The control of nutrient availability is an essential ecological function of the host organism in host-microbe systems. Although often overshadowed by macronutrients such as carbohydrates, ...micronutrient metals are known as key drivers of host-microbe interactions. The ways in which host organisms control nutrient metal availability are dictated by principles in bioinorganic chemistry. Here I ponder about the actions of metal-binding molecules from the host organism in controlling nutrient metal availability to the host microbiota. I hope that these musings will encourage new explorations into the fundamental roles of metals in the ecology of diverse host-microbe systems.
Zinc (Zn) and copper (Cu) are essential for optimal innate immune function, and nutritional deficiency in either metal leads to increased susceptibility to bacterial infection. Recently, the ...decreased survival of bacterial pathogens with impaired Cu and/or Zn detoxification systems in phagocytes and animal models of infection has been reported. Consequently, a model has emerged in which the host utilizes Cu and/or Zn intoxication to reduce the intracellular survival of pathogens. This review describes and assesses the potential role for Cu and Zn intoxication in innate immune function and their direct bactericidal function.
Formaldehyde is the simplest of all aldehydes and is highly cytotoxic. Its use and associated dangers from environmental exposure have been well documented. Detoxification systems for formaldehyde ...are found throughout the biological world and they are especially important in methylotrophic bacteria, which generate this compound as part of their metabolism of methanol. Formaldehyde metabolizing systems can be divided into those dependent upon pterin cofactors, sugar phosphates and those dependent upon glutathione. The more prevalent thiol-dependent formaldehyde detoxification system is found in many bacterial pathogens, almost all of which do not metabolize methane or methanol. This review describes the endogenous and exogenous sources of formaldehyde, its toxic effects and mechanisms of detoxification. The methods of formaldehyde sensing are also described with a focus on the formaldehyde responsive transcription factors HxlR, FrmR, and NmlR. Finally, the physiological relevance of detoxification systems for formaldehyde in bacterial pathogens is discussed.
Copper (Cu) is a potent antimicrobial agent. Its use as a disinfectant goes back to antiquity, but this metal ion has recently emerged to have a physiological role in the host innate immune response. ...Recent studies have identified iron–sulfur containing proteins as key targets for inhibition by Cu. However, the way in these effects at the molecular level translate into a global effect on cell physiology is not fully understood. Here, we provide a new insight into the way in which Cu poisons bacteria. Using a copA mutant of the obligate human pathogen Neisseria gonorrhoeae that lacks a Cu efflux pump, we showed that Cu overloading led to an increased sensitivity to hydrogen peroxide. However, instead of promoting disproportionation of H2O2 via Fenton chemistry, Cu treatment led to an increased lifetime of H2O2 in cultures as a result of a marked decrease in catalase activity. We showed that this observation correlated with a loss of intracellular heme. We further established that Cu inhibited the pathway for heme biosynthesis. We proposed that this impaired ability to produce heme during Cu stress would lead to the failure to activate hemoproteins that participate in key processes, such as the detoxification of various reactive oxygen and nitrogen species, and aerobic respiration. The impact would be a global disruption of cellular biochemistry and an amplified Cu toxicity.
CueO from Escherichia coli is a multicopper oxidase (MCO) involved in copper tolerance under aerobic conditions. It features four copper atoms that act as electron transfer (T1) and dioxygen ...reduction (T2, T3; trinuclear) sites. In addition, it displays a methionine-rich insert which includes a helix that blocks physical access to the T1 site and which provides an extra labile site T4 adjacent to the T1 center. This T4 site is required for CueO function. Like many MCOs, CueO exhibits phenol oxidase activity with broad substrate specificity. Maximal activity with model substrate 2,6-dimethoxyphenol required stoichiometric occupation of T4 by CuII (notation: CuII-CueO). This was achieved in Mops buffer which has little affinity for Cu2+. However, pH buffers that bind or precipitate Cu2+ (Tris, BisTris, and KPi) generated enzyme with a vacant T4 site (notation: ◻-CueO) which has no phenol oxidase activity. Addition of excess Cu2+ effectively generated a Cu2+ buffer and recovered the activity partially or completely, depending upon the specific pH buffer. This phenomenon allowed reliable estimation of the affinity of T4 for CuII: K D = 5.5 × 10−9 M. CueO is involved in copper tolerance and has been suggested to be a cuprous oxidase. The anion CuI(Bca)23− (Bca = bicinchoninate) acted as a novel chromophoric substrate. It is a robust reagent, being air-stable and having a CuI affinity comparable to those of periplasmic CuI binding proteins. The influences of pH buffer composition and of excess Cu2+ on cuprous oxidation were diametrically opposite to those seen for phenol oxidation, suggesting that ◻-CueO, not CuII-CueO, is the resting form of the cuprous oxidase. Steady-state kinetics demonstrated that the intact anion CuI(Bca)23−, not “free” Cu+, is the substrate that donates CuI directly to T4. The data did not follow classical Michaelis−Menten kinetics but could be fitted satisfactorily by an extension that considered the effect of free ligand Bca. The K m term consists of two components, allowing estimation of the transient affinity of T4 for CuI: K D = 1.3 × 10−13 M. It may be concluded that CuI carried by CuI(Bca)23− is oxidized only upon complete transfer of CuI to T4. The transfer is required to induce a negative shift in the copper reduction potential to allow oxidation and electron transfer to the T1 site. The results provide compelling evidence that CueO is a cuprous oxidase. The new approach will have significant application to the study of metallo-oxidase enzymes.
Copper (Cu) is a key antibacterial component of the host innate immune system and almost all bacterial species possess systems that defend against the toxic effects of excess Cu. The Cu tolerance ...system in Gram-negative bacteria is composed minimally of a Cu sensor (CueR) and a Cu export pump (CopA). The cueR and copA genes are encoded on the chromosome typically as a divergent but contiguous operon. In Escherichia coli, cueR and copA are separated by two additional genes, ybaS and ybaT, which confer glutamine (Gln)-dependent acid tolerance and contribute to the glutamate (Glu)-dependent acid resistance system in this organism. Here we show that Cu strongly inhibits growth of a ΔcopA mutant strain in acidic cultures. We further demonstrate that Cu stress impairs the pathway for Glu biosynthesis via glutamate synthase, leading to decreased intracellular levels of Glu. Addition of exogenous Glu rescues the ΔcopA mutant from Cu stress in acidic conditions. Gln is also protective but this relies on the activities of YbaS and YbaT. Notably, expression of both enzymes is up-regulated during Cu stress. These results demonstrate a link between Cu stress, acid stress, and Glu/Gln metabolism, establish a role for YbaS and YbaT in Cu tolerance, and suggest that subtle changes in core metabolic pathways may contribute to overcoming host-imposed copper toxicity.
The nasopharynx and the skin are the major oxygen-rich anatomical sites for colonization by the human pathogen Streptococcus pyogenes (group A Streptococcus GAS). To establish infection, GAS must ...survive oxidative stress generated during aerobic metabolism and the release of reactive oxygen species (ROS) by host innate immune cells. Glutathione is the major host antioxidant molecule, while GAS is glutathione auxotrophic. Here, we report the molecular characterization of the ABC transporter substrate binding protein GshT in the GAS glutathione salvage pathway. We demonstrate that glutathione uptake is critical for aerobic growth of GAS and that impaired import of glutathione induces oxidative stress that triggers enhanced production of the reducing equivalent NADPH. Our results highlight the interrelationship between glutathione assimilation, carbohydrate metabolism, virulence factor production, and innate immune evasion. Together, these findings suggest an adaptive strategy employed by extracellular bacterial pathogens to exploit host glutathione stores for their own benefit.
During infection, microbes must escape host immune responses and survive exposure to reactive oxygen species produced by immune cells. Here, we identify the ABC transporter substrate binding protein GshT as a key component of the glutathione salvage pathway in glutathione-auxotrophic GAS. Host-acquired glutathione is crucial to the GAS antioxidant defense system, facilitating escape from the host innate immune response. This study demonstrates a direct link between glutathione assimilation, aerobic metabolism, and virulence factor production in an important human pathogen. Our findings provide mechanistic insight into host adaptation that enables extracellular bacterial pathogens such as GAS to exploit the abundance of glutathione in the host cytosol for their own benefit.
In bacteria, copper (Cu) is often recognised for its potential toxicity and its antibacterial activity is now considered a key component of the mammalian innate immune system. Cu ions bound in weak ...sites can catalyse harmful redox reactions while Cu ions in strong but adventitious sites can disrupt protein or enzyme function. For these reasons, the outward transport of Cu from bacteria has received significant attention. Yet, Cu is also a bacterial nutrient, required as a cofactor by enzymes that catalyse electron transfer processes, for instance in aerobic and anaerobic respiration. To date, the inward flow of this metal ion as a nutrient and its insertion into target cuproenzymes remain poorly defined. Here we revisit the available evidence related to bacterial nutrient Cu trafficking and identify gaps in knowledge. Particularly intriguing is the evidence that bacterial cuproenzymes do not always require auxiliary metallochaperones to insert nutrient Cu into their active sites. This review outlines our effort to consolidate the available experimental data using an established energy-driven model for metalation.
There is increasing interest in the use of lipophilic copper (Cu)-containing complexes to combat bacterial infections. In this work, we showed that Cu complexes with bis(thiosemicarbazone) ligands ...Cu(btsc) exert antibacterial activity against a range of medically significant pathogens. Previous work using Neisseria gonorrhoeae showed that Cu(btsc) complexes may act as inhibitors of respiratory dehydrogenases in the electron transport chain. We now show that these complexes are also toxic against pathogens that lack a respiratory chain. Respiration in Escherichia coli was slightly affected by Cu(btsc) complexes, but our results indicate that, in this model bacterium, the complexes act primarily as agents that deliver toxic Cu ions efficiently into the cytoplasm. Although the chemistry of Cu(btsc) complexes may dictate their mechanism of action, their efficacy depends heavily on bacterial physiology. This is linked to the ability of the target bacterium to tolerate Cu and, additionally, the susceptibility of the respiratory chain to direct inhibition by Cu(btsc) complexes. The physiology of N. gonorrhoeae, including multidrug-resistant strains, makes it highly susceptible to damage by Cu ions and Cu(btsc) complexes, highlighting the potential of Cu(btsc) complexes (and Cu-based therapeutics) as a promising treatment against this important bacterial pathogen.
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