A comparison of the inhibition of translation and 50S ribosomal subunit formation in Staphylococcus aureus cells by nine different macrolide antibiotics

Nine structurally similar macrolide antibiotics were tested at a concentration of 0.5 microg/ml for their relative inhibitory effects on ribosome functions in Staphylococcus aureus cells. Eight of the compounds examined inhibited protein synthesis at this concentration. Seven of the nine compounds were also effective in blocking formation of the 50S ribosomal subunit. Roxithromycin and 14-hydroxy clarithromycin inhibited protein synthesis to a greater extent than they affected 50S subunit formation. Conversely, the compound 11, 12-carbonate-3 deoxy-clarithromycin affected 50S assembly more than translation. Only clarithromycin had any effect on 30S ribosomal subunit assembly. The decline in growth rate and cell number was proportional to the effect on ribosome formation or function by each compound. These inhibitory activities can be related to structural differences between these macrolide antibiotics.     

Inhibition of translation and 50S ribosomal subunit formation in Staphylococcus aureus cells by 11 different ketolide antibiotics

Eleven structurally similar ketolide antibiotics were tested at a concentration of 1 microg/ml for their relative inhibitory effects on growth and ribosome activities in Staphylococcus aureus cells. Ten of the compounds examined had an inhibitory effect on protein synthesis at this concentration and eight of the 11 compounds were also effective inhibitors of the formation of the 50S ribosomal subunit. All of the drugs tested inhibited protein synthesis to a greater extent than they affected 50S subunit formation. The decline in growth rate and cell number was proportional to the effect on ribosome formation and function. The growth of an ermC erythromycin-resistant strain of S. aureus was also significantly inhibited by nine ketolide compounds, suggesting that they were not inducers of methylase gene expression. These inhibitory activities can be related to structural differences between these ketolide antibiotics.     

Macrolide antibiotics inhibit 50S ribosomal subunit assembly in Bacillus subtilis and Staphylococcus aureus

Macrolide antibiotics are clinically important antibiotics which are effective inhibitors of protein biosynthesis in bacterial cells. We have recently shown that some of these compounds also inhibit 50S ribosomal subunit formation in Escherichia coli. Now we show that certain macrolides have the same effect in two gram-positive organisms, Bacillus subtilis and Staphylococcus aureus. Assembly in B. subtilis was prevented by erythromycin, clarithromycin, and azithromycin but not by oleandomycin. 50S subunit formation in S. aureus was prevented by each of seven structurally related 14-membered macrolides but not by lincomycin or two streptogramin antibiotics. Erythromycin treatment did not stimulate the breakdown of performed 50S subunits in either organism. The formation of the 30S ribosomal subunit was also unaffected by these compounds. Assembly was also inhibited in a B. subtilis strain carrying a plasmid with the ermC gene that confers macrolide resistance by rRNA methylation. These results suggest that ribosomes contain an additional site for the inhibitory functions of macrolide antibiotics.     

Ribosomal protein L15 as a probe of 50 S ribosomal subunit structure

L15, a 15 kDa protein of the large ribosomal subunit, interacts with over ten other proteins during 50 S assembly in vitro. We have probed the interaction L15 with 23 S rRNA in 50 S ribosomal subunits by chemical footprinting, and have used localized hydroxyl radical probing, generated from Fe(II) tethered to unique sites of L15, to characterize the three-dimensional 23 S rRNA environment of L15. Footprinting of L15 was done by reconstituting purified, recombinant L15 with core particles derived from Escherichia coli 50 S subunits by treatment with 2 M LiCl. The cores migrate as compact 50 S-like particles in sucrose gradients, contain 23 S and 5 S rRNA, and lack a subset of the 50 S proteins, including L15. Using both Fe(II).EDTA and dimethyl sulfate, we have identified a strong footprint for L15 in the region spanning nucleotides 572-654 in domain II of 23 S rRNA. This footprint cannot be detected when L15 is incubated with "naked" 23 S rRNA, indicating that formation of the L15 binding site requires a partially assembled particle.Protein-tethered hydroxyl radical probing was done using mutants of L15 containing single cysteine residues at amino acid positions 68, 71 and 115. The mutant proteins were derivatized with 1-[p-(bromo-acetamido)benzyl]-EDTA. Fe(II), bound to core particles, and hydroxyl radical cleavage was initiated. Distinct but overlapping sets of cleavages were obtained in the footprinted region of domain II, and in specific regions of domains I, IV and V of 23 S rRNA. These data locate L15 in proximity to several 23 S rRNA elements that are dispersed in the secondary structure, consistent with its central role in the latter stages of 50 S subunit assembly. Furthermore, these results indicate the proximity of these rRNA regions to one another, providing constraints on the tertiary folding of 23 S rRNA.     

Translocation of 60S ribosomal subunit in spreading cardiac myocytes

Cardiac myocytes in culture undergo considerable structural reorganization. The remodeling of the myofibrils and the nonmyofibrillar cytoskeleton that occurs in the spreading cardiac myocytes resembles the cellular features observed in the hypertrophying heart. In this study we examined the distribution of the large 60S ribosomal subunit in freshly isolated cardiac myocytes and during the course of attachment and spreading in culture. Initially, anti-60S immunolabeling was scattered widely throughout the sarcoplasm of the dissociated cardiac myocytes. After attachment to the substrate, the 60S ribosomal subunit attained wide sarcoplasmic localization before a sarcomere-related staining pattern appeared in the spreading cell. Double labeling experiments with alpha-actinin confirmed co-localization of the 60S ribosomal subunit with nascent and mature myofibrils. These findings demonstrate that translocation of the 60S ribosomal subunit coincides with the cytoskeletal reorganization taking place in these cells. Moreover, the close association between the myofibrils indicates a particular role for the ribosomes in maintenance and growth of the contractile apparatus. (J Histochem Cytochem 46:963-969, 1998)     

Localization of proteins HL29 and HL31 from Haloarcula marismortui within the 50 S ribosomal subunit by chemical crosslinking

Isolated 50 S ribosomal subunits from the halophilic archaebacterium Haloarcula marismortui were treated in situ with the homobifunctional and cleavable crosslinking reagent dithiobis(succinimidyl propionate) (12 A). Several crosslinked complexes were obtained. Among these were the protein pairs HmaL4-HL29 and HmaL18-HL31; HL29 and HL31 are ribosomal proteins without any equivalent in eubacterial ribosomes. The crosslinked protein pairs were isolated on a preparative scale by combining conventional ion-exchange chromatography and reverse phase high-pressure liquid chromatography. The monomeric proteins involved in crosslink formation were unambiguously identified by two-dimensional gel electrophoresis and N-terminal or internal protein sequencing. Due to the homology between HmaL4 and HmaL18 and their Escherichia coli counterparts, and the roughly known location of these proteins within the 50 S subunit, our results demonstrate that HL29 is probably located in the centre of the large subunit in the vicinity of the peptidyltransferase domain, whereas HL31 must be situated within the central protuberance close to the region of the 5 S RNA.     

Absorption of various actinomycins by Staphylococcus aureus cells

The absorption of actinomycin D by the cell suspension of Staphylococcus aureus via diffusion linearly depended on the antibiotic concentration in the suspension within the ranges of 2 to 15 micrograms/ml. The absorption of active actinomycins C2, C3 and Au6 was the same as that of actinomycin D. The Staphylococcus intact membranes limited the inlet of the actinomycins to the cells since the membranotropic substances such as gramicidin S and its derivatives and thyrocidin increased their absorption by 30-70 per cent. The absorption of a low active actinomycin D0 and inactive actinomycinic acid even after the exposure to the membranotropic substances was not detectable. These compounds did not form any complexes with DNA. The level of the absorption of the actinomycins by the cells was likely defined by their ability to complex with DNA.     

Minimal inhibition concentrations of selected antibiotics for strains of Staphylococcus aureus

Minimal inhibition concentrations (MIC) of gentamycin (Ge), neomycin (Neo), rifampicin (Rif), ampicillin (Amp), lincomycin (Lin), erythromycin (Ery), and streptomycin (STM) were determined by the agar dilution technique using 46, 130, 131, 125, 140, 139 and 142 strains of Staphylococcus aureus, respectively. The strains, selected from the collection of the authors' laboratory, were isolated from mammary gland secretions of cows affected with clinical or subclinical mastitis. The following ranges of MIC (micrograms/ml) were assessed for the antibiotics under study: Ge 0.125-0.50, Neo 0.06-0.50, Rif 0.0039-0.030, Amp 0.015-1.00, Lin 0.25-1.00, Ery 0.06-0.25, STM 0.50-64.0. Modal MIC (micrograms/ml) were as follows; Ery 0.125 (86%), Lin 0.5 (71.4%), Rif 0.007 (68.7%), Ge 0.25 (56.5%), STM 1.00 (54.2%), Neo 0.25 (53.8%), Amp 0.06 (41.6%). The order of efficiency expressed in MIC 90 (micrograms/ml) was as follows: Rif (0.015), Ery (0.125), Ge (0.25), Neo (0.25), Amp (0.5), STM (4.0).     

Preparation of a ''beheaded'' derivative of the 30S ribosomal subunit

In order to test the hypothesis that an alteration in the sex hormone milieu may underlie risk factors for myocardial infarction, fasting serum sex hormones, ie, estradiol, testosterone, free testosterone, and androstenedione, were measured in 24 hypertensive and in 19 healthy postmenopausal women. The mean serum free testosterone level (P=0.01) and the free-to-total testosterone ratio (P < 0.04) were increased in the women with hypertension. In a stepwise multiple regression analysis on the hypertensive and normotensive groups combined, with systolic blood pressure (SBP) as the dependent variable and body mass index, age, free testosterone, estradiol, insulin, and cholesterol levels as the independent variables, only free testosterone showed an independent relationship to SBP (P=0.009). The finding in the present study of an independent positive relationship of free testosterone with hypertension is consistent with a similar relationship of free testosterone with other risk factors for myocardial infarction in women found in previous studies and supports the hypothesis.     

Internalization of Staphylococcus aureus by endothelial cells induces apoptosis

The ability of Staphylococcus aureus to invade and survive within endothelial cells is believed to contribute to its propensity to cause persistent endovascular infection with endothelial destruction. In the present study, we show that following invasion of human umbilical vein endothelial cells, intracellular S. aureus organisms remain viable over a 72-h period and, as determined by transmission electron microscopic examination, that the bacteria exist within vacuoles and free within the cytoplasm. We also demonstrate that endothelial cell death following S. aureus invasion occurs at least in part by apoptosis as shown by DNA fragmentation and changes in nuclear morphology. Apoptotic changes were evident as early as 1 h after infection of endothelial cells. Internalization of S. aureus rather than adherence appears to be necessary, since use of the phagocytosis inhibitor cytochalasin D prevented apoptosis. UV-killed staphylococci, although retaining the capacity to be internalized, were not capable of inducing apoptosis, suggesting that apoptosis is dependent upon a factor associated with viable organisms. The studies demonstrate that viable intracellular S. aureus induces apoptosis of endothelial cells and that internalized staphylococci can exist free within the cytoplasm.     

Regulation of Staphylococcus aureus capsular polysaccharide type 5: CO2 inhibition in vitro and in vivo

Staphylococcus aureus capsular polysaccharide type 5 (CP5) expression was investigated in lung tissue and nasal polyps of two cystic fibrosis (CF) patients, in rats, and in vitro using ELISA and IFA. In CF tissues, S. aureus expressed protein A and teichoic acid but only 1%-5% of cells expressed CP5. When rats were challenged with CP5-positive S. aureus in the granuloma pouch model, only 1%-5% of CP5-positive cells were detectable in pouch exudates. CF and pouch isolates, however, reexpressed CP5 (70%-90% of cells) when grown in vitro with air. Addition of > or = 1% CO2 to air or to O2/N2 gas mixtures reduced CP5 expression significantly (P < .001) in a dose-dependent manner (6%-1% CP5-positive cells). The results show that S. aureus does not produce CP5 in CF airways and in rat granuloma pouches and that CO2 is an environmental signal that regulates CP5 expression.     

Gentamicin-resistant menadione and hemin auxotrophic Staphylococcus aureus persist within cultured endothelial cells

Staphylococcus aureus menadione and hemin auxotrophs, generated by in vitro gentamicin selection, demonstrated reduced hemolytic activity and enhanced intracellular survival within cultured bovine aortic endothelial cells relative to their hemolytic parent. Supplementation of the auxotrophs with exogenous menadione or hemin resulted in rapid growth, increased hemolytic activity, and reduced intracellular persistence to the level found for the hemolytic clinical parent. Aminoglycoside selection of staphylococcal menadione and hemin auxotrophs and subsequent persistence of these variants in the intracellular milieu may adapt S. aureus for evasion of host defenses and resistance to antimicrobial therapy.     

Influence of age of the donor sheep on the phagocytosis of Staphylococcus aureus subspecies anaerobius and S aureus by neutrophils

A 29-year-old man underwent a one-stage island flap urethroplasty, using perineal skin, to treat a urethral stricture. Four years following surgery he represented with symptoms from a urethral diverticulum containing a hair ball. This was at the site of the previous urethroplasty and was treated by excision.     

Increase of non-amidated muropeptides in the cell wall of vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50

BACKGROUND: In previous survey we found large socioeconomic differences in mortality among urban Swedish men which remained unexplained after controlling for smoking and standard coronary risk factors. The present analysis was undertaken in order to investigate a broader set of possible explanatory factors in another cohort of Swedish men. STUDY POPULATION AND METHODS: Occupation was coded into five occupational classes for 717 of 776 participant men from a random population sample of 1016 men who were born in 1933. All were living in G?teborg and were 50 years old at the baseline examination. After 12 years' follow-up, 68 of the 717 men had died (9.5%). RESULTS: Low occupational class was associated with a higher prevalence of smoking at baseline, but no association was found with systolic blood pressure, body mass index, waist to hip ratio, serum triglycerides or serum cholesterol. Subjects from higher socioeconomic strata were taller, had higher maximum peak respiratory flow, lower plasma fibrinogen and lower body temperature. Low occupational class was associated with low social integration, low home activity levels, low levels of activity outside home and low social activity levels (p = 0.001 for all) and with low emotional support (p = 0.018). There were also associations between low occupational class and poor self-perceived health, as well as with several cardiovascular symptoms. During 12 years' follow-up, there was a graded and inverse relationship between occupational class and mortality from all causes. The highest mortality was found among the men who could not be classified (23 per 1,000 person years) Of the men in the lowest occupational class, 12 per 1,000 died, compared to 3 per 1,000 in the highest class (relative risk 3.7 (1.4-9.8)). After controlling for smoking, the relative risk decreased to 3.2 (1.2-8.6) and after further adjustment for emotional support, self-perceived health, activity level at home, and peak expiratory flow, the relative risk was still twofold but not significantly so (RR 2.1 (0.8-5.8)). CONCLUSION: We were able to confirm earlier results as to the wide mortality differentials in urban middle-aged men in Sweden. There were also large differences in several other factors, including constitutional factors, health variables, lifestyle and social support indices, which explained important parts of the social mortality gradient, the most prominent being smoking, respiratory function, social network factors and subjective health.     

Biofilm formation of Staphylococcus aureus strains isolated from impetigo and furuncle: role of fibrinogen and fibrin

Effective utilization of spiral computed tomography (CT) technology in imaging of the thorax requires an understanding of technical parameters that affect image and scan quality. This article discusses how operator-controlled scan parameters can be optimized to achieve diagnostic and cost-effective examinations appropriate for daily clinical practice.     

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.     

Differential diagnosis of Staphylococcus aureus from Staphylococcus epidermidis and Staphylococcus saprophyticus by alphazurine A dye

Staphylococcal bacterial suspensions were streaked on Trypticase soy agar with 5% sheep blood culture plates. Paper discs containing alphazurine A, a triphenylmethane dye, were placed on the inoculated plates which were incubated at 37 degrees C for 24 hours. A wide zone of inhibition of growth of Staphylococcus aureus was present around the paper discs. Growth of Staphylococcus epidermidis and Staphylococcus saprophyticus was not inhibited.     

An agar medium for direct enumeration of Staphylococcus aureus: pork plasma medium for S. aureus (PPSA)

A selective agar medium (pork plasma medium for S. aureus (PPSA)) enables the direct enumeration of coagulase-positive staphylococci. This medium is based on the Baird-Parker agar without egg yolk and is supplemented with pig plasma. Colonies of Staphylococcus aureus are surrounded by a halo of precipitated fibrin. When foods such as dairy products contain large numbers of egg yolk-negative strains of S. aureus, the PPSA agar has the advantage over egg yolk containing media such as Baird-Parker agar that fewer suspect colonies have to be confirmed.