Self-Organization of Iron Sulfide Nanoparticles into Complex Multicompartment Supraparticles.

Self-assembled compartments from nanoscale components are found in all life forms. Their characteristic dimensions are in 50-1000 nm scale, typically assembled from a variety of bioorganic "building blocks". Among the various functions that these mesoscale compartments carry out, protection of the content from the environment is central. Finding synthetic pathways to similarly complex and functional particles from technologically friendly inorganic nanoparticles (NPs) is needed for a multitude of biomedical, biochemical, and biotechnological processes. Here, it is shown that FeS2 NPs stabilized by l-cysteine self-assemble into multicompartment supraparticles (mSPs). The NPs initially produce ≈55 nm concave assemblies that reconfigure into ≈75 nm closed mSPs with ≈340 interconnected compartments with an average size of ≈5 nm. The intercompartmental partitions and mSP surface are formed primarily from FeS2 and Fe2 O3 NPs, respectively. The intermediate formation of cup-like particles enables encapsulation of biological cargo. This capability is demonstrated by loading mSPs with DNA and subsequent transfection of mammalian cells. Also it is found that the temperature stability of the DNA cargo is enhanced compared to the traditional delivery vehicles. These findings demonstrate that biomimetic compartmentalized particles can be used to successfully encapsulate and enhance temperature stability of the nucleic acid cargo for a variety of bioapplications.

Staphylococcus epidermidis Has Growth Phase Dependent Affinity for Fibrinogen and Resulting Fibrin Clot Elasticity.

Bacterial infection and thrombosis are highly correlated, especially in patients with indwelling medical devices. Coagulase-negative staphylococci, typified by Staphylococcus epidermidis, are a common cause of medical device infections owing to their biofilm forming capacity which provides protection from antibiotics and host immune response. Attention has been drawn to the interaction between S. epidermidis and host proteins, specifically fibrinogen. However, little is known regarding the impact of the transition from planktonic to biofilm forming phenotype on this interaction. Here we investigate the growth phase dependence of bacteria-fibrinogen interaction and the resulting effect on fibrin clot formation, structure, and mechanics. Flow cytometry demonstrated growth phase dependent affinity for fibrinogen. To mimic intravascular device seeding, we quantified the adhesion of S. epidermidis to a fibrinogen coated surface under continuous flow conditions in vitro. The bacterial deposition rate onto fibrinogen was significantly greater for stationary (5,360 ± 1,776 cells/cm2s) versus exponential phase (2,212 ± 264, cells/cm2 s). Furthermore, the expression of sdrG-a cell surface adhesion protein with specificity for fibrinogen-was upregulated ∼twofold in the stationary versus the exponential phase. Rheometry and confocal microscopy demonstrated that stationary phase S. epidermidis slows clot formation and generates a more heterogeneous fibrin network structure with greater elasticity (G' = 5.7 ± 1.0 Pa) compared to sterile fibrinogen (G' = l.5 ± 0.2 Pa), while exponential phase cells had little effect. This work contributes to the current understanding of the growth phase dependent regulation of bacterial virulence factors and the correlation between bacterial infection and thrombosis.

Plasmonic nanoparticles assemblies templated by helical bacteria and resulting optical activity.

Plasmonic nanoparticles (NPs) adsorbing onto helical bacteria can lead to formation of NP helicoids with micron scale pitch. Associated chiroptical effects can be utilized as bioanalytical tool for bacterial detection and better understanding of the spectral behavior of helical self-assembled structures with different scales. Here, we report that enantiomerically pure helices with micron scale of chirality can be assembled on Campylobacter jejuni, a helical bacterium known for severe stomach infections. These organisms have right-handed helical shapes with a pitch of 1-2 microns and can serve as versatile templates for a variety of NPs. The bacteria itself shows no observable rotatory activity in the visible, red, and near-IR ranges of electromagnetic spectrum. The bacterial dispersion acquires chiroptical activity at 500-750 nm upon plasmonic functionalization with Au NPs. Finite-difference time-domain simulations confirmed the attribution of the chiroptical activity to the helical assembly of gold nanoparticles. The position of the circular dichroism peaks observed for these chiral structures overlaps with those obtained before for Au NPs and their constructs with molecular and nanoscale chirality. This work provides an experimental and computational pathway to utilize chiroplasmonic particles assembled on bacteria for bioanalytical purposes.

Anti-Biofilm Activity of Graphene Quantum Dots via Self-Assembly with Bacterial Amyloid Proteins.

Bacterial biofilms represent an essential part of Earth's ecosystem that can cause multiple ecological, technological, and health problems. The environmental resilience and sophisticated organization of biofilms are enabled by the extracellular matrix that creates a protective network of biomolecules around the bacterial community. Current anti-biofilm agents can interfere with extracellular matrix production but, being based on small molecules, are degraded by bacteria and rapidly diffuse away from biofilms. Both factors severely reduce their efficacy, while their toxicity to higher organisms creates additional barriers to their practicality. In this paper, we report on the ability of graphene quantum dots to effectively disperse mature amyloid-rich Staphylococcus aureus biofilms, interfering with the self-assembly of amyloid fibers, a key structural component of the extracellular matrix. Mimicking peptide-binding biomolecules, graphene quantum dots form supramolecular complexes with phenol-soluble modulins, the peptide monomers of amyloid fibers. Experimental and computational results show that graphene quantum dots efficiently dock near the N-terminus of the peptide and change the secondary structure of phenol-soluble modulins, which disrupts their fibrillation and represents a strategy for mitigation of bacterial communities.

Antimicrobial Microwebs of DNA-Histone Inspired from Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) are decondensed chromatin networks released by neutrophils that can trap and kill pathogens but can also paradoxically promote biofilms. The mechanism of NET functions remains ambiguous, at least in part, due to their complex and variable compositions. To unravel the antimicrobial performance of NETs, a minimalistic NET-like synthetic structure, termed "microwebs," is produced by the sonochemical complexation of DNA and histone. The prepared microwebs have structural similarity to NETs at the nanometer to micrometer dimensions but with well-defined molecular compositions. Microwebs prepared with different DNA to histone ratios show that microwebs trap pathogenic Escherichia coli in a manner similar to NETs when the zeta potential of the microwebs is positive. The DNA nanofiber networks and the bactericidal histone constituting the microwebs inhibit the growth of E. coli. Moreover, microwebs work synergistically with colistin sulfate, a common and a last-resort antibiotic, by targeting the cell envelope of pathogenic bacteria. The synthesis of microwebs enables mechanistic studies not possible with NETs, and it opens new possibilities for constructing biomimetic bacterial microenvironments to better understand and predict physiological pathogen responses.

Culture-free bacterial detection and identification from blood with rapid, phenotypic, antibiotic susceptibility testing.

The current culture-based approach for the diagnosis of bloodstreams infection is incommensurate with timely treatment and curbing the prevalence of multi-drug resistant organisms (MDROs) due to its long time-to-result. Bloodstream infections typically involve extremely low (e.g., <10 colony-forming unit (CFU)/mL) bacterial concentrations that require a labor-intensive process and as much as 72 hours to yield a diagnosis. Here, we demonstrate a culture-free approach to achieve rapid diagnosis of bloodstream infections. An immuno-detection platform with intrinsic signal current amplification was developed for the ultrasensitive, rapid detection, identification (ID) and antibiotic susceptibility testing (AST) of infections. With its capability of monitoring short-term (1-2 hours) bacterial growth in blood, the platform is able to provide 84-minute simultaneous detection and ID in blood samples below the 10 CFU/mL level and 204-minute AST. The susceptible-intermediate-resistant AST capacity was demonstrated.

Unexpected insights into antibacterial activity of zinc oxide nanoparticles against methicillin resistant Staphylococcus aureus (MRSA).

Zinc oxide nanoparticles (ZnO-NPs) are attractive as broad-spectrum antibiotics, however, their further engineering as antimicrobial agents and clinical translation is impeded by controversial data about their mechanism of activity. It is commonly reported that ZnO-NP's antimicrobial activity is associated with the production of reactive oxygen species (ROS). Here we disprove this concept by comparing the antibacterial potency of ZnO-NPs and their capacity to generate ROS with hydrogen peroxide (H2O2). Then, using gene transcription microarray analysis, we provide evidence for a novel toxicity mechanism. Exposure to ZnO-NPs resulted in over three-log reduction in colonies of methicillin resistant S. aureus with minimal increase in ROS or lipid peroxidation. The amount of ROS required for the same amount of killing by H2O2 was much greater than that generated by ZnO-NPs. In contrast to H2O2, ZnO-NP mediated killing was not mitigated by the antioxidant, N-acetylcysteine. ZnO-NPs caused significant up-regulation of pyrimidine biosynthesis and carbohydrate degradation. Simultaneously, amino acid synthesis in S. aureus was significantly down-regulated indicating a complex mechanism of antimicrobial action involving multiple metabolic pathways. The results of this study point to the importance of specific experimental controls in the interpretation of antimicrobial mechanistic studies and the need for targeted molecular mechanism studies. Continued investigation on the antibacterial mechanisms of biomimetic ZnO-NPs is essential for future clinical translation.

Antibacterial Metal Oxide Nanoparticles: Challenges in Interpreting the Literature.

Metal oxide nanoparticles (MO-NPs) are known to effectively inhibit the growth of a wide range of Gram-positive and Gram-negative bacteria. They have emerged as promising candidates to challenge the rising global issue of antimicrobial resistance. However, a comprehensive understanding of their mechanism of action and identifying the most promising NP materials for future clinical translation remain a major challenge due to variations in NP preparation and testing methods. With various types of MO-NPs being rapidly developed, a robust, standardized, in vitro assessment protocol for evaluating the antibacterial potency and efficiency of these NPs is needed. Calculating the number of NPs that actively interact with each bacterial cell is critical for assessing the dose response for toxicity. Here we discuss methods to evaluate MO-NPs antibacterial efficiency with focus on issues related to NPs in these assays. We also highlight sources of experimental variability including NP preparation, initial bacterial concentration, bacterial strains tested, culture microenvironment, and reported dose.