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PubMed1995

In Situ Detection of nDNA Fragmentation during the Differentiation of Tracheary Elements in Higher Plants.

Programmed cell death (pcd) is thought to occur during the autolysis of xylem vessels. Although several ultrastructural aspects of this differentiation process have been characterized, certain key aspects of this process remain unsolved. Here we demonstrate in pea (Pisum sativum) that nuclei of vessel elements undergoing pcd contain fragmented nDNA. This finding may provide evidence for the activation of a DNA degradation mechanism prior to the final disruption of the nucleus that occurs during the autolysis stage of this differentiation process. In situ detection of DNA fragmentation in nuclei of vessel elements undergoing pcd may therefore suggest that this death process involves the activation of a mechanism for DNA degradation, similar to that activated during apoptosis in animal cells. In addition, this differentiation process may serve as a useful positive control for the in situ detection of pcd in other developmental pathways and during the hypersensitive response of plants to avirulent pathogens.

Journal Article↗
PubMed1998

Proteasome inhibitors prevent tracheary element differentiation in zinnia mesophyll cell cultures

To determine whether proteasome activity is required for tracheary element (TE) differentiation, the proteasome inhibitors clasto-lactacystin beta-lactone and carbobenzoxy-leucinyl-leucinyl-leucinal (LLL) were used in a zinnia (Zinnia elegans) mesophyll cell culture system. The addition of proteasome inhibitors at the time of culture initiation prevented differentiation otherwise detectable at 96 h. Inhibition of the proteasome at 48 h, after cellular commitment to differentiation, did not alter the final percentage of TEs compared with controls. However, proteasome inhibition at 48 h delayed the differentiation process by approximately 24 h, as indicated by examination of both morphological markers and the expression of putative autolytic proteases. These results indicate that proteasome function is required both for induction of TE differentiation and for progression of the TE program in committed cells. Treatment at 48 h with LLL but not clasto-lactacystin beta-lactone resulted in partial uncoupling of autolysis from differentiation. Results from gel analysis of protease activity suggested that the observed incomplete autolysis was due to the ability of LLL to inhibit TE cysteine proteases.

Journal Article↗
PubMed1983

Lipase in the Lipid Bodies of Corn Scutella during Seedling Growth.

In the scutella of corn (Zea mays), lipase activity is absent in ungerminated seeds and increases during seedling growth. At the peak stage of lipolysis, about 50% of the lipase activity is recovered in the lipid body fraction after flotation centrifugation. The lipase is tightly bound to the lipid bodies, and resists solubilization by repeated washing with buffers or NaCl solutions. Isolated lipid bodies undergo autolysis of internal triacylglycerols, resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies have been extracted with diethyl ether, the lipase is recovered in the membrane fraction. The lipase has an optimal activity at pH 7.5 in the autolysis of lipid bodies, or on trilinolein or N-methylindoxylmyristate. Of the various acylglycerols examined, the enzyme is active only on acylglycerols of linoleic and oleic acids which are the major fatty acid constituents of corn oil. The activity is not greatly affected by NaCl, CaCl(2), or pretreatment of the enzyme with p-chloromercuribenzoate or mersalyl, and detergents abolish the activity. The enzyme hydrolyzes trilinolein completely to fatty acids; during the course of reaction, there is little accumulation of di- or mono-linolein.

Journal Article↗
PubMed1987

Identification of polysaccharide hydrolases involved in autolytic degradation of zea cell walls.

Cell walls of Zea mays (cv L.G.11) seedlings labeled with (14)C were treated with alpha-amylase from Bacillus subtilis to remove starch and mixed linkage glucans. These walls released arabinose, xylose, galactose, and galacturonic acid in addition to glucose when they were allowed to autolyze. Methylation analysis was performed on samples of wall which had been incubated autolytically and the results indicated that degradation of the major polymer of the wall, the glucoarabinoxylan, had occurred. A number of glycanases could be dissociated from the wall by use of 3 m LiCL. The proteins which were released were found to contain a number of exoglycosidase activities in addition to being effective in degrading the polysaccharide substrates, araban, xylan, galactan, laminarin, mannan, and polygalacturonic acid. The effects of these enzymes on the wall during autolysis appear to result from endo-activity in addition to exo-activity. The structural changes that occurred in the cell walls during autolysis were found to be related to the changes previously found to occur in cell walls during auxin induced extension.

Journal Article↗
PubMed2001

The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity.

The contribution of induced fit to enzyme specificity has been much debated, although with little experimental data. Here we probe the effect of induced fit on enzyme specificity using the trypsin(ogen) system. BPTI is known to induce trypsinogen to assume a trypsinlike conformation. Correlations are observed between BPTI affinity and the values of k(cat)/K(m) for the hydrolysis of two substrates by eight trypsin(ogen) variants. The slope of both correlations is -1.8. The crystal structures of the BPTI complexes of four variant trypsinogens were also solved. Three of these enzymes, K15A, DeltaI16V17/D194N, and DeltaI16V17/Q156K trypsinogen, are 10- to 100-fold more active than trypsinogen. The fourth variant, DeltaI16V17 trypsinogen, is the lone outlier in the correlations; its activity is lower than expected based on its affinity for BPTI. The S1 site and oxyanion hole, formed by segments 184A-194 and 216-223, are trypsinlike in all of the enzymes. These structural and kinetic data confirm that BPTI induces an active conformation in the trypsin(ogen) variants. Thus, changes in BPTI affinity monitor changes in the energetic cost of inducing a trypsinlike conformation. Although the S1 site and oxyanion hole are similar in all four variants, the N-terminal and autolysis loop (residues 142-152) segments have different interactions for each variant. These results indicate that zymogen activity is controlled by a simple conformational equilibrium between active and inactive conformations, and that the autolysis loop and N-terminal segments control this equilibrium. Together, these data illustrate that induced fit does not generally contribute to enzyme specificity.

Amino Acid Substitution↗
PubMed1999

Drug resistance mutations can effect dimer stability of HIV-1 protease at neutral pH.

The monomer-dimer equilibrium for the human immunodeficiency virus type 1 (HIV-1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in beta-sheet structure and a lower degree of ANS binding. An autolysis-resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild-type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 microM at pH 7.0 and was below the limit of measurement (approximately 100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 microM at pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer-dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug-resistant HIV-1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis-resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.

Circular Dichroism↗
PubMed1990

Full-thickness human skin explants for testing the toxicity of topically applied chemicals.

This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin. In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically. The human skin specimens were discards from a variety of surgical procedures. They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant. The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C. In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis. Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants. Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells. The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium. During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity. Incubation (for 4 or 24 h at 36 degrees C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage. This reduction was probably caused by autolysis of many of the vacuolated cells. Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type. The storage type seemed to be caused by autolysis of cell components. The toxicant type seemed to be caused by an invagination of the plasma membrane. Only toxicant-type vacuoles increased appreciably in number when skin explants were exposed to mustard, and to other toxicants.

Administration, Topical↗
PubMed2006

Proteolytic enzyme activities in Cheddar cheese juice made using lactococcal starters of differing autolytic properties.

AIMS: To determine proteolytic enzyme activities released in Cheddar cheese juice manufactured using lactococcal starter strains of differing autolytic properties. METHODS AND RESULTS: The activities of residual chymosin, cell envelope proteinase and a range of intracellular proteolytic enzymes were determined during the first 70 days of ripening when starter lactococci predominate the microbial flora. In general, in cell free extracts (CFE) of the strains, the majority of proteolytic activities was highest for Lactococcus lactis HP, intermediate for L. lactis AM2 and lowest for L. lactis 303. However, in cheese juice, as ripening progressed, released proteolytic activities were highest for the highly autolytic strain L. lactis AM2, intermediate for L. lactis 303 and lowest for L. lactis HP. CONCLUSIONS: These results indicate that strain related differences in autolysis influence proteolytic enzyme activities released into Cheddar cheese during ripening. No correlation was found between proteolytic potential of the starter strains measured in CFE prior to cheese manufacture and levels of activities released in cheese juice. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings further support the importance of autolysis of lactococcal starters in determining the levels of proteolytic activities present in cheese during initial stages of ripening.

Cheese↗
PubMed2005

Pseudomonas aeruginosa fimL regulates multiple virulence functions by intersecting with Vfr-modulated pathways.

Virulence of Pseudomonas aeruginosa involves the co-ordinate expression of a range of factors including type IV pili (tfp), the type III secretion system (TTSS) and quorum sensing. Tfp are required for twitching motility, efficient biofilm formation, and for adhesion and type III secretion (TTS)-mediated damage to mammalian cells. We describe a novel gene (fimL) that is required for tfp biogenesis and function, for TTS and for normal biofilm development in P. aeruginosa. The predicted product of fimL is homologous to the N-terminal domain of ChpA, except that its putative histidine and threonine phosphotransfer sites have been replaced with glutamine. fimL mutants resemble vfr mutants in many aspects including increased autolysis, reduced levels of surface-assembled tfp and diminished production of type III secreted effectors. Expression of vfr in trans can complement fimL mutants. vfr transcription and production is reduced in fimL mutants whereas cAMP levels are unaffected. Deletion and insertion mutants of fimL frequently revert to wild-type phenotypes suggesting that an extragenic suppressor mutation is able to overcome the loss of fimL. vfr transcription and production, as well as cAMP levels, are elevated in these revertants, while Pseudomonas quinolone signal (PQS) production is reduced. These results suggest that the site(s) of spontaneous mutation is in a gene(s) which lies upstream of vfr transcription, cAMP, production, and PQS synthesis. Our studies indicate that Vfr and FimL are components of intersecting pathways that control twitching motility, TTSS and autolysis in P. aeruginosa.

Bacterial Proteins↗
PubMed1988

The enzymatic activity of proteinase K is controlled by calcium.

The fungal proteinase K (EC 3.4.21.14) is a very potent unusually stable member of the subtilisin family. Its X-ray structure determined at 0.15-nm resolution shows two bound Ca2+ ions. Ca1 is in near-ideal pentagonal bipyramidal configuration with Asp200 carboxylate and Pro175 peptide C = O in an apical, and Val177 peptide C = O and four water molecules in an equatorial position, whereas Ca2 displays incomplete octahedral coordination with the carboxylate of Asp260, the peptide C = O of Val16 and the two water molecules. Scatchard analysis of the titration of Ca2+-free proteinase K with Ca2+ yields a single dissociation constant (7.6 +/- 2.5) x 10(-8) M associated with the tightly bound Ca1 whereas Ca2 is so weakly bound that it cannot be titrated. If proteinase K is depleted of Ca2+ by treatment with EDTA, followed by gel filtration, its enzymatic activity drops within 6 h to 20% of its original value, without autolysis. Addition of excess Ca2+ immediately raises the residual activity to 28%, but full activity is not achieved. Removal of Ca2+ triggers a conformational change of the substrate recognition site because there is a direct connection, via secondary structure hydrogen bonds, between the Ca1 binding site and the substrate-recognition site. This is indicated further by circular dichroism and fluorescence-spectroscopic data, and by reversed-phase FPLC, carried out in the presence and absence of Ca2+, but the overall structure of the enzyme is not affected. Depletion of Ca2+ also influences binding of longer peptide inhibitors of the chloromethane type, it increases the rate of autolysis after about 48 h, it reduces the thermal stability (measured by activity tests from 65 degrees C to 46 degrees C), and it enhances the deactivation by 8 M urea which inactivates to only 65%, whereas sodium dodecyl sulfate totally inactivates at a concentration of 12.5%.

Calcium↗
PubMed2006

Ca2+ activation of diffusible and bound pools of mu-calpain in rat skeletal muscle.

Skeletal muscle fibres contain ubiquitous and muscle-specific calcium-dependent proteases known as calpains. During normal activity, intracellular [Ca(2+)] in muscle fibres increases to high levels ( approximately 2-20 microm), and it is not apparent how this can be reconciled with the activation properties of the calpains. Calpains evidently do not cause widespread proteolytic damage within muscle fibres under normal circumstances, but do have a role in necrosis in dystrophic muscle fibres. In this study, we examined the in situ localization and regulation of calpains in muscle fibres in order to identify how they are attuned to normal function. The sarcolemma of individual muscle fibres of the rat was removed by microdissection (fibre 'skinning') in order to determine the compartmentalization and diffusibility of the two most Ca(2+)-sensitive calpains, mu-calpain and calpain-3, and to permit precise manipulation of cytoplasmic [Ca(2+)] under physiological in situ conditions. Passive force production in stretched fibres, which indicates the patency of the important elastic structural protein titin, was used as a sensitive assay of the amount of diffusible proteolytic activity in individual fibre segments and in muscle homogenates at set [Ca(2+)]. All calpain-3 is bound tightly within a fibre, whereas most mu-calpain ( approximately 0.2 microm) is initially freely diffusible in the cytoplasm at resting [Ca(2+)] but binds within seconds at high [Ca(2+)]. [Ca(2+)] has to be raised to >/= 2 microm for >/= 1 min to initiate detectable autolysis of mu-calpain and to activate appreciable proteolytic activity. If the [Ca(2+)] is raised sufficiently for long enough to initiate substantial autolysis of mu-calpain, the Ca(2+) sensitivity of the proteolytic activity is greatly increased, and it remains active even at 300 nm Ca(2+), with activity only ceasing if the [Ca(2+)] is decreased to approximately 50 nm Ca(2+), close to the normal resting [Ca(2+)]. These findings on the Ca(2+)- and time-dependent binding, autolytic and proteolytic properties of mu-calpain under physiological conditions demonstrate how it is precisely attuned to avoid uncontrolled proteolytic activity under normal circumstances, and indicate why it could lead to substantial proteolytic damage if resting or localized [Ca(2+)] is elevated, as is likely to occur after eccentric contraction and in dystrophic muscle.

Animals↗
PubMed2006

Action of disinfectant quaternary ammonium compounds against Staphylococcus aureus.

Mode-of-action studies concluded that alkyldimethylbenzylammonium chloride (ADBAC) (a blend of C(12), C(14) and C(16) alkyl homologues) and didecyldimethylammonium chloride (DDAC) are both membrane-active agents, possessing subtly different modes of action reflecting early cell interactions against Staphylococcus aureus. ADBAC and DDAC exhibited similar MIC behaviors from 0.4 ppm to 1.8 ppm over an inoculum range of 1 x 10(5) to 1 x 10(9) CFU/ml at 35 degrees C. For ADBAC and DDAC, an increased rapidity of killing against S. aureus (final concentration, 2 x 10(9) CFU/ml) was observed at 35 degrees C compared to 25 degrees C. Concentration exponents (eta) for killing were <2.5 for both agents, and temperature influenced the eta value. Examination of leakage and kill data suggested that a single leakage marker was not indicative of cell death. ADBAC and DDAC possessed Langmuir (L4) and high-affinity (H3/4) uptake isotherms, respectively. ADBAC molecules formed a single monolayer of coverage of cells at the end of primary uptake, and DDAC formed a double monolayer. Rapid cell leakage occurred at bactericidal concentrations, with total depletion of the intracellular potassium and 260-nm-absorbing pools released in this strict order. Autolysis was observed for ADBAC and DDAC at concentrations of 9 mug/ml (0.0278 mM and 0.0276 mM, respectively) and above, together with the depletion of approximately 30% of the internal potassium pool. Autolysis contributed to ADBAC and DDAC lethality, although high biocide concentrations may have inhibited autolytic enzyme activity.

Bacteriolysis↗
PubMed1977

Bacteriolytic action of fluoride ions.

Bacillus subtilis, Neisseria subflava, and LYT coccus were found to undergo massive lysis after growth in media containing 0.01 to 10 mM NaF. When cells of these organisms were transferred from late-exponential-phase cultures to 0.02 M sodium phosphate buffer plus 0.1 M KCl, they underwent spontaneous autolysis. Cells grown in media with fluoride were more liable to autolysis, and walls isolated from them also showed enhanced autolytic sensitivity, even though added fluoride did not directly stimulate autolysins. Sporadic or partial lysis occurred in populations of Streptococcus sanguis and Streptococcus mutans BHT or LM-7 after growth in fluoridated media. Most bacteria that were tested did not undergo fluoride-induced lysis. However, cells of all test bacteria were found to have reduced amounts of peptidoglycan per unit of cell weight when grown in the presence of fluoride. Incorporation of labeled lysine or glucosamine into peptidoglycan (Park-Hancock residue) was stimulated, instead of inhibited, by fluoride. However, fluoride also stimulated the loss of radioactivity from Park-Hancock residues of cells that had previously incorporated labeled lysine or glucosamine. Thus, fluoride appeared to enhance peptidoglycan turnover, and this turnover reduced the peptidoglycan contents of all bacteria tested, but induced lysis in only those bacteria that normally have highly active autolytic systems.

Bacteria↗
PubMed1996

Bactericidal activities of cefprozil, penicillin, cefaclor, cefixime, and loracarbef against penicillin-susceptible and -resistant Streptococcus pneumoniae in an in vitro pharmacodynamic infection model.

We examined the bactericidal activities of penicillin, cefprozil, cefixime, cefaclor, and loracarbef against three clinical isolates of Streptococcus pneumoniae which were susceptible, moderately susceptible, and resistant to penicillin. An in vitro two-compartment glass infection model was used to simulate human pharmacokinetics in the presence of bacteria. Also, changes in organism susceptibility and development of resistant subpopulations were evaluated. Simulated pediatric dosage regimens and target peak concentrations in the central compartment were as follows: penicillin V-potassium, 26 mg/kg of body weight every 6 h (q6h) and 14 micrograms/ml; cefaclor, 13.4 mg/kg q8h and 16 micrograms/ml; loracarbef, 15 mg/kg q12h and 19 micrograms/ml; cefprozil, 15 mg/kg q12h and 11 micrograms/ml; and cefixime, 8mg/kg q24h and 4 micrograms/ml. Targeted half-lives of each agent were 1 h for penicillin, cefaclor, and loracarbef; 1.3 h for cefprozil; and 3.5 h for cefixime. Growth controls were performed at two different pump rates, 0.8 and 2.0 ml/min (half-lives = 3.5 and 1 h, respectively). Each isolate demonstrated autolysis at the lower rate which was attributed to a decreased supply of fresh nutrients available to the organisms in the infection compartment. Against the susceptible isolate, the time to 99.9% killing was statistically significant between penicillin V-potassium and both cefaclor and cefixime (P < 0.029). Loracarbef never achieved a 99.9% reduction in the inoculum. At 48 h penicillin, cefprozil, and cefaclor were equivalent in extent of killing. Against the intermediately resistant isolate, cefprozil was superior to all other regimens with respect to rate of killing (P < 0.013) and extent of killing at 24 h (P < 0.0003). At 48 h penicillin, cefprozil, and cefaclor were equivalent in extent of killing. All of the regimens exhibited inferior activity against this penicillin-resistant isolate. A 99.9% kill was never obtained with any of the regimens, nor was there an appreciable decrease in the colony counts. In conclusion, it appears that cefprozil, penicillin, and cefaclor are effective therapies against sensitive and even intermediately sensitive isolates of S. pneumoniae. However, none of the oral therapies appear to be of any benefit against penicillin-resistant isolates. The in vitro model may be an effective tool in evaluating other multiple-dose therapies against this fastidious organism, since the continual supply of fresh medium maintains the viability of S. pneumoniae with minimal stationary-phase autolysis.

Cefaclor↗
PubMed2006

Two 2[5H]-furanones as possible signaling molecules in Lactobacillus helveticus.

Two 2[5H]-furanones, in association with medium-chain fatty acids, were released in whey by Lactobacillus helveticus exposed to oxidative and heat stresses. This species plays an important role in cheese technology, particularly for Swiss-type cheeses and Grana cheese. Moreover, it significantly contributes to cheese ripening by means of an early autolysis and the release of enzymes during processing. Experimental evidence of the involvement of the two 2[5H]-furanones, detected by a gas chromatography-mass spectrometry/solid-phase microextraction technique, in the autolysis phenomenon has been obtained. Zymograms performed by using renaturing sodium dodecyl sulfate-polyacrylamide gels were used to detect the bioactivity of the supernatants containing the two furanones on fresh cells of the same strain. In addition to bands corresponding to known autolysins, new autolysins were detected concomitant with the exposure of Lactobacillus helveticus to the supernatants, which can be regarded as conditioned media (CM), and to a commercial furanone, 5-ethyl-3-hydroxy-4-methyl-2[5H]-furanone (HEMFi), having spectral data similar to those of the newly described 2[5H]-furanones. Morphological changes were observed when fresh cells were exposed to CM containing the two 2[5H]-furanones and HEMFi. The two furanones produced by Lactobacillus helveticus, which met a number of criteria to be included in cell-cell signaling molecules, have a presumptive molecular mass lower than those of already known 3[2H]-furanones having an autolytic activity and being produced by gram-negative bacteria. Moreover, they present a different chemical structure with respect to the furanones already identified as products of Lactococcus lactis subsp. cremoris or to those identified in some cheeses with Lactobacillus helveticus as a starter culture.

Cheese↗
PubMed2004

Morphotypic conversion in Listeria monocytogenes biofilm formation: biological significance of rough colony isolates.

Adherence to a stainless steel surface selected isolates of Listeria monocytogenes with enhanced surface colonization abilities and a change in phenotype from the common smooth colony morphology to a succession of rough colony morphotypes. Growth in broth culture of the best-adapted, surface-colonizing rough colony morphotype gave a smooth colony revertant. Comparative analysis revealed that the smooth and rough variants had similar phenotypic and biochemical characteristics (e.g., identical growth rates and tolerances to antibiotics and environmental stressors). Rough colony isolates, however, failed to coordinate motility or induce autolysis. The defect in autolysis of rough colony isolates, which involved impaired cellular localization of several peptidoglycan-degrading enzymes, including cell wall hydrolase A (CwhA), suggested a link to a secretory pathway defect. The genetic basis for the impairment was studied at the level of the accessory secretory pathway component SecA2. DNA sequencing of the secA2 gene in smooth and rough colony isolates found no mutations in the coding or promoter regions. Analysis of SecA2 expression with an integrated secA2-FLAG tag construct found the protein to be upregulated in the rough and revertant backgrounds compared to the parental smooth colony isolate. A compensatory mechanism involving the SecA2 secretion pathway components is postulated to control smooth to rough interconversion of L. monocytogenes. Such phenotypic variation may enhance the ability of this opportunistic pathogen to colonize environments as diverse as processing surfaces, food products, and animal hosts.

Animals↗
PubMed2006

Alanine esters of enterococcal lipoteichoic acid play a role in biofilm formation and resistance to antimicrobial peptides.

Enterococcus faecalis is among the predominant causes of nosocomial infections. Surface molecules like d-alanine lipoteichoic acid (LTA) perform several functions in gram-positive bacteria, such as maintenance of cationic homeostasis and modulation of autolytic activities. The aim of the present study was to evaluate the effect of d-alanine esters of teichoic acids on biofilm production and adhesion, autolysis, antimicrobial peptide sensitivity, and opsonic killing. A deletion mutant of the dltA gene was created in a clinical E. faecalis isolate. The absence of d-alanine in the LTA of the dltA deletion mutant was confirmed by nuclear magnetic resonance spectroscopy. The wild-type strain and the deletion mutant did not show any significant differences in growth curve, morphology, or autolysis. However, the mutant produced significantly less biofilm when grown in the presence of 1% glucose (51.1% compared to that of the wild type); adhesion to eukaryotic cells was diminished. The mutant absorbed 71.1% of the opsonic antibodies, while absorption with the wild type resulted in a 93.2% reduction in killing. Sensitivity to several cationic antimicrobial peptides (polymyxin B, colistin, and nisin) was considerably increased in the mutant strain, confirming similar results from other studies of gram-positive bacteria. Our data suggest that the absence of d-alanine in LTA plays a role in environmental interactions, probably by modulating the net negative charge of the bacterial cell surface, and therefore it may be involved in the pathogenesis of this organism.

Alanine↗
PubMed1982

High-molecular-weight hemolysin of Clostridium tetani.

Clostridium tetani excretes hemolysins of two size classes, a high-molecular-weight hemolysin (HMH), which was eluted near void volume of a Sepharose 6B column, and conventional tetanolysin (molecular weight, approximately 50,000). The total hemolysin activity in the culture supernatant increased sharply with growth of bacteria and remained at a high level during autolysis. The content of HMH, however, decreased from 41% at 4 h of culture to 0.4% at the early stage of autolysis. The cell bodies also exhibited hemolytic activity, 70% of which could be solubilized and separated into HMH and the 50,000 Mr tetanolysin as extracellular hemolysins. The activity ratio of HMH to the total solubilized hemolysins was 0.45, on the average, at 6 h of culture but was 0.23 at the middle of logarithmic growth. Partially purified HMH from both sources appeared as broken pieces of cytoplasmic membranes under an electron microscope. The ratio of proteins to phospholipids in HMH was found to 3.26, a value similar to that in cell membrane. The total cell hemolytic activity decreased by 90 or 75% upon addition of chloramphenicol or anti-tetanolysin serum, respectively, into a 6-h-old culture of bacteria. It is suggested that HMH is a complex of tetanolysin with a membrane fragment and releases the conventional tetanolysin during bacterial culture.

Bacterial Toxins↗