Search

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.

Ag-doped sol-gel derived bioactive glass-ceramic particles (Ag-BG) were used to fabricate highly porous scaffolds exhibiting advanced antibacterial properties and formation of an apatite-like layer. The applied heat treatment for the development of the 3D Ag-BG scaffolds was selected after the characterization of the thermal behavior of Ag-BG particles using differential thermal analysis (DTA), thermogravimetric analysis (TGA), and hot stage microscopy (HSM). The structural characteristics of the scaffolds were studied using optical microscopy, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), micro-computerized tomography (Micro-CT), X-ray diffraction (XRD), Fourier-transformed infrared (FTIR), magic angle spinning nuclear magnetic resonance (MAS-NMR), and transmission electron microscopy (TEM) to correlate how the characteristics in the hierarchal structure of the Ag-BG scaffolds affected their antibacterial performance and apatite forming ability. Methicillin-resistant Staphylococcus aureus (MRSA) was used to evaluate the antibacterial response of the Ag-BG scaffolds. The observed characteristics make these Ag-BG scaffolds attractive candidates for biomedical applications.

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.

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.

Sulfur is an indispensable element for proliferation of bacterial pathogens. Prior studies indicated 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 previously uncharacterized five-gene locus comprising a putative ABC-transporter and 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 the Glutathione import system (GisABCD). We confirm that Ggt is capable of cleaving GSH and GSSG γ–bonds and that this process is required for their use as nutrient sulfur sources. Additionally, we find that the enzyme is cell associated. Bioinformatic analyses reveal that only Staphylococcus species closely related to S. aureus encode GisABCD-Ggt homologues. Homologues are not detected in Staphylococcus epidermidis. Consequently, we establish that GisABCD-Ggt provides a competitive advantage for S. aureus over S. epidermidis in a GSH-dependent manner. Overall, this study describes the discovery of a nutrient sulfur acquisition system in S. aureus that targets GSH and promotes competition against other staphylococci commonly associated with the human microbiota.

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.

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., Graphical abstract Image 1, Highlights • Ag-BG scaffolds exhibited anti-MRSA and bioactive behavior. • Processing significantly affected microstructural characteristics of Ag-BG scaffolds. • Nano to macroscale characterization allowed tight correlation of microstructural characteristics to macroscopic properties.

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.

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.

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.

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.

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.

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.

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.

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.

Staphylococcus aureus osteomyelitis is a common and debilitating invasive infection of bone. Treatment of osteomyelitis is confounded by widespread antimicrobial resistance and the propensity of bacteria to trigger pathological changes in bone remodeling that limit antimicrobial penetration to the infectious focus. Adjunctive therapies that limit pathogen-induced bone destruction could therefore limit morbidity and enhance traditional antimicrobial therapies. In this study, we evaluate the efficacy of the U.S. Food and Drug Administration-approved, nonsteroidal anti-inflammatory (NSAID) compound diflunisal in limiting S. aureus cytotoxicity toward skeletal cells and in preventing bone destruction during staphylococcal osteomyelitis. Diflunisal is known to inhibit S. aureus virulence factor production by the accessory gene regulator ( agr ) locus, and we have previously demonstrated that the Agr system plays a substantial role in pathological bone remodeling during staphylococcal osteomyelitis. Consistent with these observations, we find that diflunisal potently inhibits osteoblast cytotoxicity caused by S. aureus secreted toxins independently of effects on bacterial growth. Compared to commonly used NSAIDs, diflunisal is uniquely potent in the inhibition of skeletal cell death in vitro . Moreover, local delivery of diflunisal by means of a drug-eluting, bioresorbable foam significantly limits bone destruction during S. aureus osteomyelitis in vivo . Collectively, these data demonstrate that diflunisal potently inhibits skeletal cell death and bone destruction associated with S. aureus infection and may therefore be a useful adjunctive therapy for osteomyelitis.

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.

Methicillin-resistant Staphylococcus aureus (MRSA) is a threat to global health. Consequently, much effort has focused on the development of new antimicrobials that target novel aspects of S. aureus physiology. Fatty acids are required to maintain cell viability, and bacteria synthesize fatty acids using the type II fatty acid synthesis (FASII) pathway. FASII is significantly different from human fatty acid synthesis, underscoring the therapeutic potential of inhibiting this pathway. However, many Gram-positive pathogens incorporate exogenous fatty acids, bypassing FASII inhibition and leaving the clinical potential of FASII inhibitors uncertain. Importantly, the source(s) of fatty acids available to pathogens within the host environment remains unclear. Fatty acids are transported throughout the body by lipoprotein particles in the form of triglycerides and esterified cholesterol. Thus, lipoproteins, such as low-density lipoprotein (LDL), represent a potentially rich source of exogenous fatty acids for S. aureus during infection. We sought to test the ability of LDLs to serve as a fatty acid source for S. aureus and show that cells cultured in the presence of human LDLs demonstrate increased tolerance to the FASII inhibitor triclosan. Using mass spectrometry, we observed that host-derived fatty acids present in the LDLs are incorporated into the staphylococcal membrane and that tolerance to triclosan is facilitated by the fatty acid kinase A, FakA, and Geh, a triacylglycerol lipase. Finally, we demonstrate that human LDLs support the growth of S. aureus fatty acid auxotrophs. Together, these results suggest that human lipoprotein particles are a viable source of exogenous fatty acids for S. aureus during infection. IMPORTANCE Inhibition of bacterial fatty acid synthesis is a promising approach to combating infections caused by S. aureus and other human pathogens. However, S. aureus incorporates exogenous fatty acids into its phospholipid bilayer. Therefore, the clinical utility of targeting bacterial fatty acid synthesis is debated. Moreover, the fatty acid reservoir(s) exploited by S. aureus is not well understood. Human low-density lipoprotein particles represent a particularly abundant in vivo source of fatty acids and are present in tissues that S. aureus colonizes. Herein, we establish that S. aureus is capable of utilizing the fatty acids present in low-density lipoproteins to bypass both chemical and genetic inhibition of fatty acid synthesis. These findings imply that S. aureus targets LDLs as a source of fatty acids during pathogenesis.

MALDI imaging mass spectrometry is a highly sensitive and selective tool used to visualize biomolecules in tissue. However, identification of detected proteins remains a difficult task. Indirect identifications strategies have been limited by insufficient mass accuracy to confidently link ion images to proteomics data. Here we demonstrate the capabilities of MALDI FTICR MS for imaging intact proteins. MALDI FTICR IMS provides an unprecedented combination of mass resolving power (∼75,000 at m/z 5,000) and accuracy (

Summary Adaptations that enable antimicrobial resistance often pose a fitness cost to the microorganism. Resistant pathogens must therefore overcome such fitness decreases to persist within their hosts. Here we demonstrate that the reduced fitness associated with one resistance-conferring mutation can be offset by community interactions with microorganisms harboring alternative mutations or via interactions with the human microbiota. Mutations that confer antibiotic resistance in the human pathogen Staphylococcus aureus led to decreased fitness, whereas coculture or coinfection of two distinct mutants resulted in collective recovery of fitness comparable to that of wild-type. Such fitness enhancements result from the exchange of metabolites between distinct mutants, leading to enhanced growth, virulence factor production, and pathogenicity. Interspecies fitness enhancements were also identified, as members of the human microbiota can promote growth of antibiotic-resistant S. aureus. Thus, inter- and intraspecies community interactions offset fitness costs and enable S. aureus to develop antibiotic resistance without loss of virulence.

The Gram-positive pathogen Staphylococcus aureus is a leading cause of global morbidity and mortality. Like many multi-drug-resistant organisms, S. aureus contains antibiotic-modifying enzymes that facilitate resistance to a multitude of antimicrobial compounds. FosB is a Mn(2+)-dependent fosfomycin-inactivating enzyme found in S. aureus that catalyzes nucleophilic addition of either l-cysteine (l-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bactericidal properties. The three-dimensional X-ray crystal structure of FosB from S. aureus (FosB(Sa)) has been determined to a resolution of 1.15 Å. Cocrystallization of FosB(Sa) with either l-Cys or BSH results in a disulfide bond between the exogenous thiol and the active site Cys9 of the enzyme. An analysis of the structures suggests that a highly conserved loop region of the FosB enzymes must change conformation to bind fosfomycin. While two crystals of FosB(Sa) contain Zn(2+) in the active site, kinetic analyses of FosB(Sa) indicated that the enzyme is inhibited by Zn(2+) for l-Cys transferase activity and only marginally active for BSH transferase activity. Fosfomycin-treated disk diffusion assays involving S. aureus Newman and the USA300 JE2 methicillin-resistant S. aureus demonstrate a marked increase in the sensitivity of the organism to the antibiotic in either the BSH or FosB null strains, indicating that both are required for survival of the organism in the presence of the antibiotic. This work identifies FosB as a primary fosfomycin-modifying pathway of S. aureus and establishes the enzyme as a potential therapeutic target for increased efficacy of fosfomycin against the pathogen.

SummaryMycobacterium tuberculosis(Mtb) must sense and adapt to immune pressures such as acidic pH and reactive oxygen species (ROS) during pathogenesis. The goal of this study was to isolate compounds that inhibit acidic pH resistance, thus defining virulence pathways that are vulnerable to chemotherapy. Here we report that the acidic pH-dependent compound AC2P36 depletes intracellular thiol pools, sensitizes Mtb to killing by acidic pH, and potentiates the bactericidal activity of isoniazid, clofazimine, and oxidizing agents. We show that the pHdependent activity of AC2P36 is associated with metabolic stress at acidic pH and a pHdependent accumulation of intracellular ROS. Mechanism of action studies show that AC2P36 directly depletes Mtb thiol pools. These data support a model where chemical depletion of Mtb thiol pools at acidic pH enhances sensitivity to oxidative damage, resulting in bacterial killing and potentiation of antibiotics.

Staphylococcus aureus is the leading cause of skin and soft tissue infections, bacteremia, osteomyelitis, and endocarditis in the developed world. The ability of S. aureus to cause substantial disease in distinct host environments is supported by a flexible metabolism that allows this pathogen to overcome challenges unique to each host organ. One feature of staphylococcal metabolic flexibility is a branched aerobic respiratory chain composed of multiple terminal oxidases. Whereas previous biochemical and spectroscopic studies reported the presence of three different respiratory oxygen reductases (o type, bd type, and aa3 type), the genome contains genes encoding only two respiratory oxygen reductases, cydAB and qoxABCD. Previous investigation showed that cydAB and qoxABCD are required to colonize specific host organs, the murine heart and liver, respectively. This work seeks to clarify the relationship between the genetic studies showing the unique roles of the cydAB and qoxABCD in virulence and the respiratory reductases reported in the literature. We establish that QoxABCD is an aa3-type menaquinol oxidase but that this enzyme is promiscuous in that it can assemble as a bo3-type menaquinol oxidase. However, the bo3 form of QoxABCD restricts the carbon sources that can support the growth of S. aureus. In addition, QoxABCD function is supported by a previously uncharacterized protein, which we have named CtaM, that is conserved in aerobically respiring Firmicutes. In total, these studies establish the heme A biosynthesis pathway in S. aureus, determine that QoxABCD is a type aa3 menaquinol oxidase, and reveal CtaM as a new protein required for type aa3 menaquinol oxidase function in multiple bacterial genera., IMPORTANCE Staphylococcus aureus relies upon the function of two terminal oxidases, CydAB and QoxABCD, to aerobically respire and colonize distinct host tissues. Previous biochemical studies support the conclusion that a third terminal oxidase is also present. We establish the components of the S. aureus electron transport chain by determining the heme cofactors that interact with QoxABCD. This insight explains previous observations by revealing that QoxABCD can utilize different heme cofactors and confirms that the electron transport chain of S. aureus is comprised of two terminal menaquinol oxidases. In addition, a newly identified protein, CtaM, is found to be required for the function of QoxABCD. These results provide a more complete assessment of the molecular mechanisms that support staphylococcal respiration.

Summary Staphylococcus aureus is a pathogen that infects multiple anatomical sites leading to a diverse array of diseases. Although vertebrates can restrict the growth of invading pathogens by sequestering iron within haem, S. aureus surmounts this challenge by employing high-affinity haem uptake systems. However, the presence of excess haem is highly toxic, necessitating tight regulation of haem levels. To overcome haem stress, S. aureus expresses the detoxification system HrtAB. In this work, a transposon screen was performed in the background of a haem-susceptible, HrtAB-deficient S. aureus strain to identify the substrate transported by this putative pump and the source of haem toxicity. While a recent report indicates that HrtAB exports haem itself, the haem-resistant mutants uncovered by the transposon selection enabled us to elucidate the cellular factors contributing to haem toxicity. All mutants identified in this screen inactivated the menaquinone (MK) biosynthesis pathway. Deletion of the final steps of this pathway revealed that quinone molecules localizing to the cell membrane potentiate haem-associated superoxide production and subsequent oxidative damage. These data suggest a model in which membrane-associated haem and quinone molecules form a redox cycle that continuously generates semiquinones and reduced haem, both of which react with atmospheric oxygen to produce superoxide.

Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β-strand-loop-β-strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the first, second, or third repeating units were able to convert CsgA into fibers. However, mutants missing either the fourth or fifth repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2, and 4 self-assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units.

The unique redox potential of iron makes it an ideal cofactor in diverse biochemical reactions. Iron is therefore vital for the growth and proliferation of nearly all organisms, including pathogenic bacteria. Vertebrates sequester excess iron within proteins in order to alleviate toxicity and restrict the amount of free iron available for invading pathogens. Restricting the growth of infectious microorganisms by sequestering essential nutrients is referred to as nutritional immunity. In order to circumvent nutritional immunity, bacterial pathogens have evolved elegant systems that allow for the acquisition of iron during infection. The gram-positive extracellular pathogen Staphylococcus aureus is a commensal organism that can cause severe disease when it gains access to underlying tissues. Iron acquisition is required for S. aureus colonization and subsequent pathogenesis. Herein we review the strategies S. aureus employs to obtain iron through the production of siderophores and the consumption of host heme.

Curli are extracellular amyloid fibres produced by Escherichia coli that are critical for biofilm formation and adhesion to biotic and abiotic surfaces. CsgA and CsgB are the major and minor curli subunits, respectively, while CsgE, CsgF and CsgG direct the extracellular localization and assembly of curli subunits into fibres. The secretion and stability of CsgA and CsgB are dependent on the outer membrane lipoprotein CsgG. Here, we identified functional interactions between CsgG and CsgE during curli secretion. We discovered that CsgG overexpression restored curli production to a csgE strain under curli-inducing conditions. In antibiotic sensitivity and protein secretion assays, CsgG expression alone allowed translocation of erythromycin and small periplasmic proteins across the outer membrane. Coexpression of CsgE with CsgG blocked non-specific protein and antibiotic passage across the outer membrane. However, CsgE did not block secretion of proteins containing a 22-amino-acid putative outer membrane secretion signal of CsgA (A22). Finally, using purified proteins, we found that CsgE prohibited the self-assembly of CsgA into amyloid fibres. Collectively, these data indicate that CsgE provides substrate specificity to the curli secretion pore CsgG, and acts directly on the secretion substrate CsgA to prevent premature subunit assembly.

Objectives/Hypothesis: Diagnosis and treatment of gastroesophageal and laryngopharyngeal reflux disease has significantly increased over recent years. The larynx is highly sensitive to the effects of LPRD and is similarly responsive to proton pump inhibitor pharmacotherapy. The hypothesis of the study was that proton pump activity exists in the human larynx and plays a functional role in normal and/or pathological laryngeal tissue. Study Design: Pathological investigation. Methods: Two fresh human cadaveric larynges (one male and one female larynx) were obtained as part of an exempt protocol from the Human Subjects Committee and were formalin fixed and paraffin embedded. Banked human stomach tissue was also obtained for use as comparative positive and negative control specimens. Sections were immuno-stained with monoclonal antibodies reactive with both alpha and beta subunits of the H + /K + -ATPase (proton) pump. Specimens were reviewed for staining pattern and intensity. Results: Stomach parietal cells (known to produce gastric acid) exhibited strongly positive staining for both the alpha and beta subunits of the proton pump. There was no staining in stomach cells that were not morphologically consistent with the parietal celL In the human larynx there were strong focal and identical staining patterns in the serous cells and ducts of the minor seromucinous glands by both alpha and beta monoclonals to the proton pump. There was variable staining in the laryngeal epithelium that was thought to be consistent with artifact staining resulting from tissue processing. Conclusion: The H + /K + -ATPase (proton) pump is present in serous cells and ducts of submucosal glands in the human larynx. Proton pump inhibitor pharmacotherapy may have a site of action in seromucinous glands of the human larynx, with possible relevance for patients treated for chronic laryngitis with or without laryngopharyngeal reflux disease.

Amyloid fibers are filamentous protein structures commonly associated with neurodegenerative diseases. Unlike disease-associated amyloids, which are the products of protein misfolding, Escherichia coli assemble membrane-anchored functional amyloid fibers called curli. Curli fibers are composed of two proteins, CsgA and CsgB. In vivo, the polymerization of the major curli subunit protein, CsgA, is dependent on CsgB-mediated nucleation. The amyloid core of CsgA features five imperfect repeats (R1–R5), and R1 and R5 govern CsgA responsiveness to CsgB nucleation and self-seeding by CsgA fibers. Here, the specificity of bacterial amyloid nucleation was probed, revealing that certain aspartic acid and glycine residues inhibit the intrinsic aggregation propensities and nucleation responsiveness of R2, R3, and R4. These residues function as “gatekeepers” to modulate CsgA polymerization efficiency and potential toxicity. A CsgA molecule lacking gatekeeper residues polymerized in vitro significantly faster than wild-type CsgA and polymerized in vivo in the absence of the nucleation machinery, resulting in mislocalized fibers. This uncontrolled polymerization was associated with cytotoxicity, suggesting that incorrectly regulated CsgA polymerization was detrimental to the cell.

Curli are functional extracellular amyloid fibers produced by uropathogenic Escherichia coli (UPEC) and other Enterobacteriaceae. Ring-fused 2-pyridones, such as FN075 and BibC6, inhibited curli biogenesis in UPEC and prevented the in vitro polymerization of the major curli subunit protein CsgA. The curlicides FN075 and BibC6 share a common chemical lineage with other ring-fused 2-pyridones termed pilicides. Pilicides inhibit the assembly of type 1 pili, which are required for pathogenesis during urinary tract infection. Notably, the curlicides retained pilicide activities and inhibited both curli-dependent and type 1-dependent biofilms. Furthermore, pretreatment of UPEC with FN075 significantly attenuated virulence in a mouse model of urinary tract infection. Curli and type 1 pili exhibited exclusive and independent roles in promoting UPEC biofilms, and curli provided a fitness advantage in vivo. Thus, the ability of FN075 to block the biogenesis of both curli and type 1 pili endows unique anti-biofilm and anti-virulence activities on these compounds.

Amyloid fibers are filamentous proteinaceous structures commonly associated with mammalian neurodegenerative diseases. Nucleation is the rate-limiting step of amyloid propagation, and its nature remains poorly understood. Escherichia coli assembles functional amyloid fibers called curli on the cell surface using an evolved biogenesis machine. In vivo, amyloidogenesis of the major curli subunit protein, CsgA, is dependent on the minor curli subunit protein, CsgB. Here, we directly demonstrated that CsgB+ cells efficiently nucleated purified soluble CsgA into amyloid fibers on the cell surface. CsgA contains five imperfect repeating units that fulfill specific roles in directing amyloid formation. Deletion analysis revealed that the N- and C-terminal most repeating units were required for in vivo amyloid formation. We found that CsgA nucleation specificity is encoded by the N- and C-terminal most repeating units using a blend of genetic, biochemical, and electron microscopic analyses. In addition, we found that the C-terminal most repeat was most aggregation-prone and dramatically contributed to CsgA polymerization in vitro. This work defines the elegant molecular signatures of bacterial amyloid nucleation and polymerization, thereby revealing how nature directs amyloid formation to occur at the correct time and location.

Objectives: Proton pump inhibitors (PPI) used to suppress acid secretion in the stomach are among the most widely prescribed medications. There is emerging evidence of proton secretion elsewhere in the aerodigestive tract, and acidic microenvironments are integral to oral flora such as Lactobacillus. The hypothesis of this study is that the growth rate and morphology of oral Lactobacillus strains are effected by PPIs. Methods: Nineteen different strains of Lactobacilli were inoculated in microtiter plates at pH of 4.5 to 6.5 and exposed to twofold dilutions of pantoprazole at a range of 2.5 mg/mL to 2.5 μg/mL. Bacterial growth was monitored, and the minimum inhibitory concentration (MIC) of the drug was determined for the strains most sensitive to pantoprazole. Results: In the unexposed (control) group, nine Lactobacilli strains were affected by pH changes from 6.5 to 4.5. In the group exposed to pantoprazole, 9 of the 19 Lactobacilli strains were found to have an MIC below 625 μg/mL, with L. plantarum 14917 being the most sensitive (MIC = 20 μg/mL). In some strains, such as L. s. salivarius 11741, Gram-staining revealed conformational changes in the bacteria when grown in the presence of pantoprazole. Conclusion: Growth rates and morphology of oral Lactobacillus are affected by the pH of the environment. Pantoprazole at supraphysiologic doses further affects growth rates and conformation in some strains. Significance: The balance of oral flora and upper digestive tract homeostasis may be affected by unexpected targets of PPI pharmacotherapy, with possible unanticipated consequences.

Curli are functional amyloid fibers assembled by enteric bacteria such as Escherichia coli and Salmonella spp. In E. coli , the polymerization of the major curli fiber subunit protein CsgA into an amyloid fiber depends on the minor curli subunit protein, CsgB. The outer membrane-localized CsgB protein shares ≈30% sequence identity with the amyloid-forming protein CsgA, suggesting that CsgB might also have amyloidogenic properties. Here, we characterized the biochemical properties of CsgB and the molecular basis for CsgB-mediated nucleation of CsgA. Deletion analysis revealed that a CsgB molecule missing 19 amino acids from its C terminus (CsgB trunc ) was not outer membrane-associated, but secreted away from the cell. CsgB trunc was overexpressed and purified from the extracellular milieu of cells as an SDS-soluble, nonaggregated protein. Soluble CsgB trunc assembled into fibers that bound to the amyloid-specific dyes Congo red and thioflavin-T. CsgB trunc fibers were able to seed soluble CsgA polymerization in vitro . CsgB trunc displayed modest nucleator activity in vivo , as demonstrated by its ability to convert extracellular CsgA into an amyloid fiber. Unlike WT CsgB, CsgB trunc was only able to act as a nucleator when cells were genetically manipulated to secrete higher concentrations of CsgA. This work represents a unique demonstration of functional amyloid nucleation and it suggests an elegant model for how E. coli guides efficient amyloid fiber formation on the cell surface.

Few studies have explored the mechanistic basis for the apparent paradoxical effects of nitric oxide and its interrelated redox species (NO(X)) in cancer biology. Our aim was to determine the differential effects of the redox state and kinetics of nitrosative species on the key cancer processes of apoptosis. Therefore, a murine lung adenocarcinoma cell line was exposed to various NO(X) donor compounds differing in redox state and delivery kinetics. DNA strand breaks (DSBs) were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay) and correlated with cell viability by the MTT and soft agar colony assays, while caspase enzymatic activity was measured using an in vitro fluorogenic caspase assay. Finally, cDNA microarrays defined apoptosis-related gene expression alterations resultant from these NO(X) donors. Exogenous NO(X) differentially influences DSBs, and apoptosis-related cell death and expression based on the redox state and kinetics of NO(X) delivery. In our murine lung adenocarcinoma model we have demonstrated differential effects of NO(X) based on the mode of delivery and redox state. These data suggest that the development of NO(X)-based cancer chemotherapy must consider the redox state and kinetics of delivery into their logical design.

Porphyrazines (pzs), or tetraazaporphyrins, can be viewed as porphyrinic macrocycles in which the porphyrin meso (CH) groups are replaced by nitrogen atoms; as such, it can be anticipated that pzs would show similar biocompatibility and biodistribution to those of porphyrins. However, distinctive chemical and physical features of the pzs differentiate them from either the porphyrins or phthalocyanines, in particular making them excellent candidates as optical imaging/therapeutic agents. The novelty of the pzs requires that we first determine how specific structures selectively alter biological function, leading to the development of "rules" that will be used to predict future biologically functional pzs. In the first of these studies, we present here a correlation of pz charge with biocompatibility for a suite of three pzs-neutral, negative, and positive. Confocal fluorescence microscopy and proliferation/viability measurements disclose that the three pzs differ in their toxicity, uptake, and localization in A549 human lung adenocarcinoma cells and WI-38 VA13 normal cells. Interestingly, the negatively charged pz exhibits selective dark toxicity in pulmonary adenocarcinoma cells.

Recent pilot research suggested that the H+/K+-ATPase (proton pump), which is the target of pharmacotherapy for laryngopharyngeal reflux disease (LPRD), is associated with human laryngeal submucosal glands. The hypothesis of this study is that proton pump is expressed in the human larynx, and is not solely associated with the parietal cells of the stomach.Fifteen surgical larynx subjects (27 pathologic specimens) containing seromucinous glands from banked tissue were retrospectively obtained after approval from Human Subjects Committee. Banked human stomach tissue was also obtained for comparative positive and negative controls. Sections were immunostained with two monoclonal antibodies selectively reactive with alpha or beta subunits of the H+/K+-ATPase (proton) pump.In the human larynx, positive staining was seen in 14 of 15 subjects. Twenty-six specimens showed consistent staining in the seromucinous cells and ducts for the alpha subunit, and 23 specimens for the beta subunit. Stomach parietal cells exhibited strongly positive staining for both the alpha and beta subunits of the proton pump. There was no staining in stomach cells that were not morphologically consistent with the parietal cell.The H+/K+-ATPase (proton) pump is present in seromucinous cells and ducts in the human larynx, with some variable expression noted. Proton pump involvement in human laryngeal seromucinous glands may explain heightened laryngeal sensitivity in those patients with chronic laryngitis believed to have LPRD. Also, proton pump inhibitor pharmacotherapy may have a site of action in the human larynx, explaining some of the controversies attributable to LPRD.B-3.

Mycobacterium tuberculosis (Mtb) must sense and adapt to immune pressures such as acidic pH during pathogenesis. The goal of this study was to isolate compounds that inhibit acidic pH resistance, thus defining virulence pathways that are vulnerable to chemotherapy. Here, we report that the compound AC2P36 selectively kills Mtb at acidic pH and potentiates the bactericidal activity of isoniazid, clofazimine, and diamide. We show that AC2P36 activity is associated with thiol stress and causes an enhanced accumulation of intracellular reactive oxygen species at acidic pH. Mechanism of action studies demonstrate that AC2P36 directly depletes Mtb thiol pools, with enhanced depletion of free thiols at acidic pH. These findings support that Mtb is especially vulnerable to thiol stress at acidic pH and that chemical depletion of thiol pools is a promising target to promote Mtb killing and potentiation of antimicrobials.

Staphylococcus aureus is a significant cause of infections worldwide and is able to utilize aerobic respiration, anaerobic respiration, or fermentation as the means by which it generates the energy needed for proliferation. Aerobic respiration is supported by heme-dependent terminal oxidases that catalyze the final step of aerobic respiration, the reduction of O 2 to H 2 O. An inability to respire forces bacteria to generate energy via fermentation, resulting in reduced growth. Elucidating the roles of these energy-generating pathways during colonization of the host could uncover attractive therapeutic targets. Consistent with this idea, we report that inhibiting aerobic respiration by inactivating heme biosynthesis significantly impairs the ability of S. aureus to colonize the host. Two heme-dependent terminal oxidases support aerobic respiration of S. aureus , implying that the staphylococcal respiratory chain is branched. Systemic infection with S. aureus mutants limited to a single terminal oxidase results in an organ-specific colonization defect, resulting in reduced bacterial burdens in either the liver or the heart. Finally, inhibition of aerobic respiration can be achieved by exposing S. aureus to noniron heme analogues. These data provide evidence that aerobic respiration plays a major role in S. aureus colonization of the host and that this energy-generating process is a viable therapeutic target. IMPORTANCE Staphylococcus aureus poses a significant threat to public health as antibiotic-resistant isolates of this pathogen continue to emerge. Our understanding of the energy-generating processes that allow S. aureus to proliferate within the host is incomplete. Host-derived heme is the preferred source of nutrient iron during infection; however, S. aureus can synthesize heme de novo and use it to facilitate aerobic respiration. We demonstrate that S. aureus heme biosynthesis powers a branched aerobic respiratory chain composed of two terminal oxidases. The importance of having two terminal oxidases is demonstrated by the finding that each plays an essential role in colonizing distinct organs during systemic infection. Additionally, this process can be targeted by small-molecule heme analogues called noniron protoporphyrins. This study serves to demonstrate that heme biosynthesis supports two terminal oxidases that are required for aerobic respiration and are also essential for S. aureus pathogenesis.

The chlorite dismutases (C-family proteins) are a widespread family of heme-binding proteins for which chemical and biological roles remain unclear. An association of the gene with heme biosynthesis in Gram-positive bacteria was previously demonstrated by experiments involving introduction of genes from two Gram-positive species into heme biosynthesis mutant strains of Escherichia coli, leading to the gene being renamed hemQ. To assess the gene product's biological role more directly, a Staphylococcus aureus strain with an inactivated hemQ gene was generated and shown to be a slow growing small colony variant under aerobic but not anaerobic conditions. The small colony variant phenotype is rescued by the addition of exogenous heme despite an otherwise wild type heme biosynthetic pathway. The ΔhemQ mutant accumulates coproporphyrin specifically under aerobic conditions. Although its sequence is highly similar to functional chlorite dismutases, the HemQ protein has no steady state reactivity with chlorite, very modest reactivity with H2O2 or peracetic acid, and no observable transient intermediates. HemQ's equilibrium affinity for heme is in the low micromolar range. Holo-HemQ reconstituted with heme exhibits heme lysis after