The Broad-Spectrum Antimicrobial Potential of [Mn(CO)4(S2CNMe(CH2CO2H))], a Water-Soluble CO-Releasing Molecule (CORM-401): Intracellular Accumulation, Transcriptomic and Statistical Analyses, and Membrane Polarization.

AIMS: Carbon monoxide (CO)-releasing molecules (CORMs) are candidates for animal and antimicrobial therapeutics. We aimed to probe the antimicrobial potential of a novel manganese CORM. RESULTS: [Mn(CO)4S2CNMe(CH2CO2H)], CORM-401, inhibits growth of Escherichia coli and several antibiotic-resistant clinical pathogens. CORM-401 releases CO that binds oxidases in vivo, but is an ineffective respiratory inhibitor. Extensive CORM accumulation (assayed as intracellular manganese) accompanies antimicrobial activity. CORM-401 stimulates respiration, polarizes the cytoplasmic membrane in an uncoupler-like manner, and elicits loss of intracellular potassium and zinc. Transcriptomics and mathematical modeling of transcription factor activities reveal a multifaceted response characterized by elevated expression of genes encoding potassium uptake, efflux pumps, and envelope stress responses. Regulators implicated in stress responses (CpxR), respiration (Arc, Fnr), methionine biosynthesis (MetJ), and iron homeostasis (Fur) are significantly disturbed. Although CORM-401 reduces bacterial growth in combination with cefotaxime and trimethoprim, fractional inhibition studies reveal no interaction. INNOVATION: We present the most detailed microbiological analysis yet of a CORM that is not a ruthenium carbonyl. We demonstrate CO-independent striking effects on the bacterial membrane and global transcriptomic responses. CONCLUSIONS: CORM-401, contrary to our expectations of a CO delivery vehicle, does not inhibit respiration. It accumulates in the cytoplasm, acts like an uncoupler in disrupting cytoplasmic ion balance, and triggers multiple effects, including osmotic stress and futile respiration. Rebound Track: This work was rejected during standard peer review and rescued by rebound peer review (Antioxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Miguel Aon, Giancarlo Biagini, James Imlay, and Nigel Robinson. Antioxid. Redox Signal. 28, 1286-1308.

Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability.

Campylobacter jejuni, the most frequent cause of food-borne bacterial gastroenteritis worldwide, is a microaerophile that has to survive high environmental oxygen tensions, adapt to oxygen limitation in the intestine and resist host oxidative attack. Here, oxygen-dependent changes in C. jejuni physiology were studied at constant growth rate using carbon (serine)-limited continuous chemostat cultures. We show that a perceived aerobiosis scale can be calibrated by the acetate excretion flux, which becomes zero when metabolism is fully aerobic (100% aerobiosis). Transcriptome changes in a downshift experiment from 150% to 40% aerobiosis revealed many novel oxygen-regulated genes and highlighted re-modelling of the electron transport chains. A label-free proteomic analysis showed that at 40% aerobiosis, many proteins involved in host colonisation (e.g., PorA, CadF, FlpA, CjkT) became more abundant. PorA abundance increased steeply below 100% aerobiosis. In contrast, several citric-acid cycle enzymes, the peptide transporter CstA, PEB1 aspartate/glutamate transporter, LutABC lactate dehydrogenase and PutA proline dehydrogenase became more abundant with increasing aerobiosis. We also observed a co-ordinated response of oxidative stress protection enzymes and Fe-S cluster biogenesis proteins above 100% aerobiosis. Our approaches reveal key virulence factors that respond to restricted oxygen availability and specific transporters and catabolic pathways activated with increasing aerobiosis.

Hemerythrins in the microaerophilic bacterium Campylobacter jejuni help protect key iron-sulphur cluster enzymes from oxidative damage.

Microaerophilic bacteria are adapted to low oxygen environments, but the mechanisms by which their growth in air is inhibited are not well understood. The citric acid cycle in the microaerophilic pathogen Campylobacter jejuni is potentially vulnerable, as it employs pyruvate and 2-oxoglutarate:acceptor oxidoreductases (Por and Oor), which contain labile (4Fe-4S) centres. Here, we show that both enzymes are rapidly inactivated after exposure of cells to a fully aerobic environment. We investigated the mechanisms that might protect enzyme activity and identify a role for the hemerythrin HerA (Cj0241). A herA mutant exhibits an aerobic growth defect and reduced Por and Oor activities after exposure to 21% (v/v) oxygen. Slow anaerobic recovery of these activities after oxygen damage was observed, but at similar rates in both wild-type and herA strains, suggesting the role of HerA is to prevent Fe-S cluster damage, rather than promote repair. Another hemerythrin (HerB; Cj1224) also plays a protective role. Purified HerA and HerB exhibited optical absorption, ligand binding and resonance Raman spectra typical of µ-oxo-bridged di-iron containing hemerythrins. We conclude that oxygen lability and poor repair of Por and Oor are major contributors to microaerophily in C. jejuni; hemerythrins help prevent enzyme damage microaerobically or during oxygen transients.