Spectral Snapshots of Bacterial Cell-Wall Composition and the Influence of Antibiotics by Whole-Cell NMR

Gram-positive bacteria surround themselves with a thick cell wall that is essential to cell survival and is a major target of antibiotics. Quantifying alterations in cell-wall composition are crucial to evaluating drug modes of action, particularly important for human pathogens that are now resistant to multiple antibiotics such as Staphylococcus aureus. Macromolecular and whole-cell NMR spectroscopy allowed us to observe the full panel of carbon and nitrogen pools in S. aureus cell walls and intact whole cells. We discovered that one-dimensional ¹³C and ¹⁵N NMR spectra, together with spectroscopic selections based on dipolar couplings as well as two-dimensional spin-diffusion measurements, revealed the dramatic compositional differences between intact cells and cell walls and allowed the identification of cell-wall signatures in whole-cell samples. Furthermore, the whole-cell NMR approach exhibited the sensitivity to detect distinct compositional changes due to treatment with the antibiotics fosfomycin (a cell-wall biosynthesis inhibitor) and chloramphenicol (a protein synthesis inhibitor). Whole cells treated with fosfomycin exhibited decreased peptidoglycan contributions while those treated with chloramphenicol contained a higher percentage of peptidoglycan as cytoplasmic protein content was reduced. Thus, general antibiotic modes of action can be identified by profiling the total carbon pools in intact whole cells.     

An electron microscopic study of antagonism between cephalexin and erythromycin in Staphylococcus aureus.

This study concerns investigations at the cellular level of antagonism between cephalexin (CEX) and erythromycin (EM) with the aid of electron microscopes and a liquid scintillation counter. Exposure of Staphylococcus aureus 209-P to CEX and EM in combination was found to result in a marked antagonism between the two antibiotics in their effects on the growth of the organism. Observations under a scanning electron microscope revealed lysed cells in the presence of CEX alone but almost no lysis in the presence of a combination of CEX and EM. Observations under a transmission electron microscope, on the other hand, disclosed that nearly all of the cells exposed to 20 mug/ml of CEX were transformed into protoplasts with their morphological changes being most marked after 4 hr of exposure. When 1 mug/ml of EM was allowed to act alone, this exposure resulted in thickening of the cell walls. The combined use of CEX and EM, however, resulted in neither thickening of the cell walls as in the presence of EM alone nor in the formation of protoplasts as in the presence of CEX alone but merely produced the swelling of separating walls. Cellular uptake of 14C-L-lysine and N-acetylglucosamine-1-14C into the cell wall fraction and the protein fraction was affected by CEX and EM, respectively, when used alone or in combination.     

Macrolide Resistance in Staphylococcus aureus

A heat-inducible mutant, resistant to macrolide antibiotics (Mac), was isolated from Staphylococcus aureus MS537 in which Mac-resistance was induced by subinhibitory concentrations of erythromycin (EM). After induction at 42 C, this mutant acquired a high resistance to both Mac and lincomycin (LMC). Transduction and biochemical studies revealed that spiramycin (SP)-resistance in this mutant was induced by exposure to a high temperature (42 C) or by treatment with EM in broth but not in phosphate buffer. Induction did not take place when chloramphenicol (CM) was added to the induction mixture. Ribosomes from the mutant cultured at 42 C decreased their affinity for SP and consequently polypeptide synthesis on such ribosomes was not inhibited by SP, when compared with those cultured at 30 C. From these results, it was concluded that alteration of ribosomes took place after induction by exposure at high temperatures or by EM-treatment and that the mechanism of SP-resistance after induction was accounted for by a decrease in SP-binding to ribosomes.     

An Electron Microscopic Study of Antagonism between Cephalexin and Erythromycin in Staphylococcus aureus

This study concerns investigations at the cellular level of antagonism between cephalexin (CEX) and erythromycin (EM) with the aid of electron microscopes and a liquid scintillation counter. Exposure of Staphylococcus aureus 209-P to CEX and EM in combination was found to result in a marked antagonism between the two antibiotics in their effects on the growth of the organism. Observations under a scanning electron microscope revealed lysed cells in the presence of CEX alone but almost no lysis in the presence of a combination of CEX and EM. Observations under a transmission electron microscope, on the other hand, disclosed that nearly all of the cells exposed to 20 μg/ml of CEX were transformed into protoplasts with their morphological changes being most marked after 4 hr of exposure. When 1 μg/ml of EM was allowed to act alone, this exposure resulted in thickening of the cell walls. The combined use of CEX and EM, however, resulted in neither thickening of the cell walls as in the presence of EM alone nor in the formation of protoplasts as in the presence of CEX alone but merely produced the swelling of separating walls. Cellular uptake of ¹⁴C-L -lysine and N-acetylglucosamine-1-¹⁴C into the cell wall fraction and the protein fraction was affected by CEX and EM, respectively, when used alone or in combination.     

Inhibition of protein synthesis by polypeptide antibiotics. I. Inhibition in intact bacteria

Ennis, Herbert L. (St. Jude Children's Research Hospital, Memphis, Tenn.). Inhibition of protein synthesis by polypeptide antibiotics. I. Inhibition in intact bacteria. J. Bacteriol. 90:1102-1108. 1965.-The mechanism of inhibition of growth of cells by the polypeptide antibiotics of the PA 114, vernamycin, and streptogramin complexes was studied. This inhibition apparently was due to the selective inhibition of protein synthesis by these antibiotics. Ribonucleic acid synthesis was unaffected by concentrations of the antibiotics which completely inhibited protein synthesis. Deoxyribonucleic acid synthesis was slightly inhibited. These antibiotics are composed of a number of components. Mixtures of equal amounts of PA 114 A and PA 114 B or vernamycin A and Balpha were more active in stopping protein synthesis in intact cells than each of the components of the antibiotic complex alone. Mutants resistant to one of the antibiotics were resistant to all of the group and, in addition, were resistant to erythromycin and oleandomycin.     

β-Lactam and glycopeptide antibiotics: first and last line of defense?

Most infections are caused by bacteria, many of which are ever-evolving and resistant to nearly all available antibiotics. β-Lactams and glycopeptides are used to combat these infections by inhibiting bacterial cell-wall synthesis. This mechanism remains an interesting target in the search for new antibiotics in light of failed genomic approaches and the limited input of major pharmaceutical companies. Several strategies have enriched the pipeline of bacterial cell-wall inhibitors; examples include combining screening strategies with lesser-explored microbial diversity, or reinventing known scaffolds based on structure-function relationships. Drugs developed using novel strategies will contribute to the arsenal in fight against the continued emergence of bacterial resistance.     

A simple method for following the fate of alanine-containing components in murein synthesis inEscherichia coli

The fate of the alanine-containing components in murein synthesis was followed by incorporation of¹⁴C-l-alanine inE. coli under conditions allowing cell-wall synthesis while preventing protein synthesis. The components were separated by chromatography and detected by autoradiography.Spots containing murein, cell-wall precursors, alanine andd-alanyl-d-alanine were identified. A further component was probably identical to pyruvic acid. Two unidentified spots were found in the region where lipid-intermediates in cell-wall synthesis are usually found. However, the absence of turnover of these two components was at variance with the proposed properties of the lipidintermediates. d-Alanyl-d-alanine and the component which is probably identical to pyruvic acid were excreted into the medium, whereas murein and cell-wall precursors were found in the cellular fraction.The influence of the concentration of alanine, and of the number of cells per ml, on the acid-precipitable activity were studied. The latter increased during, at least, the first two hours and represented mainly lysozyme-degradable material.Significant turnover of murein could be detected neither in the presence nor in the absence of protein synthesis.A time course of the activity of the radioactive components is provided. The influence of a number of antibiotics inhibiting cell-wall synthesis on the acid-precipitable activity and on the activity of the main intermediates in murein synthesis was studied.We thank Mrs. Arna van Schijndel-van Dam and Mr. A. A. G. Verweij for excellent assistance. We thank Dr. P. E. Reynolds (University of Cambridge) for teaching one of us (E J. J. L.) several techniques in the field of bacterial cell walls, and Dr. H. J. W. Wijsman for stimulating discussions.     

Failure of tachykinins including substance P and its fragments to influence proteoglycan and protein synthesis in bovine chondrocytes in vitro.

Adult bovine articular chondrocytes were exposed to substance P, neurokinins A and B or substance P fragments, SP1-4, SP1-6 and SP7-11 in vitro. Proteoglycan synthesis was assessed by measuring proteoglycans which were released into the culture medium or incorporated into the cell layer. The intact tachykinins or substance P fragments had no direct effect on proteoglycan synthesis. Nor was total protein production affected. Gel chromatography, under dissociative conditions, revealed that sulphated proteoglycans detected in the medium or cell layer following treatment of chondrocytes with substance P, contained proteoglycans of similar molecular weight to those produced by cells exposed only to diluent controls. Therefore, we conclude that the acceleration of arthritis by substance P does not appear to be mediated through an effect on chondrocyte synthetic function.     

Minimizing the release of proinflammatory and toxic bacterial products within the host: a promising approach to improve outcome in life-threatening infections

Various bacterial components (e.g., endotoxin, teichoic and lipoteichoic acids, peptidoglycans, DNA) induce or enhance inflammation by stimulating the innate immune system and/or are directly toxic in eukariotic cells (e.g., hemolysins). When antibiotics which inhibit bacterial protein synthesis kill bacteria, smaller quantities of proinflammatory or toxic compounds are released in vitro and in vivo than during killing of bacteria by beta-lactams and other cell-wall active drugs. In general, high antibiotic concentrations liberate lower quantities of bacterial proinflammatory or toxic compounds than concentrations close to the minimum inhibitory concentration. In animal models of Escherichia coli Pseudomonas aeruginosa and Staphylococcus aureus peritonitis/sepsis and of Streptococcus pneumoniae meningitis, a lower release of proinflammatory bacterial compounds was associated with a reduced mortality or neuronal injury. Pre-treatment with a bacterial protein synthesis inhibitor reduced the strong release of bacterial products usually observed during treatment with a beta-lactam antibiotic. Data available strongly encourage clinical trials comparing antibiotic regimens with different release of proinflammatory/toxic bacterial products. The benefit of the approach to reduce the liberation of bacterial products should be greatest in patients with a high bacterial load.     

Effects of Phagocytosis by Rabbit Granulocytes on Macromolecular Synthesis and Degradation in Different Species of Bacteria

Phagocytosis and killing of gram-positive Bacillus megaterium and Micrococcus lysodeikticus by granulocytes in vitro is associated with almost immediate cessation of bacterial protein synthesis. By contrast, protein synthesis by Escherichia coli continues after ingestion and killing. After preincubation of E. coli with intact granulocytes for 15 min, when 95% or more of the bacteria can no longer multiply, induction of beta-galactosidase proceeds at rates about half of control values. With disrupted granulocytes, which kill E. coli as rapidly as intact cells, the rate of induction of beta-galactosidase does not fall until after 30 min of preincubation. We attribute the different effects of phagocytosis on the biochemical apparatus of these microorganisms to the different fates of their envelopes. Specifically labeled protein, ribonucleic acid, deoxyribonucleic acid, and lipid of all three species of bacteria and peptidoglycan of E. coli are apparently incompletely degraded during phagocytosis. However, the cell walls of M. lysodeikticus and B. megaterium undergo rapid and almost complete degradation. The resulting structural disintegration of these gram-positive microorganisms must cause extensive biochemical disorganization as well. Our evidence indicates that the E. coli envelope, on the other hand, retains sufficient structural organization to preserve integrated biochemical function for at least 1 h after the bacteria have lost the ability to multiply.     

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms.     

Resistance of Escherichia coli to tetracyclines

1. A strain of Escherichia coli highly resistant to chlortetracycline and partially cross-resistant to tetracycline has been isolated. 2. The nitro-reductase system of the resistant cells was inhibited to a smaller extent by chlortetracycline than was the corresponding enzyme of sensitive cells. 3. The incorporation of leucine in vitro into the ribosomal protein of cell-free preparations from sensitive and resistant cells was equally inhibited by chlortetracycline. 4. Resistant cells accumulated much less chlortetracycline and tetracycline than did sensitive cells when both were cultured in the presence of these drugs. 5. The uptake of tetracycline by both sensitive and resistant E. coli was dependent on the presence of glucose in the medium. 6. Fractionation of cells cultured in medium containing [¹⁴C]chlortetracycline indicated that the largest proportion of radioactivity in sensitive cells was in the fraction consisting mainly of cell-wall material. There was no concentration of radioactivity in any one fraction of the resistant cells. 7. No evidence could be obtained for a specific tetracycline-excretion system in the resistant cells. 8. The significance of these results in relation to current theories of the antibiotic action of and resistance to the tetracycline drugs is discussed.     

Chemoresistance in prostate cancer cells is regulated by miRNAs and Hedgehog pathway

Many prostate cancers relapse due to the generation of chemoresistance rendering first-line treatment drugs like paclitaxel (PTX) ineffective. The present study aims to determine the role of miRNAs and Hedgehog (Hh) pathway in chemoresistant prostate cancer and to evaluate the combination therapy using Hh inhibitor cyclopamine (CYA). Studies were conducted on PTX resistant DU145-TXR and PC3-TXR cell lines and clinical prostate tissues. Drug sensitivity and apoptosis assays showed significantly improved cytotoxicity with combination of PTX and CYA. To distinguish the presence of cancer stem cell like side populations (SP), Hoechst 33342 flow cytometry method was used. PTX resistant DU145 and PC3 cells, as well as human prostate cancer tissue possess a distinct SP fraction. Nearly 75% of the SP cells are in the G0/G1 phase compared to 62% for non-SP cells and have higher expression of stem cell markers as well. SP cell fraction was increased following PTX monotherapy and treatment with CYA or CYA plus PTX effectively reduced their numbers suggesting the effectiveness of combination therapy. SP fraction cells were allowed to differentiate and reanalyzed by Hoechst staining and gene expression analysis. Post differentiation, SP cells constitute 15.8% of total viable cells which decreases to 0.6% on treatment with CYA. The expression levels of P-gp efflux protein were also significantly decreased on treatment with PTX and CYA combination. MicroRNA profiling of DU145-TXR and PC3-TXR cells and prostate cancer tissue from the patients showed decreased expression of tumor suppressor miRNAs such as miR34a and miR200c. Treatment with PTX and CYA combination restored the expression of miR200c and 34a, confirming their role in modulating chemoresistance. We have shown that supplementing mitotic stabilizer drugs such as PTX with Hh-inhibitor CYA can reverse PTX chemoresistance and eliminate SP fraction in androgen independent, metastatic prostate cancer cell lines.     

Further similarities between chloroplast and bacterial ribosomes

Summary Protein synthesis by chloroplasts isolated under aseptic conditions from Phaseolus vulgaris leaves is inhibited by the bacterial antibiotics spectinomycin, lincomycin, and erythromycin; that by chloroplasts from Nicotiana tabacum leaves is inhibited by spectinomycin and lincomycin but not by erythromycin. Protein synthesis by cytoplasmic ribosomes from plants and animals is not inhibited by these compounds, nor is amino acid activation by the soluble fraction from bean chloroplasts. These results suggest that chloroplast ribosomes possess sites which bind several unrelated bacterial antibiotics and support the idea that chloroplasts originated from prokaryotic cells. These antibiotics may be useful in studying the process of chloroplast formation in intact cells.     

Cellular re-distribution of flavin-containing polyamine oxidase in differentiating root and mesocotyl of <Emphasis Type="Italic">Zea mays</Emphasis> L. seedlings

Plant polyamine oxidases (PAOs; EC 1.5.3.11) are hydrogen peroxide-producing enzymes supposedly involved in cell-wall differentiation processes and defence responses. Maize (Zea mays L.) PAO (MPAO) is a 53 kDa secretory glycoprotein, abundant in primary and secondary cell walls of several tissues. Using biochemical, histochemical, ultrastructural and immunocytochemical techniques, the distribution and sub-cellular compartmentalisation of MPAO in the primary root and mesocotyl of seedlings at different maturation stages or after growth under varying light conditions were analysed. In apical root tissues, MPAO immunoreactivity was mainly detected in the cytoplasmic compartment, while a lower immunoreactivity was observed in the cell walls. In the more mature, basal part of the root, intense immunogold labelling was found in the primary and secondary walls of protoxylem precursors and vessels, while endodermal cells and living metaxylem precursors were immunopositive both in their walls and in their thin cytoplasmic compartments. A re-distribution of MPAO protein from the cytoplasm toward the primary and secondary walls was also recognised when immunoreactivity of basal root tissues from 3-day-old seedlings was compared with that detected in 11-day-old tissues. Accordingly, biochemical analyses revealed MPAO entrapment in the extracellular matrix of mature tissues. In the mesocotyl, an enrichment of MPAO immunolabelling in the cell wall of protoxylem, metaxylem and epidermal tissues, as a function of light exposure, was observed. Taken together, these data support the hypothesised role of PAOs in cell-wall maturation. Moreover, the relevant intraprotoplasmic MPAO localisation observed mainly in differentiating root tissues suggests an additional role in intracellular production of hydrogen peroxide.Alessandra Cona and Sandra Moreno have contributed equally to this paper.     

Drug Resistance of Staphylococci

According to an epidemiological survey of drug resistance in Staphylococcus aureus, it was found that there are three types of cross-resistance to macrolide antibiotics (EM, erythromycin: OM, oleandomycin: LM, leucomycin: SP, spiramycin), i.e., resistance to EM, OM, LM, and SP, to EM and OM, and inducible resistance to “EM, OM”. In the inducible strains of S. aureus, EM and OM are active inducers, and the optimal concentrations of the inducers are 0.1 μg/ml and 1.0 μg/ml, respectively. The induction of high resistance (800 μg/ml or more) to both EM and OM occurred within 10 min exposure to 0.1 μg/ml of EM, and the resistance of induced cells was lost after overnight growth in the absence of inducer. After 1 to 3 hr exposure to 1.0 μg/ml of OM, the inducible strains acquired high resistance (100 μg/ml or more) to EM and to a lesser extent, resistance to OM, and the acquired resistance was lost when grown in antibiotic free media. When a known concentration of EM was mixed with the induced cells or with a crude extract from induced cells which had acquired high resistance to EM and OM, the antibiotic activity of EM was still retained in the mixture, indicating that the induced mixture or the extract from the induced cells was incapable of antibiotic (EM) inactivation under the test conditions.     

Effect of Abscisic Acid on Cell-Wall Metabolism in Guard Cells of Vicia faba L.

Cell-wall synthesis in guard cells of Vicia faba L. was examinedusing sonicated epidermal strips incubated with [¹⁴C]glucose.The cell walls of the guard cells incorporated [¹⁴C]glucoseat a lower level in the dark than in the light. Stomatal aperturein the epidermal strips was reduced by application of 1 µmabscisic acid (ABA) in the light but not in the dark. The ABAtreatment reduced the incorporation of [¹⁴C]glucose into thecell walls especially in the light. Fractionation of the labeledcell-wall components revealed that ABA inhibited the synthesisof pectic substances and cellulose, but did not affect hemicellulosesynthesis. Microautoradiographs of the cell-wall fraction ofthe epidermal strips showed that a large amount of radioactivitywas distributed at both ends of the guard cells in the absenceof ABA and that removal of pectic substances from the cell-wallfraction resulted in uniform distribution of the radioactivityin the cell walls of the guard cells. These results indicatedthat the synthesis of pectic substances was active at both endsof the guard cells and was inhibited by ABA. Measurement ofspecific activities of neutral sugars in the guard-cell wallsshowed that polymers composed of galactose underwent activeturnover and that synthesis of glucans was inhibited by ABA.These results revealed a strong correlation between the stomatalmovement and the synthesis of pectic substances and cellulosein the guard cells, suggesting that the cell-wall metabolismin the guard cells may play a role in the regulation of stomatalmovement. (Received October 9, 1987; Accepted March 9, 1988)     

In vitro stimulation of immune functions by lipids derived from macrophages exposed to bacterial peptidoglycan.

It has previously been established that the processing of Bacillus subtilis cell walls by the macrophage-like cell line RAW264 leads to the formation of peptidoglycolipids containing glycopeptides of bacterial origin. In view of the immunologic activities associated with lipophilic muramyl peptide derivatives, lipid extracts derived from macrophages exposed to bacterial cell walls were assayed for mitogenicity. The crude lipid extract derived from RAW264 cells exposed to bacterial cell walls gave a strong mitogenic response when delivered as liposomes to murine splenocytes, whereas lipids derived from macrophages not exposed to bacterial cell walls did not give a mitogenic response. Fractionation of the RAW264 lipids into neutral lipids, glycolipids, and phospholipids demonstrated that mitogenicity was associated primarily with the phospholipid fraction and thus correlated with the presence of the aforementioned peptidoglycolipids. Mitogenicity was specific to the lipids derived from macrophages exposed to bacterial cell walls and was not observed in lipids derived from macrophages that had either been nonspecifically activated by exposure to Con A or had been allowed to phagocytize latex beads. The mitogenic response was found to be dependent on the presence of macrophages in the splenocyte population and to, at least in part, involve macrophage activation.     

Mechanism of natural resistance to kirromycin-type antibiotics in actinomycetes

Abstract The sensitivity of intact cells and subcellular fractions of actinomycetes to kirromycin and pulvomycin was examined. These antibiotics block bacterial protein synthesis by acting on elongation factor Tu (EF-Tu). Two types of natural resistance were encountered in actinomycetes. Some strains were resistant to kirromycin and pulvomycin by virtue of inefficient cellular uptake of these drugs. In 3 strains, kirromycin resistance was attributable to a drug-insensitive EF-Tu. These 3 organisms produce kirromycin-type antibiotics: Streptomyces cinnamomeus, Streptomyces lactamdurans and Streptoverticillium mobaraense synthesize kirrothricin, efrotomycin and pulvomycin, respectively. In S. cinnamomeus and S. lactamdurans resistance to their own antibiotic is due to possession of a nonresponding EF-Tu factor, whereas pulvomycin resistance in Sv. mobaraense is more likely derived from the permeability properties of the cell envelope.     

Heat-induced stabilization of the nuclear matrix: A morphological and biochemical analysis in murine erythroleukemia cells

Using mouse erythroleukemia cells we performed a comprehensive morphological and biochemical study of the nuclear matrix obtained after exposure of isolated nuclei to 37 degrees C or from cells heat shocked in vivo at 43 or 45 degrees C. At the ultrastructural level it was possible to see that in the absence of a 37 degrees C incubation of purified nuclei, the final matrix lacked well-defined nucleolar remnants but a peripheral lamina was clearly visible, as well as a sparse fibrogranular network which was located at the periphery of the structures. On the contrary, after a 37 degrees C nuclear incubation, very electron-dense nucleolar remnants were observed along with an abundant meshwork dispersed throughout the interior of the structures. When intact cells were heat shocked in vivo, electron-dense residual nucleoli were present only when isolated nuclei had been exposed to 37 degrees C in vitro, whereas without such an incubation, they were not as easily distinguishable and appeared less electron-dense. In the latter case the inner network was more evenly distributed. After purified nuclei were incubated at 37 degrees C for 45 min, the high salt and DNase I resistant fraction retained about 18% of the nuclear protein whereas if the heating was omitted protein recovery dropped to 6%. An increase in the recovery of intact structures in the matrix fraction was the main reason for the higher protein recovery. Heating nuclei in vitro further increased the amount of nuclear protein present in the matrix fraction even if intact cells had been heat shocked in vivo. No major qualitative differences were seen when the polypeptide pattern of the various types of nuclear matrices was analyzed on one-dimensional polyacrylamide gels and this finding was further supported by Western blot analysis with a monoclonal antibody to lamins A and C. These results show that heating mainly stabilizes the nucleolar remnants of the matrix and to a lesser extent the inner network, but the morphology of the final structures is different depending on whether the stabilization is performed in vivo or in vitro.