Quercetin Is a Novel Inhibitor of the Choline Kinase of Streptococcus pneumoniae.

The effectiveness of current antimicrobial methods for addressing for food-borne Gram-positive pathogens has dropped with the emergence of resistant strains. Consequently, new methods for addressing Gram-positive strains have to be developed continuously. This includes establishing novel targets for antimicrobial discovery efforts. Eukaryotic choline kinases have been highly developed as drug targets for the treatment of cancer, rheumatoid arthritis, malaria and many other conditions and diseases. Recently, choline kinase (ChoK) has been proposed as a drug target for Gram-positive species generally. The aim of this work was to discover novel, natural sources of inhibitors for bacterial ChoK from tea extracts. We report the first natural bacterial ChoK inhibitor with antimicrobial activity against Streptococcus pneumoniae: quercetin.

Inhibition of Streptococcus pneumoniae growth by masarimycin.

Despite 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 bacterium Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide masarimycin as a bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 8 µM. The diamide inhibited detergent-induced autolysis in a concentration-dependent manner, indicating perturbation of peptidoglycan degradation as the mode-of-action. Cell based screening of masarimycin against a panel of autolysin mutants, identified a higher MIC against a ΔlytB strain lacking an endo-N-acetylglucosaminidase involved in cell division. Subsequent biochemical and phenotypic analyses suggested that the higher MIC was due to an indirect interaction with LytB. Further analysis of changes to the cell surface in masarimycin treated cells identified the overexpression of several moonlighting proteins, including elongation factor Tu which is implicated in regulating cell shape. Checkerboard assays using masarimycin in concert with additional antibiotics identified an antagonistic relationship with the cell wall targeting antibiotic fosfomycin, which further supports a cell wall mode-of-action.

Lactic Acid Bacteria as Antimicrobial Agents: Food Safety and Microbial Food Spoilage Prevention.

In the wake of continual foodborne disease outbreaks in recent years, it is critical to focus on strategies that protect public health and reduce the incidence of foodborne pathogens and spoilage microorganisms. Currently, there are limitations associated with conventional microbial control methods, such as the use of chemical preservatives and heat treatments. For example, such conventional treatments adversely impact the sensorial properties of food, resulting in undesirable organoleptic characteristics. Moreover, the growing consumer advocacy for safe and healthy food products, and the resultant paradigm shift toward clean labels, have caused an increased interest in natural and effective antimicrobial alternatives. For instance, natural antimicrobial elements synthesized by lactic acid bacteria (LAB) are generally inhibitory to pathogens and significantly impede the action of food spoilage organisms. Bacteriocins and other LAB metabolites have been commercially exploited for their antimicrobial properties and used in many applications in the dairy industry to prevent the growth of undesirable microorganisms. In this review, we summarized the natural antimicrobial compounds produced by LAB, with a specific focus on the mechanisms of action and applications for microbial food spoilage prevention and disease control. In addition, we provide support in the review for our recommendation for the application of LAB as a potential alternative antimicrobial strategy for addressing the challenges posed by antibiotic resistance among pathogens.

Teaching Aspects of Antibiotics and Antimicrobials to the Food Science Student through a Combination Wet Lab and In Silico Activity.

To prevent the growth of food pathogens and food spoilage organisms, antimicrobials are used to disinfect surfaces or are added to food. In light of the important role that antibiotics and antimicrobials play in food safety, food science students need a deep understanding of how these chemicals function. We describe here a combined wet lab and in silico laboratory experience designed to help students visualize two biochemical concepts explaining antibiotic activity: (i) bacteriolytic versus bacteriostatic activity and (ii) competitive versus noncompetitive inhibition. This laboratory experience was implemented for students enrolled in the Introduction to Food Science course in Family and Consumer Sciences at North Carolina A&T State University.

Autolysis and Cell Death Is Affected by pH in L. reuteri DSM 20016 Cells.

A key obstacle to the successful delivery of a probiotic to the consumer is maintaining viability of the live cells during storage, a challenge for the beneficial Lactibacillus reuteri. Three processes play a role in the reduction of viability: autolysis, cell death, and cell weakening. Using a phosphate induction model of autolysis, the initial aim of this project was to discover novel molecular determinants of autolysis in L. reuteri, with the long -term goal of elucidating new strategies for increasing viability. We employed a 2D Native/SDS-Page method to monitor changes in protein expression over time; however, the result was that excess phosphate did not induce noticeable changes in expression patterns. On the other hand, we found that pH affects both the rate of total viability and autolysis, as seen with other species of LAB. In addition, we found that the phosphate model of autolysis may not be sufficient to explain how autolysis is triggered in L. reuteri. Two parameters appear to modulate the pH in media containing L. reuteri cells: overall buffering capacity and the presence of a carbon source. Ultimately, phosphate sources appear to facilitate autolysis by maintaining pH in the media via a higher buffering capacity. In addition, the alkaline sugar free almond drink appears to be a promising possible preservative for L. reuteri.

Choline Kinase: An Unexpected Journey for a Precision Medicine Strategy in Human Diseases.

Choline kinase (ChoK) is a cytosolic enzyme that catalyzes the phosphorylation of choline to form phosphorylcholine (PCho) in the presence of ATP and magnesium. ChoK is required for the synthesis of key membrane phospholipids and is involved in malignant transformation in a large variety of human tumours. Active compounds against ChoK have been identified and proposed as antitumor agents. The ChoK inhibitory and antiproliferative activities of symmetrical bispyridinium and bisquinolinium compounds have been defined using quantitative structure-activity relationships (QSARs) and structural parameters. The design strategy followed in the development of the most active molecules is presented. The selective anticancer activity of these structures is also described. One promising anticancer compound has even entered clinical trials. Recently, ChoKα inhibitors have also been proposed as a novel therapeutic approach against parasites, rheumatoid arthritis, inflammatory processes, and pathogenic bacteria. The evidence for ChoKα as a novel drug target for approaches in precision medicine is discussed.

Tackling Airborne Virus Threats in the Food Industry: A Proactive Approach.

The current SARS-COVID-19 crisis has demonstrated the dangers that airborne virus (AV) pandemics pose to the health of all workers (particularly in the meat processing industry), the economic health of the food industry, and food security. The impact that the current pandemic has had on the food industry points to the need for a proactive rather than reactive approach towards preventing future AV outbreaks. Such a proactive approach should be based on empirical assessments of current AV food safety practices and the development of more robust practices tailored to the culture and needs of the food industry. Moreover, a proactive approach is necessary in order to better prepare the food industry for future AV outbreaks, protect the health of workers, reduce disparities in AV occupational health risks, and enhance the safety of the food supply chain. The aim of this review is to make the case for a new food safety research paradigm that incorporates the intensive study of airborne viruses under conditions that simulate food industry work environments.

Identification and validation of novel and more effective choline kinase inhibitors against Streptococcus pneumoniae.

Streptococcus pneumoniae choline kinase (sChoK) has previously been proposed as a drug target, yet the effectiveness of the first and only known inhibitor of sChoK, HC-3, is in the millimolar range. The aim of this study was thus to further validate sChoK as a potential therapeutic target by discovering more powerful sChoK inhibitors. LDH/PK and colorimetric enzymatic assays revealed two promising sChoK inhibitor leads RSM-932A and MN58b that were discovered with IC50 of 0.5 and 150 µM, respectively, and were shown to be 2-4 magnitudes more potent than the previously discovered inhibitor HC-3. Culture assays showed that the minimum inhibitory concentration (MIC) of RSM-932A and MN58b for S. pneumoniae was 0.4 µM and 10 µM, respectively, and the minimum lethal concentration (MLC) was 1.6 µM and 20 µM, respectively. Western blot monitoring of teichoic acid production revealed differential patterns in response to each inhibitor. In addition, both inhibitors possessed a bacteriostatic mechanism of action, and neither interfered with the autolytic effects of vancomycin. Cells treated with MN58b but not RSM-932A were more sensitive to a phosphate induced autolysis with respect to the untreated cells. SEM studies revealed that MN58b distorted the cell wall, a result consistent with the apparent teichoic acid changes. Two novel and more highly potent putative inhibitors of sChoK, MN58b and RSM-932A, were characterized in this study. However, the effects of sChoK inhibitors can vary at the cellular level. sChoK inhibition is a promising avenue to follow in the development of therapeutics for treatment of S. pneumoniae.

A modified reinforced clostridial medium for the isolation and enumeration of Lactobacillus delbrueckii ssp. bulgaricus in a mixed culture.

In this study, we modified reinforced clostridial medium (RCM) to selectively enumerate and isolate Lactobacillus delbrueckii ssp. bulgaricus, a probiotic and important starter culture in the dairy industry. The disparity in the reported carbohydrate fermentation pattern of L. delbrueckii ssp. bulgaricus was used to develop a growth medium not only selective for L. delbrueckii ssp. bulgaricus but significantly inhibitory to the growth of other lactic acid bacteria. A recently modified RCM (mRCM) was optimized for this study by the addition of 0.5% fructose, 0.5% dextrose, 1% maltose, and 0.25% sodium pyruvate while replacing lactose as a carbohydrate source. The cell recovery and bacterial counts of L. delbrueckii ssp. bulgaricus in tested products (pure L. delbrueckii ssp. bulgaricus strains, starter culture, probiotic supplements, and yogurt) using our mRCM with sodium pyruvate (mRCM-PYR) were significantly higher than in the recently modified RCM and the common de Man, Rogosa, and Sharpe (MRS) culture medium. The growth of other lactic acid bacteria (Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus reuteri) and Bifidobacteria was retarded in this modified medium compared with their growth in MRS and mRCM. This result is a significant improvement in the enumeration and differentiation of L. delbrueckii ssp. bulgaricus in mRCM-PYR compared with the results in MRS and mRCM where the high background growth of similar species interferes with the accuracy of bacterial population counts. Our results thus suggest that mRCM-PYR could be recommended as a reliable alternative growth medium for the selective enumeration and isolation of L. delbrueckii ssp. bulgaricus in a mixed culture.

Amoxicillin Inactivation by Thiol-Catalyzed Cyclization Reduces Protein Haptenation and Antibacterial Potency.

Serum and cellular proteins are targets for the formation of adducts with the ß-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that several thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. Increased diketopiperazine conversion is also observed with human serum albumin but not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other ß-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions.

A comparative study of extraction techniques for maximum recovery of ß-galactosidase from the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus.

The study reported in this research communication evaluates the chemical (solvents) and mechanical (sonication, bead-beater) extraction methods to determine the maximum recovery of ß-galactosidase from L. bulgaricus spp. Among all extraction techniques, sonication-assisted extraction yielded the highest amounts of enzyme activity (between 1892-2156 Miller Units) in cell-free extract (supernatant). Interestingly, solvent extracted enzyme activities were found to be very low (between 83-153 Miller Units) in supernatant. SDS-polyacrylamide gel electrophoresis and the total protein determination showed that mechanical methods can completely lyse the cells. Our results thus demonstrated that the mechanical extraction method of sonication is the best one for recovering the maximum amount of lactase from L. bulgaricus strains.

Choline Kinase Emerges as a Promising Drug Target in Gram-Positive Bacteria.

Both nosocomial pathogens, such as Streptococcus pneumoniae and Haemophilus influenzae and food-borne pathogens, such as Bacillus cereus and Clostridium perfringens are known to be detrimental to human and animal health. The effectiveness of currently used treatments for these pathogens becomes limited as resistant strains emerge. Therefore, new methods for eliminating bacterial pathogens must be developed continuously. This includes establishing novel targets to which drug discovery efforts could be focused. A promising method for discovering new drug targets in prokaryotes is to take advantage of the information available regarding the enzymatic pathways that have been established as drug targets in eukaryotic systems and explore the analogous pathways found in bacterial systems. This is an efficient strategy because the same inhibitors developed at considerable expense to block these pathways in eukaryotic systems could also be employed in prokaryotes. Drugs that are used to prevent diseases involving eukaryotic cells could be repurposed as antibiotics and antimicrobials for the control of bacteria pathogens. This strategy could be pursued whenever the primary and tertiary structures of a target are are conserved between eukaryotic and prokaryotes. A possible novel target fitting these parameters is choline kinase (ChoK), whose active site sequences are conserved (Figure 1) and whose tertiary structure (Figure 2) is maintained. Here, we describe why ChoK is a putative drug target by describing its role in the growth and pathogenesis of Gram-positive bacteria S. pneumoniae and the Gram-negative bacteria H. influenzae. Using S. pneumoniae as a model, we also present promising preliminary information that repurposing of drugs known to inhibit the human isoform of ChoK (hChoK), is a promising strategy for blocking the growth of S. pneumoniae cells and inhibiting the activity of the S. pneumoniae isoform of ChoK (sChok), with downstream physiological effects on the cell wall.

Parallel Colorimetric Quantification of Choline and Phosphocholine as a Method for Studying Choline Kinase Activity in Complex Mixtures.

Choline kinase (Chok) is an enzyme found in eukaryotes and Gram-positive bacteria. Chok catalyzes the production of phosphocholine from choline and ATP. This enzyme has been validated as a drug target in Streptococcus pneumonia, but the role Chok enzymatic activity plays in bacterial cell growth and division is not well understood. Phosphocholine production by Chok and its attenuation by inhibitors in the context of complex samples such as cell extracts can currently be quantified by several methods. These include choline depletion measurements, radioactive methods, mass-spectrometry, and nuclear magnetic resonance. The first does not measure phosphocholine directly, the second requires elaborate safety procedures, and the third and fourth require significant capital investments and technical expertise. For these reasons, a less expensive, higher throughput, more easily accessible assay is needed to facilitate further study in Gram-positive Choks. Here, we present the development of a triiodide/activated charcoal/molybdenum blue system for detecting and quantifying choline and phosphocholine in parallel. We demonstrate that this system can reliably quantify changes in choline and phosphocholine concentrations over time in Chok enzymatic assays using cell extracts as the source of the enzyme. This is an easily accessible, convenient, robust, and economical method for studying Chok activity in complex samples. The triiodide/activated charcoal/molybdenum blue system opens new doors into the study choline kinase in Gram-positive pathogens.

Choline Kinase, A Novel Drug Target for the Inhibition of Streptococcus pneumoniae.

Gram-positive pathogens, such as Streptococcuspneumoniae, can have deleterious effects on both human and animal health. Antibiotics and antimicrobials have been developed to treat infections caused by such pathogens and to prevent food contamination. However, these strategies have been increasingly thwarted by the emergence of resistant bacteria strains. Thus, new methods for controlling Gram-positive pathogen growth need to be continuously developed. Choline analogs, such as Hemicholinium-3 (HC-3), have been shown to be useful in blocking cell division in eukaryotic cells through the inhibition of choline kinase, an enzyme which catalyzes the production of phosphocholine from choline and ATP. In some Gram-positive pathogens, choline kinase is an important enzyme in the production of the cell wall element, lipoteichoic acid. However, it is not known if inhibiting this enzyme has any effect on cell division in Gram-positive bacteria. Using the R6 strain as a model, we tested the ability of HC-3 to block the activity of choline kinase in S. pneumoniae and inhibit cell growth. Mass-spectrometry measurements of crude extracts revealed that HC-3 blocked choline kinase activity. Turbidity measurements and population counts showed that HC-3 inhibited cell growth. Competition assays with choline suggested that HC-3 also blocked choline transporters. Western blots showed that lipoteichoic acid production was blocked in the presence of HC-3, and autolytic assays showed that this decrease in lipoteichoic acids caused cells to be more resistant to autolysis. Scanning electron microscopy revealed that HC-3 distorted the cell wall. This study thus establishes choline kinase as a novel drug target for S. pneumoniae.

Autolyse the cell in order to save it? Inducing, then blocking, autolysis as a strategy for delaying cell death in the probiotic Lactobacillus reuteri.

OBJECTIVE: To examine whether choline and its derivatives can be used to preserve viable cells of Lactobacillus reuteri in autolytic models. RESULTS: A phosphate-induced autolytic model in de Man, Rogosa and Sharpe medium (MRS) was used. Viable cell counts were determined by plated on MRS-agar. Choline and hemicholinium-3 (HC-3) significantly blocked autolysis of L. reuteri at 360 mM and 4 mM, respectively. Viable cell counts corroborated these observations. Importantly, autolytically induced cells treated with choline and hemicholinium-3 were significantly more viable then even non-induced cells. Over-production of a known autolytic protein, spirosin, was not attenuated in the presence of choline and hemicholinium-3. CONCLUSION: Inducing autolysis and then blocking it with choline and its analogs is a promising approach for retaining the viability of L. reuteri cells.

Molecular Interactions and Implications of Aldose Reductase Inhibition by PGA1 and Clinically Used Prostaglandins.

Aldose reductase (AKR1B1) is a critical drug target because of its involvement in diabetic complications, inflammation, and tumorigenesis. However, to date, development of clinically useful inhibitors has been largely unsuccessful. Cyclopentenone prostaglandins (cyPGs) are reactive lipid mediators that bind covalently to proteins and exert anti-inflammatory and antiproliferative effects in numerous settings. By pursuing targets for modification by cyPGs we have found that the cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 adduct was observed, both by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by SDS-PAGE using biotinylated PGA1 (PGA1-B). Insight into the molecular interactions between AKR1B1 and PGA1 was advanced by molecular modeling. This anticipated the addition of PGA1 to active site Cys298 and the potential reversibility of the adduct, which was supported experimentally. Indeed, loss of biotin label from the AKR1B1-PGA1-B adduct was favored by glutathione, indicating a retro-Michael reaction, which unveils new implications of cyPG-protein interaction. PGA1 elicited only marginal inhibition of aldehyde reductase (AKR1A1), considered responsible for the severe adverse effects of many AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) inhibited the enzyme, including non-electrophilic PGE1 and PGE2, currently used in clinical practice. Moreover, both PGA1 and PGE1 reduced the formation of sorbitol in an ex-vivo model of diabetic cataract to an extent comparable to that attained by the known AKR inhibitor epalrestat. Taken together, these results highlight the role of PGs as AKR1B1 inhibitors and the interest in PG-related molecules as leads for the development of novel pharmacological tools.

Antiplasmodial activity and mechanism of action of RSM-932A, a promising synergistic inhibitor of Plasmodium falciparum choline kinase.

We have investigated the mechanism of action of inhibition of the choline kinase of P. falciparum (p.f.-ChoK) by two inhibitors of the human ChoKα, MN58b and RSM-932A, which have previously been shown to be potent antitumoral agents. The efficacy of these inhibitors against p.f.-ChoK is investigated using enzymatic and in vitro assays. While MN58b may enter the choline/phosphocholine binding site, RSM-932A appears to have an altogether novel mechanism of inhibition and is synergistic with respect to both choline and ATP. A model of inhibition for RSM-932A in which this inhibitor traps p.f.-ChoK in a phosphorylated intermediate state blocking phosphate transfer to choline is presented. Importantly, MN58b and RSM-932A have in vitro inhibitory activity in the low nanomolar range and are equally effective against chloroquine-sensitive and chloroquine-resistant strains. RSM-932A and MN58b significantly reduced parasitemia and induced the accumulation of trophozoites and schizonts, blocking intraerythrocytic development and interfering with parasite egress or invasion, suggesting a delay of the parasite maturation stage. The present data provide two new potent structures for the development of antimalarial compounds and validate p.f.-ChoK as an accessible drug target against the parasite.

Design, synthesis, and functional evaluation of leukocyte function associated antigen-1 antagonists in early and late stages of cancer development.

The integrin leukocyte function associated antigen 1 (LFA-1) binds the intercellular adhesion molecule 1 (ICAM-1) by its α(L)-chain inserted domain (I-domain). This interaction plays a key role in cancer and other diseases. We report the structure-based design, small-scale synthesis, and biological activity evaluation of a novel family of LFA-1 antagonists. The design led to the synthesis of a family of highly substituted homochiral pyrrolidines with antiproliferative and antimetastatic activity in a murine model of colon carcinoma, as well as potent antiadhesive properties in several cancer cell lines in the low micromolar range. NMR analysis of their binding to the isolated I-domain shows that they bind to the I-domain allosteric site (IDAS), the binding site of other allosteric LFA-1 inhibitors. These results provide evidence of the potential therapeutic value of a new set of LFA-1 inhibitors, whose further development is facilitated by a synthetic strategy that is versatile and fully stereocontrolled.

Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry.

Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.

Utilization of I-domain of LFA-1 to Target Drug and Marker Molecules to Leukocytes.

The long-term objective of this project is to utilize the I-domain protein for the α-subunit of LFA-1 to target drugs to lymphocytes by binding to ICAM receptors on the cell surface. The short-term goal is to provide proof-of-concept that I-domain conjugated to small molecules can still bind to and uptake by ICAM-1 on the surface of lymphocytes (i.e., Raji cells). To accomplish this goal, the I-domain protein was labeled with FITC at several lysine residues to produce the FITC-I-domain and CD spectroscopy showed that the FITC-I-domain has a secondary structure similar to that of the parent I-domain. The FITC-I-domain was taken up by Raji cells via receptor-mediated endocytosis and its uptake can be blocked by anti-I-domain mAb but not by its isotype control. Antibodies to ICAM-1 enhance the binding of I-domain to ICAM-1, suggesting it binds to ICAM-1 at different sites than the antibodies. The results indicate that fluorophore modification does not alter the binding and uptake properties of the I-domain protein. Thus, I-domain could be useful as a carrier of drug to target ICAM-1-expressing lymphocytes.