Inactivation of the Pta-AckA Pathway Causes Cell Death in Staphylococcus aureus

During growth under conditions of glucose and oxygen excess, Staphylococcus aureus predominantly accumulates acetate in the culture medium, suggesting that the phosphotransacetylase-acetate kinase (Pta-AckA) pathway plays a crucial role in bacterial fitness. Previous studies demonstrated that these conditions also induce the S. aureus CidR regulon involved in the control of cell death. Interestingly, the CidR regulon is comprised of only two operons, both encoding pyruvate catabolic enzymes, suggesting an intimate relationship between pyruvate metabolism and cell death. To examine this relationship, we introduced ackA and pta mutations in S. aureus and tested their effects on bacterial growth, carbon and energy metabolism, cid expression, and cell death. Inactivation of the Pta-AckA pathway showed a drastic inhibitory effect on growth and caused accumulation of dead cells in both pta and ackA mutants. Surprisingly, inactivation of the Pta-AckA pathway did not lead to a decrease in the energy status of bacteria, as the intracellular concentrations of ATP, NAD⁺, and NADH were higher in the mutants. However, inactivation of this pathway increased the rate of glucose consumption, led to a metabolic block at the pyruvate node, and enhanced carbon flux through both glycolysis and the tricarboxylic acid (TCA) cycle. Intriguingly, disruption of the Pta-AckA pathway also induced the CidR regulon, suggesting that activation of alternative pyruvate catabolic pathways could be an important survival strategy for the mutants. Collectively, the results of this study demonstrate the indispensable role of the Pta-AckA pathway in S. aureus for maintaining energy and metabolic homeostasis during overflow metabolism.     

Structure of the 70S ribosome from human pathogen Staphylococcus aureus

Comparative structural studies of ribosomes from various organisms keep offering exciting insights on how species-specific or environment-related structural features of ribosomes may impact translation specificity and its regulation. Although the importance of such features may be less obvious within more closely related organisms, their existence could account for vital yet species-specific mechanisms of translation regulation that would involve stalling, cell survival and antibiotic resistance. Here, we present the first full 70S ribosome structure from Staphylococcus aureus, a Gram-positive pathogenic bacterium, solved by cryo-electron microscopy. Comparative analysis with other known bacterial ribosomes pinpoints several unique features specific to S. aureus around a conserved core, at both the protein and the RNA levels. Our work provides the structural basis for the many studies aiming at understanding translation regulation in S. aureus and for designing drugs against this often multi-resistant pathogen.     

A systems biology approach to investigate the antimicrobial activity of oleuropein

Oleuropein and its hydrolysis products are olive phenolic compounds that have antimicrobial effects on a variety of pathogens, with the potential to be utilized in food and pharmaceutical products. While the existing research is mainly focused on individual genes or enzymes that are regulated by oleuropein for antimicrobial activities, little work has been done to integrate intracellular genes, enzymes and metabolic reactions for a systematic investigation of antimicrobial mechanism of oleuropein. In this study, the first genome-scale modeling method was developed to predict the system-level changes of intracellular metabolism triggered by oleuropein in Staphylococcus aureus, a common food-borne pathogen. To simulate the antimicrobial effect, an existing S. aureus genome-scale metabolic model was extended by adding the missing nitric oxide reactions, and exchange rates of potassium, phosphate and glutamate were adjusted in the model as suggested by previous research to mimic the stress imposed by oleuropein on S. aureus. The developed modeling approach was able to match S. aureus growth rates with experimental data for five oleuropein concentrations. The reactions with large flux change were identified and the enzymes of fifteen of these reactions were validated by existing research for their important roles in oleuropein metabolism. When compared with experimental data, the up/down gene regulations of 80% of these enzymes were correctly predicted by our modeling approach. This study indicates that the genome-scale modeling approach provides a promising avenue for revealing the intracellular metabolism of oleuropein antimicrobial properties.     

Characterization of the type 2 NADH:menaquinone oxidoreductases from Staphylococcus aureus and the bactericidal action of phenothiazines

Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple drug resistant bacterial pathogens causing serious infections, many of which are life-threatening. Consequently, new therapeutic targets are required to combat such infections. In the current work, we explore the type 2 Nicotinamide adenine dinucleotide reduced form (NADH) dehydrogenases (NDH-2s) as possible drug targets and look at the effects of phenothiazines, known to inhibit NDH-2 from Mycobacterium tuberculosis. NDH-2s are monotopic membrane proteins that catalyze the transfer of electrons from NADH via flavin adenine dinucleotide (FAD) to the quinone pool. They are required for maintaining the NADH/Nicotinamide adenine dinucleotide (NAD⁺) redox balance and contribute indirectly to the generation of proton motive force. NDH-2s are not present in mammals, but are the only form of respiratory NADH dehydrogenase in several pathogens, including S. aureus. In this work, the two putative ndh genes present in the S. aureus genome were identified, cloned and expressed, and the proteins were purified and characterized. Phenothiazines were shown to inhibit both of the S. aureus NDH-2s with half maximal inhibitory concentration (IC₅₀) values as low as 8 μM. However, evaluating the effects of phenothiazines on whole cells of S. aureus was complicated by the fact that they are also acting as uncouplers of oxidative phosphorylation. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Metabolic Footprint Analysis Uncovers Strain Specific Overflow Metabolism and D-Isoleucine Production of Staphylococcus Aureus COL and HG001

During infection processes, Staphylococcus aureus is able to survive within the host and to invade tissues and cells. For studying the interaction between the pathogenic bacterium and the host cell, the bacterial growth behaviour and its metabolic adaptation to the host cell environment provides first basic information. In the present study, we therefore cultivated S. aureus COL and HG001 in the eukaryotic cell culture medium RPMI 1640 and analyzed the extracellular metabolic uptake and secretion patterns of both commonly used laboratory strains. Extracellular accumulation of D-isoleucine was detected starting during exponential growth of COL and HG001 in RPMI medium. This non-canonical D-amino acid is known to play a regulatory role in adaptation processes. Moreover, individual uptake of glucose, accumulation of acetate, further overflow metabolites, and intermediates of the branched-chain amino acid metabolism constitute unique metabolic footprints. Altogether these time-resolved footprint analyses give first metabolic insights into staphylococcal growth behaviour in a culture medium used for infection related studies.     

Elucidating the inhibition of peptidoglycan biosynthesis in Staphylococcus aureus by albocycline,a macrolactone isolated from Streptomyces maizeus

Antibiotic resistance is a serious threat to global public health, and methicillin-resistant Staphylococcus aureus (MRSA) is a poignant example. The macrolactone natural product albocycline, derived from various Streptomyces strains, was recently identified as a promising antibiotic candidate for the treatment of both MRSA and vancomycin-resistant S. aureus (VRSA), which is another clinically relevant and antibiotic resistant strain. Moreover, it was hypothesized that albocycline’s antimicrobial activity was derived from the inhibition of peptidoglycan (i.e., bacterial cell wall) biosynthesis. Herein, preliminary mechanistic studies are performed to test the hypothesis that albocycline inhibits MurA, the enzyme that catalyzes the first step of peptidoglycan biosynthesis, using a combination of biological assays alongside molecular modeling and simulation studies. Computational modeling suggests albocycline exists as two conformations in solution, and computational docking of these conformations to an ensemble of simulated receptor structures correctly predicted preferential binding to S. aureus MurA—the enzyme that catalyzes the first step of peptidoglycan biosynthesis—over Escherichia coli (E. coli) MurA. Albocycline isolated from the producing organism (Streptomyces maizeus) weakly inhibited S. aureus MurA (IC₅₀ of 480?μM) but did not inhibit E. coli MurA. The antimicrobial activity of albocycline against resistant S. aureus strains was superior to that of vancomycin, preferentially inhibiting Gram-positive organisms. Albocycline was not toxic to human HepG2 cells in MTT assays. While these studies demonstrate that albocycline is a promising lead candidate against resistant S. aureus, taken together they suggest that MurA is not the primary target, and further work is necessary to identify the major biological target.     

Antibacterial activity of hypocrellin A against Staphylococcus aureus

The antibacterial activity and acting mechanism of hypocrellin A (HA) were conducted regarding in vitro activity of HA on Staphylococcus aureus GZ86 by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with HA. The experimental results indicated 1.5?mg/l HA could completely inhibit the growth of 10^7?CFU/ml S. aureus cells in liquid beef extract-peptone medium under a halogen?Ctungsten lamp for 120?min. Meanwhile, HA resulted in the leakage of reducing sugars and proteins and induced the respiratory chain dehydrogenases into inactive state, suggesting that HA were able to destroy the permeability of the bacterial membranes. When the cells of S. aureus were exposed to 2.5?mg/l HA under a halogen?Ctungsten lamp for 120?min, many pits and gaps were observed in bacterial cells by scanning electron microscopy, and the cell wall was fragmentary, indicating the bacterial cells were damaged severely. The experiments strongly confirmed the contribution of multiform reactive oxygen species (ROS) to bactericidal effect. In conclusion, the combined results suggested that ROS may damage the structure of bacterial cell wall and depress the activity of some membranous enzymes, which cause S. aureus bacteria to die eventually.     

A novel series of enoyl reductase inhibitors targeting the ESKAPE pathogens, Staphylococcus aureus and Acinetobacter baumannii

S. aureus and A. baumannii are among the ESKAPE pathogens that are increasingly difficult to treat due to the rise in the number of drug resistant strains. Novel therapeutics targeting these pathogens are much needed. The bacterial enoyl reductase (FabI) is as potentially significant drug target for developing pathogen-specific antibiotics due to the presence of alternate FabI isoforms in many other bacterial species. We report the identification and development of a novel N-carboxy pyrrolidine scaffold targeting FabI in S. aureus and A. baumannii, two pathogens for which FabI essentiality has been established. This scaffold is unrelated to other known antibiotic families, and FabI is not targeted by any currently approved antibiotic. Our data shows that this scaffold displays promising enzyme inhibitory activity against FabI from both S. aureus and A. baumannii, as well as encouraging antibacterial activity in S. aureus. Compounds also display excellent synergy when combined with colistin and tested against A. baumannii. In this combination the MIC of colistin is reduced by 10-fold. Our first generation compound displays promising enzyme inhibition, targets FabI in S. aureus with a favorable selectivity index (ratio of cytotoxicity to MIC), and has excellent synergy with colistin against A. baumannii, including a multidrug resistant strain.     

Identification of the Immunodominant Regions of Staphylococcus aureus Fibronectin-Binding Protein A

Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of human diseases and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. The fibronectin binding protein A (FnBPA) of S. aureus is one of multifunctional ‘microbial surface components recognizing adhesive matrix molecules'' (MSCRAMMs). It is one of the most important adhesin molecules involved in the initial adhesion steps of S. aureus infection. It has been studied as potential vaccine candidates. However, FnBPA is a high-molecular-weight protein of 106 kDa and difficulties in achieving its high-level expression in vitro limit its vaccine application in S. aureus infection diseases control. Therefore, mapping the immunodominant regions of FnBPA is important for developing polyvalent subunit fusion vaccines against S. aureus infections. In the present study, we cloned and expressed the N-terminal and C-terminal of FnBPA. We evaluated the immunogenicity of the two sections of FnBPA and the protective efficacy of the two truncated fragments vaccines in a murine model of systemic S. aureus infection. The results showed recombinant truncated fragment F1_30-500 had a strong immunogenicity property and survival rates significantly increased in the group of mice immunized with F1_30-500 than the control group. We futher identified the immunodominant regions of FnBPA. The mouse antisera reactions suggest that the region covering residues 110 to 263 (F1B_110-263) is highly immunogenic and is the immunodominant regions of FnBPA. Moreover, vaccination with F1B_110-263 can generate partial protection against lethal challenge with two different S. aureus strains and reduced bacterial burdens against non-lethal challenge as well as that immunization with F1_30-500. This information will be important for further developing anti- S. aureus polyvalent subunit fusion vaccines.     

Development of pooled suppression subtractive hybridization to analyze the pangenome of Staphylococcus aureus

We describe the development and application of a Pooled Suppression Subtractive Hybridization (PSSH) method to describe differences between the genomic content of a pool of clinical Staphylococcus aureus isolates and a sequenced reference strain. In comparative bacterial genomics, Suppression Subtractive Hybridization (SSH) is normally utilized to compare genomic features or expression profiles of one strain versus another, which limits its ability to analyze communities of isolates. However, a PSSH approach theoretically enables the user to characterize the entirety of gene content unique to a related group of isolates in a single reaction. These unique fragments may then be linked to individual isolates through standard PCR. This method was applied to examine the genomic diversity found in pools of S.aureus isolates associated with complicated bacteremia infections leading to endocarditis and osteomyelitis. Across four pools of 10 isolates each, four hundred and twenty seven fragments not found in or significantly divergent from the S. aureus NCTC 8325 reference genome were detected. These fragments could be linked to individual strains within its pool by PCR. This is the first use of PSSH to examine the S. aureus pangenome. We propose that PSSH is a powerful tool for researchers interested in rapidly comparing the genomic content of multiple unstudied isolates.     

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system¹. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm^2,3. This pathway has been dissected through numerous in vitro studies^4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models^8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.     

Heterogeneity of Host TLR2 Stimulation by Staphylocoocus aureus Isolates

High lipoprotein expression and potent activation of host Toll-like receptor-2 (TLR2) are characteristic features of the staphylococcal species. Expression of TLR2 in the host is important for clearance of Staphylococcus aureus infection and host survival. Thus, we hypothesized that bacterial regulation of its intrinsic TLR2-stimulatory capacity could represent a means for immune evasion or host adaptation. We, therefore, compared clinical S. aureus isolates in regards to their TLR2 activation potential and assessed the bacterial factors that modulate TLR2-mediated recognition. S. aureus isolates displayed considerable variability in TLR2-activity with low to absent TLR2-activity in 64% of the isolates tested (68/106). Notably, strain-specific TLR2-activity was independent of the strain origin, e.g. no differences were found between strains isolated from respiratory specimen from cystic fibrosis patients or those isolated from invasive disease specimen. TLR2-activity correlated with protein A expression but not with the agr status. Capsule expression and small colony variant formation had a negative impact on TLR2-activity but any disruption of cell wall integrity enhanced TLR2 activation. Altogether, heterogeneity in host TLR2-activity reflects differences in metabolic activity and cell wall synthesis and/or remodeling.     

Evaluation of metabolism of azo dyes and their effects on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> metabolome

Dyes containing one or more azo linkages are widely applied in cosmetics, tattooing, food and drinks, pharmaceuticals, printing inks, plastics, leather, as well as paper industries. Previously we reported that bacteria living on human skin have the ability to reduce some azo dyes to aromatic amines, which raises potential safety concerns regarding human dermal exposure to azo dyes such as those in tattoo ink and cosmetic colorant formulations. To comprehensively investigate azo dye-induced toxicity by skin bacteria activation, it is very critical to understand the mechanism of metabolism of the azo dyes at the systems biology level. In this study, an LC/MS-based metabolomics approach was employed to globally investigate metabolism of azo dyes by Staphylococcus aureus as well as their effects on the metabolome of the bacterium. Growth of S. aureus in the presence of Sudan III or Orange II was not affected during the incubation period. Metabolomics results showed that Sudan III was metabolized to 4-(phenyldiazenyl) aniline (48%), 1-[(4-aminophenyl) diazenyl]-2-naphthol (4%) and eicosenoic acid Sudan III (0.9%). These findings indicated that the azo bond close to naphthalene group of Sudan III was preferentially cleaved compared with the other azo bond. The metabolite from Orange II was identified as 4-aminobenzene sulfonic acid (35%). A much higher amount of Orange II (~90×) was detected in the cell pellets from the active viable cells compared with those from boiled cells incubated with the same concentration of Orange II. This finding suggests that Orange II was primarily transported into the S. aureus cells for metabolism, instead of the theory that the azo dye metabolism occurs extracellularly. In addition, the metabolomics results showed that Sudan III affected energy pathways of the S. aureus cells, while Orange II had less noticeable effects on the cells. In summary, this study provided novel information regarding azo dye metabolism by the skin bacterium, the effects of azo dyes on the bacterial cells and the important role on the toxicity and/or inactivation of these compounds due to microbial metabolism.