Antimicrobial and anticoagulant activities of N-chlorotaurine, N,N-dichloro-2,2-dimethyltaurine, and N-monochloro-2,2-dimethyltaurine in human blood.

The aim of this study was to determine the potential application of N-chlorotaurine (NCT), N,N-dichloro-2,2-dimethyltaurine (NVC-422), and N-monochloro-2,2-dimethyltaurine (NVC-612) as catheter lock solutions for the prevention of catheter blockage and catheter-related bloodstream infections by testing their anticoagulant and broad-spectrum antimicrobial activities in human blood. NCT, NVC-422, NVC-612, and control compounds were serially diluted in fresh human blood to evaluate the effects on prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen, and direct thrombin inhibition. Quantitative killing assays against pathogens, including methicillin-resistant Staphylococcus aureus, Escherichia coli, and Candida albicans, were performed in the presence of heparin and human blood. NCT and NVC-612 (1.38 mM each) and 1.02 mM NVC-422 prolonged prothrombin time (Quick value, 17 to 30%), activated partial thromboplastin time 3- to 4-fold to 76 to 125 s, and thrombin time 2- to 4-fold to 34 to 68 s. Fibrinogen decreased from 258 to 283 mg/dl (range of controls) to <40 mg/dl. No direct thrombin inhibition was observed by NVC-422 or NVC-612. Heparin did not influence the bactericidal activity of NCT. The microbicidal activities of NCT, NVC-422, and NVC-612 were maintained in diluted human blood. NCT, NVC-612, and NVC-422 have broad-spectrum antimicrobial activity in blood and anticoagulant activity targeting both intrinsic and extrinsic pathways of the coagulation system. These properties support their application as catheter lock solutions.

Biosynthesis of D-alanyl-lipoteichoic acid: the tertiary structure of apo-D-alanyl carrier protein.

The D-alanylation of lipoteichoic acid (LTA) allows the Gram-positive organism to modulate its surface charge, regulate ligand binding, and control the electromechanical properties of the cell wall. The incorporation of D-alanine into LTA requires the D-alanine:D-alanyl carrier protein ligase (AMP-forming) (Dcl) and the carrier protein (Dcp). The high-resolution solution structure of the 81-residue (8.9 kDa) Dcp has been determined by multidimensional heteronuclear NMR. An ensemble of 30 structures was calculated using the torsion angle dynamics approach of DYANA. These calculations utilized 3288 NOEs containing 1582 unique nontrivial NOE distance constraints. Superposition of residues 4-81 on the mean structure yields average atomic rmsd values of 0.43 +/- 0.08 and 0.86 +/- 0.09 A for backbone and non-hydrogen atoms, respectively. The solution structure is composed of three alpha-helices in a bundle with additional short 3(10)- and alpha-helices in intervening loops. Comparisons of the three-dimensional structure with the acyl carrier proteins involved in fatty acid, polyketide, and nonribosomal peptide syntheses support the conclusion that Dcp is a homologue in this family. While there is conservation of the three-helix bundle fold, Dcp has a higher enthalpy of unfolding and no apparent divalent metal binding site(s), features that distinguish it from the fatty acid synthase acyl carrier protein of Escherichia coli. This three-dimensional structure also provides insights into the D-alanine ligation site recognized by Dcl, as well as the site which may bind the poly(glycerophosphate) acceptor moiety of membrane-associated LTA.

Biosynthesis of the glycolipid anchor in lipoteichoic acid of Staphylococcus aureus RN4220: role of YpfP, the diglucosyldiacylglycerol synthase.

In Staphylococcus aureus RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety. The gene (ypfP) which encodes diglucosyldiacylglycerol synthase was recently cloned from Bacillus subtilis and expressed in Escherichia coli (P. Jorasch, F. P. Wolter, U. Zahringer, and E. Heinz, Mol. Microbiol. 29:419-430, 1998). To define the role of ypfP in this strain of S. aureus, a fragment of ypfP truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate ypfP. Chloramphenicol-resistant (ypfP::cat) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol. Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in S. aureus providing glycolipid for LTA assembly. In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol. Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed. This inhibition was antagonized by either L- or D-alanine. Moreover, viability of the mutant at 37 degrees C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to <0.1%. Addition of 0.1% D-glucose to the phosphate-saline ensured viability under these conditions. The autolysis of the ypfP::cat mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain. Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells. Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol.

Defects in D-alanyl-lipoteichoic acid synthesis in Streptococcus mutans results in acid sensitivity.

In the cariogenic organism, Streptococcus mutans, low pH induces an acid tolerance response (ATR). To identify acid-regulated proteins comprising the ATR, transposon mutagenesis with the thermosensitive plasmid pGh9:ISS1 was used to produce clones that were able to grow at neutral pH, but not in medium at pH 5.0. Sequence analysis of one mutant (IS1A) indicated that transposition had created a 6.3-kb deletion, one end of which was in dltB of the dlt operon encoding four proteins (DltA-DltD) involved in the synthesis of D-alanyl-lipoteichoic acid. Inactivation of the dltC gene, encoding the D-alanyl carrier protein (Dcp), resulted in the generation of the acid-sensitive mutant, BH97LC. Compared to the wild-type strain, LT11, the mutant exhibited a threefold-longer doubling time and a 33% lower growth yield. In addition, it was unable to initiate growth below pH 6.5 and unadapted cells were unable to survive a 3-h exposure in medium buffered at pH 3.5, while a pH of 3.0 was required to kill the wild type in the same time period. Also, induction of the ATR in BH97LC, as measured by the number of survivors at a pH killing unadapted cells, was 3 to 4 orders of magnitude lower than that exhibited by the wild type. While the LTA of both strains contained a similar average number of glycerolphosphate residues, permeabilized cells of BH97LC did not incorporate D-[(14)C]alanine into this amphiphile. This defect was correlated with the deficiency of Dcp. Chemical analysis of the LTA purified from the mutant confirmed the absence of D-alanine-esters. Electron micrographs showed that BH97LC is characterized by unequal polar caps and is devoid of a fibrous extracellular matrix present on the surface of the wild-type cells. Proton permeability assays revealed that the mutant was more permeable to protons than the wild type. This observation suggests a mechanism for the loss of the characteristic acid tolerance response in S. mutans.

Biosynthesis of lipoteichoic acid in Lactobacillus rhamnosus: role of DltD in D-alanylation.

The dlt operon (dltA to dltD) of Lactobacillus rhamnosus 7469 encodes four proteins responsible for the esterification of lipoteichoic acid (LTA) by D-alanine. These esters play an important role in controlling the net anionic charge of the poly (GroP) moiety of LTA. dltA and dltC encode the D-alanine-D-alanyl carrier protein ligase (Dcl) and D-alanyl carrier protein (Dcp), respectively. Whereas the functions of DltA and DltC are defined, the functions of DltB and DltD are unknown. To define the role of DltD, the gene was cloned and sequenced and a mutant was constructed by insertional mutagenesis of dltD from Lactobacillus casei 102S. Permeabilized cells of a dltD::erm mutant lacked the ability to incorporate D-alanine into LTA. This defect was complemented by the expression of DltD from pNZ123/dlt. In in vitro assays, DltD bound Dcp for ligation with D-alanine by Dcl in the presence of ATP. In contrast, the homologue of Dcp, the Escherichia coli acyl carrier protein (ACP), involved in fatty acid biosynthesis, was not bound to DltD and thus was not ligated with D-alanine. DltD also catalyzed the hydrolysis of the mischarged D-alanyl-ACP. The hydrophobic N-terminal sequence of DltD was required for anchoring the protein in the membrane. It is hypothesized that this membrane-associated DltD facilitates the binding of Dcp and Dcl for ligation of Dcp with D-alanine and that the resulting D-alanyl-Dcp is translocated to the primary site of D-alanylation.

Insertional inactivation of genes responsible for the D-alanylation of lipoteichoic acid in Streptococcus gordonii DL1 (Challis) affects intrageneric coaggregations.

Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci. Two genes relevant to these intrageneric coaggregations were identified by transposon Tn916 mutagenesis of Streptococcus gordonii DL1 (Challis). A 626-bp sequence flanking the left end of the transposon was homologous to dltA and dltB of Lactobacillus rhamnosus ATCC 7469 (formerly called Lactobacillus casei). A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of S. gordonii DL1. This DNA encoded D-alanine-D-alanyl carrier protein ligase that was expressed in Escherichia coli from the fosmid clone. The cloned streptococcal dltA was disrupted by inserting an ermAM cassette, and then it was linearized and transformed into S. gordonii DL1 for allelic replacement. Erythromycin-resistant transformants containing a single insertion in dltA exhibited a loss of D-alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation. This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C. J. Whittaker, D. L. Clemans, and P. E. Kolenbrander, Infect. Immun. 64:4137-4142, 1996). The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations. The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs. The natural DNA transformation frequency was reduced 10-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the dltA probe revealed the presence of this gene in most viridans streptococci. Thus, it is hypothesized that D-alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin to mediate intrageneric coaggregation.

The D-Alanyl carrier protein in Lactobacillus casei: cloning, sequencing, and expression of dltC.

The incorporation of D-alanine into membrane-associated D-alanyl-lipoteichoic acid in Lactobacillus casei requires the 56-kDa D-alanine-D-alanyl carrier protein ligase (Dcl) and the 8.9-kDa D-alanyl carrier protein (Dcp). To identify and isolate the gene encoding Dcp, we have cloned and sequenced a 4.3-kb chromosomal fragment that contains dcl (dltA). In addition to this gene, the fragment contains three other genes, dltB, d1tC, and a partial dltD gene. dltC (246 nucleotides) was subcloned from this region and expressed in Escherichia coli. The product was identified as apo-Dcp lacking the N-terminal methionine (8,787.9 Da). The in vitro conversion of the recombinant apo-Dcp to holo-Dcp by recombinant E. coli holo-ACP synthase provided Dcp which accepts activated D-alanine in the reaction catalyzed by Bcl. The recombinant D-alanyl-Dcp was functionally identical to native D-alanyl-Dcp in the incorporation of D-alanine into lipoteichoic acid. L. casei Dcp is 46% identical to the putative product of dltC in the Bacillus subtilis dlt operon (M. Perego, P. Glaser, A. Minutello, M. A. Strauch, K. Leopold, and W. Fischer, J. Biol. Chem. 270:15598-15606, 1995), and therefore, this gene also encodes Dcp. Comparisons of the primary sequences and predicted secondary structures of the L. casei and B. subtilis Dcps with that of the E. coli acyl carrier protein (ACP) were undertaken together with homology modeling to identify the functional determinants of the donor and acceptor specificities of Dcp. In the region of the phospho-pantetheine attachment site, significant similarity between Dcps and ACPs was observed. This similarity may account for the relaxed acceptor specificity of the Dcps and ACPs in the ligation Of D-alanine catalyzed by Dcl. In contrast, two Dcp consensus sequences, KXXVLDXLA and DXVKXNXD, share little identity with the rest of the ACP family and, thus, may determine the donor specificity of D-alanyl-Dcp in the D-alanylation of membrane-associated D-alanyl-lipoteichoic acid.

The dlt operon in the biosynthesis of D-alanyl-lipoteichoic acid in Lactobacillus casei.

The D-alanine incorporation system allows Lactobacillus casei to modulate the chemical properties of lipoteichoic acid (LTA) and hence control its proposed functions, i.e., regulation of autolysin action, metal ion binding, and the electromechanical properties of the cell wall. The system requires the D-alanine-D-alanyl carrier protein ligase (Dcl) and the D-alanyl carrier protein (Dcp). Our results indicate that the genes for these proteins are encoded in the dlt operon and that this operon contains at least 2 other genes, dltB and dltD. The aim of this paper is to describe the genetic organization of the operon, the role of the D-alanyl carrier protein, and the function of the putative protein encoded by dltB in the intramembranal translocation of the activated D-alanine.

[Heterologous secretion in the Escherichia coli system]. / Geterologichnaia sekretsiia v sisteme Escherichia coli.

The mechanisms of protein secretion in Escherichia coli are considered. Vector systems intended for secretion of eukaryotic proteins both into the periplasmic space and the culture medium are discussed.