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.
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.
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.
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.
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.
MOPS and coxsackievirus B3 stability Carson, Steven D; Hafenstein, Susan; Lee, Hyunwook
Virology (New York, N.Y.),
01/2017, Letnik:
501
Journal Article
Recenzirano
Odprti dostop
Abstract 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.
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 (Δ
) and glutamate dehydrogenase (Δ
), reduced the ability of the bacteria to proliferate within a skin abscess, suggesting that peptides and free amino acids are important for
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
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
has the ability to detect peptides in the environment.
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
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
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
senses a nutrient-limiting environment and induces factors that function to acquire and utilize carbon from host-derived sources.
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-Å resolution 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-Å resolution and shows significant rearrangement of internal features and symmetric interactions with the RNA genome.
This report presents new information about receptor use by picornaviruses and highlights the importance of attaining at least an ∼9-Å resolution 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.
Acinetobacter baumannii is an emerging bacterial pathogen of considerable health care concern. Nonetheless, relatively little is known about the organism's virulence factors or their regulatory ...networks. Septicemia and ventilator-associated pneumonia are two of the more severe forms of A. baumannii disease. To identify virulence factors that may contribute to these disease processes, genetically diverse A. baumannii clinical isolates were evaluated for the ability to proliferate in human serum. A transposon mutant library was created in a strain background that propagated well in serum and screened for members with decreased serum growth. The results revealed that disruption of A. baumannii phospholipase D (PLD) caused a reduction in the organism's ability to thrive in serum, a deficiency in epithelial cell invasion, and diminished pathogenesis in a murine model of pneumonia. Collectively, these results suggest that PLD is an A. baumannii virulence factor.
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 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.
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