Crystal structure of the NanB sialidase from Streptococcus pneumoniae

The Streptococcus pneumoniae genomes encode up to three sialidases (or neuraminidases), NanA, NanB and NanC, which are believed to be involved in removing sialic acid from host cell surface glycans, thereby promoting colonization of the upper respiratory tract. Here, we present the crystal structure of NanB to 1.7 Å resolution derived from a crystal grown in the presence of the buffer Ches (2-N-cyclohexylaminoethanesulfonic acid). Serendipitously, Ches was found bound to NanB at the enzyme active site, and was found to inhibit NanB with a K_i of ∼ 0.5 mM. In addition, we present the structure to 2.4 Å resolution of NanB in complex with the transition-state analogue Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetyl neuraminic acid), which inhibits NanB with a K_i of ∼ 0.3 mM. The sulphonic acid group of Ches and carboxylic acid group of Neu5Ac2en interact with the arginine triad of the active site. The cyclohexyl group of Ches binds in the hydrophobic pocket of NanB occupied by the acetamidomethyl group of Neu5Ac2en. The topology around the NanB active site suggests that the enzyme would have a preference for α2,3-linked sialoglycoconjugates, which is confirmed by a kinetic analysis of substrate binding. NMR studies also confirm this preference and show that, like the leech sialidase, NanB acts as an intramolecular trans-sialidase releasing Neu2,7-anhydro5Ac. All three pneumoccocal sialidases possess a carbohydrate-binding domain that is predicted to bind sialic acid. These studies provide support for a possible differential role for NanB compared to NanA in pneumococcal virulence.     

链球菌神经氨酸酶的作用机制及酶活性的测定技术

神经氨酸酶不仅存在于流感病毒,在细菌中也有分布。细菌的神经氨酸酶可裂解宿主体内糖结合物末端的神经氨酸残基,有助于细菌实现在宿主体内的定殖、穿透和扩散,是细菌重要的毒力因子之一。链球菌是自然界广泛存在的人畜共患的病原菌,在多种链球菌中均可检测出神经氨酸酶。肺炎链球菌的神经氨酸酶研究最为透彻,该菌可产生3种神经氨酸酶(NanA,NanB,NanC),NanA不但可以发挥酶的催化作用,分解唾液酸残基,暴露细菌的黏附受体,还能不依赖酶活基团,辅助细菌感染宿主细胞;NanB催化后产物可作为细菌的碳源;NanC可辅助细菌入侵脑部。在无乳链球菌和猪链球菌中,神经氨酸酶的活性一直未得到确切的验证,可能是由于它们的荚膜均含有神经氨酸,所以其神经氨酸酶的活性逐渐在进化中丧失。另外一些链球菌,例如化脓链球菌和C、G、L群链球菌,其神经氨酸酶的底物偏好相近,均对唾液类黏蛋白的催化活性较强,利于链球菌在含唾液类黏蛋白的组织中扩散。在口腔链球菌和血链球菌中,神经氨酸酶破坏血液成分中的神经氨酸链。由此可见,神经氨酸酶的特异性催化作用与链球菌在宿主体内的定植部位密切相关。此外,随着科技的发展,对神经氨酸酶的活性检测,也由早期的硫代巴比妥法,转为现在的荧光值和吸光度的测定,更为便捷和敏感。本文旨在对链球菌的神经氨酸酶的作用机制、与毒力关系及酶活测定方法等研究进展作一综述,为从事相关研究的科学工作者提供参考。     

Structural basis for Streptococcus pneumoniae NanA inhibition by influenza antivirals zanamivir and oseltamivir carboxylate

The human pathogen Streptococcus pneumoniae is the major cause of bacterial meningitis, respiratory tract infection, septicemia, and otitis media. The bacterium expresses neuraminidase (NA) proteins that contribute to pathogenesis by cleaving sialic acids from host glycoconjugates, thereby enhancing biofilm formation and colonization. Recent in vivo experiments have shown that antiviral compounds, widely used in clinics and designed to inhibit influenza NA, significantly reduce biofilm formation and nasopharyngeal colonization of S. pneumoniae in mice. Here, we present the structural basis for the beneficial effect of these compounds against pneumococcal infection. Crystal structures of pneumococcal NanA in complex with zanamivir and oseltamivir carboxylate are discussed, correlated with measured inhibitory constants K_i, and compared with the binding modes of the inhibitors in the viral enzyme. Inhibitor structures show for the first time how clinically approved anti-influenza compounds interact with an NA of the human pathogen S. pneumoniae and give a rational explanation for their antibacterial effects.     

Growth of Streptococcus pneumoniae on human glycoconjugates is dependent upon the sequential activity of bacterial exoglycosidases

In the human host, Streptococcus pneumoniae encounters a variety of glycoconjugates, including mucin, host defense molecules, and glycans associated with the epithelial surface. S. pneumoniae is known to encode a number of glycosidases that may modify these glycoconjugates in vivo. Three exoglycosidases, a neuraminidase (NanA), β-galactosidase (BgaA), and N-acetylglucosaminidase (StrH), have been previously demonstrated to sequentially deglycosylate N-linked glycans on host defense molecules, which coat the pneumococcal surface in vivo. This cleavage is proposed to alter the clearance function of these molecules, allowing pneumococci to persist in the airway. However, we propose that the exoglycosidase-dependent liberation of monosaccharides from these glycoconjugates in close proximity to the pneumococcal surface provides S. pneumoniae with a convenient source of fermentable carbohydrate in vivo. In this study, we demonstrate that S. pneumoniae is able to utilize complex N-linked human glycoconjugates as a sole source of carbon to sustain growth and that efficient growth is dependent upon the sequential deglycosylation of the glycoconjugate substrate by pneumococcal exoglycosidases. In addition to demonstrating a role for NanA, BgaA, and StrH, we have identified a function for the second pneumococcal neuraminidase, NanB, in the deglycosylation of host glycoconjugates and have demonstrated that NanB activity can partially compensate for the loss or dysfunction of NanA. To date, all known functions of pneumococcal neuraminidase have been attributed to NanA. Thus, this study describes the first proposed role for NanB by which it may contribute to S. pneumoniae colonization and pathogenesis.     

Biochemical characterization and evaluation of potent inhibitors of the Pseudomonas aeruginosa PA01 acetohydroxyacid synthase

Microbes and plants synthesize essential branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohydroxyacid synthase (AHAS, EC 4.1.3.18). Recently, AHAS was identified as a potential anti bacterial target. To help find an effective inhibitor that could act as an antibacterial compound, we cloned and characterized the catalytic subunit (CSU) of Pseudomonas aeruginosa AHAS, and found four potent inhibitors through chemical library screening. The ilvI gene of P. aeruginosa encodes a 65-kDa AHAS protein, consistent with the size of the purified enzyme on SDS-PAGE. Enzyme kinetics showed that the enzyme has a K_m of 14.2 mM and a specific activity of 0.12 U/mg. Enzyme activity was optimum at a temperature of 37 °C and a pH of 7.5. The K_d for thiamine diphosphate (ThDP) was 89.92 ± 17.9 μM, as determined by fluorescence quenching. The cofactor activation constants (K_s) for ThDP and (K_c) for Mg²⁺ were 0.6 ± 0.1 and 560.8 ± 7.4 μM, respectively. Further, we determined that AVS2087, AVS2093, AVS2236, and AVS2387 compounds are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These compounds inhibit nearly 100% of AHAS activity, with IC₅₀ values of 1.19 μM, 5.0 nM, 25 nM, and 13 nM, respectively. Compound AVS2093 showed growth inhibition with a minimal inhibitory concentration (MIC) of 742.9 μg/ml against P. aeruginosa strain ATCC 9027. Furthermore, these findings were supported by molecular docking studies with the AVS compounds against P. aeruginosa AHAS in which AVS2093 showed minimum binding energy (−7.8 kJ/mol) by interacting with the receptor through a single hydrogen bond of 2.873 Å. Correlation of AVS2093 activity with P. aeruginosa AHAS cell growth inhibition suggested that AHAS might serve as a target protein for the development of novel antibacterial therapeutics. Results of the current study provide an impetus to further evaluate the potency of these inhibitors against pathogenic P. aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors.     

Parameters determining the relative efficacy of hydroxy-naphthoquinone inhibitors of the cytochrome bc1 complex

Hydroxy-naphthoquinones are competitive inhibitors of the cytochrome bc₁ complex that bind to the ubiquinol oxidation site between cytochrome b and the iron-sulfur protein and presumably mimic a transition state in the ubiquinol oxidation reaction catalyzed by the enzyme. The parameters that affect efficacy of binding of these inhibitors to the bc₁ complex are not well understood. Atovaquone®, a hydroxy-naphthoquinone, has been used therapeutically to treat Pneumocystis carinii and Plasmodium infections. As the pathogens have developed resistance to this drug, it is important to understand the molecular basis of the drug resistance and to develop new drugs that can circumvent the drug resistance. We previously developed the yeast and bovine bc₁ complexes as surrogates to model the interaction of atovaquone with the bc₁ complexes of the target pathogens and human host. As a first step to identify new cytochrome bc₁ complex inhibitors with therapeutic potential and to better understand the determinants of inhibitor binding, we have screened a library of 2-hydroxy-naphthoquinones with aromatic, cyclic, and non-cyclic alkyl side-chain substitutions at carbon-3 on the hydroxy-quinone ring. We found a group of compounds with alkyl side-chains that effectively inhibit the yeast bc₁ complex. Molecular modeling of these into the crystal structure of the yeast cytochrome bc₁ complex provides structural and quantitative explanations for their binding efficacy to the target enzyme. In addition we also identified a 2-hydroxy-naphthoquinone with a branched side-chain that has potential for development as an anti-fungal and anti-parasitic therapeutic.     

Potential of Lecanicillium spp. for management of insects, nematodes and plant diseases

Fungi in the genus Lecanicillium (formerly classified as the single species Verticillium lecanii) are important pathogens of insects and some have been developed as commercial biopesticides. Some isolates are also active against phytoparasitic nematodes or fungi. Lecanicillium spp. use both mechanical forces and hydrolytic enzymes to directly penetrate the insect integument and the cell wall of the fungal plant pathogen. In addition to mycoparasitism of the plant pathogen, the mode of action is linked to colonization of host plant tissues, triggering an induced systemic resistance. Recently it was demonstrated that development of Lecanicillium hybrids through protoplast fusion may result in strains that inherit parental attributes, thereby allowing development of hybrid strains with broader host range and other increased benefits, such as increased viability. Such hybrids have demonstrated increased virulence against aphids, whiteflies and the soybean cyst nematode. Three naturally occurring species of Lecanicillium, L. attenuatum, L. longisporum, and an isolate that could not be linked to any presently described species based on rDNA sequences have been shown to have potential to control aphids as well as suppress the growth and spore production of Sphaerotheca fuliginea, the causal agent of cucumber powdery mildew. These results suggest that strains of Lecanicillium spp. may have potential for development as a single microbial control agent effective against several plant diseases, pest insects and plant parasitic nematodes due to its antagonistic, parasitic and disease resistance inducing characteristics. However, to our knowledge, no Lecanicillium spp. have been developed for control of phytopathogens or phytoparasitic nematodes.     

Crystal structure of FliC flagellin from Pseudomonas aeruginosa and its implication in TLR5 binding and formation of the flagellar filament

Pseudomonas aeruginosa is one of leading opportunistic pathogens in humans and its movement is driven by a flagellar filament that is constituted through the polymerization of a single protein, FliC flagellin (paFliC). paFliC is an essential virulence factor for the colonization of P. aeruginosa. paFliC activates innate immune responses via its recognition by Toll-like receptor 5 (TLR5) and adaptive immunity in the host. Thus, paFliC has been a vaccine candidate to prevent P. aeruginosa infection, particularly for cystic fibrosis patients. To provide structural information on paFliC and its flagellar filament, we have determined the crystal structure of paFliC, which contains the conserved D1 and variable D2 domains, at 2.1 Å resolution. As observed for Salmonella FliC, the paFliC D1 domain is folded into a rod-shaped structure, and paFliC was demonstrated by gel filtration and native PAGE analyses to directly interact with TLR5. Moreover, a structural model of the paFliC-TLR5 complex suggests that paFliC D1 would provide major TLR5-binding sites, similar to Salmonella FliC. In contrast to the D1 domain, the paFliC D2 domain exhibits a unique structure of two β-sheets and one α-helix that has not been found in other flagellins. An in silico construction of a flagellar filament based on the packing of paFliC in the crystal suggests that the D2 domain would be exposed to solution and could play an important role in immunogenicity. Our biophysical and structure-based modeling study on paFliC, the paFliC-TLR5 complex, and the paFliC filament could contribute to the improvement of vaccine design to control P. aeruginosa infection.     

Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry⁻ with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.     

Structural basis for the recognition of lysozyme by MliC, a periplasmic lysozyme inhibitor in Gram-negative bacteria

Lysozymes are an important component of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall constituent. Many bacteria have contrived various means of dealing with this bactericidal enzyme, one of which is to produce lysozyme inhibitors. Recently, a novel family of bacterial lysozyme inhibitors was identified in various Gram-negative bacteria, named MliC (membrane bound lysozyme inhibitor of C-type lysozyme). Here, we report the crystal structure of Pseudomonas aeruginosa MliC in complex with chicken egg white lysozyme. Combined with mutational study, the complex structure demonstrates that the invariant loop of MliC plays a crucial role in the inhibition of the lysozyme by its insertion to the active site cleft of the lysozyme, where the loop forms hydrogen and ionic bonds with the catalytic residues. Since MliC family members have been implicated as putative colonization or virulence factors, the structures and mechanism of action of MliC will be of relevance to the control of bacterial growth in animal hosts.     

A multicenter pilot external quality assessment programme to assess the quality of molecular detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae

An external quality assessment (EQA) panel consisting of a total of 13 samples in broncho alveolar lavage (BAL) or transport medium was prepared to assess the proficiency of laboratories in the correct detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by nucleic acid amplification techniques (NAATs) (6 samples containing various concentrations (4.9-490 inclusion forming units (IFU)/ml) of C. pneumoniae, 5 samples containing various concentrations (20-5000 color-changing units (CCU)/ml) of M. pneumoniae and 2 samples negative for both).Seventy-nine laboratories from 18 countries participated in this EQA study. Sixty-four datasets were returned for C. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 43 real-time in-house, and n = 2 SDA). Sixty-seven datasets were obtained for M. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 46 real-time in-house, and n = 2 strand displacement amplification (SDA)). For the total panels, correct results per sample varied between 95.3% and 100% for C. pneumoniae and between 53.7% and 95.5% for M. pneumoniae. In general, commercial conventional NAATs showed possible sensitivity issues when compared to conventional in-house NAATs for both organisms. On the other hand, real-time commercial NAATs scored better than real-time in-house assays in terms of sensitivity for both organisms. For C. pneumoniae and M. pneumoniae, 0.8% and 2.2% true false-positive results and 1.9% and 2.0% false positives were reported in the samples spiked with the other organism.Analysis of the data for C. pneumoniae showed that the concentrations used were easily detectable by the vast majority of participants. The percentage of correct qualitative results for M. pneumoniae demonstrated that the concentrations included in this panel proved challenging for a number of participants.     

Peppermint (Mentha piperita) inhibits microbial biofilms in vitro

Microbial biofilms have become increasingly problematic in the food processing and medical industries where they cause food and surface contamination. Biofilms have also been implicated as the cause of serious infections in humans as their occurrence makes it difficult to treat common infections and the likelihood of recurrent infections is high. Due to emerging resistance, conventional control methods are fast becoming ineffective. In this study, the use of a selection of commercial plant extracts is investigated. The inhibitory effects of eight herbal extracts on the development of microbial biofilms was investigated against clinical and reference strains of Pseudomonas aeruginosa and Candida albicans. The antimicrobial activity was investigated on the planktonic forms using the minimum inhibitory concentration assay. The extracts that showed the highest antimicrobial activity against the two test organisms were Echinacea angustifolia (cone flower), Mentha piperita (peppermint) and Rosmarinus officinalis (rosemary) with minimum inhibitory concentration values between 0.38 and 2.5 mg/ml. The crystal violet assay was used to assess the effect of pre-treating a surface with plant extracts on cell attachment and the extent of biofilm development following exposure to extracts (biofilm biomass). Most of the extracts reduced microbial colonization by at least 50%. In contrast, preformed biofilms were less responsive to the majority of extracts, thus growth inhibition was more difficult to achieve. Mentha piperita was the only extract that showed some antibiofilm activity against both pathogens.     

Antibiotics LL-Z1272 identified as novel inhibitors discriminating bacterial and mitochondrial quinol oxidases

To counter antibiotic-resistant bacteria, we screened the Kitasato Institute for Life Sciences Chemical Library with bacterial quinol oxidase, which does not exist in the mitochondrial respiratory chain. We identified five prenylphenols, LL-Z1272β, γ, δ, ? and ζ, as new inhibitors for the Escherichia coli cytochrome bd. We found that these compounds also inhibited the E. coli bo-type ubiquinol oxidase and trypanosome alternative oxidase, although these three oxidases are structurally unrelated. LL-Z1272β and ? (dechlorinated derivatives) were more active against cytochrome bd while LL-Z1272γ, δ, and ζ (chlorinated derivatives) were potent inhibitors of cytochrome bo and trypanosome alternative oxidase. Thus prenylphenols are useful for the selective inhibition of quinol oxidases and for understanding the molecular mechanisms of respiratory quinol oxidases as a probe for the quinol oxidation site. Since quinol oxidases are absent from mammalian mitochondria, LL-Z1272β and δ, which are less toxic to human cells, could be used as lead compounds for development of novel chemotherapeutic agents against pathogenic bacteria and African trypanosomiasis.     

Oscillations in continuous culture populations of Streptococcus pneumoniae: population dynamics and the evolution of clonal suicide

Agents that kill or induce suicide in the organisms that produce them or other individuals of the same genotype are intriguing puzzles for ecologists and evolutionary biologists. When those organisms are pathogenic bacteria, these suicidal toxins have the added appeal as candidates for the development of narrow spectrum antibiotics to kill the pathogens that produce them. We show that when clinical as well as laboratory strains of Streptococcus pneumoniae are maintained in continuous culture (chemostats), their densities oscillate by as much as five orders of magnitude with an apparently constant period. This dynamic, which is unanticipated for single clones of bacteria in chemostats, can be attributed to population-wide die-offs and recoveries. Using a combination of mathematical models and experiments with S. pneumoniae, we present evidence that these die-offs can be attributed to the autocatalytic production of a toxin that lyses or induces autolysis in members of the clone that produces it. This toxin, which our evidence indicates is a protein, appears to be novel; S. pneumoniae genetic constructs knocked out for lytA and other genes coding for known candidates for this agent oscillate in chemostat culture. Since this toxin lyses different strains of S. pneumoniae as well as other closely related species of Streptococcus, we propose that its ecological role is as an allelopathic agent. Using a mathematical model, we explore the conditions under which toxins that kill members of the same clone that produces them can prevent established populations from invasion by different strains of the same or other species. We postulate that the production of the toxin observed here as well as other bacteria-produced toxins that kill members of the same genotype, ‘clonal suicide’, evolved and are maintained to prevent colonization of established populations by different strains of the same and closely related species.     

South African Lippia herbal infusions: Total phenolic content,antioxidant and antibacterial activities

Lippia javanica and Lippia scaberrima are used as herbal remedies and are commercially traded as health teas in southern Africa under the brands “Mosukujane” and “Musukudu”, respectively. This study evaluates the relationship between the presence of phenolic compounds and the antioxidant activities of infusions prepared from four Lippia species (L. javanica, L. scaberrima, L. rehmannii and L. wilmsii) indigenous to South Africa. The antioxidant activities of the infusions, determined by the 2,2-diphenylpycrylhydrazyl (DPPH) method, were also compared to those of popular black, green and herbal tea brands. Of the four indigenous species, infusions of L. javanica and L. wilmsii exhibited the highest antioxidant activities (EC₅₀: 358 and 525 µg/ml, respectively) and contained the most phenolic compounds (14.8 and 14.5 mg/ml of dry weight gallic acid equivalent, respectively). Antibacterial activities of methanolic extracts of the four Lippia species were determined against four human pathogens (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa). The extract of L. javanica was the most active against all the pathogens tested. Those Lippia species (L. javanica and L. wilmsii) previously reported to produce higher levels of the pharmacologically active phenylethanoid glycosides verbascoside and isoverbascoside, portrayed stronger antioxidant and antibacterial activities. This study gives credence to the use of infusions of these Lippia species for their general health benefits.     

Commensal pathogens, with a focus on Streptococcus pneumoniae, and interactions with the human host

Many important pathogens have humans as their normal ecological niche where healthy carriage dominates over disease. The ability of these commensal pathogens, such as Streptococcus pneumoniae, to cause disease depends on a series of microbial factors as well as of genetic and environmental factors in the human host affecting the clearing capacity mediated by the innate and adaptive immune system. This delicate interplay between microbe and host affects not only the likelihood for a commensal pathogen to cause disease, but also disease type and disease severity.     

An optimized method for suicide vector-based allelic exchange in Klebsiella pneumoniae

Klebsiella pneumoniae is an important and versatile bacterium that can be found in diverse environments and is also a frequent cause of human infections. Limited data exists on the mechanisms of interaction between K. pneumoniae and the human host and of adaptations to other environments. Coupled with the high genetic diversity of this species, these factors highlight the necessity for substantial further K. pneumoniae-focused molecular genetics studies.In this report we describe a simple and efficient experimental protocol for suicide vector-based allelic exchange in K. pneumoniae. The protocol has been validated by mutating multiple loci in four distinct K. pneumoniae strains, including highly capsulated and/or multi-antibiotic resistant clinical isolates. Three key enhancements are reported:(1) Use of pDS132-derived conjugative plasmids carrying improved cloning sites, (2) Performance of sacB counterselection at 25 °C as opposed to higher temperatures, and (3) Exploitation of Flp-recombinase-mediated deletion of FRT (Flp recombinase target) flanked resistance cassettes to allow for reiterative manipulations with a single selectable marker. This study also highlights a problem that may be encountered when the aacC1 gentamicin resistance marker is used in K. pneumoniae and suggests alternative markers.The protocol developed in this study will help investigate the plethora of uncharacterized genes present in the K. pneumoniae pan-genome and shed further light upon clinically and industrially important phenotypes observed in this ubiquitous species.     

Endogenous H2O2 produced by Streptococcus pneumoniae controls FabF activity

FabF elongation condensing enzyme is a critical factor in determining the spectrum of products produced by the FASII pathway. Its active site contains a critical cysteine-thiol residue, which is a plausible target for oxidation by H₂O₂. Streptococcus pneumoniae produces exceptionally high levels of H₂O₂, mainly through the conversion of pyruvate to acetyl-P via pyruvate oxidase (SpxB). We present evidence showing that endogenous H₂O₂ inhibits FabF activity by specifically oxidizing its active site cysteine-thiol residue. Thiol trapping methods revealed that one of the three FabF cysteines in the wild-type strain was oxidized, whereas in an spxB mutant, defective in H₂O₂ production, none of the cysteines was oxidized, indicating that the difference in FabF redox state originated from endogenous H₂O₂. In vitro exposure of the spxB mutant to various H₂O₂ concentrations further confirmed that only one cysteine residue was susceptible to oxidation. By blocking FabF active site cysteine with cerulenin we show that the oxidized cysteine was the catalytic one. Inhibition of FabF activity by either H₂O₂ or cerulenin resulted in altered membrane fatty acid composition. We conclude that FabF activity is inhibited by H₂O₂ produced by S. pneumoniae.     

Tuning of functional heme reduction potentials in Shewanella fumarate reductases

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.