L4 - Metals and microbes in the context of infection Flashcards
What is the importance of iron?
- Iron is an essential co-factor for numerous basic metabolic pathways in both the mammalian host and the microorganism
- Iron is a critical component of cytochromes and iron-sulfur proteins that function in electron transport reactions
- The competition for iron between pathogens and host is of critical importance for pathogenesis
The dominant oxidation state of iron (Fe3+)
The dominant oxidation state of iron (Fe3+) is highly insoluble.
Iron is essential for all life-forms, and can exist in two oxidation states:
Fe3+ (ferric) dominates under oxygenated environments & at neutral pH
Fe2+ (ferrous) dominates in anaerobic environments & at low pH
However, solubility of ferric iron is extremely low compared to ferrous
0.1M for Fe2+ compared to 10-17M for Fe3+ (at pH 7)
Presents a problem for microorganisms with an aerobic lifestyle
Bacteria typically require iron concentrations of 10-5-10-7 M, and so they either:
reduce ferric iron to the more soluble ferrous form
employ ferric iron chelators as solubilising agents
Iron sequestration
Iron sequestration as an innate immune defence.
A first line of defence against infection is the withholding of nutrients to prevent pathogen growth
The majority of iron in the mammalian host is intracellular, either:
sequestered by the iron storage protein, ferritin
complexed with the porphyrin ring of haem as a cofactor of haemoglobin or myoglobin. Myoglobin is an iron- and oxygen-binding protein found in the muscle tissue of vertebrates in general and in almost all mammals. It is related to haemoglobin, which is the iron- and oxygen-binding protein in blood, specifically in the red blood cells. Myoglobin is only found in the bloodstream after muscle injury.
Extracellular iron (e.g. in serum) is typically in the insoluble ferric form due to the aerobic environment & neutral pH Iron limitation in the extracellular environment is further enhanced by high affinity iron-binding proteins (e.g. transferrin, lactoferrin).
Microbicidal activity of the phagosome
Acidification of the phagosome
V-ATPase complex translocates H+ across the membrane
Reactive oxygen & nitrogen species
NADPH oxidase & iNOS create ROS and RNS respectively
Antimicrobial peptides
Cationic peptides that bind to the negatively-charged bacterial cell
Sequestration of essential nutrients
e.g. action of metal transporters and metal binding proteins
Microbicidal activity of the phagosome (More extensive information)
The phagosome is the vesicle within phagocytic cells in which the ingested material (e.g. bacterial cell) is found following phagocytosis. The phagosome contains numerous bactericidal activities that aim to kill the ingested bacterium.
The V-ATPase complex used energy derived from hydrolysis of ATP to translocate protons (H+) across the membrane into the phagosome, resulting in acidification. The acidic pH is damaging to the microbe, as well as boosting the activity of enzymes within the phagosome whose function is optimal at acidic pH.
A major killing mechanism is through reactive oxygen species (ROS) that are generated directly or indirectly by the NADPH oxidase complex. The oxidase releases O2(-) into the lumen, which can dismutate to hydrogen peroxide. This can then react with O2(-) to generate hydroxyl radicals, and can also be converted into hypochlorous acid by myeloperoxidase (MPO). Collectively, these ROS are highly toxic, and effectively kill microorganisms.
Reactive nitrogen species are also produced, largely by inducible nitric oxide synthase (iNOS). Nitric oxide is produced by iNOS on the cytoplasmic side of the phagosome, and then diffuses across the membrane into the phagosome. The nitric oxide can then undergo spontaneous or catalytic conversion to a range of RNS including nitrogen dioxide and peroxynitrite. ROS and RNS synergize to exert highly toxic effects.
Antimicrobial peptides are small (12-50 amino acids) peptides with potent antimicrobial activity. They are positively-charged (cationic) and interact with the negatively-charged bacterial membrane. They disrupt the membrane, causing cell death.
All organisms require metal for growth and survival, as metals are essential cofactors for numerous enzymes. The phagosome attempts to starve the ingested bacterium of such metals by either pumping the metals out of the phagosome or by sequestering the metals within the phagosome. The diagram above shows this process for iron, but certain other metals are similarly treated, including manganese.
(Picture in notes)
What does iron sequestration also protects against?
Iron sequestration also protects against toxicity.
Under aerobic conditions, iron can be extremely toxic through its interaction with reactive oxygen species
Iron reduction: O2- + Fe3+ → Fe2+ + O2 Fenton reaction: Fe2+ + H2O2 + H+ → Fe3+ + HO˙ + H2O
The hydroxyl radical (HO˙) is highly reactive, resulting in protein denaturation, DNA breaks & lipid peroxidation
Therefore, all aspects of iron homeostasis are tightly coordinated to protect against toxicity.
Superoxide and hydrogen peroxide are only mildly reactive. In contrast, the hydroxyl radical (HO˙) generated through interaction with iron in the Fenton reaction is highly damaging and toxic to cells.
Define the antimicrobial activity of host iron-binding proteins
Lactoferrin is an iron-binding protein that is abundant in various secretions, including breast milk, saliva, tears and airway secretions
Present in the mg/ml concentration range
Name the iron uptake strategies of Pseudomonas aeruginosa
Numerous strategies exist by which P. aeruginosa can acquire iron:
- Via the production of extracellular Fe3+ chelating molecules termed siderophores
- Via the uptake of haem from host haemoproteins
- Via the extracellular reduction of Fe3+ to Fe2+ and the subsequent uptake of Fe2+ via the Feo system
Critical components for ferric iron uptake
The TonB complex consists of three proteins – TonB, ExbB & ExbD. Together, ExbB & ExbD couple the activity of TonB to the proton gradient of the cytoplasmic membrane. The periplasmic C-terminal domain of TonB interacts with both the outer membrane receptor and the PBP
(Picture in notes)
The critical role of the TonB complex
In bacterial cells, energy is generated via the proton gradient across the cytoplasmic membrane
However, the outer membrane of Gram-negative bacteria lacks such a proton gradient and thus energy (ATP)
Iron transport is energy dependent
The TonB complex couples the proton motive force of the cytoplasmic membrane to the outer membrane
Siderophores of Pseudomonas aeruginosa
P. aeruginosa produces two siderophores:
1. Pyoverdines
Considered the primary siderophore
Very high affinity for iron; able to displace iron from transferrin
Three types of pyoverdines identified in P. aeruginosa, differing in their peptide side chains.
- Pyochelin
Lower affinity than pyoverdines, but considered metabolically less costly
Possibly produced first, with a switch to pyoverdines only when iron concentrations are really low
What is the fate of iron-siderophore complexes?
“Ferrisiderophore” is used to describe the iron-siderophore complex
Although poorly defined, evidence suggests three distinct mechanisms by which iron is extracted from ferrisiderophores:
- Hydrolysis of the siderophore
- Modification of the siderophore scaffold
- Reduction of the bound iron from Fe3+ to Fe2+
The above processes can occur in the cytoplasm or in the periplasm
In P. aeruginosa:
- Ferripyochelin is transported into the cytoplasm
- Evidence suggests that iron is released from ferripyoverdines via Fe3+ reduction in the periplasm.
The affinity of siderophores is much lower for Fe2+, thus reduction of the iron centre results in liberation of the iron from the siderophore complex. Evidence suggests that iron is released from ferripyoverdines by reduction in the periplasm. The intact pyoverdine is then pumped out from the periplasm to the extracellular environment to chelate further iron.
Haem uptake by Pseudomonas aeruginosa
Two distinct mechanisms exist for haem uptake in P. aeruginosa:
- Has system
P. aeruginosa secretes a haemophore
Haemophore extract haem
Haemophore-haem complex is recognised by a TBDR - Phu system
Haem is directly extracted by a TBDR from the host haemoprotein within the cytoplasm, haem oxygenase degrades the molecule, liberating the iron.
TBDR = TonB-dependent receptor
Following transport across the outer membrane, haem is bound by a periplasmic binding protein & transported into cytoplasm by an ABC transporter. There, haem is bound by a haem chaperone (PhuS) that delivers the haem to a haem oxygenase.
Uptake of Fe2+ by the Feo system of P. aeruginosa
Fe2+ is soluble and present under anaerobic conditions or in microaerobic conditions at low pH
When Fe2+ is present, it can be taken up directly by the Feo system:
Fe2+ diffuses through the outer membrane
Transported across the cytoplasmic membrane by FeoABC
FeoB is the major component, acting as a permease across the membrane.
Permeases are membrane transport proteins that facilitate the diffusion of a specific molecule in or out of a cell by passive transport.
Exoproducts of P. aeruginosa can aid iron uptake
Proteases:
- Protease secreted by P. aeruginosa can degrade host iron-binding proteins such as lactoferrin & transferrin
- Iron is released and subsequently bound by siderophores.
Haemolysins:
- Haemolysins promote access to host haemoproteins
Phenazines:
- Phenazines are redox-active secondary metabolites
- Phenazine-1-carboxylic acid (PCA) is precursor of pyocyanin
- PCA (and to a lesser extent pyocyanin) reduces Fe3+ to Fe2+, promoting uptake by the Feo system
