A comprehensive synthetic library of poly-N-acetyl glucosamines enabled vaccine against lethal challenges of Staphylococcus aureus.

Poly-ß-(1-6)-N-acetylglucosamine (PNAG) is an important vaccine target, expressed on many pathogens. A critical hurdle in developing PNAG based vaccine is that the impacts of the number and the position of free amine vs N-acetylation on its antigenicity are not well understood. In this work, a divergent strategy is developed to synthesize a comprehensive library of 32 PNAG pentasaccharides. This library enables the identification of PNAG sequences with specific patterns of free amines as epitopes for vaccines against Staphylococcus aureus (S. aureus), an important human pathogen. Active vaccination with the conjugate of discovered PNAG epitope with mutant bacteriophage Qß as a vaccine carrier as well as passive vaccination with diluted rabbit antisera provides mice with near complete protection against infections by S. aureus including methicillin-resistant S. aureus (MRSA). Thus, the comprehensive PNAG pentasaccharide library is an exciting tool to empower the design of next generation vaccines.

Tailored Coatings for Enhanced Performance of Zinc-Magnesium Alloys in Absorbable Implants.

Absorbable metals exhibit potential for next-generation temporary medical implants, dissolving safely in the body during tissue healing and regeneration. Their commercial incorporation could substantially diminish the need for additional surgeries and complications that are tied to permanent devices. Despite extensive research on magnesium (Mg) and iron (Fe), achieving the optimal combination of mechanical properties, biocompatibility, and controlled degradation rate for absorbable implants remains a challenge. Zinc (Zn) and Zn-based alloys emerged as an attractive alternative for absorbable implants, due to favorable combination of in vivo biocompatibility and degradation behavior. Moreover, the development of suitable coatings can enhance their biological characteristics and tailor their degradation process. In this work, four different biodegradable coatings (based on zinc phosphate (ZnP), collagen (Col), and Ag-doped bioactive glass nanoparticles (AgBGNs)) were synthesized by chemical conversion, spin-coating, or a combination of both on Zn-3Mg substrates. This study assessed the impact of the coatings on in vitro degradation behavior, cytocompatibility, and antibacterial activity. The ZnP-coated samples demonstrated controlled weight loss and a decreased corrosion rate over time, maintaining a physiological pH. Extracts from the uncoated, ZnP-coated, and Col-AgBGN-coated samples showed higher cell viability with increasing concentration. Bacterial viability was significantly impaired in all coated samples, particularly in the Col-AgBGN coating. This study showcases the potential of a strategic material-coating combination to effectively tackle multiple challenges encountered in current medical implant technologies by modifying the properties of absorbable metals to tailor patient treatments.

Silver-releasing bioactive glass nanoparticles for infected tissue regeneration.

Bacterial infections represent a formidable challenge, often leaving behind significant bone defects post-debridement and necessitating prolonged antibiotic treatments. The rise of antibiotic-resistant bacterial strains further complicates infection management. Bioactive glass nanoparticles have been presented as a promising substitute for bone defects and as carriers for therapeutic agents against microorganisms. Achieving consistent incorporation of ions into BGNs has proven challenging and restricted to a maximum ion concentration, especially when reducing the particle size. This study presents a notable achievement in the synthesis of 10 nm-sized Ag-doped bioactive glass nanoparticles (Ag-BGNs) using a modified yet straightforward Stöber method. The successful incorporation of essential elements, including P, Ca, Al, and Ag, into the glass structure at the intended concentrations (i.e., CaO wt% above 20 %) was confirmed by EDS, signifying a significant advancement in nanoscale biomaterial engineering. While exhibiting a spherical morphology and moderate dispersity, these nanoparticles tend to form submicron-sized aggregates outside of a solution state. The antibacterial effectiveness against MRSA was established across various experimental conditions, with Ag-BGNs effectively sterilizing planktonic bacteria without the need for antibiotics. Remarkably, when combined with oxacillin or fosfomycin, Ag-BGNs demonstrated a potent synergistic effect, restoring antibacterial capabilities against MRSA strains resistant to these antibiotics when used alone. Ag-BGNs exhibited potential in promoting human mesenchymal stromal cell proliferation, inducing the upregulation of osteoblast gene markers, and significantly contributing to bone regeneration in mice. This innovative synthesis protocol holds substantial promise for the development of biomaterials dedicated to the regeneration of infected tissue.

The glutathione import system satisfies the Staphylococcus aureus nutrient sulfur requirement and promotes interspecies competition.

Sulfur is an indispensable element for bacterial proliferation. Prior studies demonstrated that the human pathogen Staphylococcus aureus utilizes glutathione (GSH) as a source of nutrient sulfur; however, mechanisms of GSH acquisition are not defined. Here, we identify a five-gene locus comprising a putative ABC-transporter and predicted γ-glutamyl transpeptidase (ggt) that promotes S. aureus proliferation in medium supplemented with either reduced or oxidized GSH (GSSG) as the sole source of nutrient sulfur. Based on these phenotypes, we name this transporter operon the glutathione import system (gisABCD). Ggt is encoded within the gisBCD operon, and we show that the enzyme is capable of liberating glutamate using either GSH or GSSG as substrates, demonstrating it is a bona fide γ-glutamyl transpeptidase. We also determine that Ggt is expressed in the cytoplasm, representing only the second example of cytoplasmic Ggt localization, the other being Neisseria meningitidis. Bioinformatic analyses revealed that Staphylococcus species closely related to S. aureus encode GisABCD-Ggt homologs. However, homologous systems were not detected in Staphylococcus epidermidis. Consequently, we establish that GisABCD-Ggt provides a competitive advantage for S. aureus over S. epidermidis in a GSH- and GSSG-dependent manner. Overall, this study describes the discovery of a nutrient sulfur acquisition system in S. aureus that targets GSSG in addition to GSH and promotes competition against other staphylococci commonly associated with the human microbiota.

Crucial Role for Lipoteichoic Acid Assembly in the Metabolic Versatility and Antibiotic Resistance of Staphylococcus aureus.

Staphylococcus aureus is a public health threat due to the prevalence of antibiotic resistance and the capacity of this organism to infect numerous organs in vertebrates. To generate energy needed to proliferate within tissues, S. aureus transitions between aerobic respiration and fermentation. Fermentation results in a distinct colony morphology called the small-colony variant (SCV) due to decreased membrane potential and ATP production. These traits promote increased resistance to aminoglycoside antibiotics. Consequently, SCVs are associated with persistent infections. We hypothesize that dedicated physiological pathways support fermentative growth of S. aureus that represent potential targets for treatment of resistant infections. Lipoteichoic acid (LTA) is an essential component of the Gram-positive cell envelope that functions to maintain ion homeostasis, resist osmotic stress, and regulate autolytic activity. Previous studies revealed that perturbation of LTA reduces viability of metabolically restricted S. aureus, but the mechanism by which LTA supports S. aureus metabolic versatility is unknown. Though LTA is essential, the enzyme that synthesizes the modified lipid anchor, YpfP, is dispensable. However, ypfP mutants produce altered LTA, leading to elongation of the polymer and decreased cell association. We demonstrate that viability of ypfP mutants is significantly reduced upon environmental and genetic induction of fermentation. This anaerobic viability defect correlates with decreased membrane potential and is restored upon cation supplementation. Additionally, ypfP suppressor mutants exhibiting restored anaerobic viability harbor compensatory mutations in the LTA biosynthetic pathway that restore membrane potential. Overall, these results demonstrate that LTA maintains membrane potential during fermentative proliferation and promotes S. aureus metabolic versatility.

Unraveling the mechanisms of inhibition of silver-doped bioactive glass-ceramic particles.

Infections are a major concern in orthopedics. Antibacterial agents such as silver ions are of great interest as broad-spectrum biocides and have been incorporated into bioactive glass-ceramic particles to control the release of ions within a therapeutic concentration and provide tissue regenerative properties. In this work, the antibacterial capabilities of silver-doped bioactive glass (Ag-BG) microparticles were explored to reveal the unedited mechanisms of inhibition against methicillin-resistant Staphylococcus aureus (MRSA). The antibacterial properties were not limited to the delivery of silver ions but rather a combination of antibacterial degradation by-products. For example, nano-sized debris punctured holes in bacteria membranes, osmotic effects, and reactive oxygen species causing oxidative stress and almost 40% of the inhibition. Upon successive Ag-BG treatments, MRSA underwent phenotypic and genomic mutations which were not only insufficient to develop resistance but instead, the clones became more sensitive as the treatment was re-delivered. Additionally, the unprecedented restorative functionality of Ag-BG allowed the effective use of antibiotics that MRSA resists. The synergy mechanism was mainly identified for combinations targeting cell-wall activity and their action was proven in biofilm-like and virulent conditions. Unraveling these mechanisms may offer new insights into how to tailor healthcare materials to prevent or debilitate infections and join the fight against antibiotic resistance in clinical cases.

The 27th Annual Midwest Microbial Pathogenesis Conference in the Age of COVID.

Michigan State University was honored to host in-person the 27th Annual Midwest Microbial Pathogenesis Conference from 17 to 19 September 2021 in East Lansing, MI. Here, we report the precautions that were used to host a safe, in-person meeting during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) pandemic and the research on microbial pathogenesis that was presented at the meeting. One of the most significant impacts of the SARS-CoV2 pandemic on the scientific community is the cancelation of many in-person scientific conferences. This has limited the ability of scientists, especially those who are early in their careers, to present their research and establish scientific networks and collaborations. Using a series of safety precautions, we describe here how we implemented a highly successful in-person meeting of 280 attendees in September 2021. Six of the research projects presented at this meeting are being published together in this issue of the Journal of Bacteriology.

Materials for restoring lost Activity: Old drugs for new bugs.

The escalation of bacterial resistance to conventional medical antibiotics is a serious concern worldwide. Improvements to current therapies are urgently needed to address this problem. The synergistic combination of antibiotics with other agents is a strategic solution to combat multi-drug-resistant bacteria. Although these combinations decrease the required high dosages and therefore, reduce the toxicity of both agents without compromising the bactericidal effect, they cannot stop the development of further resistance. Recent studies have shown certain elements restore the ability of antibiotics to destroy bacteria that have acquired resistance to them. Due to these synergistic activities, organic and inorganic molecules have been investigated with the goal of restoring antibiotics in new approaches that mitigate the risk of expanding resistance. Herein, we summarize recent studies that restore antibiotics once thought to be ineffective, but have returned to our armamentarium through innovative, combinatorial efforts. A special focus is placed on the mechanisms that allow the synergistic combinations to combat bacteria. The promising data that demonstrated restoration of antimicrobials, supports the notion to find more combinations that can combat antibiotic-resistant bacteria.

A Resourceful Race: Bacterial Scavenging of Host Sulfur Metabolism during Colonization.

Sulfur is a requirement for life. Therefore, both the host and colonizing bacteria must regulate sulfur metabolism in a coordinated fashion to meet cellular demands. The host environment is a rich source of organic and inorganic sulfur metabolites that are utilized in critical physiological processes such as redox homeostasis and cellular signaling. As such, modulating enzymes dedicated to sulfur metabolite biosynthesis plays a vital role in host fitness. This is exemplified from a molecular standpoint through layered regulation of this machinery at the transcriptional, translational, and posttranslational levels. With such a diverse metabolite pool available, pathogens and symbionts have evolved multiple mechanisms to exploit sulfur reservoirs to ensure propagation within the host. Indeed, characterization of sulfur transporters has revealed that bacteria employ multiple tactics to acquire ideal sulfur sources, such as cysteine and its derivatives. However, bacteria that employ acquisition strategies targeting multiple sulfur sources complicate in vivo studies that investigate how specific sulfur metabolites support proliferation. Furthermore, regulatory systems controlling the bacterial sulfur regulon are also multifaceted. This too creates an interesting challenge for in vivo work focused on bacterial regulation of sulfur metabolism in response to the host. This review examines the importance of sulfur at the host-bacterium interface and the elegant studies conducted to define this interaction.

Interaction between bacteria and cholesterol crystals: Implications for endocarditis and atherosclerosis.

BACKGROUND: The interaction between pathogenic bacteria and cholesterol crystals (CCs) has not been investigated. However, CCs are found extensively in atherosclerotic plaques and sclerotic cardiac valves. Interactions between pathogenic bacteria and CCs could provide insights into destabilization of atherosclerotic plaques and bacterial adhesion to cardiac valves. METHODS: Staphylococcus aureus and Pseudomonas aeruginosa were used to assess in vitro bacterial adhesion to CCs and proliferation in the presence of CCs compared to plastic microspheres and glass shards as controls. Ex vivo studies evaluated bacterial adhesion to atherosclerotic rabbit arteries compared to normal arteries and human atherosclerotic carotid plaques compared to normal carotid arteries. Scanning electron microscopy (SEM) was used to visualize bacterial adhesion to CCs and confocal microscopy was used to detect cholesterol binding to bacteria grown in the presence or absence of CCs. RESULTS: In vitro, S. aureus and P. aeruginosa displayed significantly greater adhesion, 36% (p<0.0001) and 89% (p<0.0001), respectively, and growth upon exposure to CCs compared to microspheres or glass shards. Rabbit and human atherosclerotic arteries contained significantly greater bacterial burdens compared to controls (4× (p<0.0004); 3× (p<0.019), respectively. SEM demonstrated that bacteria adhered and appeared to degrade CCs. Consistent with this, confocal microscopy indicated increased cholesterol bound to the bacterial cells. CONCLUSIONS: This study is the first to demonstrate an interaction between bacteria and CCs showing that bacteria dissolve and bind to CCs. This interaction helps to elucidate adhesion of bacteria to sclerotic valves and atherosclerotic plaques that may contribute to endocarditis and plaque destabilization.

Quantifying Staphylococcus aureus Membrane Potential Using Flow Cytometry.

Quantifying fluorescent markers in cell populations using flow cytometry has been a powerful technological advance. Fluorescent properties of cyanine dyes coupled with flow cytometry allow investigators to monitor the membrane potential (MP), an important component of the proton motive force (PMF). MP (or ΔΨ) is the electrical potential across the cell membrane. The other component of the PMF is ΔpH, or the difference in interior and exterior proton concentrations. MP plays a critical role in bacterial physiology. In Staphylococcus aureus, MP is required for generation of ATP, regulating autolytic activity, maintaining ion homeostasis, and resistance to some classes of antibiotics. This protocol exploits unique spectral and physical properties of the cyanine-based molecule diethyloxacarbocyanine iodide, or DiOC, and flow cytometry technology to quantify MP in S. aureus. This assay has been used by researchers to define the electron transport chain of S. aureus as well as determine how intrinsic and extrinsic factors affect MP.

Silver-doped bioactive glass particles for in vivo bone tissue regeneration and enhanced methicillin-resistant Staphylococcus aureus (MRSA) inhibition.

Infection is a significant risk factor for failed healing of bone and other tissues. We have developed a sol-gel (solution-gelation) derived bioactive glass doped with silver ions (Ag-BG), tailored to provide non-cytotoxic antibacterial activity while significantly enhancing osteoblast-lineage cell growth in vitro and bone regeneration in vivo. Our objective was to engineer a biomaterial that combats bacterial infection while maintaining the capability to promote bone growth. We observed that Ag-BG inhibits bacterial growth and potentiates the efficacy of conventional antibiotic treatment. Ag-BG microparticles enhance cell proliferation and osteogenic differentiation in human bone marrow stromal cells (hBMSC) in vitro. Moreover, in vivo tests using a calvarial defect model in mice demonstrated that Ag-BG microparticles induce bone regeneration. This novel system with dual biological and advanced antibacterial properties is a promising therapeutic for combating resistant bacteria while triggering new bone formation.

3D printed bioactive and antibacterial silicate glass-ceramic scaffold by fused filament fabrication.

The fused filament fabrication (FFF) technique was applied for the first time to fabricate novel 3D printed silicate bioactive and antibacterial Ag-doped glass-ceramic (Ag-BG) scaffolds. A novel filament consisting primarily of polyolefin and Ag-BG micro-sized particles was developed and its thermal properties characterized by thermogravimetric analysis (TGA) to define the optimum heat treatment with minimal macrostructural deformation during thermal debinding and sintering. Structural characteristics of the Ag-BG scaffolds were evaluated from macro- to nanoscale using microscopic and spectroscopic techniques. The compressive strength of the Ag-BG scaffolds was found to be in the range of cancellous bone. Bioactivity of the 3D printed Ag-BG scaffolds was evaluated in vitro through immersion in simulated body fluid (SBF) and correlated to the formation of an apatite-like phase. Methicillin-resistant Staphylococcus aureus (MRSA) inoculated with the Ag-BG scaffolds exhibited a significant decrease in viability underscoring a potent anti-MRSA effect. This study demonstrates the potential of the FFF technique for the fabrication of bioactive 3D silicate scaffolds with promising characteristics for orthopedic applications.

Sol-Gel-Derived Bioactive and Antibacterial Multi-Component Thin Films by the Spin-Coating Technique.

Although metallic alloys commonly used as prosthetics are durable and mechanically strong, they are often bioinert and lack antibacterial properties. Implementing a bioactive glass material with antibacterial properties as a coating on a metallic substrate provides mechanical strength and bioactivity, as well as antibacterial properties. Many coating methods have been extensively investigated; however, most of them can be expensive, are difficult to scale up, or do not form thin films, which could prevent their translation to clinical practice. The formation of thin films by spin-coating multi-component solution-gelation (sol-gel)-derived glass with antibacterial and bioactive properties has not been achieved previously. For this study, stainless steel 316L substrates were spin-coated with a sol-gel-derived bioactive and antibacterial glass coating in SiO2 58.3-P2O5 7.1-CaO 25.6-Al2O5 5.4-Ag2O 2.1-Na2O 1.5 wt% system (Ag-BG). A sol-gel processing condition that avoids elemental separation upon spin-coating when sintering happens at below the calcination temperature (500 °C) has been developed. This work demonstrates that silver reduction occurs when the concentrations of other cations such as Ca2+ and Na+ in the solution increase. Increasing the stirring duration time prior to the increase of cations, Ag+ ions are stabilized by aluminum tetrahedra, and their reduction to metallic silver does not occur. This study also shows that large dilution ratios (water:tetraethyl orthosilicate) greater than 25:1, accompanied by long stirring durations, produce morphologically homogeneous coatings. Using this strategy, thin films were formed with antibacterial properties against methicillin-resistant Staphylococcus aureus (MRSA) biofilm and biological responses that promote eukaryotic cell adhesion and proliferation. In total, the improved synthesis strategy opens new avenues for the development of novel bioactive and antibacterial thin-film coatings, as it reveals the processing characteristics that control the physicochemical and morphological properties of the formed films.

mSphere of Influence: a Controlled Burn-Pathogen Manipulation of the Dynamic Chemistry That Results from Inflammation.

Neal Hammer works in the field of bacterial pathogenesis, metabolism, and antibiotic resistance. In this mSphere of Influence article, he reflects on how "Gut inflammation provides a respiratory electron acceptor for Salmonella" by Winter and colleagues (S. E. Winter, P. Thiennimitr, M. G. Winter, B. P. Butler, et al., Nature 467:426-429, 2010, https://doi.org/10.1038/nature09415) made an impact on him by demonstrating that Salmonella enterica serotype Typhimurium metabolism is uniquely suited to exploit the chemical by-products that result from the host's inflammatory response.

The Staphylococcus aureus Cystine Transporters TcyABC and TcyP Facilitate Nutrient Sulfur Acquisition during Infection.

Staphylococcus aureus is a significant human pathogen due to its capacity to cause a multitude of diseases. As such, S. aureus efficiently pillages vital nutrients from the host; however, the molecular mechanisms that support sulfur acquisition during infection have not been established. One of the most abundant extracellular sulfur-containing metabolites within the host is cysteine, which acts as the major redox buffer in the blood by transitioning between reduced and oxidized (cystine) forms. We therefore hypothesized that S. aureus acquires host-derived cysteine and cystine as sources of nutrient sulfur during systemic infection. To test this hypothesis, we used the toxic cystine analogue selenocystine to initially characterize S. aureus homologues of the Bacillus subtilis cystine transporters TcyABC and TcyP. We found that genetic inactivation of both TcyA and TcyP induced selenocystine resistance. The double mutant also failed to proliferate in medium supplemented with cystine, cysteine, or N-acetyl cysteine as the sole sulfur source. However, only TcyABC was necessary for proliferation in defined medium containing homocystine as the sulfur source. Using a murine model of systemic infection, we observed tcyP-dependent competitive defects in the liver and heart, indicating that this sulfur acquisition strategy supports proliferation of S. aureus in these organs. Phylogenetic analyses identified TcyP homologues in many pathogenic species, implying that this sulfur procurement strategy is conserved. In total, this study is the first to experimentally validate sulfur acquisition systems in S. aureus and establish their importance during pathogenesis.

Resurrection of antibiotics that methicillin-resistant Staphylococcus aureus resists by silver-doped bioactive glass-ceramic microparticles.

This work describes a novel strategy to combat methicillin-resistant Staphylococcus aureus (MRSA) via the reactivation of inert antibiotics. This strategy exploits a multifunctional system consisting of bioactive glass-ceramic microparticles with antibacterial properties combined with various antibiotics to kill MRSA. Specifically, sol-gel derived silver-doped bioactive glass-ceramic microparticles (Ag-BG) combined with antibiotics that MRSA resists such as oxacillin or fosfomycin, significantly decreased the viability of MRSA. Ag-BG also potentiated the activity of vancomycin on static bacteria, which are typically resistant to this antibiotic. Notably, the synergistic activity is restricted to cell-envelope acting antibiotics as Ag-BG supplementation did not increase the efficacy of gentamicin. Bacteria viability assays and electron microscopy images demonstrate that Ag-BG synergizes to restore antibacterial activity to antibiotics that MRSA resists. The low cytotoxicity previously studied against oral bacteria, together with the known regenerative properties presented in previous studies, and the unique antibacterial properties observed in this work when they are combined with antibiotics, make this multifunctional system a promising approach for healing infected tissue. STATEMENT OF SIGNIFICANCE: This study addresses a very significant issue in the field of antibiotic resistance presenting an innovative way to clear MRSA, by utilizing bioactive glass-ceramic microparticles in combination with antibiotics. Multifunctional glass-ceramic microparticles doped with silver ions (Ag-BG) have been previously observed to exhibit bioactive and antibacterial properties. In this study Ag-BG microparticles were observed to synergize with antibiotics restoring their sensitivity against MRSA. This research work presents a novel approach to resurrect ineffective antibiotics and render them effective against MRSA. Cytotoxicity to eukaryotic cells is not anticipated, as it has been previously observed that these microparticles can trigger hard and soft dental tissue regeneration, when they are utilized in certain concentrations. This study opens a new avenue in the treatment of multidrug resistance bacteria.

Fabrication and multiscale characterization of 3D silver containing bioactive glass-ceramic scaffolds.

In this work, we fabricated and characterized bioactive 3D glass-ceramic scaffolds with inherent antibacterial properties. The sol-gel (solution-gelation) technique and the sacrificial template method were applied for the fabrication of 3D highly porous scaffolds in the 58.6SiO2 - 24.9CaO - 7.2P2O5 - 4.2Al2O3 - 1.5Na2O -1.5K2O - 2.1Ag2O system (Ag-BG). This system is known for its advanced bioactive and antibacterial properties. The fabrication of 3D scaffolds has potential applications that impact tissue engineering. The study of the developed scaffolds from macro-characteristics to nano-, revealed a strong correlation between the macroscale properties such as antibacterial action, bioactivity with the microstructural characteristics such as elemental analysis, crystallinity. Elemental homogeneity, morphological, and microstructural characteristics of the scaffolds were studied by scanning electron microscopy associated with energy dispersive spectroscopy (SEM-EDS), transmittance electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and UV-visible spectroscopy methods. The compressive strength of the 3D scaffolds was measured within the range of values for glass-ceramic scaffolds with similar compositions, porosity, and pore size. The capability of the scaffolds to form an apatite-like phase was tested by immersing the scaffolds in simulated body fluid (SBF) and the antibacterial response against methicillin-resistant Staphylococcus aureus (MRSA) was studied. The formation of an apatite phase was observed after two weeks of immersion in SBF and the anti-MRSA effect occurs after both direct and indirect exposure.

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids.

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

Nutrient sulfur acquisition strategies employed by bacterial pathogens.

Pathogens have evolved elegant mechanisms to acquire essential nutrients from host environments. Sulfur is a requirement for bacterial growth and inorganic and organic sulfur-containing metabolites are abundant within the host-pathogen interface. A growing body of evidence suggests that pathogens are capable of scavenging both types of sulfur sources to fulfill the nutritional requirement. While therapeutic strategies focusing on inhibiting inorganic sulfate assimilation and cysteine synthesis show promise in vitro, in vivo efficacy maybe limited due to the diversity of host-derived sulfur sources and the fact that most pathogens are capable of acquiring multiple sources of sulfur.