Inhibition of Streptococcus pneumoniae autolysins highlight distinct differences between chemical and genetic inactivation

AbstractDespite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive pathogen Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide fgkc as a narrow spectrum bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 7.8 μM. The diamide inhibited detergent-induced autolysis in a concentration dependent manner indicating peptidoglycan degradation as the mode-of-action. Genetic screening of autolysin mutants suggested LytB, an endo-N-acetylglucosaminidase, involved in cell division as the potential target. Surprisingly, biochemical, and phenotypic analysis contradicted the genetic screen results. Phenotypic studies with the Δlytb strain illustrate the difference between genetic and chemical inactivation of autolysins. These findings suggest that meta-phenotypes including autolytic activity, cell morphology, and genetic screening can be the result of the complex interaction of one or more possible pathways that are connected to cell wall metabolism.

Download Full-text

L-Lysine: A Modulator for the Relative Activity of High Molecular Weight (HMW) and Low Molecular Weight(LHW) Urokinase(UK)

10.1055/s-0039-1687222 ◽

1979 ◽

Author(s):

L.L. Shen ◽

W.H. Holleman

Keyword(s):

Molecular Weight ◽

Caproic Acid ◽

Fibrin Clot ◽

Relative Activity ◽

Test Tube ◽

Clot Lysis ◽

Dependent Manner ◽

Plasma Clot ◽

Concentration Dependent Manner ◽

The Difference

L-Lysine(Lys), in a concentration dependent manner, progressively inhibited UK-activated lysis of human plasma clots as demonstrated by Ploug test-tube method and elastometric measurements. Lys was more effective with HMW UK than LMW UK, and the effect of Lys with LMW UK from tissue culture and urine sources was the same. Epsilon amino caproic acid(EACA) and tranexamic acid(TXA) were stronger inhibitors but inhibited HMW and LMW UK-induced lysis to the same degree. Elastometric measurements showed that Lys inhibition was not due to its interference with the initial clotting process nor to the reduction of clot rigidity. Amidolytic assays using chromogenic substrates showed that Lys had no direct effect, on UK, and that Lys enhanced the activation of the native Glu-plasminogen(Pg) by LMW UK, but not the activation by HMW UK. When the substrate was human fibrin clots, Lys enhanced the lysis induced by LMW UK while the lysis induced by HMW UK was inhibited; however, the extent of enhancement and inhibition was limited. We concluded that the mode of Lys action is not identical to that of EACA or TXA, and that the stronger Lys inhibition of plasma clot lysis as compared to fibrin clot lysis is due to the potentiation of plasma fibrinolytic inhibitors by Lys. The difference In effect of Lys on HMW and LMW UK-induced lyels is likely due to a partial conformation change of Glu-Pg molecule upon Lys binding. The relatively moderate interaction of Lys with Glu-Fg results In a mildly modified UK substrate which reacts preferentially with the enzyme smaller in size.

Download Full-text

Comparative Intracellular (THP-1 Macrophage) and Extracellular Activities of β-Lactams, Azithromycin, Gentamicin, and Fluoroquinolones against Listeria monocytogenes at Clinically Relevant Concentrations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.7.2095-2103.2002 ◽

2002 ◽

Vol 46 (7) ◽

pp. 2095-2103 ◽

Cited By ~ 78

Author(s):

Stéphane Carryn ◽

Françoise Van Bambeke ◽

Marie-Paule Mingeot-Leclercq ◽

Paul M. Tulkens

Keyword(s):

Listeria Monocytogenes ◽

Conventional Therapy ◽

The Other ◽

Intracellular Bacteria ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cellular Accumulation ◽

Intracellular Activities ◽

The Difference ◽

In Cells

ABSTRACT The activities of ampicillin, meropenem, azithromycin, gentamicin, ciprofloxacin, and moxifloxacin against intracellular hemolysin-positive Listeria monocytogenes were measured in human THP-1 macrophages and were compared with the extracellular activities observed in broth. All extracellular concentrations were adjusted to explore ranges that are clinically achievable in human serum upon conventional therapy. In broth, ampicillin, meropenem, and azithromycin were only bacteriostatic, whereas gentamicin, ciprofloxacin, and moxifloxacin were strongly bactericidal in a concentration-dependent manner. In cells, ampicillin, meropenem, azithromycin, and ciprofloxacin were slightly bactericidal (0.3- to 0.8-log CFU reductions), moxifloxacin was strongly bactericidal (2.1-log CFU reduction), and gentamicin was virtually inactive. The difference in the efficacies of moxifloxacin and ciprofloxacin in cells did not result from a difference in levels of accumulation in cells (6.96 ± 1.05 versus 7.75 ± 1.03) and was only partially explainable by the difference in the MICs (0.58 ± 0.04 versus 1.40 ± 0.17 mg/liter). Further analysis showed that intracellular moxifloxacin expressed only approximately 1/7 of the activity demonstrated against extracellular bacteria and ciprofloxacin expressed only 1/15 of the activity demonstrated against extracellular bacteria. Gentamicin did not increase the intracellular activities of the other antibiotics tested. The data suggest (i) that moxifloxacin could be of potential interest for eradication of the intracellular forms of L. monocytogenes, (ii) that the cellular accumulation of an antibiotic is not the only determinant of its intracellular activity (for fluoroquinolones, it is actually a self-defeating process as far as activity is concerned), and (iii) that pharmacodynamics (activity-to-concentration relationships) need to be considered for the establishment of efficacy against intracellular bacteria, just as they are for the establishment of efficacy against extracellular infections.

Download Full-text

In vitroandin vivoactivities of a bi-aryl oxazolidinone RBx 11760 against Gram positive bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00453-16 ◽

2016 ◽

pp. AAC.00453-16 ◽

Cited By ~ 4

Author(s):

Tarani Kanta Barman ◽

Manoj Kumar ◽

Tarun Mathur ◽

Tridib Chaira ◽

G. Ramkumar ◽

...

Keyword(s):

Staphylococcus Epidermidis ◽

High Dose ◽

Dependent Manner ◽

Gram Positive ◽

Concentration Dependent Manner ◽

Gram Positive Bacteria ◽

Initial Control ◽

The Difference ◽

Time Kill ◽

Groin Abscess

RBx 11760, a bi-aryl oxazolidinone was investigated for antibacterial activity against Gram positive bacteria. The MIC90(mg/L) of RBx 11760 and linezolid againstStaphylococcus aureuswere: 2 and 4,Staphylococcus epidermidis: 0.5 and 2,Enterococcus: 1 and 4, respectively. Similarly againstStreptococcus pneumoniaeMIC90was: 0.5 and 2, respectively. In time-kill studies, RBx 11760, tedizolid and linezolid exhibited bacteriostatic effect exceptS. pneumoniae. RBx 11760 showed 2-log10kill at 4 X MIC while tedizolid and linezolid showed 2 log10and 1.4-log10kill at 16 X MIC, respectively against MRSA H-29. AgainstS. pneumoniae5051, RBx 11760 showed bactericidal activity with 4.6 log10kill at 4 X MIC compared to 2.42 log10and 1.95 log10kill of tedizolid and linezolid at 16 X MIC. RBx 11760 showed 3 h post antibiotic effects (PAE) at 4 mg/L against MRSA H-29 and linezolid showed same effect at 16mg/L. RBx 11760 inhibited the biofilm production against MRSE ATCC 35984 in concentration dependent manner. In foreign body model, linezolid and rifampicin resulted in no advantage over stasis, while same dose of RBx 11760 demonstrated a significant killing from initial control againstS. aureus(*p<0.05) and MRSE (**p<0.01). The difference in killing was statistically significant for the lower dose of RBx 11760 (*p<0.05) versus high dose of linezolid (nsp>0.05) in groin abscess model. In neutropenic mouse thigh infection, RBx 11760 showed stasis at 20 mg/kg whereas tedizolid showed same effect at 40 mg/kg. These data support the RBx 11760 as a promising investigational candidate.

Download Full-text

Fish-Derived Antifreeze Proteins and Antifreeze Glycoprotein Exhibit a Different Ice-Binding Property with Increasing Concentration

Biomolecules ◽

10.3390/biom10030423 ◽

2020 ◽

Vol 10 (3) ◽

pp. 423 ◽

Cited By ~ 1

Author(s):

Sakae Tsuda ◽

Akari Yamauchi ◽

N. M.-Mofiz Uddin Khan ◽

Tatsuya Arai ◽

Sheikh Mahatabuddin ◽

...

Keyword(s):

Concentration Dependence ◽

Thermal Hysteresis ◽

Antifreeze Proteins ◽

Biochemical Properties ◽

Binding Property ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Ice Binding ◽

Binding Function ◽

The Difference

The concentration of a protein is highly related to its biochemical properties, and is a key determinant for its biotechnological applications. Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are structurally diverse macromolecules that are capable of binding to embryonic ice crystals below 0 °C, making them useful as protectants of ice-block formation. In this study, we examined the maximal solubility of native AFP I–III and AFGP with distilled water, and evaluated concentration dependence of their ice-binding property. Approximately 400 mg/mL (AFP I), 200 mg/mL (AFP II), 100 mg/mL (AFP III), and >1800 mg/mL (AFGP) of the maximal solubility were estimated, and among them AFGP’s solubility is much higher compared with that of ordinary proteins, such as serum albumin (~500 mg/mL). The samples also exhibited unexpectedly high thermal hysteresis values (2–3 °C) at 50–200 mg/mL. Furthermore, the analysis of fluorescence-based ice plane affinity showed that AFP II binds to multiple ice planes in a concentration-dependent manner, for which an oligomerization mechanism was hypothesized. The difference of concentration dependence between AFPs and AFGPs may provide a new clue to help us understand the ice-binding function of these proteins.

Download Full-text

Proteomic Adaptation of Streptococcus pneumoniae to the Human Antimicrobial Peptide LL-37

Microorganisms ◽

10.3390/microorganisms8030413 ◽

2020 ◽

Vol 8 (3) ◽

pp. 413 ◽

Cited By ~ 3

Author(s):

Pierre-Alexander Mücke ◽

Sandra Maaß ◽

Thomas P. Kohler ◽

Sven Hammerschmidt ◽

Dörte Becher

Keyword(s):

Streptococcus Pneumoniae ◽

Drug Targets ◽

High Resolution Mass Spectrometry ◽

Teichoic Acid ◽

Antimicrobial Effect ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Bacterial Membranes ◽

Adaptation Mechanisms ◽

Pore Complexes

Secreted antimicrobial peptides (AMPs) are an important part of the human innate immune system and prevent local and systemic infections by inhibiting bacterial growth in a concentration-dependent manner. In the respiratory tract, the cationic peptide LL-37 is one of the most abundant AMPs and capable of building pore complexes in usually negatively charged bacterial membranes, leading to the destruction of bacteria. However, the adaptation mechanisms of several pathogens to LL-37 are already described and are known to weaken the antimicrobial effect of the AMP, for instance, by repulsion, export or degradation of the peptide. This study examines proteome-wide changes in Streptococcus pneumoniae D39, the leading cause of bacterial pneumonia, in response to physiological concentrations of LL-37 by high-resolution mass spectrometry. Our data indicate that pneumococci may use some of the known adaptation mechanisms to reduce the effect of LL-37 on their physiology, too. Additionally, several proteins seem to be involved in resistance to AMPs which have not been related to this process before, such as the teichoic acid flippase TacF (SPD_1128). Understanding colonization- and infection-relevant adaptations of the pneumococcus to AMPs, especially LL-37, could finally uncover new drug targets to weaken the burden of this widespread pathogen.

Download Full-text

Capsule Independent Antimicrobial Activity Induced by Nanochitosan against Streptococcus pneumoniae

Polymers ◽

10.3390/polym13172924 ◽

2021 ◽

Vol 13 (17) ◽

pp. 2924

Author(s):

Fulwah Y. Alqahtani ◽

Fadilah S. Aleanizy ◽

Eram El Tahir ◽

Hessa Alowais ◽

Assalh Binkelaib ◽

...

Keyword(s):

Antimicrobial Activity ◽

Streptococcus Pneumoniae ◽

Polymeric Nanoparticles ◽

Wide Spectrum ◽

A549 Cells ◽

Community Acquired Pneumonia ◽

High Morbidity ◽

Dependent Manner ◽

Hemolysis Assay ◽

Concentration Dependent Manner

Background: Streptococcus pneumoniae remains a major cause of community-acquired pneumonia, meningitis, and other diseases, contributing significantly to high morbidity and mortality worldwide. Although it responds to antibiotics, their use is becoming limited due to the rise in antibiotic resistance, which necessitates the development of new therapeutics. Nanotechnology is used to counteract antimicrobial resistance. In this regard, polymeric nanoparticles (NPs) made of natural, biodegradable, biocompatible, and cationic polymers such as Chitosan (CNPs) exhibit wide-spectrum antimicrobial activity. Therefore, this study aimed to prepare CNPs, characterize their physiochemical characteristics: particle size (PZ), polydispersity index (PDI), and zeta potential (ZP), and investigate their antimicrobial activity against Streptococcus pneumoniae TIGR4 (virulent serotype 4) and its capsular mutant (∆cps). Methods: CNPs were prepared at 1, 2.5, and 5 mg/mL concentrations using the ion gelation method. Then, PZ, PDI, and ZP were characterized using a Zetasizer. Transmission electron microscopy (TEM) was used to visualize the CNP’s morphology. Broth and agar dilution methods were used to assess their antimicrobial activity. Cytotoxicity of prepared NPs on A549 cells and their effect on pneumococcal hemolysis were also investigated. Results: Spherical CNPs were produced with PZ ranging from 133.3 nm ± 0.57 to 423 nm ± 12.93 PDI < 0.35, and ZP from 19 ± 0.115 to 27 ± 0.819. The prepared CNPs exhibited antibacterial activity against TIGR4 and its capsule mutant with a minimum inhibitory concentration (MIC90) of 0.5 to 2.5 mg/mL in a non-acidic environment. The hemolysis assay results revealed that CNPs reduced bacterial hemolysis in a concentration-dependent manner. Their mammalian cytotoxicity results indicated that CNPs formed from low concentrations of Chitosan (Cs) were cytocompatible. Conclusion: Nanochitosan particles showed anti-pneumococcal activity regardless of the presence of capsules. They resulted in a concentration-dependent reduction in bacterial hemolysis and were cytocompatible at a lower concentration of Cs. These findings highlight the potential of CNPs in the treatment of pneumococcal diseases.

Download Full-text

The Involvement of HAb18G/CD147 in Regulation of Store-operated Calcium Entry and Metastasis of Human Hepatoma Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m108291200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 46870-46877 ◽

Cited By ~ 90

Author(s):

Jian Li Jiang ◽

Qing Zhou ◽

Mei Kuen Yu ◽

Lok Sze Ho ◽

Zhi Nan Chen ◽

...

Keyword(s):

Inhibitory Effect ◽

Hepatoma Cells ◽

Inhibitory Response ◽

Dependent Manner ◽

Human Hepatoma ◽

Human Hepatoma Cells ◽

Concentration Dependent Manner ◽

No Donor ◽

Store Operated Calcium Entry ◽

The Difference

The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca2+mobilization and metastatic process of human hepatoma cells. HAb18G/CD147 cDNA was transfected into human 7721 hepatoma cells to obtain a cell line stably expressing HAb18G/CD147, T7721, as demonstrated by Northern blot and immunocytochemical studies. 8-Bromo-cGMP (cGMP) inhibited the thapsigargin-induced Ca2+entry in a concentration-dependent manner in 7721 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1 μm). However, expression of HAb18G/CD147 in T7721 cells decreased the inhibitory response to cGMP. A similar concentration-dependent inhibitory effect on the Ca2+entry was observed in 7721 cells in response to a NO donor, (±)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca2+entry was significantly reduced in HAb18G/CD147-expressing T7721 cells, indicating a role for HAb18G/CD147 in NO/cGMP-regulated Ca2+entry. Experiments investigating metastatic potentials demonstrated that HAb18G/CD147-expressing T7721 cells attached to the Matrigel-coated culture plates and invaded through Matrigel-coated permeable filters at the rate significantly greater than that observed in 7721 cells. Both the attachment and invasion rates could be suppressed by SNAP, and the inhibitory effect of SNAP could be reversed by NO inhibitor,NG-nitro-l-arginine methyl ester. The sensitivity of the attachment and invasion rates to cGMP was significantly reduced in T7721 cells as compared with 7721 cells when cells were pretreated with thapsigargin. The difference in the sensitivity between the two cells could be abolished by a Ca2+channel blocker, Ni2+(3 mm). These results suggest that HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca2+entry by NO/cGMP.

Download Full-text

Evidence for the presence of essential histidine and cysteine residues in platelet cGMP-inhibited phosphodiesterase

Biochemical Journal ◽

10.1042/bj3170495 ◽

1996 ◽

Vol 317 (2) ◽

pp. 495-501 ◽

Cited By ~ 13

Author(s):

Faika A. GHAZALEH ◽

George A. OMBURO ◽

Robert W. COLMAN

Keyword(s):

Active Site ◽

Difference Spectrum ◽

Cysteine Residue ◽

Enzymic Activity ◽

Cysteine Residues ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Histidine Residues ◽

The Difference ◽

Rate Profile

cAMP is a major regulator of platelet function. cGMP-inhibited phosphodiesterase (cGI-PDE) is the predominant platelet enzyme hydrolysing cAMP. The pH–rate profile plot for this enzyme yields pKa values of 6.5 and 9.0, consistent with histidine and cysteine residues respectively. Diethyl pyrocarbonate (DEP) inactivates cGI-PDE in a time- and concentration-dependent manner, and this effect was rapidly reversed by hydroxylamine. It was estimated that 2 mol of histidine residues per mol of enzyme were responsible for the loss of catalytic activity, as deduced from the correlation of the difference spectrum at 240 nm of the DEP-modified cGI-PDE with the enzymic activity. N-Ethylmaleimide (NEM) and 5,5´-dithiobis-(2-nitrobenzoic acid) (DTNB) inactivate cGI-PDE in a time- and concentration-dependent manner, suggesting the selective modification of a cysteine residue. AMP protects the enzyme against DEP, NEM and DTNB, suggesting the presence of histidine and cysteine residues at the active site of cGI-PDE. [14C]DEP incorporation in the presence of AMP or cGMP indicates the protection of two histidine residues by each nucleotide. These residues are different for each agent, since the combination of AMP and cGMP protects four histidine residues. [3H]NEM incorporation showed that 1 mol of cysteine per mol of cGI-PDE was protected by AMP, but not by cGMP. We conclude that cGI-PDE possesses two essential histidine residues for activity, two additional histidines for cGMP inhibition, and one cysteine residue at the active site.

Download Full-text

Application of multi-omics technology for the elucidation of anti-pneumococcal activity of 3-acyl-2-phenylamino-1,4-dihydroquinolin-4-one (APDQ) derivative against Streptococcus pneumoniae

Scientific Reports ◽

10.1038/s41598-020-77694-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Sang-Yeop Lee ◽

Hayoung Lee ◽

Sung Ho Yun ◽

Sangmi Jun ◽

Yujeong Lee ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Underlying Mechanism ◽

Differentially Expressed ◽

Dependent Manner ◽

Rna Seq ◽

Concentration Dependent Manner ◽

Peptidoglycan Biosynthesis ◽

Omics Analysis ◽

Electron Microscopy Analysis ◽

Microscopy Analysis

AbstractStreptococcus pneumoniae is one of Gram-positive pathogen that causes invasive pneumococcal disease. Nowadays, many S. pneumoniae strains are resistant to commonly used antibiotics such as β-lactams and macrolides. 3-Acyl-2-phenylamino-1,4-dihydroquinolin-4-one (APDQ) derivatives are known as novel chemicals having anti-pneumococcal activity against S. pneumoniae. The underlying mechanism of the anti-pneumococcal activity of this inhibitor remains unknown. Therefore, we tried to find the anti-pneumococcal mechanism of APDQ230122, one of the APDQ derivatives active against S. pneumoniae. We performed transcriptomic analysis (RNA-Seq) and proteomic analysis (LC–MS/MS analysis) to get differentially expressed genes (DEG) and differentially expressed proteins (DEP) of S. pneumoniae 521 treated with sub-inhibitory concentrations of APDQ230122 and elucidated the comprehensive expression changes of genes and proteins using multi-omics analysis. As a result, genes or proteins of peptidoglycan biosynthesis and DNA replication were significantly down-regulated. Electron microscopy analysis revealed that the structure of peptidoglycan was damaged by APDQ230122 in a chemical concentration-dependent manner. Therefore, we suggest peptidoglycan biosynthesis is a major target of APDQ230122. Multi-omics analysis can provide us useful information to elucidate anti-pneumococcal activity of APDQ230122.

Download Full-text

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000195 ◽

2014 ◽

Vol 84 (1-2) ◽

pp. 79-91 ◽

Cited By ~ 7

Author(s):

Amin F. Majdalawieh ◽

Hyo-Sung Ro

Keyword(s):

Cholesterol Efflux ◽

Transcriptional Activity ◽

Molecular Mechanisms ◽

Foam Cell ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Foam Cell Formation ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamins anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

Download Full-text