Characterization of the transport system for beta-lactam antibiotics and dipeptides in rat renal brush-border membrane vesicles by photoaffinity labeling

The uptake of the alpha-aminocephalosporin cephalexin into brush-border membrane vesicles from rat renal cortex was independent on an inward H+-gradient in contrast to the intestinal transport system. The transport system could be irreversibly inhibited by photoaffinity labeling. Two binding polypeptides for beta-lactam antibiotics and dipeptides with apparent molecular weights 130,000 and 95,000 were identified by photoaffinity labeling with [3H]benzylpenicillin and N-(4-azido[3,5-3H]benzoyl) derivatives of cephalexin and glycyl-L-proline. The uptake of cephalexin and the labeling of the respective binding proteins was inhibited by beta-lactam antibiotics and dipeptides as with intestinal brush-border membranes. These data indicate that the transport systems for beta-lactam antibiotics and dipeptides in the brush-border membrane from rat kidney and small intestine are similar but not identical.     

Inactivation of the intestinal uptake system for beta-lactam antibiotics by diethylpyrocarbonate

The uptake system for beta-lactam antibiotics in the rabbit small intestine was investigated using brush-border membrane vesicles. After treatment of membrane vesicles with the reagent diethylpyrocarbonate (DEP), the uptake of orally active beta-lactam antibiotics with an alpha-amino group in the substituent at position 6 or 7 of the penam or cephem nucleus was significantly inhibited, whereas DEP-treatment had no inhibitory effect on the uptake of beta-lactam antibiotics without an alpha-amino group. The kinetic analysis revealed an apparent competitive inhibition indicating a decreased affinity of the transport system for alpha-amino-beta-lactam antibiotics. Substrates of the intestinal dipeptide transport system - dipeptides and alpha-amino-beta-lactam antibiotics - could protect the transport system from irreversible inhibition by DEP, whereas beta-lactam antibiotics without an alpha-amino group as well as amino acids or bile acids had no effect. Incubation of DEP-treated vesicles with hydroxylamine led to a partial restoration of the transport activity indicating that DEP may have led to a modification of a histidine residue of the transport protein. From the data presented we conclude that a specific interaction of the alpha-amino group in the substituent at position 6 or 7 of the penam or cephem nucleus presumably with a histidine residue of the transport protein is involved in the translocation process of orally active alpha-amino-beta-lactam antibiotics across the intestinal brush-border membrane.     

Isolation of two new sulfur-containing amino acids from the urine of a cystathioninuric patient.

Two new sulfur-containing amino acids are isolated from the urine of a cystathioninuric patient. They are presumed to be cystathionine sulfoxide and perhydro-1,4-thiazepine-3,5-dicarboxylic acid.     

Resistance to beta-lactam antibiotics and its mediation by the sensor domain of the transmembrane BlaR signaling pathway in Staphylococcus aureus

Staphylococci, a leading cause of infections worldwide, have devised two mechanisms for resistance to beta-lactam antibiotics. One is production of beta-lactamases, hydrolytic resistance enzymes, and the other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by beta-lactam antibiotics. The beta-lactam sensor-transducer (BlaR), an integral membrane protein, binds beta-lactam antibiotics on the cell surface and transduces the information to the cytoplasm, where gene expression is derepressed for both beta-lactamase and penicillin-binding protein 2a. The gene for the sensor domain of the sensor-transducer protein (BlaR(S)) of Staphylococcus aureus was cloned, and the protein was purified to homogeneity. It is shown that beta-lactam antibiotics covalently modify the BlaR(S) protein. The protein was shown to contain the unusual carboxylated lysine that activates the active site serine residue for acylation by the beta-lactam antibiotics. The details of the kinetics of interactions of the BlaR(S) protein with a series of beta-lactam antibiotics were investigated. The protein undergoes acylation by beta-lactam antibiotics with microscopic rate constants (k(2)) of 1-26 s(-1), yet the deacylation process was essentially irreversible within one cell cycle. The protein undergoes a significant conformational change on binding with beta-lactam antibiotics, a process that commences at the preacylation complex and reaches its full effect after protein acylation has been accomplished. These conformational changes are likely to be central to the signal transduction events when the organism is exposed to the beta-lactam antibiotic.     

Simultaneous determination of urinary cystathionine, lanthionine, and their cyclic compounds using liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization

A measurement system for cystathionine (Cysta) lanthionine (LT), and (AEC), and reduced products of their ketimines, perhydro-1,4-thiazapine-3,5-dicarboxylic acid (PHTZDC), 1,4-thiomorpholine-3,5-dicarboxylic acid (TMDA) and 1,4-thiomorpholine-3-carboxylic acid (TMA) in the urine samples of a patient with cystathioninuria and normal human subjects has been developed, using column liquid chromatography-mass spectrometry. The recoveries were about 90–105% for Cysta, LT and AEC, and about 77–87% for PHTZDC, TMDA and TMA after ion-exchange treatment. The concentrations of Cysta and PHTZDC in the urine of a patient with cystathioninuria were much higher compared with those in the urine of normal human subjects. The concentrations of AEC and TMDA were almost the same. LT and TMA could not be detected in the urine samples by this method. This method proved useful for the determination of sulfur-containing amino acids and their cyclic compounds in biological samples.     

Uptake of dipeptide and beta-lactam antibiotics by the basolateral membrane vesicles prepared from rat kidney

The transport of dipeptides and beta-lactam antibiotics across the rat renal basolateral membrane was examined. The initial uptake of glycylsarcosine and cefadroxil by rat renal basolateral membrane vesicles was inhibited by the presence of all the di- and tripeptides and beta-lactam antibiotics that were tested in this study. However, the uptake of both substrates was not inhibited by glycine, an amino acid. The initial uptake of zwitterionic beta-lactam antibiotics, cefadroxil, cephradine, and cephalexin, was stimulated by preloaded glycylsarcosine (countertransport effect). On the other hand, the uptake of dianionic beta-lactam antibiotics, ceftibuten and cefixime, was not affected. A concentration-dependent initial uptake of glycylsarcosine and cefadroxil suggested the existence of a carrier-mediated mechanism, whereas the transport of ceftibuten did not show any saturated uptake. The transporter that participates in the permeation of dipeptides and beta-lactam antibiotics across basolateral membranes showed lower affinity than did PEPT1 and PEPT2. This is the first study that showed an evidence for a peptide transporter, expressed in the rat renal basolateral membrane, that recognizes zwitterionic beta-lactam antibiotics using basolateral membrane vesicles isolated from normal rat kidney.     

Insights into the reaction of beta-lactam antibiotics with copper(II) ions in aqueous and micellar media: kinetic and spectrometric studies

Degradation of beta-lactam antibiotics by means of metallic cations seems to have a very complex chemistry, involving not only the catalytic effect of the metal ion but also complex formation. Many different compounds, such as methylpyrazines, oxazolones, penicilloic, penicillenic, and penicillonic acids, have been reported as degradation products of such antibiotics, although not many details about the progress of the reaction can be found in the literature. Two novel fluorimetric and spectrophotometric methods previously published by the authors, as well as kinetic studies, have been used to propose a possible reaction mechanism for the ampicillin degradation in the presence of copper(II) ions. Likewise, we have proposed the chemical structure required by the beta-lactam antibiotics to develop absorption or fluorescence properties. Kinetics in micellar and aqueous media shows that the copper-ampicillin reaction proceeds through different pathways depending on the reaction medium.     

Diffusion of beta-lactam antibiotics through oligomeric or monomeric porin channels of some gram-negative bacteria

The penetration of anionic beta-lactam antibiotics through porins was evaluated as a mechanism of drug resistance. The major proteins with porin activity were purified from the outer membranes of six bacteria. Three of the six porins were oligomeric porins. The molecular weights of their monomers were 37 kDa from Photobacterium damsela, 42 kDa from Serratia liquefaciens, and 36 kDa from E. coli B. The other three porins were heat-modifiable monomeric porins with molecular weights of 43 kDa from Porphyromonas asaccharolytica and Acinetobacter baumannii, and 37 kDa from Escherichia coli K12.Comparison of the six porin proteins revealed that, independent of their aggregation state, their amino acid content is similar but not identical. All have double the amount of negatively charged amino acids compared with positively charged amino acids. They have a similar polarity and polarity index. Two of the six tested bacteria do not produce beta-lactamase. These two bacteria were sensitive to the different beta-lactams tested. The other four bacteria were resistant to all or to several beta-lactams.A modified liposome swelling method was used for determining the rate of penetration of charged beta-lactam antibiotics. Zwitterionic beta-lactams were found to penetrate into liposomes at a rate that more or less fits their molecular weight, whether the porins are monomeric or oligomeric. The penetration rates of negatively charged beta-lactams are different for oligomeric and monomeric porins. Negatively charged beta-lactams penetrate through oligomeric porins better than estimated by their molecular weight, whereas monomeric porins are less penetrable to negatively charged beta-lactams than estimated by their molecular weight. The contribution of all types of porins to the susceptibility of bacteria to beta-lactam antibiotics (zwitterionic or negatively charged) is apparently doubtful. The porins may decrease or increase bacterial penetration rates to beta-lactams, and only the existence of a potential beta-lactamase that can destroy the penetrating drug will cause resistance.     

Replacement of the essential penicillin-binding protein 5 by high-molecular mass PBPs may explain vancomycin-β-lactam synergy in low-level vancomycin-resistant Enterococcus faecium D366

The mechanism of synergy between vancomycin and penicillin, as well as other beta-lactam antibiotics, was examined in a penicillin-resistant E. faecium (D366) expressing an inducible low-level resistance to vancomycin. It was demonstrated that penicillin per se was not able to reduce the inducible expression of the 39.5-kDa protein (VANB) or the carboxypeptidase activity which are involved in the mechanism of vancomycin resistance of this strain. Assays of competition between 3H-benzylpenicillin and diverse beta-lactam antibiotics suggested as the most likely explanation of the synergy that, once vancomycin resistance has been induced, the high-molecular mass penicillin-binding proteins (PBPs), and possibly PBP1 in particular, which have a high affinity for beta-lactam antibiotics, take over the role of the low-affinity PBP5 which is, in the non-induced strain, responsible for beta-lactam resistance.     

beta-lactam antibiotics as substrates for OCTN2, an organic cation/carnitine transporter

Therapeutic use of cephaloridine, a beta-lactam antibiotic, in humans is associated with carnitine deficiency. A potential mechanism for the development of carnitine deficiency is competition between cephaloridine and carnitine for the renal reabsorptive process. OCTN2 is an organic cation/carnitine transporter that is responsible for Na(+)-coupled transport of carnitine in the kidney and other tissues. We investigated the interaction of several beta-lactam antibiotics with OCTN2 using human cell lines that express the transporter constitutively as well as using cloned human and rat OCTN2s expressed heterologously in human cell lines. The beta-lactam antibiotics cephaloridine, cefoselis, cefepime, and cefluprenam were found to inhibit OCTN2-mediated carnitine transport. These antibiotics possess a quaternary nitrogen as does carnitine. Several other beta-lactam antibiotics that do not possess this structural feature did not interact with OCTN2. The interaction of cephaloridine with OCTN2 is competitive with respect to carnitine. Interestingly, many of the beta-lactam antibiotics that were not recognized by OCTN2 were good substrates for the H(+)-coupled peptide transporters PEPT1 and PEPT2. In contrast, cephaloridine, cefoselis, cefepime, and cefluprenam, which were recognized by OCTN2, did not interact with PEPT1 and PEPT2. The interaction of cephaloridine with OCTN2 was Na(+)-dependent, whereas the interaction of cefoselis and cefepime with OCTN2 was largely Na(+)-independent. Furthermore, the Na(+)-dependent, OCTN2-mediated cellular uptake of cephaloridine could be demonstrated by direct uptake measurements. These studies show that OCTN2 plays a crucial role in the pharmacokinetics and therapeutic efficacy of certain beta-lactam antibiotics such as cephaloridine and that cephaloridine-induced carnitine deficiency is likely to be due to inhibition of carnitine reabsorption in the kidney.     

Novel imidazole substituted 6-methylidene-penems as broad-spectrum beta-lactamase inhibitors

Beta-lactamases are serine and metallo-dependent enzymes produced by the bacteria in defense against beta-lactam antibiotics. Production of class-A, class-B, and class-C enzymes by the bacteria make the use of beta-lactam antibiotics ineffective in certain cases. To overcome resistance to beta-lactam antibiotics, several beta-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are widely used in the clinic in combination with beta-lactam antibiotics. However, single point mutations within these enzymes have allowed bacteria to overcome the inhibitory effect of the commercially approved beta-lactamase inhibitors. Although the commercially available beta-lactamase inhibitor/beta-lactam antibiotic combinations are effective against class-A producing bacteria and many extended spectrum beta-lactamase (ESBL's) producing bacteria they are less effective against class-C enzymes expressing bacteria. To circumvent this problem, based on modeling studies several novel imidazole substituted 6-methylidene-penem derivatives were synthesized and tested against various beta-lactamase producing isolates. The present paper deals with the synthesis and structure-activity relationships (SAR) of these compounds.     

Intestinal absorption of dipeptides and beta-lactam antibiotics. II. Purification of the binding protein for dipeptides and beta-lactam antibiotics from rabbit small intestinal brush border membranes

By photoaffinity labeling of brush border membrane vesicles from rabbit small intestine with photoreactive derivatives of beta-lactam antibiotics and dipeptides, a binding protein for dipeptides and beta-lactam antibiotics with an apparent molecular weight of 127,000 was labeled. The labeled 127 kDa polypeptide could be solubilized with the non-ionic detergents Triton X-100, n-octyl glucoside or CHAPS. If the vesicles were solubilized prior to photoaffinity labeling, no clear incorporation of radioactivity into the 127 kDa polypeptide occurred indicating a loss of binding ability upon solubilization. By affinity chromatography of solubilized brush border membrane proteins on an agarose wheat germ lectin column, the binding protein for dipeptides and beta-lactam antibiotics of Mr 127,000 was retained on the column. With N-acetyl-D-glucosamine the photolabeled binding protein for beta-lactam antibiotics and dipeptides was eluted together with the brush border membrane-bound enzyme aminopeptidase N. Separation from aminopeptidase N and final purification was achieved by anion-exchange chromatography on DEAE-sephacel. Polyclonal antibodies against the purified binding protein were raised in guinea pigs. The photolabeled 127 kDa protein could be precipitated from solubilized brush border membranes with these antibodies. Incubation of brush border membrane vesicles with antiserum prior to photoaffinity labeling significantly reduced the extent of labeling of the 127 kDa protein. Treatment of brush border membrane vesicles with antiserum significantly inhibited the efflux of the alpha-aminocephalosporin cephalexin from the brush border membrane vesicles compared to vesicles treated with preimmune serum. These studies indicate that the binding protein for dipeptides and beta-lactam antibiotics of apparent molecular weight 127,000 in the brush border membrane of rabbit small intestinal enterocytes is directly involved in the uptake process of small peptides and orally active beta-lactam antibiotics across the enterocyte brush border membrane.     

Identification of NAc-HCPC and NAc-beta-CEC, and qualitative analyses of sulphur amino acids in the urine of a patient with cystathioninuria using liquid chromatography/atmospheric pressure ionization mass spectrometry.

Standard sulphur amino acids and various cystathionine metabolites in the urine of a patient with cystathioninuria were analysed using liquid chromatography/mass spectrometry with an atmospheric pressure ionization interface system. Very intense quasi-molecular ions ([M + H]+) of synthetic cystathionine, N-monoacetylcystathionine, perhydro-1,4-thiazepine-3,5-dicarboxylic acid, S-(3-hydroxy-3-carboxy-n-propyl)cysteine, S-(2-carboxyethyl) cysteine, S-(2-hydroxy-2-carboxyethyl)homocysteine, S-(carboxymethyl)homocysteine, N-acetyl-S-(3-hydroxy-3-carboxy-n-propyl)cysteine and N-acetyl-S-(2-carboxyethyl)cysteine were observed by this method. Quasi-molecular ions ([M + H]+) of these sulphur amino acids were observed in the urine sample of the patient with cystathioninuria, and N-acetyl-HCPC and N-acetyl-beta-CEC as N-substituted sulphur amino acids were also identified in the urine of the same patient.     

Intestinal uptake of dipeptides and beta-lactam antibiotics. I. The intestinal uptake system for dipeptides and beta-lactam antibiotics is not part of a brush border membrane peptidase

The uptake of beta-lactam antibiotics into small intestinal enterocytes occurs by the transport system for small peptides. The role of membrane-bound peptidases in the brush border membrane of enterocytes from rabbit and pig small intestine for the uptake of small peptides and beta-lactam antibiotics was investigated using brush border membrane vesicles. The enzymatic activity of aminopeptidase N was inhibited by beta-lactam antibiotics in a non-competitive manner whereas dipeptidylpeptidase IV was not affected. The peptidase inhibitor bestatin led to a strong competitive inhibition of aminopeptidase N whereas the uptake of cephalexin into brush border membrane vesicles was only slightly inhibited at high bestatin concentrations (greater than 1 mM). Modification of brush border membrane vesicles with the histidine-modifying reagent diethyl pyrocarbonate led to a strong irreversible inhibition of cephalexin uptake whereas the activity of aminopeptidase N remained unchanged. A modification of serine residues with diisopropyl fluorophosphate completely inactivated dipeptidylpeptidase IV whereas the transport activity for cephalexin and the enzymatic activity of aminopeptidase N were not influenced. With polyclonal antibodies raised against aminopeptidase N from pig renal microsomes the aminopeptidase N from solubilized brush border membranes from pig small intestine could be completely precipitated; the binding protein for beta-lactam antibiotics and oligopeptides of apparent Mr 127,000 identified by direct photoaffinity labeling with [3H]benzylpenicillin showed no crossreactivity with the aminopeptidase N anti serum and was not precipitated by the anti serum. These results clearly demonstrate that peptidases of the brush border membrane like aminopeptidase N and dipeptidylpeptidase IV are not directly involved in the intestinal uptake process for small peptides and beta-lactam antibiotics and are not a constituent of this transport system. This suggests that a membrane protein of Mr 127,000 is (a part of) the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of small intestinal enterocytes.     

Pseudomonas aeruginosa chromosomal beta-lactamase in patients with cystic fibrosis and chronic lung infection. Mechanism of antibiotic resistance and target of the humoral immune response

The intensive antibiotic treatment of cystic fibrosis (CF) patients with chronic lung infection with Pseudomonas aeruginosa has improved the survival rate and the clinical condition of Danish patients. Acquirement of resistance to anti-pseudomonal antibiotics is one of the main drawbacks of this therapeutic strategy and our results showed the development of resistance of P. aeruginosa to several antibiotics during 25 years of intensive antibiotic treatment. Our studies have been concentrating on the development of resistance to beta-lactam antibiotics. We have shown an association between the development of resistance to beta-lactam antibiotics and the occurrence of high beta-lactamase producing strains and between the MIC of the beta-lactams and the levels of beta-lactamase expression. Partially derepressed mutants, characterized by high basal levels of beta-lactamase with the possibility of induction to even higher levels during treatment with beta-lactam antibiotics, were the most frequent phenotype found among resistant Danish P. aeruginosa CF isolates. We have also shown that the high alginate producing P. aeruginosa isolates, that characterize the chronic lung infection in CF patients, are more susceptible to antibiotics and produce less beta-lactamase than the non-mucoid paired isolates. We propose that the non-mucoid isolates are exposed to a relatively higher antibiotic pressure than the mucoid isolates and therefore, they become easily antibiotic resistant and in consequence produce high levels of beta-lactamase. The beta-lactamase produced by the non-mucoid isolates might play a protective role in the biofilm, defending the mucoid isolates from the action of beta-lactam antibiotics and helping them to maintain their antibiotic susceptibility. We have also shown that beta-lactamase, which is a periplasmic enzyme, can be secreted extracellulary packed in membrane vesicles liberated by high beta-lactamase-producing P. aeruginosa. The continuos presence in the CF lungs of bacteria producing high basal levels of beta-lactamase (partial derepressed) induces a humoral immune response to beta-lactamase. We have shown that antibodies against the chromosomally encoded beta-lactamase (a beta ab) might be considered a marker of the development of resistance to beta-lactam antibiotics. We investigated the humoral immune response to beta-lactamase by quantifying a beta ab specific IgG and IgG subclass antibodies, by investigating the influence of the allotypes on the IgG subclass response and by measuring the avidity of the IgG a beta ab. We found that CF patients with good lung function had in the early stages of the chronic lung infection higher titers of a beta ab of good avidity than patients with poor lung function. Therefore, we raised the hypothesis that some of the a beta ab might have beta-lactamase neutralizing effect, playing a beta-lactamase inhibitor role and improving the effect of the treatment with beta-lactam antibiotics. Finally, we tested our hypothesis in the rat model of chronic lung infection by assessing the effect of a beta ab raised by vaccination with purified chromosomal beta-lactamase on the outcome of the treatment with ceftazidime of bacteria resistant to beta-lactam antibiotics. Our results showed that significantly lower bacterial load and better lung pathology were found in rats with neutralizing antibodies compared to non-immunized rats or rats without neutralizing antibodies. Our findings might be of potential importance for the improvement of the treatment with beta-lactam antibiotics of resistant P. aeruginosa hyperproducing chromosomal beta-lactamase that represent a threat especially for patients with CF and chronic lung infection.     

Identification of identical binding polypeptides for cephalosporins and dipeptides in intestinal brush-border membrane vesicles by photoaffinity labeling

The uptake of a photolabile derivative of the orally effective cephalosporin cephalexin, N-(4-azidobenzoyl)cephalexin, was investigated in brush-border membrane vesicles. The compound was taken up into the intravesicular space and inhibited the active uptake of cephalexin in a concentration-dependent manner. Therefore, this probe interacts with the transport system shared by alpha-aminocephalosporins and dipeptides. Photoaffinity labeling of brush-border membrane vesicles from rat small intestine with N-(4-azido[3,5-3H]benzoyl) derivatives of the cephalosporin cephalexin and the dipeptide glycyl-L-proline resulted in the covalent incorporation of radioactivity into membrane polypeptides with apparent molecular weights of 127,000, 100,000, 94,000 and 86,000, the polypeptide of molecular weight 127,000 being predominantly labeled. The specificity of labeling was demonstrated by a decrease in the labeling of the polypeptide of apparent molecular weight 127,000 in the presence of beta-lactam antibiotics and dipeptides, whereas glucose, taurocholate or amino acids had no effect on the labeling pattern. These data demonstrate an interaction of cephalosporins and dipeptides with a common membrane protein of molecular weight 127,000, which could be a component of the intestinal transport system(s) responsible for the uptake of orally effective cephalosporins and dipeptides.     

Characterization of the beta-lactam antibiotic sensor domain of the MecR1 signal sensor/transducer protein from methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved two mechanisms for resistance to beta-lactam antibiotics. One is production of a beta-lactamase, and the other is that of penicillin-binding protein 2a (PBP 2a). The expression of these two proteins is regulated by the bla and mec operons, respectively. BlaR1 and MecR1 are beta-lactam sensor/signal transducer proteins, which experience acylation by beta-lactam antibiotics on the cell surface and transduce the signal into the cytoplasm. The C-terminal surface domain of MecR1 (MecRS) has been cloned, expressed, and purified to homogeneity. This protein has been characterized by documenting that it has a critical and unusual Nzeta-carboxylated lysine at position 394. Furthermore, the kinetics of interactions with beta-lactam antibiotics were evaluated, a process that entails conformational changes for the protein that might be critical for the signal transduction event. Kinetics of acylation of MecRS are suggestive that signal sensing may be the step where the two systems are substantially different from one another.     

Interaction of beta-lactam antibiotics with penicillin-binding proteins from Bacillus megaterium

The binding properties of 25 beta-lactam antibiotics to Bacillus megaterium membranes have been studied. The affinities of the antibiotics for the penicillin-binding proteins (PBPs) are also reported. We found that PBP 4 has the highest affinity for nearly all the antibiotics studied whereas PBP 5 has the lowest affinity. Both PBP 4 and PBP 5 appear to be dispensable for the maintenance of bacterial growth and survival and appear to be DD-carboxypeptidases. Only the beta-lactam cefmetazol bound preferentially to PBP 5 and has been used to study the inhibition of DD-carboxypeptidase. Comparative studies with beta-lactam that simultaneously result in (a) binding to PBPs 1 and 3, (b) inhibition of cell growth and (c) lysis, stressed the importance of PBPs 1 and 3 for cell growth and survival.     

Diversity of penicillin-binding proteins. Resistance factor FmtA of Staphylococcus aureus

Antibiotic-resistant Staphylococcus aureus is a major concern to public health. Methicillin-resistant S. aureus strains are completely resistant to all beta-lactams antibiotics. One of the main factors involved in methicillin resistance in S. aureus is the penicillin-binding protein, PBP2a. This protein is insensitive to inactivation by beta-lactam antibiotics such as methicillin. Although other proteins are implicated in high and homogeneous levels of methicillin resistance, the functions of these other proteins remain elusive. Herein, we report for the first time on the putative function of one of these proteins, FmtA. This protein specifically interacts with beta-lactam antibiotics forming covalently bound complexes. The serine residue present in the sequence motif Ser-X-X-Lys (which is conserved among penicillin-binding proteins and beta-lactamases) is the active-site nucleophile during the formation of acyl-enzyme species. FmtA has a low binding affinity for beta-lactams, and it experiences a slow acylation rate, suggesting that this protein is intrinsically resistant to beta-lactam inactivation. We found that FmtA undergoes conformational changes in presence of beta-lactams that may be essential to the beta-lactam resistance mechanism. FmtA binds to peptidoglycan in vitro. Our findings suggest that FmtA is a penicillin-binding protein, and as such, it may compensate for suppressed peptidoglycan biosynthesis under beta-lactam induced cell wall stress conditions.     

Formation and degradation of dicarboxylic acids in relation to alterations in fatty acid oxidation in rats.

Dicarboxylic acids are excreted in urine when fatty acid oxidation is increased (ketosis) or inhibited (defects in beta-oxidation) and in Reye's syndrome. omega-Hydroxylation and omega-oxidation of C6-C12 fatty acids were measured by mass spectrometry in rat liver microsomes and homogenates, and beta-oxidation of the dicarboxylic acids in liver homogenates and isolated mitochondria and peroxisomes. Medium-chain fatty acids formed large amounts of medium-chain dicarboxylic acids, which were easily beta-oxidized both in vitro and in vivo, in contrast to the long-chain C16-dicarboxylic acid, which was toxic to starved rats. Increment of fatty acid oxidation in rats by starvation or diabetes increased C6:C10 dicarboxylic acid ratio in rats fed medium-chain triacylglycerols, and increased short-chain dicarboxylic acid excretion in urine in rats fed medium-chain dicarboxylic acids. Valproate, which inhibits fatty acid oxidation and may induce Reye like syndromes, caused the pattern of C6-C10-dicarboxylic aciduria seen in beta-oxidation defects, but only in starved rats. It is suggested, that the origin of urinary short-chain dicarboxylic acids is omega-oxidized medium-chain fatty acids, which after peroxisomal beta-oxidation accumulate as C6-C8-dicarboxylic acids. C10-C12-dicarboxylic acids were also metabolized in the mitochondria, but did not accumulate as C6-C8-dicarboxylic acids, indicating that beta-oxidation was completed beyond the level of adipyl CoA.