay-accelerating factor (DAF) (CD55) and CAR. A new strain, CVB3/RDVa, has been isolated from RD cells chronically infected with CVB3/RD and binds multiple molecules in addition to DAF and CAR. The capsid proteins of CVB3/RD differ from those of CVB3/28, a cloned strain that binds only CAR, by only four amino acids, including a glutamate/glutamine dimorphism in the DAF-binding region of the capsid. The capsid proteins of CVB3/RD and CVB3/RDVa differ by seven amino acids. The ability of CVB3/RDVa to bind ligands in addition to CAR and DAF may be attributed to lysine residues near the icosahedral 5-fold axes of symmetry. Considered with differences in the stability of the CVB3 strains, these traits suggest that in vitro selection in a CAR-limited environment selects for virus populations that can associate with molecules on the cell surface and survive until CAR becomes available to support infection.

Differentiation and transplantation of human embryonic stem cell-derived hepatocytes.

BACKGROUND & AIMS: The ability to obtain unlimited numbers of human hepatocytes would improve the development of cell-based therapies for liver diseases, facilitate the study of liver biology, and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, potentially can differentiate into any cell type, and therefore could be developed as a source of human hepatocytes. METHODS: To generate human hepatocytes, human embryonic stem cells were differentiated by sequential culture in fibroblast growth factor 2 and human activin-A, hepatocyte growth factor, and dexamethasone. Functional hepatocytes were isolated by sorting for surface asialoglycoprotein-receptor expression. Characterization was performed by real-time polymerase chain reaction, immunohistochemistry, immunoblot, functional assays, and transplantation. RESULTS: Embryonic stem cell-derived hepatocytes expressed liver-specific genes, but not genes representing other lineages, secreted functional human liver-specific proteins similar to those of primary human hepatocytes, and showed human hepatocyte cytochrome P450 metabolic activity. Serum from rodents given injections of embryonic stem cell-derived hepatocytes contained significant amounts of human albumin and alpha1-antitrypsin. Colonies of cytokeratin-18 and human albumin-expressing cells were present in the livers of recipient animals. CONCLUSIONS: Human embryonic stem cells can be differentiated into cells with many characteristics of primary human hepatocytes. Hepatocyte-like cells can be enriched and recovered based on asialoglycoprotein-receptor expression and potentially could be used in drug discovery research and developed as therapeutics.

Endogenous low-level expression of the coxsackievirus and adenovirus receptor enables coxsackievirus B3 infection of RD cells.

Cells in which the appropriate viral receptor cannot be detected may paradoxically act as a host to the virus. For example, RD cells are often considered to be non-permissive for infection with coxsackievirus and adenovirus receptor (CAR)-dependent group B coxsackieviruses (CVB), insofar as inoculated cell monolayers show little or no cytopathic effect (CPE) and immunohistological assays for CAR have been consistently negative. Supernatants recovered from RD cells exposed to CVB, however, contained more virus than was added in the initial inoculum, indicating that productive virus replication occurred in the monolayer. When infected with a recombinant CVB type 3 (CVB3) chimeric strain expressing S-Tag within the viral polyprotein, 4-11 % of RD cells expressed S-Tag over 48 h. CAR mRNA was detected in RD cells by RT-PCR, and CAR protein was detected on Western blots of RD lysates; both were detected at much lower levels than in HeLa cells. Receptor blockade by an anti-CAR antibody confirmed that CVB3 infection of RD cells was mediated by CAR. These results show that some RD cells in the culture population express CAR and can thereby be infected by CVB, which explains the replication of CAR-dependent CVB in cell types that show little or no CPE and in which CAR has not previously been detected. Cells within cultures of cell types that have been considered non-permissive may express receptor transiently, leading to persistent replication of virus within the cultured population.

Coxsackievirus B3 infection and type 1 diabetes development in NOD mice: insulitis determines susceptibility of pancreatic islets to virus infection.

Group B coxsackieviruses (CVB) are believed to trigger some cases of human type 1 diabetes (T1D), although the mechanism by which this may occur has not been shown. We demonstrated previously that inoculation of young nonobese diabetic (NOD) mice with any of several different CVB strains reduced T1D incidence. We also observed no evidence of CVB replication within islets of young NOD mice, suggesting no role for CVB in T1D induction in the NOD mouse model. The failure to observe CVB replication within islets of young NOD mice has been proposed to be due to interferon expression by insulin-producing beta cells or lack of expression of the CVB receptor CAR. We found that CAR protein is detectable within islets of young and older NOD mice and that a CVB3 strain, which expresses murine IL-4, can replicate in islets. Mice inoculated with the IL-4 expressing CVB3 chimeric strain were better protected from T1D onset than were mock-infected control mice despite intraislet viral replication. Having demonstrated that CVB can replicate in healthy islets of young NOD mice when the intraislet environment is suitably altered, we asked whether islets in old prediabetic mice were resistant to CVB infection. Unlike young mice in which insulitis is not yet apparent, older NOD mice demonstrate severe insulitis in all islets. Inoculating older prediabetic mice with different pathogenic CVB strains caused accelerated T1D onset relative to control mice, a phenomenon that was preceded by detection of virus within islets. Together, the results suggest a model for resolving conflicting data regarding the role of CVB in human T1D etiology.

Coxsackievirus and adenovirus receptor (CAR) is modified and shed in membrane vesicles.

Vesicles shed by U87-MG cells contain coxsackievirus and adenovirus receptor (CAR) protein that has been posttranslationally modified. Relative to full-length CAR, migration of the vesicle-associated soluble CAR antigen (CARd6) on SDS-polyacrylamide gels indicated a loss of approximately 6 kDa. HeLa and END-HHV6 cells also shed a similar vesicle-associated CAR protein. Vesicles shed by U87-MG cells following stimulation with calcium and A23187 contained CARd6 similar to that present in vesicles shed constitutively. RD cells transfected to express full-length CAR produced CARd6, but cells that expressed CAR with a truncated cytoplasmic domain produced no equivalent to CARd6. In U87-MG cells, calpain activity was required for release of CARd6 with shed vesicles, and accumulation of CARd6 in cells that rounded up and released from the plastic substrate in response to A23187 treatment was blocked by N-ethylmaleimide. These experiments show that CAR, posttranslationally modified in the cytoplasmic domain, can be released with vesicles shed by cells. Posttranslational modification of the CAR cytoplasmic domain occurs during cell rounding and release from the culture substrate. This modified, vesicle-associated CAR was the principal form of soluble CAR released by the cells.

Monoclonal antibody against mouse CAR following genetic immunization.

To broaden our repertoire of monoclonal antibodies against CAR (coxsackievirus and adenovirus receptor), we inoculated mice with an expression vector containing the cDNA encoding human CAR extracellular and transmembrane sequence, and boosted the response by inoculation with soluble human CAR protein produced in E. coli. Of the hybridomas obtained following this immunization protocol, one secreted IgG with exceptional reactivity against mouse CAR. Since CAR has been shown to form dimers, expression of human CAR in cells that express mouse CAR may have stimulated the host immune system to recognize endogenous CAR in heterodimers.

Caspase-3 activation and ERK phosphorylation during CVB3 infection of cells: influence of the coxsackievirus and adenovirus receptor and engineered variants.

Caspase activation and MAP kinase signaling have been implicated in coxsackievirus B3 (CVB3) pathogenesis, and both have been demonstrated late in the virus life cycle. We studied activation of caspase-3, an effector protease of apoptosis, and ERK phosphorylation, indicative of MAPK signaling pathway activation, following CVB3 infection of cells that express the coxsackievirus and adenovirus receptor (CAR) or CAR constructs lacking the cytoplasmic domain, and cells which express no detectable CAR. These experiments showed that a burst of caspase-3 activity preceded lysis of CVB3-infected cells expressing CAR, irrespective of the CAR cytoplasmic domain. In RD cells, which were infected in the absence of detectable CAR, caspase-3 activity increased progressively over 52 h with no apparent burst. ERK phosphorylation also occurred late in the virus life cycle, preceding caspase-3 activation, and occurred in cells expressing full-length CAR but not in RD. These results show that ERK phosphorylation precedes caspase-3 activation, both occur late in the infection, and both are influenced by the presence of CAR.