A Tale of Three Species: Adaptation of Sodalis glossinidius to Tsetse Biology, Wigglesworthia Metabolism, and Host Diet.

The tsetse fly is the insect vector for the Trypanosoma brucei parasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium, Sodalis glossinidius The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model of S. glossinidius metabolism. The model enabled the design and experimental verification of a defined medium that supports S. glossinidius growth ex vivo This has been used subsequently to analyze in vitro aspects of S. glossinidius metabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomer N-acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence on l-glutamate as a source of carbon and nitrogen and by the ability to rescue a predicted l-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiont Wigglesworthia glossinidia, reveals an intersymbiont dependence. The reductive evolution of S. glossinidius to exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibit S. glossinidius growth and aid the reduction of trypanosomal transmission.IMPORTANCE Human African trypanosomiasis is caused by the Trypanosoma brucei parasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential for T. brucei life cycle progression and transmission. The tsetse's mutualistic endosymbiont Sodalis glossinidius has a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model of S. glossinidius metabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects of S. glossinidius metabolism. We present S. glossinidius as uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally, S. glossinidius has adapted to the tsetse's obligate symbiont Wigglesworthia glossinidia by scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission.

Conserved Histidine Adjacent to the Proximal Cluster Tunes the Anaerobic Reductive Activation of Escherichia coli Membrane-Bound [NiFe] Hydrogenase-1.

[NiFe] hydrogenases are electrocatalysts that oxidize H2 at a rapid rate without the need for precious metals. All membrane-bound [NiFe] hydrogenases (MBH) possess a histidine residue that points to the electron-transfer iron sulfur cluster closest ("proximal") to the [NiFe] H2-binding active site. Replacement of this amino acid with alanine induces O2 sensitivity, and this has been attributed to the role of the histidine in enabling the reversible O2-induced over-oxidation of the [Fe4S3Cys2] proximal cluster possessed by all O2-tolerant MBH. We have created an Escherichia coli Hyd-1 His-to-Ala variant and report O2-free electrochemical measurements at high potential that indicate the histidine-mediated [Fe4S3Cys2] cluster-opening/closing mechanism also underpins anaerobic reactivation. We validate these experiments by comparing them to the impact of an analogous His-to-Ala replacement in Escherichia coli Hyd-2, a [NiFe]-MBH that contains a [Fe4S4] center.

The Antimicrobial Activity of a Carbon Monoxide Releasing Molecule (EBOR-CORM-1) Is Shaped by Intraspecific Variation within Pseudomonas aeruginosa Populations.

Carbon monoxide releasing molecules (CORMs) have been suggested as a new synthetic class of antimicrobials to treat bacterial infections. Here we utilized a novel EBOR-CORM-1 ([NEt4][MnBr2(CO)4]) capable of water-triggered CO-release, and tested its efficacy against a collection of clinical Pseudomonas aeruginosa strains that differ in infection-related virulence traits. We found that while EBOR-CORM-1 was effective in clearing planktonic and biofilm cells of P. aeruginosa strain PAO1 in a concentration dependent manner, this effect was less clear and varied considerably between different P. aeruginosa cystic fibrosis (CF) lung isolates. While a reduction in cell growth was observed after 8 h of CORM application, either no effect or even a slight increase in cell densities and the amount of biofilm was observed after 24 h. This variation could be partly explained by differences in bacterial virulence traits: while CF isolates showed attenuated in vivo virulence and growth compared to strain PAO1, they formed much more biofilm, which could have potentially protected them from the CORM. Even though no clear therapeutic benefits against a subset of isolates was observed in an in vivo wax moth acute infection model, EBOR-CORM-1 was more efficient at reducing the growth of CF isolate co-culture populations harboring intraspecific variation, in comparison with efficacy against more uniform single isolate culture populations. Together these results suggest that CORMs could be effective at controlling genetically diverse P. aeruginosa populations typical for natural chronic CF infections and that the potential benefits of some antibiotics might not be observed if tested only against clonal bacterial populations.

Retuning the Catalytic Bias and Overpotential of a [NiFe]-Hydrogenase via a Single Amino Acid Exchange at the Electron Entry/Exit Site.

The redox chemistry of the electron entry/exit site in Escherichia coli hydrogenase-1 is shown to play a vital role in tuning biocatalysis. Inspired by nature, we generate a HyaA-R193L variant to disrupt a proposed Arg-His cation-π interaction in the secondary coordination sphere of the outermost, "distal", iron-sulfur cluster. This rewires the enzyme, enhancing the relative rate of H2 production and the thermodynamic efficiency of H2 oxidation catalysis. On the basis of Fourier transformed alternating current voltammetry measurements, we relate these changes in catalysis to a shift in the distal [Fe4S4]2+/1+ redox potential, a previously experimentally inaccessible parameter. Thus, metalloenzyme chemistry is shown to be tuned by the second coordination sphere of an electron transfer site distant from the catalytic center.

Re-engineering a NiFe hydrogenase to increase the H2 production bias while maintaining native levels of O2 tolerance.

Naturally occurring oxygen tolerant NiFe membrane bound hydrogenases have a conserved catalytic bias towards hydrogen oxidation which limits their technological value. We present an Escherichia coli Hyd-1 amino acid exchange that apparently causes the catalytic rate of H2 production to double but does not impact the O2 tolerance.

Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases.

Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH.

Inhibition of the ferric uptake regulator by peptides derived from anti-FUR peptide aptamers: coupled theoretical and experimental approaches.

The FUR protein (ferric uptake regulator) is an iron-dependent global transcriptional regulator. Specific to bacteria, FUR is an attractive antibacterial target since virulence is correlated to iron bioavailability. Recently, four anti-FUR peptide aptamers, composed of 13 amino acid variable loops inserted into a thioredoxinA scaffold, were identified, which were able to interact with Escherichia coli FUR (EcFUR), inhibit its binding to DNA and to decrease the virulence of pathogenic E. coli in a fly infection model. The first characterization of anti-FUR linear peptides (pF1 6 to 13 amino acids) derived from the variable part of the F1 anti-FUR peptide aptamer is described herein. Theoretical and experimental approaches, in original combination, were used to study interactions of these peptides with FUR in order to understand their mechanism of inhibition. After modeling EcFUR by homology, docking with Autodock was combined with molecular dynamics simulations in implicit solvent to take into account the flexibility of the partners. All calculations were cross-checked either with other programs or with experimental data. As a result, reliable structures of EcFUR and its complex with pF1 are given and an inhibition pocket formed by the groove between the two FUR subunits is proposed. The location of the pocket was validated through experimental mutation of key EcFUR residues at the site of proposed peptide interaction. Cyclisation of pF1, mimicking the peptide constraint in F1, improved inhibition. The details of the interactions between peptide and protein were analyzed and a mechanism of inhibition of these anti-FUR molecules is proposed.

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase.

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe-3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function.