Anthony W. Smith

Expression of the virulence‐related Sca (Mn2+) permease in Streptococcus gordonii is regulated by a diphtheria toxin metallorepressor‐like protein ScaR

The acquisition of transition metal ions by pathogenic bacteria is crucial to their growth and survival within the human host, however, the mechanisms of metal ion homeostasis in streptococci are unknown. The scaCBA operon in the human oral bacterium Streptococcus gordonii encodes the components of an ABC‐type transporter for manganese (Mn2+). Production of substrate‐binding lipoprotein ScaA was increased approximately fivefold in cells cultured in low Mn2+ medium (< 0.1 µM Mn2+), but not in iron (Fe2+/Fe3+)‐limited medium, and was enhanced in the presence of human saliva or serum. mRNA analysis revealed that under low Mn2+ conditions, levels of scaCBA transcript (2.6 kb) were increased > 20‐fold. The Mn2+‐responsive transcriptional regulator of the sca operon was purified and characterized as a 215‐amino‐acid residue polypeptide, designated ScaR, with 26% identity to the Corynebacterium diphtheriae diphtheria toxin repressor (DtxR). Inactivation of scaR in S. gordonii DL1 (Challis) resulted in constitutive derepression of sca operon transcription. Expression of tpx, located immediately downstream of scaA and encoding a putative thiol peroxidase, was not subject to ScaR regulation. Purified ScaR protein bound to the scaC promoter region in vitro in the presence of Mn2+ (Kd∼ 80 nM) and, to a lesser extent, in the presence of Ni2+ or Zn2+. The metalloregulator protein binding region was localized by DNA protection analysis to a 46 bp sequence encompassing the −35 and −10 promoter signatures. This sequence was well conserved within the promoters of corresponding virulence‐related permease operons in other streptococci. The results identify a new Mn2+‐sensing regulator of Mn2+ transport in streptococci, important for Mn2+ homeostasis during infection of the human host.

Interactions of some common pathogenic bacteria with Acanthamoeba polyphaga

Protozoan grazing is a major trophic pathway whereby the biomass re-enters the food web. Nonetheless, not all bacteria are digested by protozoa and the number known to evade digestion, resulting in their environmental augmentation, is increasing. We investigated the interactions of Bacillus cereus, Enterococcus faecalis, Enteropathogenic Escherichia coli (EPEC), Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and methicillin-sensitive Staphylococcus aureus (MSSA), with the amoeba, Acanthamoeba polyphaga. There was evidence of predation of all bacterial species except L. monocytogenes and S. aureus, where extracellular numbers were significantly higher when cultured with amoebae compared with growth in the absence of amoebae. Intracellular growth kinetic experiments and fluorescent confocal microscopy suggest that S. aureus survived and may even multiply within A. polyphaga, whereas there was no apparent intra-amoebal replication of L. monocytogenes and higher numbers were likely sustained on metabolic waste products released during coculture.

Oxidative stress tolerance is manganese (Mn2+) regulated in Streptococcus gordonii

The Sca permease in the oral bacterium Streptococcus gordonii is a member of a family of ATP-binding cassette (ABC)-type transporters for manganese (Mn(2+)) and related cations that are associated with streptococcal virulence in a number of infection models. Since Mn(2+) has a protective function against oxidative damage in a variety of bacteria, we have investigated the role of Sca permease in oxidative stress tolerance in Streptococcus gordonii. A single Mn(2+)-dependent superoxide dismutase (SOD), encoded by sodA, is expressed by S. gordonii and was >10-fold up-regulated under oxidative stress conditions. Inactivation of sodA resulted in increased susceptibility of S. gordonii cells to growth inhibition by dioxygen (O(2)), and to killing by paraquat (a superoxide anion generator) and by hydrogen peroxide (H(2)O(2)). Expression of thiol peroxidase, encoded by the tpx gene located immediately downstream of the scaCBA operon, was also up-regulated under oxidative conditions. Inactivation of tpx led to increased susceptibility of cells to H(2)O(2), but not to O(2) or paraquat. In low-Mn(2+) medium (0.01 micro M Mn(2+)) sodA and tpx genes were transcriptionally down-regulated, SOD activity was reduced and cells were more sensitive to growth inhibition by O(2). A Sca permease-deficient (scaC) mutant showed further reduced SOD activity and hypersensitivity to O(2) in medium containing <0.1 micro M Mn(2+). These results demonstrate that the Sca (Mn(2+)) permease in S. gordonii is essential for protection against oxidative stress.

Siderophore-specific induction of iron uptake in Pseudomonas aeruginosa

Pseudomonas aeruginosa has two siderophore-based high-affinity iron-uptake systems utilizing pyoverdin and pyochelin. Using strain IA1, a mutant deficient in production of both siderophores, we have shown that addition of purified siderophore to the growth medium induces expression of specific iron-regulated outer-membrane proteins and increases 55Fe-siderophore transport. Addition of pyoverdin from the parent strain PAO1 or from a clinical strain 0:12 induced expression of an 85 kDa IROMP and increased the rate of 55Fe-pyoverdin transport. Transport rates for 55Fe-PAO1 pyoverdin increased from 1.27 to 3.57 pmol Fe min-1 per 10(9) cells. Addition of purified pyochelin induced expression of a 75 kDa IROMP accompanied with increased 55Fe-pyochelin uptake without affecting 55Fe-pyoverdin transport. 55Fe-pyochelin transport increased from 0.3 to 10.6 pmol min-1 per 10(9) cells. Addition of pyoverdin from the parent strain or a chromatographically distinct pyoverdin caused increased reactivity with an anti-85 kDa mAb in Western blotting, indicating that the same receptor is being induced. These results suggest that P. aeruginosa can respond specifically to the presence of siderophore and moreover that not only can the pyoverdin receptor transport its cognate ferri-pyoverdin but also different ferri-pyoverdins, albeit at a reduced rate.

Burkholderia cepacia is resistant to the antimicrobial activity of airway epithelial cells.

There has been much interest recently in the antimicrobial properties of cationic peptides called beta-defensins from epithelial cells. Human beta-defensin (hBD)-1 and -2 have been particularly implicated in cystic fibrosis (CF) patients, where their inhibition by high salt concentrations may explain in part the susceptibility of the CF lung to bacterial infection. In this work, we have employed a simple co-culture system using the 16-HBE human bronchial epithelial cell line to assess growth inhibitory activity against Pseudomonas aeruginosa and Burkholderia cepacia. In medium alone, P. aeruginosa proliferated more than 100,000-fold, whereas in the presence of 16-HBE cells or 16-HBE-conditioned medium, bacterial proliferation was less than 100-fold. Raising the salt concentration of cell-free 16-HBE conditioned medium to approximately 200 mM significantly reduced this growth inhibitory activity. In contrast, there was no evidence of epithelial-derived growth inhibitory activity against two strains of B. cepacia. RT-PCR analysis indicated expression of the hBD-2 mRNA in 16-HBE cells, but not hBD-1. These data demonstrate for the first time that B. cepacia is resistant to epithelial-derived antimicrobial substances and argue against them being important in the defense against this organism in the lung.

Synthesis and iron binding studies of myo-inositol 1,2,3-trisphosphate and (±)-myo-inositol 1,2-bisphosphate, and iron binding studies of all myo-inositol tetrakisphosphates☆

The first syntheses of the natural products myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate are described. The protected key intermediates 4,5,6-tri-O-benzoyl-myo-inositol and (+/-)-3,4,5,6-tetra-O-benzyl-myo-inositol were phosphorylated with dibenzyl N,N-di-isopropylphosphoramidite in the presence of 1H-tetrazole and subsequent oxidation of the phosphite. The crystal structures of the synthetic intermediates (+/-)-1-O-(tert-butyldiphenylsilyl)-2,3,O-cyclohexylidene-myo-inos itol and (+/-)-4,5,6-tri-O-benzoyl-1-O-(tert-butyldiphenylsilyl)-2,3-O-cycl ohexylidene- myo-inositol are reported. myo-Inositol 1,2,3-trisphosphate, (+/-)-myo-inositol 1,2-bisphosphate, and all isomeric myo-inositol tetrakisphosphates were evaluated for their ability to alter HO. production in the iron-catalysed Haber-Weiss reaction. The results demonstrated that a 1,2,3-grouping of phosphates in myo-inositol was necessary for inhibition, also that (+/-)-myo-inositol 1,2-bisphosphate potentiated HO. production. myo-Inositol 1,2,3-trisphosphate resembled myo-inositol hexakisphosphate (phytic acid) in its ability to act as a siderophore by promoting iron-uptake into Pseudomonas aeruginosa.

Nitric oxide-induced potentiation of the killing of Burkholderia cepacia by reactive oxygen species: implications for cystic fibrosis

Burkholderia (formerly Pseudomonas) cepacia has emerged as an important pulmonary pathogen in cystic fibrosis, and survives within the lung despite a vigorous neutrophil-dominated immune response. Nitric oxide (NO) contributes to the antimicrobial activity of reactive oxygen species in the normal lung, but recent evidence suggests that inducible NO synthase is not expressed in the airway epithelial cells of cystic fibrosis (CF) patients. This may explain the failure of the neutrophil response to eliminate B. cepacia. To test this hypothesis, the present study examined the combined effect of NO, superoxide and H2O2 against B. cepacia. There was no killing of a highly transmissible strain by either superoxide or NO alone, but their combination reduced the bacterial count by >1000-fold over 75 min. This bactericidal activity was not sensitive to addition of superoxide dismutase, but was abrogated completely by catalase, suggesting that NO and hydrogen peroxide were the bactericidal mediators. Increased killing by NO in combination with H2O2 was seen for seven of a further 11 strains examined. The lack of NO in the lungs of CF patients may contribute to the survival of B. cepacia.

Characterisation of a siderophore from Acinetobacter calcoaceticus.

The Gram-negative bacterium Acinetobacter calcoaceticus was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron chelator, 2,3-dihydroxybenzoic acid was identified in the culture supernatant by 1H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). A group of outer membrane proteins between 80 and 85 kDa were induced under iron restriction.

Antimicrobiol agents and biofilms

INTRODUCTION: THE PROBLEMS There is increasing concern over the role played by microbial biofilms in infection. These include well-known examples of medical device-related infections such as those associated with artificial joints, prosthetic heart valves, and catheters. Indeed, recent surveys indicate that catheter-associated bacteraemia, consequent from catheter-related infection, is by far the leading cause of nosocomial bloodstream infection in intensive care units (Brub-Buisson, 2001). Many chronic infections, not related to medical devices, are now recognised to be due to bacteria either not growing and relatively dormant or growing slowly as biomasses or adherent biofilms on mucosal surfaces. Thus, the question of how to treat biofilm infections extends to many aspects of medicine. Indeed, the issue of biofilm eradication extends way beyond the infected patient, since bacteria in the environment typically exist as biofilms. These are commonly complex multispecies ecosystems associated with protozoa (Brown and Barker, 1999). The biofilm mode of growth greatly enhances the survival of the constituent microbes. Growth as a biofilm almost always leads to a large increase in resistance to antimicrobial agents, including antibiotics, biocides, and preservatives, compared with cultures grown in suspension (planktonic) in conventional liquid media (Gilbert, Collier, and Brown, 1990; Stewart and Costerton, 2001). However, a recent paper with high density planktonic cultures indicated similar resistance to antimicrobials as did biofilm cultures (Spoering and Lewis, 2001). Currently, there is no generally agreed mechanism to account for the broad resistance to chemical agents.

Inositol polyphosphate-mediated iron transport in Pseudomonas aeruginosa

It has previously been shown that myo‐inositol hexakisphosphate (myo‐InsP6) mediates iron transport into Pseudomonas aeruginosa and overcomes iron‐dependent growth inhibition. In this study, the iron transport properties of myo‐inositol trisphosphate and tetrakisphosphate regio‐isomers were studied. Pseudomonas aeruginosa accumulated iron (III) at similar rates whether complexed with myo‐Ins(1,2,3)P3 or myo‐InsP6. Iron accumulation from other compounds, notably d/lmyo‐Ins(1,2,4,5)P4 and another inositol trisphosphate regio‐isomer, d‐myo‐Ins(1,4,5)P3, was dramatically increased. Iron transport profiles from myo‐InsP6 into mutants lacking the outer membrane porins oprF, oprD and oprP were similar to the wild‐type, indicating that these porins are not involved in the transport process. The rates of reduction of iron (III) to iron (II) complexed to any of the compounds by a Ps. aeruginosa cell lysate were similar, suggesting that a reductive mechanism is not the rate‐determining step.