Organisms were cultured in triplicate in 25 ml of TH broth at 37C for up to 24 h with antimicrobial compounds

Organisms were cultured in triplicate in 25 ml of TH broth at 37C for up to 24 h with antimicrobial compounds. of these organisms and their toxic products, including and enterotoxins (SEs), are select brokers of bioterrorism. cause infections which range from relatively benign to life-threatening, such as toxic shock syndrome (TSS) (1C7). For example, secretes the superantigen TSS toxin 1 (TSST-1), which is the predominant cause of menstrual TSS and up to 50% of nonmenstrual TSS (8, 9). Staphylococcal enterotoxin superantigens, notably serotypes B and C, cause the remaining 50% of nonmenstrual TSS cases. Staphylococcal superantigens cause TSS through overproduction of cytokines (4, 9C11). We as well as others have identified Gram-positive two-component systems (TCS) that affect virulence and ability to interact with the external environment (12C14). We hypothesized that one or more of these crucial TCS may be regulated by menaquinone. Part of the basis for this hypothesis is usually that many compounds we have tested, including positively charged hemoglobin peptides (15), clindamycin (16), glycerol monolaurate (GML) (17, 18), and chitosan (19), although disparate in properties, appear in part to target and bacterial YHO-13177 TCS signaling, with the consequence being reduction of growth and/or exotoxin production. This suggests that it may be possible to find menaquinone analogs that also inhibit Gram-positive bacterial growth and exotoxin production, forming the IKK-gamma antibody basis for our studies. Although we primarily tested effects of menaquinone analogs on Gram-positive bacteria, studies were performed also with four Gram-negative bacteria. MATERIALS AND METHODS Bacteria. MN8 and CDC587 are common methicillin-sensitive menstrual TSS isolates (9). MNPE is usually a methicillin-sensitive nonmenstrual TSS isolate from a case of fatal postinfluenza pneumonia (20); this organism has a wild-type alpha-toxin gene. All three of these organisms belong to clonal group USA200. Community-associated methicillin-resistant (CA-MRSA) MW2 (clonal group USA400) was isolated from a fatal case of necrotizing pneumonia in the upper Midwest (21). CA-MRSA LAC (clonal group USA300) was isolated from a skin contamination (22). Ten recent clinical isolates included 2 methicillin-sensitive (MSSA) isolates (USA200) and 8 CA-MRSA isolates (2 USA200, 3 USA300, and 3 USA400). Sterne was purchased from the Colorado Serum Company, Denver, CO. strain MNWA is an M3 isolate from a TSS patient. strain MNSI is an isolate from a patient with neonatal sepsis. serotype YHO-13177 Typhimurium, were clinical isolates maintained in the laboratory. A complete deletion of MN8 via PCR with the primer set EcoRIsrrAupF (5-AAAGAATTCCCTGTTGGTCGTTTAGACTATGATA-3) and srrAupRev (5-CATGCGTTCTGAAATAACTATATTCAATTCACACATGCTTTTCTTTACAAAAGT-3) and downstream of with the primer set srrBdnF (5-AATTGAATATAGTTATTTCAGAACGCATG-3) and KpnIsrrBdnRev (5-AAGGTACCCGCCACTACCTACAACATTG-3). The fragments were then spliced together by use of YHO-13177 overlapping PCR with the primer set EcoRIsrrAupF and KpnIsrrBdnRev, digested with EcoRI and KpnI, and inserted into pJB38, a derivative of pKOR1 (23). The resulting plasmid, henceforth referred to as pJB38-DH5, with selection of plasmid by growth in 100 g/ml of ampicillin in Luria-Bertani broth. Plasmid was isolated with use of a mini-plasmid isolation kit (IBI Scientific, Peosta, IA) and transformed into strain RN4220, with selection with 10 g/ml of chloramphenicol. The plasmid was then transformed into MN8, and selection was performed according to the original pKOR1 protocol (23). Deletions of were detected using external primers to the deletion construct and confirmed by the decrease in band size equal to the gene pair. Loss of the plasmid was confirmed by streaking colonies with confirmed deletion on Todd-Hewitt (TH) broth plates containing 10 g/ml of chloramphenicol. Menaquinone and analogs. Menaquinone and analogs (menadione, 1,4-naphthoquinone, phylloquinone, coenzymes Q1 to Q3, and coenzyme Q10) were purchased from Sigma-Aldrich, St. Louis, MO. The compounds were studied in dose-response experiments with concentrations ranging from 0 to 200 g/ml. Menaquinone analog effects on and exotoxin production. For aerobic studies of.Staphylococcal and streptococcal superantigen exotoxins. and biosynthetic machinery YHO-13177 to interfere with two-component systems, respiration, and macromolecular synthesis. These compounds represent a novel class of potential topical therapeutic agents. INTRODUCTION Gram-positive bacteria cause large numbers of infections; some of these organisms and their toxic products, including and enterotoxins (SEs), are select agents of bioterrorism. cause infections which range from relatively benign to life-threatening, such as toxic shock syndrome (TSS) (1C7). For example, secretes the superantigen TSS toxin 1 (TSST-1), which is the predominant cause of menstrual TSS and up to 50% of nonmenstrual TSS (8, 9). Staphylococcal enterotoxin superantigens, notably serotypes B and C, cause the remaining 50% of nonmenstrual TSS cases. Staphylococcal superantigens cause TSS through overproduction of cytokines (4, 9C11). We and others have identified Gram-positive two-component systems (TCS) that affect virulence and ability to interact with the external environment (12C14). We hypothesized that one or more of these critical TCS may be regulated by menaquinone. Part of the basis for this hypothesis is that many compounds we have tested, including positively charged hemoglobin peptides (15), clindamycin (16), glycerol monolaurate (GML) (17, 18), and chitosan (19), although disparate in properties, appear in part to target and bacterial TCS signaling, with the consequence being reduction of growth and/or exotoxin production. This suggests that it may be possible to find menaquinone analogs that also inhibit Gram-positive bacterial growth and exotoxin production, forming the basis for our studies. Although we primarily tested effects of menaquinone analogs on Gram-positive bacteria, studies were performed also with four Gram-negative bacteria. MATERIALS AND METHODS Bacteria. MN8 and CDC587 are typical methicillin-sensitive menstrual TSS isolates (9). MNPE is a methicillin-sensitive nonmenstrual TSS isolate from a case of fatal postinfluenza pneumonia (20); this organism has a wild-type alpha-toxin gene. All three of these organisms belong to clonal group USA200. Community-associated methicillin-resistant (CA-MRSA) MW2 (clonal group USA400) was isolated from a fatal case of necrotizing pneumonia in the upper Midwest (21). CA-MRSA LAC (clonal group USA300) was isolated from a skin infection (22). Ten recent clinical isolates included 2 methicillin-sensitive (MSSA) isolates (USA200) and 8 CA-MRSA isolates (2 USA200, 3 USA300, and 3 USA400). Sterne was purchased from the Colorado Serum Company, Denver, CO. strain MNWA is an M3 isolate from a TSS patient. strain MNSI is an isolate from a patient with neonatal sepsis. serotype Typhimurium, were clinical isolates maintained in the laboratory. A complete deletion of MN8 via PCR with the primer set EcoRIsrrAupF (5-AAAGAATTCCCTGTTGGTCGTTTAGACTATGATA-3) and srrAupRev (5-CATGCGTTCTGAAATAACTATATTCAATTCACACATGCTTTTCTTTACAAAAGT-3) and downstream of with the primer set srrBdnF (5-AATTGAATATAGTTATTTCAGAACGCATG-3) and KpnIsrrBdnRev (5-AAGGTACCCGCCACTACCTACAACATTG-3). The fragments were then spliced together by use of overlapping PCR with the primer set EcoRIsrrAupF and KpnIsrrBdnRev, digested with EcoRI and KpnI, and inserted into pJB38, a derivative of pKOR1 (23). The resulting plasmid, henceforth referred to as pJB38-DH5, with selection of plasmid by growth in 100 g/ml of ampicillin in Luria-Bertani broth. Plasmid was isolated with use of a mini-plasmid isolation kit (IBI Scientific, Peosta, IA) and transformed into strain RN4220, with selection with 10 g/ml of chloramphenicol. The plasmid was then transformed into MN8, and selection was performed according to the original pKOR1 protocol (23). Deletions of were detected using external primers to the deletion construct and confirmed by the decrease in band size equal to the gene pair. Loss of the plasmid was confirmed by streaking colonies with confirmed deletion on Todd-Hewitt (TH) broth plates containing 10 g/ml of chloramphenicol. Menaquinone and analogs. Menaquinone and analogs (menadione, 1,4-naphthoquinone, phylloquinone, coenzymes Q1 to Q3, and coenzyme Q10) were purchased from Sigma-Aldrich, St. Louis, MO. The compounds were studied in dose-response experiments with concentrations ranging from 0 to 200 g/ml. Menaquinone analog effects on and exotoxin production. For aerobic studies of knockout mutant), CDC587, MNPE, MW2, and LAC and 10 recent clinical isolates (starting inocula of 106 to 107/ml) were cultured in triplicate in 25 ml of TH broth for 1 to 24 h with shaking (200 revolutions/min) in the presence.Microbiol. 86:1376C1392 [PMC free article] [PubMed] [Google Scholar] 30. systems, respiration, and macromolecular synthesis. These compounds represent a novel class of potential topical therapeutic agents. INTRODUCTION Gram-positive bacteria cause large numbers of infections; some of these organisms and their toxic products, including and enterotoxins (SEs), are select agents of bioterrorism. cause infections which range from relatively benign to life-threatening, such as toxic shock syndrome (TSS) (1C7). For example, secretes the superantigen TSS toxin 1 (TSST-1), which is the predominant cause of menstrual TSS and up to 50% of nonmenstrual TSS (8, 9). Staphylococcal enterotoxin superantigens, notably serotypes B and C, cause the remaining 50% of nonmenstrual TSS instances. Staphylococcal superantigens cause TSS through overproduction of cytokines (4, 9C11). We while others have recognized Gram-positive two-component systems (TCS) that impact virulence and ability to interact with the external environment (12C14). We hypothesized that one or more of these essential TCS may be controlled by menaquinone. Part of the basis for this hypothesis is definitely that many compounds we have tested, including positively charged hemoglobin peptides (15), clindamycin (16), glycerol monolaurate (GML) (17, 18), and chitosan (19), although disparate in properties, appear in part to target and bacterial TCS signaling, with the result being reduction of growth and/or exotoxin production. This suggests that it may be possible to find menaquinone analogs that also inhibit Gram-positive bacterial growth and exotoxin production, forming the basis for our studies. Although we primarily tested effects of menaquinone analogs on Gram-positive bacteria, studies were performed also with four Gram-negative bacteria. MATERIALS AND METHODS Bacteria. MN8 and CDC587 are standard methicillin-sensitive menstrual TSS isolates (9). MNPE is definitely a methicillin-sensitive nonmenstrual TSS isolate from a case of fatal postinfluenza pneumonia (20); this organism has a wild-type alpha-toxin gene. All three of these organisms belong to clonal group USA200. Community-associated methicillin-resistant (CA-MRSA) MW2 (clonal group USA400) was isolated from a fatal case of necrotizing pneumonia in the top Midwest (21). CA-MRSA LAC (clonal group USA300) was isolated from a pores and skin illness (22). Ten recent medical isolates included 2 methicillin-sensitive (MSSA) isolates (USA200) and 8 CA-MRSA isolates (2 USA200, 3 USA300, and 3 USA400). Sterne was purchased from your Colorado Serum Organization, Denver, CO. strain MNWA is an M3 isolate from a TSS individual. strain MNSI is an isolate from a patient with neonatal sepsis. serotype Typhimurium, were clinical isolates managed in the laboratory. A complete deletion of MN8 via PCR with the primer arranged EcoRIsrrAupF (5-AAAGAATTCCCTGTTGGTCGTTTAGACTATGATA-3) and srrAupRev (5-CATGCGTTCTGAAATAACTATATTCAATTCACACATGCTTTTCTTTACAAAAGT-3) and downstream of with the primer arranged srrBdnF (5-AATTGAATATAGTTATTTCAGAACGCATG-3) and KpnIsrrBdnRev (5-AAGGTACCCGCCACTACCTACAACATTG-3). The fragments were then spliced collectively by use of overlapping PCR with the primer arranged EcoRIsrrAupF and KpnIsrrBdnRev, digested with YHO-13177 EcoRI and KpnI, and put into pJB38, a derivative of pKOR1 (23). The producing plasmid, henceforth referred to as pJB38-DH5, with selection of plasmid by growth in 100 g/ml of ampicillin in Luria-Bertani broth. Plasmid was isolated with use of a mini-plasmid isolation kit (IBI Scientific, Peosta, IA) and transformed into strain RN4220, with selection with 10 g/ml of chloramphenicol. The plasmid was then transformed into MN8, and selection was performed according to the unique pKOR1 protocol (23). Deletions of were detected using external primers to the deletion create and confirmed by the decrease in band size equal to the gene pair. Loss of the plasmid was confirmed by streaking colonies with confirmed deletion on Todd-Hewitt (TH) broth plates comprising 10 g/ml of chloramphenicol. Menaquinone and analogs. Menaquinone and analogs (menadione, 1,4-naphthoquinone, phylloquinone, coenzymes Q1 to Q3, and coenzyme Q10) were purchased from Sigma-Aldrich, St. Louis, MO. The compounds were analyzed in dose-response experiments with concentrations ranging from 0 to 200 g/ml. Menaquinone analog effects on and exotoxin production. For aerobic studies of knockout mutant), CDC587, MNPE, MW2, and LAC and 10 recent medical isolates (starting inocula of 106 to 107/ml) were cultured in triplicate in 25 ml of TH broth for 1 to 24 h with shaking (200 revolutions/min) in the presence and absence of potential antimicrobial compounds. After incubation, a sample of tradition.Quinones while the redox transmission for the arc two-component system of bacteria. benign to life-threatening, such as toxic shock syndrome (TSS) (1C7). For example, secretes the superantigen TSS toxin 1 (TSST-1), which is the predominant cause of menstrual TSS and up to 50% of nonmenstrual TSS (8, 9). Staphylococcal enterotoxin superantigens, notably serotypes B and C, cause the remaining 50% of nonmenstrual TSS instances. Staphylococcal superantigens cause TSS through overproduction of cytokines (4, 9C11). We while others have recognized Gram-positive two-component systems (TCS) that impact virulence and ability to interact with the external environment (12C14). We hypothesized that one or more of these essential TCS may be controlled by menaquinone. Part of the basis for this hypothesis is definitely that many compounds we have tested, including positively charged hemoglobin peptides (15), clindamycin (16), glycerol monolaurate (GML) (17, 18), and chitosan (19), although disparate in properties, appear in part to target and bacterial TCS signaling, with the result being reduction of growth and/or exotoxin production. This suggests that it may be possible to find menaquinone analogs that also inhibit Gram-positive bacterial growth and exotoxin production, forming the basis for our studies. Although we primarily tested effects of menaquinone analogs on Gram-positive bacteria, studies were performed also with four Gram-negative bacteria. MATERIALS AND METHODS Bacteria. MN8 and CDC587 are standard methicillin-sensitive menstrual TSS isolates (9). MNPE is definitely a methicillin-sensitive nonmenstrual TSS isolate from a case of fatal postinfluenza pneumonia (20); this organism has a wild-type alpha-toxin gene. All three of these organisms belong to clonal group USA200. Community-associated methicillin-resistant (CA-MRSA) MW2 (clonal group USA400) was isolated from a fatal case of necrotizing pneumonia in the top Midwest (21). CA-MRSA LAC (clonal group USA300) was isolated from a pores and skin illness (22). Ten recent medical isolates included 2 methicillin-sensitive (MSSA) isolates (USA200) and 8 CA-MRSA isolates (2 USA200, 3 USA300, and 3 USA400). Sterne was purchased from your Colorado Serum Organization, Denver, CO. strain MNWA is an M3 isolate from a TSS individual. strain MNSI is an isolate from a patient with neonatal sepsis. serotype Typhimurium, were clinical isolates managed in the laboratory. A complete deletion of MN8 via PCR with the primer arranged EcoRIsrrAupF (5-AAAGAATTCCCTGTTGGTCGTTTAGACTATGATA-3) and srrAupRev (5-CATGCGTTCTGAAATAACTATATTCAATTCACACATGCTTTTCTTTACAAAAGT-3) and downstream of with the primer arranged srrBdnF (5-AATTGAATATAGTTATTTCAGAACGCATG-3) and KpnIsrrBdnRev (5-AAGGTACCCGCCACTACCTACAACATTG-3). The fragments were then spliced collectively by use of overlapping PCR with the primer arranged EcoRIsrrAupF and KpnIsrrBdnRev, digested with EcoRI and KpnI, and put into pJB38, a derivative of pKOR1 (23). The producing plasmid, henceforth referred to as pJB38-DH5, with selection of plasmid by growth in 100 g/ml of ampicillin in Luria-Bertani broth. Plasmid was isolated with use of a mini-plasmid isolation kit (IBI Scientific, Peosta, IA) and transformed into strain RN4220, with selection with 10 g/ml of chloramphenicol. The plasmid was then transformed into MN8, and selection was performed according to the unique pKOR1 protocol (23). Deletions of were detected using external primers to the deletion create and confirmed by the reduction in music group size add up to the gene set. Lack of the plasmid was verified by streaking colonies with verified deletion on Todd-Hewitt (TH) broth plates formulated with 10 g/ml of chloramphenicol. Menaquinone and analogs. Menaquinone and analogs (menadione, 1,4-naphthoquinone, phylloquinone, coenzymes Q1 to Q3, and coenzyme Q10) had been bought from Sigma-Aldrich, St. Louis, MO. The substances were examined in dose-response tests with concentrations which range from 0 to 200 g/ml. Menaquinone analog results on and exotoxin creation. For aerobic research of knockout mutant), CDC587, MNPE, MW2, and LAC and 10 latest scientific isolates (beginning inocula of 106 to 107/ml) had been cultured in triplicate in 25 ml of TH broth for 1 to 24 h with shaking (200 revolutions/min) in the existence and lack of potential antimicrobial substances. After incubation, an example of lifestyle was utilized to determine CFU/ml, and examples of MN8, an knockout mutant of MN8, CDC587, and MNPE with capacity to produce.