Iain S. Hunter

Azole antifungals are potent inhibitors of cytochrome P450 mono-oxygenases and bacterial growth in mycobacteria and streptomycetes

The genome sequence of Mycobacterium tuberculosis has revealed the presence of 20 different cytochrome P450 mono-oxygenases (P450s) within this organism, and subsequent genome sequences of other mycobacteria and of Streptomyces coelicolor have indicated that these actinomycetes also have large complements of P450s, pointing to important physiological roles for these enzymes. The actinomycete P450s include homologues of 14alpha-sterol demethylases, the targets for the azole class of drugs in yeast and fungi. Previously, this type of P450 was considered to be absent from bacteria. When present at low concentrations in growth medium, azole antifungal drugs were shown to be potent inhibitors of the growth of Mycobacterium smegmatis and of Streptomyces strains, indicating that one or more of the P450s in these bacteria were viable drug targets. The drugs econazole and clotrimazole were most effective against M. smegmatis (MIC values of <0.2 and 0.3 micro M, respectively) and were superior inhibitors of mycobacterial growth compared to rifampicin and isoniazid (which had MIC values of 1.2 and 36.5 micro M, respectively). In contrast to their effects on the actinomycetes, the azoles showed minimal effects on the growth of Escherichia coli, which is devoid of P450s. Azole drugs coordinated tightly to the haem iron in M. tuberculosis H37Rv P450s encoded by genes Rv0764c (the sterol demethylase CYP51) and Rv2276 (CYP121). However, the azoles had a higher affinity for M. tuberculosis CYP121, with K(d) values broadly in line with the MIC values for M. smegmatis. This suggested that CYP121 may be a more realistic target enzyme for the azole drugs than CYP51, particularly in light of the fact that an S. coelicolor DeltaCYP51 strain was viable and showed little difference in its sensitivity to azole drugs compared to the wild-type. If the azole drugs prove to inhibit a number of important P450s in M. smegmatis and S. coelicolor, then the likelihood of drug resistance developing in these species should be minimal. This suggests that azole drug therapy may provide a novel antibiotic strategy against strains of M. tuberculosis that have already developed resistance to isoniazid and other front-line drugs.

Efficacy of common hospital biocides with biofilms of multi-drug resistant clinical isolates

The hospital environment is particularly susceptible to contamination by bacterial pathogens that grow on surfaces in biofilms. The effects of hospital biocides on two nosocomial pathogens, meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, growing as free-floating (planktonic) and adherent biofilm populations (sessile) were examined. Clinical isolates of MRSA and P. aeruginosa were grown as biofilms on discs of materials found in the hospital environment (stainless steel, glass, polyethylene and Teflon) and treated with three commonly used hospital biocides containing benzalkonium chloride (1 % w/v), chlorhexidine gluconate (4 % w/v) and triclosan (1 % w/v). Cell viability following biocide treatment was determined using an XTT assay and the LIVE/DEAD BacLight Bacterial Viability kit. The minimum bactericidal concentration (MBC) of all biocides for planktonic populations of both organisms was considerably less than the concentration recommended for use by the manufacturer. However, when isolates were grown as biofilms, the biocides were ineffective at killing bacteria at the concentrations recommended for use. Following biocide treatment, 0-11 % of cells in MRSA biofilms survived, and up to 80 % of cells in P. aeruginosa biofilms survived. This study suggests that although biocides may be effective against planktonic populations of bacteria, some biocides currently used in hospitals are ineffective against nosocomial pathogens growing as biofilms attached to surfaces and fail to control this reservoir for hospital-acquired infection.

The Structure and Mechanism of the Type II Dehydroquinase from Streptomyces coelicolor

The structure of the type II DHQase from Streptomyces coelicolor has been solved and refined to high resolution in complexes with a number of ligands, including dehydroshikimate and a rationally designed transition state analogue, 2,3-anhydro-quinic acid. These structures define the active site of the enzyme and the role of key amino acid residues and provide snap shots of the catalytic cycle. The resolution of the flexible lid domain (residues 21-31) shows that the invariant residues Arg23 and Tyr28 close over the active site cleft. The tyrosine acts as the base in the initial proton abstraction, and evidence is provided that the reaction proceeds via an enol intermediate. The active site of the structure of DHQase in complex with the transition state analog also includes molecules of tartrate and glycerol, which provide a basis for further inhibitor design.

Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae

ABSTRACT Forty-seven strains representing 14 differentBacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derivedBacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex andB. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate ofB. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus,B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with theB. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.

Production of diarrheal enterotoxins and other potential virulence factors by veterinary isolates of bacillus species associated with nongastrointestinal infections.

ABSTRACT With the exceptions of Bacillus cereus and Bacillus anthracis, Bacillus species are generally perceived to be inconsequential. However, the relevance of other Bacillus species as food poisoning organisms and etiological agents in nongastrointestinal infections is being increasingly recognized. Eleven Bacillus species isolated from veterinary samples associated with severe nongastrointestinal infections were assessed for the presence and expression of diarrheagenic enterotoxins and other potential virulence factors. PCR studies revealed the presence of DNA sequences encoding hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T (BceT) in five B. cereus strains and in Bacilluscoagulans NB11. Enterotoxin HBL was also harbored by Bacilluspolymyxa NB6. After 18 h of growth in brain heart infusion broth, all seven Bacillus isolates carrying genes encoding enterotoxin HBL produced this toxin. Cell-free supernatant fluids from all 11 Bacillus isolates demonstrated cytotoxicity toward human HEp-2 cells; only one Bacilluslicheniformis strain adhered to this test cell line, and none of the Bacillus isolates were invasive. This study constitutes the first demonstration that Bacillus spp. associated with serious nongastrointestinal infections in animals may harbor and express diarrheagenic enterotoxins traditionally linked to toxigenic B. cereus.

Genetics of Streptomyces rimosus, the Oxytetracycline Producer

SUMMARY From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term.

Improved oxytetracycline production in Streptomyces rimosus M4018 by metabolic engineering of the G6PDH gene in the pentose phosphate pathway

The aromatic polyketide antibiotic, oxytetracycline (OTC), is produced by Streptomyces rimosus as an important secondary metabolite. High level production of antibiotics in Streptomycetes requires precursors and cofactors which are derived from primary metabolism; therefore it is exigent to engineer the primary metabolism. This has been demonstrated by targeting a key enzyme in the oxidative pentose phosphate pathway (PPP) and nicotinamide adenine dinucleotide phosphate (NADPH) generation, glucose-6-phosphate dehydrogenase (G6PDH), which is encoded by zwf1 and zwf2. Disruption of zwf1 or zwf2 resulted in a higher production of OTC. The disrupted strain had an increased carbon flux through glycolysis and a decreased carbon flux through PPP, as measured by the enzyme activities of G6PDH and phosphoglucose isomerase (PGI), and by the levels of ATP, which establishes G6PDH as a key player in determining carbon flux distribution. The increased production of OTC appeared to be largely due to the generation of more malonyl-CoA, one of the OTC precursors, as observed in the disrupted mutants. We have studied the effect of zwf modification on metabolite levels, gene expression, and secondary metabolite production to gain greater insight into flux distribution and the link between the fluxes in the primary and secondary metabolisms.

Ablation of the otcC Gene Encoding a Post-polyketide Hydroxylase from the Oxytetracyline Biosynthetic Pathway in Streptomyces rimosus Results in Novel Polyketides with Altered Chain Length

Oxytetracycline (OTC) is a 19-carbon polyketide antibiotic made by Streptomyces rimosus. The otcC gene encodes an anhydrotetracycline oxygenase that catalyzes a hydroxylation of the anthracycline structure at position C-6 after biosynthesis of the polyketide backbone is completed. A recombinant strain of S. rimosus that was disrupted in the genomic copy of otcC synthesized a novel C-17 polyketide. This result indicates that the absence of the otcC gene product significantly influences the ability of the OTC “minimal” polyketide synthase to make a polyketide product of the correct chain length. A mutant copy of otcC was made by site-directed mutagenesis of three essential glycine codons located within the putative NADPH-binding domain. The mutant gene was expressed in Escherichia coli, and biochemical analysis confirmed that the gene product was catalytically inactive. When the mutant gene replaced the ablated gene in the chromosome of S. rimosus, the ability to make a 19-carbon backbone was restored, indicating that OtcC is an essential partner in the quaternary structure of the synthase complex.

A novel biotransformation of benzofurans and related compounds catalysed by a chloroperoxidase

Abstract The oxidation of 3-alkyl benzofurans, indoles, and a benzothiophene by the chloroperoxidase from Caldariomyces fumago has been investigated. Under conditions in which the catalase activity of chloroperoxidase was minimised in the presence of chloride and hydrogen peroxide, 3-methylbenzothiophene was oxidised at sulfur but the indoles (5-9) and benzofurans (1-4) gave 2,3-diols as initial products. In the case of N-unsubstituted indoles, these tautomerised to give the corresponding lactam. In contrast, the diols (predominantly trans) formed from the benzofurans were sufficiently stable for isolation and full characterisation. This novel reaction has the potential to be developed into a useful synthetic biotransformation.

Cardiolipin synthase is required for Streptomyces coelicolor morphogenesis

The fluid mosaic model has recently been amended to account for the existence of membrane domains enriched in certain phospholipids. In rod‐shaped bacteria, the anionic phospholipid cardiolipin is enriched at the cell poles but its role in the morphogenesis of the filamentous bacterium Streptomyces coelicolor is unknown. It was impossible to delete clsA (cardiolipin synthase; SCO1389) unless complemented by a second copy of clsA elsewhere in the chromosome. When placed under the control of an inducible promoter, clsA expression, phospholipid profile and morphogenesis became inducer dependent. TLC analysis of phospholipid showed altered profiles upon depletion of clsA expression. Analysis of cardiolipin by mass spectrometry showed two distinct cardiolipin envelopes that reflected differences in acyl chain length; the level of the larger cardiolipin envelope was reduced in concert with clsA expression. ClsA‐EGFP did not localize to specific locations, but cardiolipin itself showed enrichment at hyphal tips, branch points and anucleate regions. Quantitative analysis of hyphal dimensions showed that the mycelial architecture and the erection of aerial hyphae were affected by the expression of clsA. Overexpression of clsA resulted in weakened hyphal tips, misshaped aerial hyphae and anucleate spores and demonstrates that cardiolipin synthesis is a requirement for morphogenesis in Streptomyces.