It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media and when growing in vivo during infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C. Interestingly, we found similar results in the BCFA-sufficient parental strain, supported by the fact that the incorporation of C18:1Δ9 into the membrane increased membrane fluidity in both strains. We show that the incorporation of C18:1Δ9 and its elongation product C20:1Δ11 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol and diglycosyldiacylglycerol lipid classes revealed major impacts of C18:1Δ9 and temperature on lipid species. Growth at 12°C in the presence of C18:1Δ9 also led to increased production of the carotenoid pigment staphyloxanthin. The enhancement of growth by C18:1Δ9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1Δ9 and other SCUFAs in various forms.
OBJECTIVE: We show that Staphylococcus aureus can use its known ability to incorporate exogenous fatty acids to enhance its growth at low temperatures. Individual species of phosphatidylglycerols and diglycosyldiacylglycerols bearing one or two degrees of unsaturation derived from the incorporation of C18:1Δ9 at 12°C are described for the first time. In addition, enhanced production of the carotenoid staphyloxanthin occurs at low temperatures. The studies describe a biochemical reality underlying membrane biophysics. This is an example of homeoviscous adaptation to low temperatures utilizing exogenous fatty acids over the regulation of the biosynthesis of endogenous fatty acids. The studies have likely relevance to food safety in that unsaturated fatty acids may enhance the growth of S. aureus in the food environment.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a great health threat to humans. Looking for compounds that could reduce the resistance of S. aureus towards methicillin is an effective way to alleviate the antimicrobial resistance crisis.
RESULTS: Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), Time-killing growth curve, staphyloxanthin and penicillin-binding protein 2a (PBP2a) were detected. A quantitative polymerase chain reaction was used to measure the effect of BBH on the gene transcription profiles of MRSA. The MIC of MRSA-ST59-t437 towards oxacillin was 8 µg/ml, and MBC was 128 µg/ml. After adding a sub-inhibitory concentration of BBH, the MIC and MBC of MRSA-ST59-t478 towards oxacillin went down to 0.125 and 32 µg/ml respectively. The amount of PBP2a and staphyloxanthin were reduced after treatment with BBH. Moreover, the transcription levels of sarA, mecA and fni genes were downregulated.
CONCLUSIONS: It is for the first time reported that BBH could inhibit staphyloxanthin synthesis by inhibiting fni gene. Moreover, fni might be the target gene of sarA, and there might be another regulatory pathway to inhibit staphyloxanthin biosynthesis. BBH could effectively reduce the methicillin resistance of MRSA-ST59-t437 by downregulating fni, sarA and mecA genes.

Background Staphyloxanthin, a carotenoid pigment found in Staphylococcus aureus, serves not only to impart color but also functions as a crucial antioxidant contributing to virulence. Traditionally, milk agar has been employed to enhance staphyloxanthin production, however, no alternative media have been explored. Objectives This study aims to enhance staphyloxanthin production in Staphylococcus aureus using beetroot and carrot formulations. Methods To assess the efficacy of the media, we utilized filter paper, slide spot tests, and microscopic visualization as preliminary identification techniques. Ultraviolet-visible (UV-Vis) spectroscopy and paper chromatography were employed for characterization. Pigment quantification was conducted using microtiter plate assays, and genotypical detection was performed using Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR). Results Beetroot agar exhibited the highest pigment intensity, followed by beetroot with carrot agar, milk agar, carrot agar, and nutrient agar with the lowest intensity. These novel media formulations increased staphyloxanthin synthesis yield, resulting in spectrum shifts ranging from 450 nm (yellow) of milk agar to 470 nm (carrot agar) /480 nm (orange) of beetroot agar. Conclusion This study demonstrates that beetroot and carrot agar can effectively enhance staphyloxanthin production in Staphylococcus aureus. Furthermore, we propose the potential for large-scale cultivation of these pigments in future studies for various industrial applications, such as integration into paints, fabrics, and sunscreen lotions, due to their antioxidant properties.

This work aimed to evaluate the effects of 4 selected essential oils on planktonic cells and microbial biofilms of the Staphylococcus aureus strain (MRSA ATCC 33591). The antibacterial activities of the four essential oils Geranium (Pelargonium graveolens), PgEO, Tea Tree (Melaleuca alternifolia) MaEO, Lemon peel (Citrus limon) ClEO and Peppermint (Mentha piperita) MpEO had MICs ranging from 1.56 to 12.5 µl/ml. The evaluation of the antibiofilm activities of the 4 EOs revealed that they had antiadhesive activities against S. aureus MRSA biofilms; the activity reached 60% (the EO of MpEO peppermint at a concentration of 3.12 µl/ml), and the eradication activity was 80% (the EO of PgEO and MpEO at 3.12 µl/ml). The antibiofilm activity of S. aureus has been explained by the binding of several essential oil bioactive molecules to the SarA protein, the main target protein involved in biofilm formation. The synthesis of the virulence factor staphyloxanthin by S. aureus MRSA ATCC 33591 was significantly inhibited in the presence of PgEO at a concentration of MIC/2. This inhibition was explained by the binding of the main PgEO molecules (β-citronellol and geraniol) to the CrTM protein involved in the staphyloxanthin synthesis pathway. There is evidence that these essential oils could be used as potential anti-virulents to control Staphylococcus biofilm formation.

OBJECTIVE: At present, the inhibition of staphyloxanthin biosynthesis has emerged as a prominent strategy in combating methicillin-resistant Staphylococcus aureus (MRSA) infection. Nonetheless, there remains a limited understanding regarding the bio-structural characteristics of staphyloxanthin biosynthetic enzymes, as well as the molecular mechanisms underlying the interaction between inhibitors and proteins. Furthermore, the functional scope of these inhibitors is relatively narrow.
METHODS: In this study, we address these limitations by harnessing the power of deep learning techniques to construct the 3D structure of diapophytoene desaturase (CrtN). We perform efficient virtual screening and unveil alnustone as a potent inhibitor of CrtN. Further investigations employing molecular modelling, site-directed mutagenesis and biolayer interferometry (BLI) confirmed that alnustone binds to the catalytic active site of CrtN. Transcriptomic analysis reveals that alnustone significantly down-regulates genes associated with staphyloxanthin, histidine and peptidoglycan biosynthesis.
RESULTS: Under the effects of alnustone, MRSA strains exhibit enhanced sensitivity to various antibiotics and the host immune system, accompanied by increased cell membrane permeability. In a mouse model of systemic MRSA infection, the combination of alnustone and antibiotics exhibited a significant therapeutic effect, leading to reduced bacterial colony counts and attenuated pathological damage.
CONCLUSIONS: Alnustone, as a natural inhibitor targeting CrtN, exhibits outstanding antibacterial properties that are single-targeted yet multifunctional. This finding provides a novel strategy and theoretical basis for the development of drugs targeting staphyloxanthin producing bacteria.

Staphylococcus aureus is a highly prevalent and aggressive human pathogen causing a wide range of infections. This study aimed to explore the potential of Patuletin, a rare natural flavone, as an anti-virulence agent against S. aureus. At a sub-inhibitory concentration (1/4 MIC), Patuletin notably reduced biofilm formation by 27 % and 23 %, and decreased staphyloxanthin production by 53 % and 46 % in Staphylococcus aureus isolate SA25923 and clinical isolate SA1, respectively. In order to gain a more comprehensive understanding of the in vitro findings, several in silico analyses were conducted. Initially, a 3D-flexible alignment study demonstrated a favorable structural similarity between Patuletin and B70, the co-crystallized ligand of CrtM, an enzyme that plays a pivotal role in the biosynthesis of staphyloxanthin. Molecular docking highlighted the strong binding of Patuletin to the active site of CrtM, with a high affinity of -20.95 kcal/mol. Subsequent 200 ns molecular dynamics simulations, along with MM-GBSA, ProLIF, PLIP, and PCAT analyses, affirmed the stability of the Patuletin-CrtM complex, revealing no significant changes in CrtM\'s structure upon binding. Key amino acids crucial for binding were also identified. Collectively, this study showcased the effective inhibition of CrtM activity by Patuletin in silico and its attenuation of key virulence factors in vitro, including biofilm formation and staphyloxanthin production. These findings hint at Patuletin\'s potential as a valuable therapeutic agent, especially in combination with antibiotics, to counter antibiotic-resistant Staphylococcus aureus infections.

Staphylococcus aureus is an opportunistic pathogen that is considered a global health threat. This microorganism can adapt to hostile conditions by regulating membrane lipid composition in response to external stress factors such as changes in pH and ionic strength. S. aureus synthesizes and incorporates in its membrane staphyloxanthin, a carotenoid providing protection against oxidative damage and antimicrobial agents. Staphyloxanthin is known to modulate the physical properties of the bacterial membranes due to the rigid diaponeurosporenoic group it contains. In this work, preparative thin layer chromatography and liquid chromatography mass spectrometry were used to purify staphyloxanthin from S. aureus and characterize its structure, identifying C15, C17 and C19 as the main fatty acids in this carotenoid. Changes in the biophysical properties of models of S. aureus membranes containing phosphatidylglycerol, cardiolipin, and staphyloxanthin were evaluated. Infrared spectroscopy shows that staphyloxanthin reduces the liquid-crystalline to gel phase transition temperature in the evaluated model systems. Interestingly, these shifts are not accompanied by strong changes in trans/gauche isomerization, indicating that chain conformation in the liquid-crystalline phase is not altered by staphyloxanthin. In contrast, headgroup spacing, measured by Laurdan GP fluorescence spectroscopy, and lipid core dynamics, measured by DPH fluorescence anisotropy, show significant shifts in the presence of staphyloxanthin. The combined results show that staphyloxanthin reduces lipid core dynamics and headgroup spacing without altering acyl chain conformations, therefore decoupling these normally correlated effects. We propose that the rigid diaponeurosporenoic group in staphyloxanthin and its positioning in the membrane is likely responsible for the results observed.

Bacterial infections are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus cause chronic co-infections, which are more problematic than mono-species infections. Understanding the mechanisms of their interactions is crucial for treating co-infections. Staphyloxanthin (STX), a yellow pigment synthesized by the S. aureus crt operon, promotes S. aureus resistance to oxidative stress and neutrophil-mediated killing. We found that STX production by S. aureus, either as surface-grown macrocolonies or planktonic cultures, was elevated when exposed to the P. aeruginosa exoproduct, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). This was observed with both mucoid and non-mucoid P. aeruginosa strains. The induction phenotype was found in a majority of P. aeruginosa and S. aureus clinical isolates examined. When subjected to hydrogen peroxide or human neutrophils, P. aeruginosa survival was significantly higher when mixed with wild-type (WT) S. aureus, compared to P. aeruginosa alone or with an S. aureus crt mutant deficient in STX production. In a murine wound model, co-infection with WT S. aureus, but not the STX-deficient mutant, enhanced P. aeruginosa burden and disease compared to mono-infection. In conclusion, we identified a role for P. aeruginosa HQNO mediating polymicrobial interactions with S. aureus by inducing STX production, which consequently promotes resistance to the innate immune effectors H2O2 and neutrophils. These results further our understanding of how different bacterial species cooperatively cause co-infections.

Antibiotic-free approaches are more important than ever to address the rapidly growing problem of the antibiotic resistance crisis. The photolysis of the bacterial virulence factor staphyloxanthin using blue light at 460 nm (BL460 nm) has been found to effectively attenuate Staphylococcus aureus to chemical and physical agents. However, phototherapy using BL640 nm still needs to be investigated in detail for its safety in eradicating Staphylococcus aureus in vitro and in vivo. In this study, we employed a 460 nm continuous-wavelength LED source and a low concentration of hydrogen peroxide to treat S. aureus under a culturing condition and a wound abrasion mouse model. The results demonstrated the safety of the combined therapy when it did not modify the bacterial virulence factors or the susceptibility to widely used antibiotics. In addition, the results of the mouse model also showed that the combined therapy was safe to apply to mouse skin since it did not cause adverse skin irritation. More importantly, the therapy can aid in healing S. aureus-infected wounds with an efficacy comparable to that of the topical antibiotic Fucidin. The aforementioned findings indicate that the concurrent application of BL460 nm and hydrogen peroxide can be used safely as an alternative or adjunct to antibiotics in treating S. aureus-infected wounds.

UNASSIGNED: Staphylococcus capitis naturally colonizes the human skin but as an opportunistic pathogen, it can also cause biofilm-associated infections and bloodstream infections in newborns. Previously, we found that two strains from the subspecies S. capitis subsp. capitis produce yellow carotenoids despite the initial species description, reporting this subspecies as non-pigmented. In Staphylococcus aureus, the golden pigment staphyloxanthin is an important virulence factor, protecting cells against reactive oxygen species and modulating membrane fluidity.
UNASSIGNED: In this study, we used two pigmented (DSM 111179 and DSM 113836) and two non-pigmented S. capitis subsp. capitis strains (DSM 20326T and DSM 31028) to identify the pigment, determine conditions under which pigment-production occurs and investigate whether pigmented strains show increased resistance to ROS and temperature stress.
UNASSIGNED: We found that the non-pigmented strains remained colorless regardless of the type of medium, whereas intensity of pigmentation in the two pigmented strains increased under low nutrient conditions and with longer incubation times. We were able to detect and identify staphyloxanthin and its derivates in the two pigmented strains but found that methanol cell extracts from all four strains showed ROS scavenging activity regardless of staphyloxanthin production. Increased survival to cold temperatures (-20°C) was detected in the two pigmented strains only after long-term storage compared to the non-pigmented strains.
UNASSIGNED: The identification of staphyloxanthin in S. capitis is of clinical relevance and could be used, in the same way as in S. aureus, as a possible target for anti-virulence drug design.