SaeR as a novel target for antivirulence therapy against Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen responsible for a wide range of clinical infections. SaeRS is one of the two-component systems in S. aureus that modulate multiple virulence factors. Although SaeR is required for S. aureus to develop an infection, inhibitors have not been reported. Using an in vivo knockdown method, we demonstrated that SaeR is targetable for the discovery of antivirulence agent. HR3744 was discovered through a high-throughput screening utilizing a GFP-Lux dual reporter system driven by saeP1 promoter. The antivirulence efficacy of HR3744 was tested using Western blot, Quantitative Polymerase Chain Reaction, leucotoxicity, and haemolysis tests. In electrophoresis mobility shift assay, HR3744 inhibited SaeR-DNA probe binding. WaterLOGSY-NMR test showed HR3744 directly interacted with SaeR's DNA-binding domain. When SaeR was deleted, HR3744 lost its antivirulence property, validating the target specificity. Virtual docking and mutagenesis were used to confirm the target's specificity. When Glu159 was changed to Asn, the bacteria developed resistance to HR3744. A structure-activity relationship study revealed that a molecule with a slight modification did not inhibit SaeR, indicating the selectivity of HR3744. Interestingly, we found that SAV13, an analogue of HR3744, was four times more potent than HR3744 and demonstrated identical antivirulence properties and target specificity. In a mouse bacteraemia model, both HR3744 and SAV13 exhibited in vivo effectiveness. Collectively, we identified the first SaeR inhibitor, which exhibited in vitro and in vivo antivirulence properties, and proved that SaeR could be a novel target for developing antivirulence drugs against S. aureus infections.

Antivirulence Agent as an Adjuvant of ß-Lactam Antibiotics in Treating Staphylococcal Infections.

Staphylococcus aureus can cause a plethora of life-threatening infections. Antibiotics have been extensively used to treat S. aureus infections. However, when antibiotics are used at sub-inhibitory concentrations, especially for ß-lactam antibiotics, they may enhance staphylococcal pathogenicity and exacerbate the infection. The combination of antivirulence agents and antibiotics may be a novel approach to controlling antibiotic-induced S. aureus pathogenicity. We have illustrated that under in vitro conditions, antivirulence agent M21, when administered concurrently with ampicillin, suppressed the expression and production of virulence factors induced by ampicillin. In a mouse peritonitis model, M21 reduced bacterial load irrespective of administration of ampicillin. In a bacteremia model, combinatorial treatment consisting of ampicillin or ceftazidime and M21 increased the survival rate of mice and reduced cytokine abundance, suggesting the suppression of antibiotic-induced virulence by M21. Different from traditional antibiotic adjuvants, an antivirulence agent may not synergistically inhibit bacterial growth in vitro, but effectively benefit the host in vivo. Collectively, our findings from this study demonstrated the benefits of antivirulence-antibiotic combinatorial treatment against S. aureus infections and provide a new perspective on the development of antibiotic adjuvants.

Subinhibitory Concentrations of Antibiotics Exacerbate Staphylococcal Infection by Inducing Bacterial Virulence.

Antibiotics are widely used for the treatment of bacterial infections. However, injudicious use of antibiotics based on an empirical method may lead to the emergence of resistant strains. Despite appropriate administration of antibiotics, their concentrations may remain subinhibitory in the body, due to individual variations in tissue distribution and metabolism rates. This may promote bacterial virulence and complicate the treatment strategies. To investigate whether the administration of certain classes of antibiotics will induce bacterial virulence and worsen the infection under in vivo conditions. Different classes of antibiotics were tested in vitro for their ability to induce virulence in a methicillin-resistant S. aureus strain Mu3 and clinical isolates. Antibiotic-induced pathogenicity was assessed in vivo using mouse peritonitis and bacteremia models. In vitro, ß-lactam antibiotics and tetracyclines induced the expression of multiple surface-associated virulence factors as well as the secretion of toxins. In peritonitis and bacteremia models, mice infected with MRSA and treated with ampicillin, ceftazidime, or tetracycline showed enhanced bacterial pathogenicity. The release of induced virulence factors in vivo was confirmed in a histological examination. Subinhibitory concentrations of antibiotics belonging to ß-lactam and tetracycline aggravated infection by inducing staphylococcal virulence in vivo. Thus, when antibiotics are required, it is preferable to employ combination therapy and to initiate the appropriate treatment plan, following diagnosis. Our findings emphasize the risks associated with antibiotic-based therapy and underline the need for alternative therapeutic options. IMPORTANCE Antibiotics are widely applied to treat infectious diseases. Empirically treatment with incorrect antibiotics, or even correct antibiotics always falls into subinhibitory concentrations, due to dosing, distribution, or secretion. In this study, we have systematically evaluated in vitro virulence induction effect of antibiotics and in vivo exacerbated infection. The major highlight of this work is to prove the ß-lactam and tetracyclines antibiotics exacerbated disease is due to their induction effect on staphylococcal virulence. This phenomenon is common and suggests that if ß-lactam antibiotics remain the first line of defense during empirical therapy, we either need to increase patient reliability or the treatment approach may improve in the future when paired with anti-virulence drugs.

PDGFRb+ mesenchymal cells, but not NG2+ mural cells, contribute to cardiac fat.

Understanding cellular origins of cardiac adipocytes (CAs) can offer important implications for the treatment of fat-associated cardiovascular diseases. Here, we perform lineage tracing studies by using various genetic models and find that cardiac mesenchymal cells (MCs) contribute to CAs in postnatal development and adult homeostasis. Although PDGFRa+ and PDGFRb+ MCs both give rise to intramyocardial adipocytes, PDGFRb+ MCs are demonstrated to be the major source of intramyocardial adipocytes. Moreover, we find that PDGFRb+ cells are heterogenous, as PDGFRb is expressed not only in pericytes and smooth muscle cells (SMCs) but also in some subendocardial, pericapillary, or adventitial PDGFRa+ fibroblasts. Dual-recombinase-mediated intersectional genetic lineage tracing reveals that PDGFRa+PDGFRb+ double-positive periendothelial fibroblasts contribute to intramyocardial adipocytes. In contrast, SMCs and NG2+ pericytes do not contribute to CAs. These in vivo findings demonstrate that PDGFRb+ MCs, but not NG2+ coronary vascular mural cells, are the major source of intramyocardial adipocytes.

In vivo antifungal activity of dipyrithione against Trichophyton rubrum on guinea pig dermatophytosis models.

The treatment of dermatophytosis has improved considerably over the past several decades following the introduction of the oral antifungals such as azoles and amphotericin B. However, these drugs have had limited success because the treated fungi often develop drug resistance, resulting in recurrence when applied in various topical formulations. Thus, there are constant needs for new topical agents that are effective against dermatophytosis. Dipyrithione is an attractive candidate to become an antifungal agent due to its broad spectrum of antimicrobial activities. In this study, we determined that dipyrithione could potently inhibit the growth of Trichophyton rubrum, which is the most common cause of dermatophytosis. The MIC50 value of dipyrithione against T. rubrum was measured as 6.03 µM, as compared with miconazole (MIC50: 1.38 µM). Additionally, the compound caused morphological changes in the fungi, which was examined using the morphological interference assay. The in vivo experiment further revealed that dipyrithione had a healing effect on the skin of guinea pigs infected with T. rubrum. Our studies have demonstrated that dipyrithione had a potent antifungal activity in vitro and in vivo, suggesting that it could be formulated as a potential antifungal lead compound in search for novel therapeutic agents against dermatophytosis.

The π-Tetrel Bond and its Influence on Hydrogen Bonding and Proton Transfer.

The positive region that lies above the plane of F2 TO (T=C and Si) interacts with malondialdehyde (MDA), which contains an intramolecular H-bond. The T atom of F2 TO can lie either in the MDA molecular plane, forming a T⋅⋅⋅O tetrel bond, or F2 TO can stack directly above MDA in a parallel arrangement. The former structure is more stable than the latter, and in either case, F2 SiO engages in a much stronger interaction than does F2 CO, reaching nearly 200 kJ mol-1 . The π-tetrel bond strengthens/weakens the MDA H-bond when the bond is formed to the hydroxyl/carbonyl group of MDA, and causes an accompanying inhibition/promotion of proton transfer within this H-bond; this effect is stronger for F2 SiO. These same aspects can be tuned by substituents placed on any of the C atoms of MDA, although their effects are not fully correlated with the electron-withdrawing or electron-releasing properties of the substituent. A new type of π-π tetrel bond occurs when the π-hole on the T atom of F2 TO approaches the middle carbon atom of MDA from above, and a similar configuration is also found between F2 TO and benzene. Evidence for extensive C⋅⋅⋅C π-π tetrel bonding in crystal materials is presented.

Influence of the protonation of pyridine nitrogen on pnicogen bonding: competition and cooperativity.

Ab initio MP2/aug-cc-pVTZ calculations were performed to investigate the pnicogen-bonded complexes of PyZX2 (Py = pyridine, Z = P and As, X = H and F) and their protonated analogues. The selected Lewis bases include H2S, PH3, H2O, NH3, and H2CO. The relative stability of pnicogen-bonded complexes is related to the nature of PyZX2 and bases. When the nitrogen atom of the pyridine ring in PyZX2 is protonated, the protonated complexes are more stabilized than their neutral counterparts, with the interaction energies increased by 8.5-34.6 kJ mol(-1) and the binding distances shortened by 0.050-0.574 Å. Protonation strengthens the pnicogen bond, from a weak interaction to one of moderate strength. In the neutral complexes of PyZX2 and H2O, the formation of a N···H-O hydrogen bond is favorable compared to the pnicogen bond. Such inclination is more prominent in the complexes of protonated PyZX2 and NH3. In H2O···PyZX2···H2O, pnicogen bonding is strengthened by hydrogen bonding due to positive synergistic effects; however, in NH3···H(+)-PyZX2···NH3, pnicogen bonding is weakened by hydrogen bonding due to negative synergistic effects.