CD18-dependent activation of the neutrophil NADPH oxidase during phagocytosis of Escherichia coli or Staphylococcus aureus is regulated by class III but not class I or II PI3Ks

Phagocytosis and activation of the NADPH oxidase are important mechanisms by which neutrophils and macrophages engulf and kill microbial pathogens. We investigated the role of PI3K signaling pathways in the regulation of the oxidase during phagocytosis of Staphylococcus aureus and Escherichia coli by mouse and human neutrophils, a mouse macrophage-like cell line and a human myeloid-like cell line. Phagocytosis of these bacteria was promoted by serum, independent of serum-derived antibodies, and effectively abolished in mouse neutrophils lacking the beta(2)-integrin common chain, CD18. A combination of PI3K isoform-selective inhibitors, mouse knock-outs, and RNA-interference indicated CD18-dependent activation of the oxidase was independent of class I and II PI3Ks, but substantially dependent on the single class III isoform (Vps34). Class III PI3K was responsible for the synthesis of PtdIns( 3) P on phagosomes containing either bacteria. The use of mouse neutrophils carrying an appropriate knock-in mutation indicated that PtdIns(3) P binding to the PX domain of their p40(phox) oxidase subunit is important for oxidase activation in response to both S aureus and E coli. This interaction does not, however, account for all the PI3K sensitivity of these responses, particularly the oxidase response to E coli, suggesting that additional mechanisms for PtdIns( 3) P-regulation of the oxidase must exist. ( Blood. 2008; 112: 5202-5211)

Functional and biochemical characterisation of the <em>Escherichia coliem> major facilitator superfamily multidrug transporter MdtM

Multidrug resistance (MDR) occurs when bacteria simultaneously acquire resistance to a broad spectrum of structurally dissimilar compounds to which they have not previously been exposed. MDR is principally a consequence of the active transport of drugs out of the cell by proteins that are integral membrane transporters. We characterised and purified the putative Escherichia coli MDR transporter, MdtM, a 410 amino acid residue protein that belongs to the large and ubiquitous major facilitator superfamily. Functional characterisation of MdtM using growth inhibition and whole cell transport assays revealed its role in intrinsic resistance of E. coli cells to the antimicrobials ethidium bromide and chloramphenicol. Site-directed mutagenesis studies implied that the MdtM aspartate 22 residue and the highly conserved arginine at position 108 play a role in proton recognition. MdtM was homologously overexpressed and purified to homogeneity in dodecyl maltopyranoside detergent solution and the oligomeric state and stability of the protein in a variety of detergent solutions was investigated using size-exclusion HPLC. Purified MdtM is monomeric and stable in dodecyl maltopyranoside solution and binds chloramphenicol with nanomolar affinity in the same detergent. This work provides a firm foundation for structural studies on this class of multidrug transporter protein.

Antibacterial activities of naturally occurring compounds against antibiotic-resistant Bacillus cereus vegetative cells and spores, Escherichia coli, and Staphylococcus aureus

After demonstrating the lack of effectiveness of standard antibiotics against the acquired antibiotic resistance of Bacillus cereus (NCTC 10989), Escherichia coli (NCTC 1186), and Staphylococcus aureus (ATCC 12715), we showed that the following natural substances were antibacterial against these resistant pathogens: cinnamon oil, oregano oil, thyme oil, carvacrol, (S)-perillaldehyde, 3,4-dihydroxybenzoic acid (beta-resorcylic acid), and 3,4-dihydroxyphenethylamine (dopamine). Exposure of the three pathogens to a dilution series of the test compounds showed that oregano oil was the most active substance. The oils and pure compounds exhibited exceptional activity against B. cereus vegetative cells, with oregano oil being active at nanogram, per milliliter levels. In contrast, activities against B. cereus spores were very low. Activities of the test compounds were in the following approximate order: oregano oil > thyme oil approximate to carvacrol > cinnamon oil > perillaldehyde > dopamine > beta-resorcylic acid. The order of susceptibilities of the pathogens to inactivation was as follows: B. cereus (vegetative) much greater than S. aureus approximate to E. coli much greater than B. cereus (spores). Some of the test substances may be effective against antibiotic-resistant bacteria in foods and feeds.

COMPARISON OF THE GROWTH INHIBITORY AND BACTERIOCIDAL EFFECTS OF TAUROLIDINE AND TAURULTAM ON A CLINICAL ISOLATE OF ESCHERICHIA-COLI

To investigate the mode of action of Taurolin, an antimicrobial preparation, the growth inhibitory and bacteriocidal effects of taurolidine and taurultam solutions on Escherichia coli isolated from a diagnosed urinary tract infection were examined at 37-degrees-C. The inhibitory effects of taurolidine solutions were observed to be greater than those of taurultam solutions at comparative concentrations; however, the presence of sublethal concentrations of formaldehyde (methylene glycol) associated with taurolidine was sufficient to account for this. The bacteriocidal activity of taurolidine (2.0% w/v) was greater than that of taurultam (4.5% w/v). Both compounds produced biphasic death rates with dissimilar initial slopes, suggested to be due to the presence of formaldehyde in taurolidine solutions. These observations indicate that the growth inhibitory and bacteriocidal effects of Taurolin solutions are primarily due to taurultam, however, the presence of sublethal concentrations of formaldehyde is significant in the expression of this activity.

Role of charge in the radioprotection of E. coli by thiols.

The role of net charge (Z) of thiols in their ability to radioprotect cells has been investigated in a glutathione (GSH)-deficient strain of E. coli. This strain, 7, is deficient in the enzyme gamma-glutamylcysteine synthetase and allows the effects of added low molecular weight thiols to be studied. Using the gas explosion system it is possible to measure the chemical repair of the free-radical precursors of lethal lesions by thiols in intact cells. The first-order chemical repair rate in strain 7 is 280s(-1) in comparison with 1100s(-1) in the wild-type strain 1157. From the measured difference in the intracellular concentration of GSH between the wild-type and the mutant, this gives a second-order repair rate, k(r)'s of 1.23 +/- 0.3 X 10(5) dm(3)mol(-1)s(-1). Measurement of intracellular thiol levels after addition of various low molecular weight thiols showed that uptake was rapid, leading to stable thiol levels within 1 min. The ratios of the intracellular to extracellular concentrations (C-in/C-out) were 0.74 for 3-mercaptopropionic acid (Z=-1), 0.56 for 2-mercaptoethanol (Z=0), 1.47 for cysteamine (Z=+1) and 1.04 for WR1065 (Z=+2). The k(r)'s for these thiols were 1.3 +/- 0.5 X 10(5) dm(3)mol(-1)s(-1) for 30-mercaptopropionic acid, 3.3 +/- 1.6 x 10(5) dm(3)mol(-1)s(-1) for 2-mercaptoethanol, 3.9 +/- 1.1 X 10(5) dm(3)mol(-1)s(-1) for cysteamine and 2.7 +/- 1.1 X 10(6) dm(3)mol(-1)s(-1) for WR1065. These are lower and increase less with charge than previously published values for chemical repair in isolated pBR322 DNA, probably because of the association of nucleoproteins and polyamines with the cellular DNA of E. coli. However, the approximate three-fold increase in k(r) per unit increase in Z shows that the counter-ion condensation and co-ion depletion are important in determining the effectiveness of charged thiols in the radioprotection of E. coli.

Synergistic phage-antibiotic combinations for the control of Escherichia coli biofilms in vitro.

The potential application of phage therapy for the control of bacterial biofilms has received increasing attention as resistance to conventional antibiotic agents continues to increase. The present study identifies antimicrobial synergy between bacteriophage T4 and a conventional antibiotic, cefotaxime, via standard plaque assay and, importantly, in the in vitro eradication of biofilms of the T4 host strain Escherichia coli 11303. Phage-antibiotic synergy (PAS) is defined as the phenomenon whereby sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacteria's production of virulent phage. Increasing sub-lethal concentrations of cefotaxime resulted in an observed increase in T4 plaque size and T4 concentration. The application of PAS to the T4 one-step growth curve also resulted in an increased burst size and reduced latent period. Combinations of T4 bacteriophage and cefotaxime significantly enhanced the eradication of bacterial biofilms when compared to treatment with cefotaxime alone. The addition of medium (10(4) PFU mL(-1) ) and high (10(7) PFU mL(-1) ) phage titres reduced the minimum biofilm eradication concentration value of cefotaxime against E. coli ATCC 11303 biofilms from 256 to 128 and 32 µg mL(-1) , respectively. Although further investigation is needed to confirm PAS, this study demonstrates, for the first time, that synergy between bacteriophage and conventional antibiotics can significantly improve biofilm control in vitro.

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx(EcO157)) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx(EcO157) consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.

Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus

Escherichia coli K-12 WcaJ and the Caulobacter crescentus HfsE, PssY, and PssZ enzymes are predicted to initiate the synthesis of colanic acid (CA) capsule and holdfast polysaccharide, respectively. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-1-phosphate with undecaprenyl phosphate (Und-P). In this study, in vivo complementation assays of an E. coli K-12 wcaJ mutant demonstrated that WcaJ and PssY can complement CA synthesis. Furthermore, WcaJ can restore holdfast production in C. crescentus. In vitro transferase assays demonstrated that both WcaJ and PssY utilize UDP-glucose but not UDP-galactose. However, in a strain of Salmonella enterica serovar Typhimurium deficient in the WbaP O antigen initiating galactosyltransferase, complementation with WcaJ or PssY resulted in O-antigen production. Gas chromatography-mass spectrometry (GC-MS) analysis of the lipopolysaccharide (LPS) revealed the attachment of both CA and O-antigen molecules to lipid A-core oligosaccharide (OS). Therefore, while UDP-glucose is the preferred substrate of WcaJ and PssY, these enzymes can also utilize UDP-galactose. This unexpected feature of WcaJ and PssY may help to map specific residues responsible for the nucleotide diphosphate specificity of these or similar enzymes. Also, the reconstitution of O-antigen synthesis in Salmonella, CA capsule synthesis in E. coli, and holdfast synthesis provide biological assays of high sensitivity to examine the sugar-1-phosphate transferase specificity of heterologous proteins.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

Functional analysis of the large periplasmic loop of the Escherichia coli K-12 WaaL O-antigen ligase

WaaL is a membrane enzyme implicated in ligating undecaprenyl-diphosphate (Und-PP)-linked O antigen to lipid A-core oligosaccharide. We determined the periplasmic location of a large (EL5) and small (EL4) adjacent loops in the Escherichia coli K-12 WaaL. Structural models of the EL5 from the K-12, R1 and R4 E. coli ligases were generated by molecular dynamics. Despite the poor amino acid sequence conservation among these proteins, the models afforded similar folds consisting of two pairs of almost perpendicular alpha-helices. One alpha-helix in each pair contributes a histidine and an arginine facing each other, which are highly conserved in WaaL homologues. Mutations in either residue rendered WaaL non-functional, since mutant proteins were unable to restore O antigen surface expression. Replacements of residues located away from the putative catalytic centre and non-conserved residues within the centre itself did not affect ligation. Furthermore, replacing a highly conserved arginine in EL4 with various amino acids inactivates WaaL function, but functionality reappears when the positive charge is restored by a replacement with lysine. These results lead us to propose that the conserved amino acids in the two adjacent periplasmic loops could interact with Und-PP, which is the common component in all WaaL substrates.

Functional analysis of predicted coiled-coil regions in the Escherichia coli K-12 O-antigen polysaccharide chain length determinant Wzz

Wzz is a membrane protein that determines the chain length distribution of the O-antigen lipopolysaccharide by an unknown mechanism. Wzz proteins consist of two transmembrane helices separated by a large periplasmic loop. The periplasmic loop of Escherichia coli K-12 Wzz (244 amino acids from K65 to A308) was purified and found to be a monomer with an extended conformation, as determined by gel filtration chromatography and analytical ultracentrifugation. Circular dichroism showed that the loop has a 60% helical content. The Wzz periplasmic loop also contains three regions with predicted coiled coils. To probe the function of the predicted coiled coils, we constructed amino acid replacement mutants of the E. coli K-12 Wzz protein, which were designed so that the coiled coils could be separate without compromising the helicity of the individual molecules. Mutations in one of the regions, spanning amino acids 108 to 130 (region I), were associated with a partial defect in O-antigen chain length distribution, while mutants with mutations in the region spanning amino acids 209 to 223 (region III) did not have an apparent functional defect. In contrast, mutations in the region spanning amino acids 153 to 173 (region II) eliminated the Wzz function. This phenotype was associated with protein instability, most likely due to conformational changes caused by the amino acid replacements, which was confirmed by limited trypsin proteolysis. Additional mutagenesis based on a three-dimensional model of region I demonstrated that the amino acids implicated in function are all located at the same face of a predicted alpha-helix, suggesting that a coiled coil actually does not exist in this region. Together, our results suggest that the regions predicted to be coiled coils are important for Wzz function because they maintain the native conformation of the protein, although the existence of coiled coils could not be demonstrated experimentally.