Adaptive Laboratory Evolution of Staphylococcus aureus Resistance to Vancomycin and Daptomycin: Mutation Patterns and Cross-Resistance.

Vancomycin and daptomycin are first-line drugs for the treatment of complicated methicillin-resistant Staphylococcus aureus (MRSA) infections, including bacteremia. However, their effectiveness is limited not only by their resistance to each antibiotic but also by their associated resistance to both drugs. It is unknown whether novel lipoglycopeptides can overcome this associated resistance. Resistant derivatives from five S. aureus strains were obtained during adaptive laboratory evolution with vancomycin and daptomycin. Both parental and derivative strains were subjected to susceptibility testing, population analysis profiles, measurements of growth rate and autolytic activity, and whole-genome sequencing. Regardless of whether vancomycin or daptomycin was selected, most of the derivatives were characterized by a reduced susceptibility to daptomycin, vancomycin, telavancin, dalbavancin, and oritavancin. Resistance to induced autolysis was observed in all derivatives. Daptomycin resistance was associated with a significant reduction in growth rate. Resistance to vancomycin was mainly associated with mutations in the genes responsible for cell wall biosynthesis, and resistance to daptomycin was associated with mutations in the genes responsible for phospholipid biosynthesis and glycerol metabolism. However, mutations in walK and mprF were detected in derivatives selected for both antibiotics.

Ultrafast Collective Excited-State Dynamics of a Virus-Supported Fluorophore Antenna.

Radiation brightening was recently observed in a multifluorophore-conjugated brome mosaic virus (BMV) particle at room temperature under pulsed excitation. On the basis of its nonlinear dependence on the number of chromophores, the origins of the phenomenon were attributed to a collective relaxation. However, the mechanism remains unknown. We present ultrafast transient absorption and fluorescence spectroscopic studies which shed new light on the collective nature of the relaxation dynamics in such radiation-brightened, multifluorophore particles. Our findings indicate that the emission dynamics is consistent with a superradiance mechanism. The ratio between the rates of competing radiative and nonradiative relaxation pathways depends on the number of chromophores per virus. The findings suggest that small icosahedral virus shells provide a unique biological scaffold for developing nonclassical, deep subwavelength light sources and may open new avenues for the development of photonic probes for medical imaging applications.

Hydrophobic Cargo Encapsulation into Virus Protein Cages by Self-Assembly in an Aprotic Organic Solvent.

While extensive studies of virus capsid assembly in environments mimicking in vivo conditions have led to an understanding of the thermodynamic driving forces at work, applying this knowledge to virus assembly in other solvents than aqueous buffers has not been attempted yet. In this study, Brome mosaic virus (BMV) capsid proteins were shown to preserve their self-assembly abilities in an aprotic polar solvent, dimethyl sulfoxide (DMSO). This facilitated protein cage encapsulation of nanoparticles and dye molecules that favor organic solvents, such as ß-NaYF4-based upconversion nanoparticles and BODIPY dye. Assembly was found to be robust relative to a surprisingly broad range of DMSO concentrations. Cargos with poor initial stability in aqueous solutions were readily encapsulated at high DMSO concentrations and then transferred to aqueous solvents, where they remained stable and preserved their function for months.

Subset of Fluorophores Is Responsible for Radiation Brightening in Viromimetic Particles.

In certain conditions, dye-conjugated icosahedral virus shells exhibit suppression of concentration quenching. The recently observed radiation brightening at high fluorophore densities has been attributed to coherent emission, i.e., to a cooperative process occurring within a subset of the virus-supported fluorophores. Until now, the distribution of fluorophores among potential conjugation sites and the nature of the active subset remained unknown. With the help of mass spectrometry and molecular dynamics simulations, we found which conjugation sites in the brome mosaic virus capsid are accessible to fluorophores. Reactive external surface lysines but also those at the lumenal interface where the coat protein N-termini are located showed virtually unrestricted access to dyes. The third type of labeled lysines was situated at the intercapsomeric interfaces. Through limited proteolysis of flexible N-termini, it was determined that dyes bound to them are unlikely to be involved in the radiation brightening effect. At the same time, specific labeling of genetically inserted cysteines on the exterior capsid surface alone did not lead to radiation brightening. The results suggest that lysines situated within the more rigid structural part of the coat protein provide the chemical environments conducive to radiation brightening, and we discuss some of the characteristics of these environments.

Virus Assembly Pathways: Straying Away but Not Too Far.

Non-enveloped RNA viruses pervade all domains of life. In a cell, they co-assemble from viral RNA and capsid proteins. Virus-like particles can form in vitro where virtually any non-cognate polyanionic cargo can be packaged. How only viral RNA gets selected for packaging in vivo, in presence of myriad other polyanionic species, has been a puzzle. Through a combination of charge detection mass spectrometry and cryo-electron microscopy, it is determined that co-assembling brome mosaic virus (BMV) coat proteins and nucleic acid oligomers results in capsid structures and stoichiometries that differ from the icosahedral virion. These previously unknown shell structures are strained and less stable than the native one. However, they contain large native structure fragments that can be recycled to form BMV virions, should a viral genome become available. The existence of such structures suggest the possibility of a previously unknown regulatory pathway for the packaging process inside cells.

The emergence of hypervirulent blaNDM-1-positive Klebsiella pneumoniae sequence type 395 in an oncology hospital.

Fifteen hypermucoviscous isolates (13 blaNDM-1-positive) obtained from 11 oncology patients were analyzed by whole genome sequencing, and selected isolates were assessed in a murine model of sepsis. ST395/K2 isolates harboring rmpA, rmpA2, peg-344, aerobactin, enterobactin, yersiniabactin, type I fimbriae, etc. displayed maximal virulence in the mouse lethality assay (LD50 = 102 CFU). ST147/K20 isolates lacking yersiniabactins were relatively less virulent (LD50 = 104 CFU), ST395/K2 isolates lacking rmpA, rmpA2, peg-344, and aerobactin, but harboring yersiniabactin demonstrated minimal virulence (LD50 = 105 CFU). Isolates represent various paths and stages of evolution directed towards convergence of multidrugresistant classical Klebsiella pneumoniae and hypervirulent K. pneumoniae.

Disassembly Intermediates of the Brome Mosaic Virus Identified by Charge Detection Mass Spectrometry.

Capsid disassembly and genome release are critical steps in the lifecycle of a virus. However, their mechanisms are poorly understood, both in vivo and in vitro. Here, we have identified two in vitro disassembly pathways of the brome mosaic virus (BMV) by charge detection mass spectrometry and transmission electron microscopy. When subjected to a pH jump to a basic environment at low ionic strength, protein-RNA interactions are disrupted. Under these conditions, BMV appears to disassemble mainly through a global cleavage event into two main fragments: a near complete capsid that has released the RNA and the released RNA complexed to a small number of the capsid proteins. Upon slow buffer exchange to remove divalent cations at neutral pH, capsid protein interactions are disrupted. The BMV virions swell but there is no measurable loss of the RNA. Some of the virions break into small fragments, leading to an increase in the abundance of species with masses less than 1 MDa. The peak attributed to the BMV virion shifts to a higher mass with time. The mass increase is attributed to additional capsid proteins associating with the disrupted capsid protein-RNA complex, where the RNA is presumably partially exposed. It is likely that this pathway is more closely related to how the capsid disassembles in vivo, as it offers the advantage of protecting the RNA with the capsid protein until translation begins.

Multicenter study of serotype distribution of Streptococcus pneumoniae nasopharyngeal isolates from healthy children in the Russian Federation after introduction of PCV13 into the National Vaccination Calendar.

Russia introduced PCV13 in 2014. We studied the serotype composition of S. pneumoniae isolated from the nasopharynx of healthy children younger than 6 years in St. Petersburg, Smolensk, Perm, Krasnoyarsk, Khanty-Mansiysk and Khabarovsk, between 2016 and 2018. 2.4% of children had completed a 3-dose course of PCV13, while 25.6% had received 1 or 2 doses. Pneumococcal DNA detection by PCR demonstrated S. pneumoniae in 37.2% of samples with regional variation between sites (27.3 to 56.9%). There was little difference between vaccinated, partially vaccinated and un-vaccinated children. Children who had received at least 1 dose of PCV13 had lower carriage rates of vaccine serotypes than their unvaccinated peers (49.9 vs. 61.4%; p < 0.001). Children who had received at least 1 dose of PCV13 showed increased carriage rates of non-vaccine serotypes (50 vs 38.6%; P < 0.001). Especially serogroup 15AF was more prevalent among fully immunized children than among their peers (12.5 vs 2.7%; P < 0.05).

Radiation Brightening from Virus-like Particles.

Concentration quenching is a well-known challenge in many fluorescence imaging applications. Here, we show that the optical emission from hundreds of chromophores confined onto the surface of a 28 nm diameter virus particle can be recovered under pulsed irradiation. We have found that as one increases the number of chromophores tightly bound to the virus surface, fluorescence quenching ensues at first, but when the number of chromophores per particle is nearing the maximum number of surface sites allowable, a sudden brightening of the emitted light and a shortening of the excited-state lifetime are observed. This radiation brightening occurs only under short pulse excitation; steady-state excitation is characterized by conventional concentration quenching for any number of chromophores per particle. The observed suppression of fluorescence quenching is consistent with efficient, collective relaxation at room temperature. Interestingly, radiation brightening disappears when the emitters' spatial and/or dynamic heterogeneity is increased, suggesting that the template structural properties may play a role that could be instrumental in developing virus-enabled imaging vectors that have optical properties qualitatively different than those of state-of-the-art biophotonic agents.

In Vitro Ceftaroline Resistance Selection of Methicillin-Resistant Staphylococcus aureus Involves Different Genetic Pathways.

The pathways in the development of ceftaroline resistance of methicillin-resistant Staphylococcus aureus (MRSA) isolates belonging to the ST8, ST239, and ST228 were evaluated. Ceftaroline-resistant derivatives were isolated through selection during 40 passages. Ceftaroline MIC measurements and whole-genome sequencing were performed after 5, 20, and 40 passages. In two ST8 derivative isolates, ceftaroline MIC increased up to 128 mg/L. Mutations were acquired in gdpP and graS in one isolate after 20 passages and in gdpP in another after 40 passages. MIC for two ST239 derivatives increased to 128 mg/L. Substitutions in Pbp4 and polymorphisms in the upstream region of pbp4 were identified in both derivatives after 40 passages. In one isolate, additional mutation in gdpP and deletion in graR were detected. In an ST228 derivative, MIC increased to 32 mg/L with one mutation in penicillin-binding protein 2a (Y446N) detected after five passages and a second (E447K) after 20 passages. Three pathways in the development of ceftaroline resistance were identified. For ST8 and ST239 derivatives mutations were detected in gdpP and pbp4, respectively, whereas in ST228 - in mecA. Most derivatives harbored additional mutations whose potential role in the development of resistance has not been determined.

In Vitro Assembly of Virus-Derived Designer Shells Around Inorganic Nanoparticles.

Nanoparticle-templated assembly of virus shells provides a promising approach to the production of hybrid nanomaterials and a potential avenue toward new mechanistic insights in virus phenomena originating in many-body effects, which cannot be understood from examining the properties of molecular subunits alone. This approach complements the successful molecular biology perspective traditionally used in virology, and promises a deeper understanding of viruses and virus-like particles through an expanded methodological toolbox. Here we present protocols for forming a virus coat protein shell around functionalized inorganic nanoparticles.

Defects and Chirality in the Nanoparticle-Directed Assembly of Spherocylindrical Shells of Virus Coat Proteins.

Virus coat proteins of small isometric plant viruses readily assemble into symmetric, icosahedral cages encapsulating noncognate cargo, provided the cargo meets a minimal set of chemical and physical requirements. While this capability has been intensely explored for certain virus-enabled nanotechnologies, additional applications require lower symmetry than that of an icosahedron. Here, we show that the coat proteins of an icosahedral virus can efficiently assemble around metal nanorods into spherocylindrical closed shells with hexagonally close-packed bodies and icosahedral caps. Comparison of chiral angles and packing defects observed by in situ atomic force microscopy with those obtained from molecular dynamics models offers insight into the mechanism of growth, and the influence of stresses associated with intrinsic curvature and assembly pathways.

Structural and functional characterization of endothelial microparticles released by cigarette smoke.

Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers.

Toward Virus-Like Surface Plasmon Strain Sensors.

The strong configuration dependence of collective surface plasmon resonances in an array of metal nanoparticles provides an opportunity to develop a bioinspired tool for sensing mechanical deformations in soft matter at the nanoscale. We study the feasibility of a strain sensor based on an icosahedral array of nanoparticles encapsulated by a virus capsid. When the system undergoes deformation, the optical scattering cross-section spectra as well as the induced electric field profile change. By numerical simulations, we examine how these changes depend on the symmetry and extent of the deformation and on both the propagation direction and polarization of the incident radiation. Such a sensor could prove useful in studies of the mechanisms of nanoparticle or virus translocation in the confines of a host cell.

Examining the Heterogeneous Genome Content of Multipartite Viruses BMV and CCMV by Native Mass Spectrometry.

Since the concept was first introduced by Brian Chait and co-workers in 1991, mass spectrometry of proteins and protein complexes under non-denaturing conditions (native MS) has strongly developed, through parallel advances in instrumentation, sample preparation, and data analysis tools. However, the success rate of native MS analysis, particularly in heterogeneous mega-Dalton (MDa) protein complexes, still strongly depends on careful instrument modification. Here, we further explore these boundaries in native mass spectrometry, analyzing two related endogenous multipartite viruses: the Brome Mosaic Virus (BMV) and the Cowpea Chlorotic Mottle Virus (CCMV). Both CCMV and BMV are approximately 4.6 megadalton (MDa) in mass, of which approximately 1 MDA originates from the genomic content of the virion. Both viruses are produced as mixtures of three particles carrying different segments of the genome, varying by approximately 0.1 MDA in mass (~2%). This mixture of particles poses a challenging analytical problem for high-resolution native MS analysis, given the large mass scales involved. We attempt to unravel the particle heterogeneity using both Q-TOF and Orbitrap mass spectrometers extensively modified for analysis of very large assemblies. We show that manipulation of the charging behavior can provide assistance in assigning the correct charge states. Despite their challenging size and heterogeneity, we obtained native mass spectra with resolved series of charge states for both BMV and CCMV, demonstrating that native MS of endogenous multipartite virions is feasible. Graphical Abstract ᅟ.

Segmented GFP-like aptamer probes for functional imaging of viral genome trafficking.

Recently developed GFP-like RNA aptamers harbor a few unique potential benefits for in vivo RNA imaging applications, including co-packaging of viral genomes. Here we examine them in the context of co-packaging of RNA strands during virion assembly and trafficking. The approach is applicable both in vitro and in vivo, thus bridging an existing methodological gap. We have found that splitting the aptamer sequence in the loop region into two separate parts allows for subsequent self-assembly into a functional unit, which preserves the dye-binding pocket. In presence of the dye, virus-like particles encapsulating segmented GFP-like aptamers provided bright fluorescence emission and showed negligible bleaching due to continuous chromophore exchange: two desirable characteristics for real-time in vivo single particle studies requiring a broader dynamic range than currently available. Proof-of-principle in vivo imaging experiments confirmed detectability of aptamer-loaded virus-like particles in barley root cells even in presence of significant autofluorescence background.

Role of charge regulation and size polydispersity in nanoparticle encapsulation by viral coat proteins.

Nanoparticles can be encapsulated by virus coat proteins if their surfaces are functionalized to acquire a sufficiently large negative charge. A minimal surface charge is required to overcome (i) repulsive interactions between the positively charged RNA-binding domains on the proteins and (ii) the loss of mixing and translational entropy of RNA and capsid coat proteins. Here, we present a model describing the encapsulation of spherical particles bearing weakly acidic surface groups and investigate how charge regulation and size polydispersity impact upon the encapsulation efficiency of gold nanoparticles by model coat proteins. We show that the surface charge density of these particles cannot be assumed fixed, but that it adjusts itself to minimize electrostatic repulsion between the charges on them and maximize the attractive interaction with the RNA binding domains on the proteins. Charge regulation in combination with the natural variation of particle radii has a large effect on the encapsulation efficiency: it makes it much more gradual despite its inherently cooperative nature. Our calculations rationalize recent experimental observations on the coassembly of gold nanoparticles by brome mosaic virus coat proteins.

Encapsulation of nanoparticles in virus protein shells.

The self-assembly of virus-like particles may lead to materials which combine the unique characteristics of viruses, such as precise size control and responsivity to environmental cues, with the properties of abiotic cargo. For a few different viruses, shell proteins are amenable to the in vitro encapsulation of non-genomic cargo in a regular protein cage. In this chapter we describe protocols of high-efficiency in vitro self-assembly around functionalized gold nanoparticles for three examples of icosahedral and non-icosahedral viral protein cages derived from a plant virus, an animal virus, and a human retrovirus. These protocols can be readily adapted with small modifications to work for a broad variety of inorganic and organic nanoparticles.

Measurement of Nanoparticle Adlayer Properties by Photothermal Microscopy.

Many nanoparticle applications require molecular adlayers that impart desirable interfacial characteristics. Such characteristics are crucial in controlling the interaction of the nanoparticle with the environment or other nanoparticles; however, departures from bulk values are expected for adlayer properties and in situ methods to evaluate the magnitude of these departures, preferably on the scale of a single nanoparticle, are needed. Here we investigate the potential of single-particle photothermal microscopy for measuring the thermal properties of nanoparticle-supported, layer-by-layer grown polyelectrolytes. We show that nanometer changes in adlayer thickness can be detected this way, and the water content of the nanoparticle-supported adlayers can be estimated.

Fusion of mApple and Venus fluorescent proteins to the Sindbis virus E2 protein leads to different cell-binding properties.

Fluorescent proteins (FPs) are widely used in real-time single virus particle studies to visualize, track and quantify the spatial and temporal parameters of viral pathways. However, potential functional differences between the wild type and the FP-tagged virus may specifically affect particular stages in the virus life-cycle. In this work, we genetically modified the E2 spike protein of Sindbis virus (SINV) with two FPs. We inserted mApple, a red FP, or Venus, a yellow FP, at the N-terminus of the E2 protein of SINV to make SINV-Apple and SINV-Venus. Our results indicate that SINV-Apple and SINV-Venus have similar levels of infectivity and are morphologically similar to SINV-wild-type by negative stain transmission electron microscopy. Both mutants are highly fluorescent and have excellent single-particle tracking properties. However, despite these similarities, when measuring cell entry at the single-particle level, we found that SINV-Apple and SINV-Venus are different in their interaction with the cell surface and FPs are not always interchangeable. We went on to determine that the FP changes the net surface charge on the virus particles, the folding of the spike proteins, and the conformation of the spikes on the virus particle surface, ultimately leading to different cell-binding properties between SINV-Apple and SINV-Venus. Our results are consistent with recent findings that FPs may alter the biological and cellular localization properties of bacterial proteins to which they are fused.