54

BACTERIA, IRON AND PATHOGENICITY

ANN J M MESSENGER and RAYMOND BARCLAY

Department of Biochemistry
University o f Hull
Hull, UK

Introduction Iron exists in both ferrous (Fe 2+) and ferric (Fe 3+) oxidation states and this property confers on
it the ability to act as a catalyst in biological systems. However, in aqueous medium at physio-
logical pH it exists in the form of an insoluble polymer Fe(OH)3 which has a solubility constant
(Ks) o f ~ 4 x 10-36

[Fe] [OHI a
K s - lEe(OH)a]

From this it is calculated that any free Fe 3÷ in excess of ~ 2.5 x 10-18 M would be precipi-
tated as Fe(OH)a, so that bacteria and indeed all living cells must use special mechanisms in order
to solubilize and assimilate this iron. These methods must be efficient enough to compete against
other micro-organisms in the same environment. In the case of pathogenic bacteria the situation is
further complicated because these bacteria must acquire iron from the host in which it is usually
tightly bound in such complexes as transferrin, lactoferrin or haemoglobin. In many cases the
efficiency of iron acquisition by the bacteria under these circumstances can be important in
determining their pathogenicity. Organisms secreting high affinity iron-binding compounds which
can sequester iron from their host transferrin and other iron proteins are obviously at an advan-
tage. The role of iron in pathogenicity is discussed in more detail later in this review.

Role o f iron in Iron has numerous, diverse functions in bacterial cells. It influences cell composition, inter-
bacterial cells mediary metabolism, secondary metabolism, enzyme activity and host cell interactions which
would include pathogenicity (see Table 1).

Table 1 Roles of iron in bacteria

Affected Area Effects

Cell composition Iron deficiency can cause: growth inhibition, decrease

Intermediary metabolism
Metabolic products
Proteins and enzymes requiring iron
in RNA and DNA synthesis, inhibition of sporulation,
changes in morphology of cells
Processes requiring iron: tricarboxylic acid cycle,
electron transport, oxidative phosphorylation, nitrogen
fixation, aromatic biosynthesis
The following products are among those regulated
ie increased or decreased by iron: porphyrins, toxins,
vitamins, antibiotics, hydroxamates, cytochromes,
pigments, siderophores, aromatic compounds, DNA
and RNA
Peroxidase, superoxide dismutase, nitrogenase, hydro-
genase, glutamate synthase.
Cytochromes, ferridoxins, flavoproteins, ferritin.

The main functions of iron are in a catalytic rather than a regulatory role. Enzymes necessary
for the oxidative metabolism of the tricarboxylic acid cycle eg aconitase, and the cytochromes
and non-haem iron electron carriers of the electron transport chain require iron for their activity.
Since glycolytic enzymes do not require iron, aerobic and facultative organisms can overcome
any deficiency of iron by shifting their metabolism in favour of glycolysis rather than the tri-
carboxylic acid cycle. Thus, their main energy source becomes substrate-level phosphorylation
rather than oxidative phosphorylation.

B I O C H E M I C A L E D U C A T I O N 11(2) 1983
 55

The use of oxygen as electron acceptor in bacteria requires the protection of the cells against
peroxide radicals. Peroxidases, catalases and certain forms of superoxide dismutase, which
destroy the harmful radicals, contain iron and so it has an important role to play in the mainten-
ance of the cell.
Iron, besides being an obvious co-factor for various proteins, can influence other components
of the cell. For example, under iron-deficient conditions Mycobacterium smegmatis shows de-
creased DNA and RNA levels. Iron is also needed in Escherichia coli for the functioning of ribo-
nucleotide diphosphate reductase subunit B2 which is responsible for the synthesis of the deoxy-
ribotides required for DNA synthesis. In Bacillus subtilis iron starvation also blocks DNA synthe-
sis. Structural changes in certain tRNA molecules during iron-deficiency have also been found.
Iron is necessary in many stages of nitrogen assimilation. The prokaryotic nitrogenase enzyme
complex responsible for the reduction of dinitrogen to ammonia consists of an iron-molybdenum
protein and an iron-protein. The electrons required for the reduction reaction are carried by the
protein ferredoxin which also contains iron. The ammonia formed by the reduction of dinitrogen
is assimilated into glutamine which can then act as a source of amino nitrogen in the formation
of glutamate from a-ketoglutarate. Glutamate synthase, the enzyme responsible for this reaction,
also requires iron. Recent results have shown that iron is required in aromatic biosynthesis in
E coli. The first enzyme of the aromatic pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate
(DAHP) synthase, contains one atom of iron per molecule of enzyme.
Unique amongst living systems with regard to their demand for iron are the lactobacilli. Many
attempts to measure their iron content have proved negative. However, it has been reported ~
that substantial amounts of iron are found in these bacteria but it is uncertain whether this iron
is functional or merely an impurity. Lactobacteria do not contain haem compounds, cyto-
chromes or peroxidases. For a key enzyme such as ribonucleotide diphosphate reductase they use
a cobalt containing vitamin B~ 2 form instead of the iron containing one found in other organ-
isms (see above). Under iron deficient conditions, many bacteria produce siderophores, ie low
molecular weight iron-sequestering compounds (see below) which function in the solubilizing and
transport of iron, but none have been discovered in the lactobacteria. However, it is thought that
if these micro-organisms do require iron they may solubilize it with the acid they secrete during
growth.
As well as its catalytic function iron has a structural role in a diverse group of aquatic bacteria.
They contain iron in the form of magnetite (Fe3 O4) which confers on them magnetotactic
characteristics so that they swim in a preferred direction in weak magnetic fields.
The function of iron in bacteria has been discussed to a greater extent in recent literature. 1,2
Production of Siderophores, formerly termed siderochromes or sideramines, are low molecular weight ( 6 0 0 -
siderophores 1000) iron-binding compounds produced by micro-organisms under iron-deficient growth con-
ditions. Their production is strongly repressed by the presence of iron. They have a high affinity
for ferric ions (stability constants are in the range 103°-10 s°) but have a very low affinity for
ferrous iron. This low affinity for reduced iron may be important in the removal of iron from the
siderophore once in the cell (see later). Siderophores, which are usually produced extracellularly,
solubilize any available iron in the culture medium (or in the milieu of the organism) and make it
available to the organism. They therefore must be able to hold iron in solution against compet-
ing anions such as phosphate, and it is on this criterion thay many so-called siderophores may fall
down when examined more critically.
Siderophores can be divided into two main structural groups: the hydroxamates and the cate-
chols or phenolates (Fig 1). For a recent review of the structures of various bacterial siderophores
see Neilands and Ratledge (1982). 3
(a) Hydroxamate type siderophores are most common in fungi although they are also produced
by some bacteria. The prototype of this group is ferrichrome (Fig la) produced by the smut
fungus Ustilago sphaerogena, which contains three residues of acetylated N-hydroxy-L-ornithine.
Although it is not produced in bacteria it can be used by them to transport iron. The secondary
o OH
hydroxamate group -~-/~-R' where R is an amino acid or a derivative of one, is responsible for
iron chelation. In most hydroxamate compounds three hydroxamate groups co-ordinately bind
with trivalent iron to yield a neutral chelate. However, mycobactins, (Fig lb) produced by the
mycobacteria are dihydroxamate compounds, the third pair of chelating groups is provided by
the N atom of a oxazoline ring and the HO group of salicylic acid. These siderophores differ from
other hydroxamates in their high lipid solubility due to the presence of a long alkyl chain.

B I O C H E M I C A L E D U C A T I O N 11(2) 1983
56

H\
H 0 [ b) H,.,C//C-(CHz)~CH~
(Q} N-~-"~ ~
/
O.C~
(iHe)~
~
NH ~o=~
HCH ~'~
/CH ~HO-I~
I
/ f~
~0-
•

~__(c.~l~"\ ~CH~
HN HsC-C~o~
I :0~ q'O ~CH~
o.~ ~CH~
O=C
~ ~ / ,~<, ,c.,
HCH~ CHz-CHz- CHz~CH C~ %~--NH--~
HN C=O
~-~ ~-o
~e ~ ~
~ . ~H

(c)
OOH COOH OHO ,6" ~
I1[ I I II
CH]C-N(CH~)~NHCOCHeCOHCH~
CONH(CHz)SN-- CCH~ H
(el
~c.~.. "cr.~
(o) /N~H ~c~ 0J I
~-0
~0
/ (CHz)
5 (CHz)z~
-\
/ \
~ / ~
~.,o-,~/ . I/NH ~ )~ NH.£

/ k
CHz)s CH~
NHz ~ ~

Figure 1 Structure of siderophores

Hydroxamate derivatives of citric acid are produced for iron transport in certain bacteria eg
schizokinen (Fig lc) in Bacillus megaterium and aerobactin in Aerobacter aerogenes. These are
dihydroxamate compounds linked to a citrate group. Because they only contain two hydrox-
amate functions the central carboxyl and hydroxy groups of citrate form the third pair of chelat-
ing groups.
Species of Streptomyces and Nocardia produce ferrioxamine type hydroxamates. These are
built on the repeating unit of w-N-hydroxy-l-amino-alkane and succinic acid. The most common
of this group is ferrioxamine B (Fig ld).
(b) Phenolates. Enterochelin (Fig le), the cyclic triester of 2,3-dihydroxybenzoylserine has been
found in E coli, Salmonella typhimurium and Klebsiella pneumoniae. 4,s Like other phenolate
siderophores each enterochelin molecule complexes 1 atom of ferric iron whereby a hexadentate
complex is formed between the iron and the six phenolic hydroxy groups. The compounds 2,3-
dihydroxybenzoate and 2,3-dihydroxybenzoylserine, which are also active in iron transport, are
found in these organisms as breakdown products of enterochelin. Other amino acid conjugates of
2,3-dihydroxybenzoate are found in other bacteria. A threonine conjugate has been found in
Klebsiella and E coli. Strains ofB subtilis contain a glycine conjugate.

Mechanisms o f Iron is solubilized in aqueous solution by the formation of complexes with siderophores. The
iron transport ferric-siderophore complexes have a higher affinity for the cell surface than the desferri (without
iron) siderophore and so function in the transport of iron across the cell envelope. Once inside
the cell the iron is released from the siderophore and becomes available for utilization by the cell
(see later section).
The mechanisms of iron transport into the cell are best understood in E coll. Here we describe
these transport mechanisms and compare them with those of a very different group of bacteria,
the mycobacteria, within whose group is included the tubercle bacillus, M tuberculosis and
several other pathogenic species. These two systems of iron transport are given to illustrate the
range and diversity of the processes which occur in bacteria and, of course, in other micro-
organisms.

B I O C H E M I C A L E D U C A T I O N 11(2) 1983
 57

(a) Mechanisms in E coli.

The importance of iron to bacterial cells is reflected in the numerous ways they have devel-
oped to acquire their iron supply. In E cell ferric enterochelin (Fig le) serves in the transport of
iron across the cell membrane. There are specific proteins within the membrane which are appar-
ently concerned in iron transport as they are absent from cells grown under iron-sufficient con-
ditions when iron transport systems become repressed. Mutants that lack these proteins turn out
to be defective in iron transport even when grown under iron-deficient conditions.
Many of the membrane receptor proteins needed for iron transport in E coli also serve as
receptors for the attachment of killing agents such as bacteriophages and colicins (small proteins
produced by bacteria which are usually bacteriocidal to related species). The ferric enterochelin
system requires a specific outer membrane receptor designated (Fep A) which serves for the bind-
ing of the enterochelin complex as well as colicins. Another specific protein, a permease, is also
necessary for transport across the cytoplasmic membrane. The Ton B receptor protein (desig-
nated from T one phage receptor) found in the cytoplasmic membrane is essential for uptake of
iron from enterochelin as well as from other ferric siderophore complexes. This receptor protein
is also necessary for transport of vitamin B12, the irreversible adsorption of T1 and ~80 phages
and for the action of certain colicins.
The role of the Ton B protein is uncertain. Active transport systems require a source of energy
and the Ton B protein may function by the coupling of this energy source to the various trans-
port systems with which it is associated. It is also possible that it acts by regulating or modifying
the permeases involved in these uptake systems. The various hypotheses for the function of the
ton B gene are discussed in a recent review by Wookey (1982). 6 The role of receptor proteins in
iron transport in E coli is reviewed by Braun and Hantke (1981). 7
E coli possesses several iron transport systems besides that using enterochelin. The natural
breakdown products of enterochelin, the linear dirner and trimer of 2,3-dihydroxybenzoyl-
serine (DBS), are efficiently transported in a similar way to enterochelin. However, transport
via 2,3-dihydroxybenzoate (DHB) uses a separate uptake system which bypasses the proteins
necessary for iron uptake from enterochelin.
Although ferrichrome is not synthesized by E coli this organism does have a transport system
enabling it to acquire iron from ferrichrome. The ferrichrome complex is initially bound to an
outer membrane protein receptor termed Fhu A and transported into the cell under the control
of t h e / h u B and tonB genes. There is competition for the outer membrane protein between
ferrichrome and T5 phage. In the ferrichrome-mediated iron transport system the iron-ferri-
chrome complex is not carried into the cytoplasm but instead iron is released from the complex
whilst it is still in the cell membrane. This release is by the reduction of the iron to Fe(II) which
has a low affinity for ferrichrome. The free iron-binding site thus created is then acetylated and
this modified product then excreted from the cell. s,9
Under certain conditions citric acid can solubilize iron and aid its transport into the cell. At
physiological pH, when citrate and iron are in solution at equimolar concentrations a high mole-
cular weight, insoluble ferric citrate complex is formed. However, once citrate and iron are at a
molar ratio of 20:1 respectively a soluble ferric dicitrate complex is formed. Iron from this
complex can be assimilated by E coli. Frost and Rosenberg have shown that uptake of iron
occurs when iron is present at 1 /aM and citrate at 1 mM. This system is induced by growth of
E coli in the presence of 1 mM citrate. 10 Under these circumstances an outer membrane protein
is also induced which is necessary only for this transport system. Binding of the ferric citrate
complex to the outer membrane is enhanced by this protein, designated Fec A. The ferric
dicitrate complex does not enter the cell and so the transfer from the complex into the cell must
occur at the outer membrane. 11 Again this release is probably (but not yet established) by a
reductive mechanism.
Another system of iron uptake in virulent strains o f E coli has recently been reported which is
coded for by a plasmid (Col V) which also codes for the production of the specific antibacterial
colicin V protein. Early in the growth phase a cell associated hydroxamate is produced in such
cells and later it is also present in the supematant. ~2 This system is independent of colicin V
synthesis and activity, but the receptor for the uptake of aerobactin also functions as receptor
for the bacteriocin cloacin DF13. ~3 Acquisition of this system by the bacterium enhances its
pathogenicity because the organism now has an increased tolerance to the bacteriocidal effects
of serum ~4 (see pathogenicity section). This system also requires the chromosome coded Ton B
protein mentioned previously. The hydroxamate produced by these cells has been identified by

B I O C H E M I C A L E D U C A T I O N 11(2) 1983
58
Braun as aerobactin which was originally isolated from Aerobacter aerogenes (now Klebsiella
pneumoniae ).
As well as these high affinity transport systems in E coli there is also a low affinity system for
iron uptake. Little is known about this system whereby uncomplexed iron is transported at a
very low rate into the cells. It may be that this represents a simple diffusion system for the
uptake of free iron.
The iron transport mechanisms o f E coli are summarised in Fig 2a.
( O) CYTOPLASM CELL ENVELOPE EXTERIOR

H~ghA f finity_..~Systems ( b ) CYTOPLASM CELL ENVELOPE EXTERIOR

Fe3.
7 "~ Fe3. ~,~
/,~1~mycobact in~ ~,F'e 3-*sali¢~l~at e

~
3
DHB and2,3 DBS Fe3~
* Fe t DHB and Fe3-*DBS Fe 3.

? ~esterase ~pe3t mycobactin~I W~salicylate

glutat~ Fene"3_*enterocheli n Fe3-+enterochelin

salicylat e t
?~ctase
~'~,,~ Fe3- Low exochelin conc.
I~ ~enterochelin :) enteroc helin (up to - 10~/.M)
Fe2tglutathione Fe3-* exochelin ~ Fe3texochetin
.,. Fe3-* dicit rate
Fe3 - ~
exochelin • exoc~lin Fe3*
Fe3~ -~cit rate

f errichrome H.~gh exochelin cone.

Fe2-*ferrichro rn•
~¢
Fe -*ferrlchr ome
(above 10 #M_)
FeJ ~ . . ~ ~.-~mycobac t i n , - , ~ ~ Fe~-ex°c helin
re2~. "~'~rorne
acetylated - - ~ acetylated
~"..~e~o~,~i,,'
~ F'e3*_rny c o b ac t in~1~
~"
~
ferr'ichrome ferrichrome
~e3~dicitrate
aerobactin ) aerobactin

Pe~
LOW Af flmty__System
Fe3+,~ p2÷
~it~.~,~ "~"
Figure 2 Postulated mechanisms of bacterial iron transport. (a) E. coli (b ) M. smegmatis
(b ) Mycobacterial mechanisms
Iron transport in these acid-fast bacteria presents problems because they possess a high
amount of lipid in their cell walls which gives these organisms many of their outward character-
istics including their insensitivity to many chemotherapeutic agents. Not only do the mycobac-
teria still need to solubilize iron in aqueous medium but they must be able to transport it across
the thick lipoidal cell wall. Mycobacteria overcome this problem by producing a family of lipid-
soluble, iron binding compounds known as mycobactins. These are found in the cell envelope
rather than the external medium when the organisms are grown under iron deficient conditions.
Since the location of mycobactin is restricted to the cell envelope it cannot function in the solu-
bilization of iron in the aqueous medium. Mycobacteria do, however, produce other water-
soluble iron-binding compounds, the exochelins.
Two types of exochelins have so far been recognized: one found in M smegrnatis, M vaccae
and M neoaurum which is water soluble and cannot be extracted into organic solvents and
another type found in M tuberculosis, M bovis, M intracellulare, M avium, and other pathogenic
species which is extractable as the ferri-complex from water into chloroform.
Structural analysis of the main exochelin from M smegmatis suggest that it is similar to the
hydroxamate siderophores, since it contains a lysine derivative, probably e-N-hydroxylysine, and
other amino acids such as/3-alanine and threonine. Two iron-transport systems are mediated by
exochelins in M smegmatis. One process is saturable at about 10/aM and is inhibited by metabolic
inhibitors. This system is an active transport process and functions independently of mycobactin.
The other process operates at higher concentrations of iron and is neither saturable (ie the rate of
uptake continually increases as the concentration of ferri-exochelin presented to the cells is
increased) nor inhibited by metabolic inhibitors. This process may involve mycobactin as the
acceptor of the iron) s

BIOCHEMICAL EDUCATION 11(2) 1983

 59

Another system for the acquisition of iron may be through the mediation of salicylic acid,
which mycobacteria secrete. This process only functions in the absence of phosphate (which pre-
cipitates the iron) and does involve the participation of mycobactin as the intracellular acceptor
molecule.
Yet another iron transport system which involves citrate is also present in mycobacteria, t 6
As with the system in E coli the citrate to iron ratio must be high for it to operate. The system
in mycobactefia differs from that in E coli in that it is constitutive and not induced by growth on
citrate. The citrate system in mycobacteria is not inhibited by metabolic inhibitors.
The iron transport systems of mycobacteria are summarised in Fig 2b.

I r o n release from Once iron has been taken into the cell in the form of a siderophore complex it must be released
siderophores from the carrier to become metabolically functional.
In most cases the redox potential of iron bound in these complexes is within the physio-
logical range for redox systems (NADH -~ NAD ÷ etc) and so the iron is probably released by
reduction. The low affinity of the siderophores for Fe(II) facilitates this removal as it is envisaged
that the various acceptor molecules for the iron (porphyrins, non-haem iron, apoproteins) will
react with Fe(II) rather than Fe(III).
Ferrisiderophore reductases have been observed in many micro-organisms. 17 Highest activity
is usually found when cell-free extracts are tested under anaerobic conditions with NADH or
NADPH as a reductant. The reaction can be summarised as follows:
Fe(III)-siderophore + NAD(P)H -* Fe(II)-siderophore + NAD(P) ÷
Work with B rnegaterium and M smegrnatis suggests that more than one enzyme may have
this activity and it may be one of the physiological roles of multifunctional reductases. In B sub-
tills, where schizokinen (see Fig lc) is the natural siderophore, this activity is associated with an
enzyme complex containing chorismate synthase and dehydroquinate synthase. 1 a
In the case of ferrienterochelin, however, it is possible that the complex must be hydrolysed
to three bidentate ligands before release of iron by reduction can occur. Enterochelin esterase
which carries out this reaction has been found in E coli but there is controversy about whether
the substrate is the ferric complex or the free ligand (enterochelin itself). Because esterase
activity would destroy the enterochelin molecule during iron release, meaning it could not be
re-utilised, this proposition seems rather unattractive. The strongest evidence for the role of the
esterase in iron release comes from mutants lacking this enzyme and which are unable to dissoci-
ate the enterochelin complex. 19 Further evidence exists in that the enzyme is only induced
under low iron growth conditions. However, B subtilis can release iron from synthetic entero-
chelin analogues which cannot be hydrolysed2° but these experiments still await to be tried with
E coli. The redox potential of Fe(III)-enterochelin is reported to be much lower than physio-
logical reductants and so, at first sight, it seems unlikely that iron could be released by reduction.
However at pH values below 6 the protonation of a catechol oxygen atom (see Fig le) may alter
the co-ordination of the complex in such a way as to facilitate the reduction of iron without the
breakdown of the enterochelin molecule. Indeed, it has recently been shown that at these pH
values glutathione, which is present at high levels in E coli, can remove iron from enterochelin
and form a stable Fe(II) glutathione complex. ~ 1

Specificity o f If a bacterial siderophore is active in the transport of iron in the producing species alone it may
siderophore function confer a high survival value on that organism when grown in a mixed population under iron-
deficient conditions: the binding of iron t o this siderophore could render it unavailable to
competing organisms. However, the ability of an organism to use siderophores produced by other
organisms would give it a survival advantage. Some bacteria can utilize foreign siderophores to
acquire iron. Thus the ferrichrome uptake system in E coli, which has already been mentioned
(Fig 2a) involves the siderophore from fungi being actively taken up by the bacterium. Similarly
mycobacteria show an ability to use each others exochelins.2: However, certain molecules do
seem to be specific for this process. Agrobactin from Agrobacterium tumeofaciens and para-
bactin from Paracoccus dinitrificans possess a stereochemical arrangement making them useful to
their producers but not to other species. Ferric citrate is utilized by E coli and M smegmatis but
not by S typhimuriutn.
The amount of specificity and interchangeability in the use of siderophores probably depends
ultimately on the habitat of the organism concerned and the amount of competition it faces in

B I O C H E M I C A L E D U C A T I O N 11(2) 1983
60

acquiring its iron. Having said this, it is interesting to note that the ferrichrome system in E coli
involves a gut organism utilizing a siderophore produced by a soil fungus.

Iron and pathogenicity There are many ways in which man and animals can avoid becoming hosts to large numbers of
bacteria. One relatively simple and non-specific way of doing this is to starve any bacteria that
enter the body of nutrients which are essential for their growth. This often effective defence
mechanism is known, for obvious reasons, as nutritional immunity (Kochan, 1976). 23 Nutri-
tional immunity is important to the host because it not only presents the invading organisms
with an immediate barrier which has to be overcome but also allows time for other, slower-
acting, defence mechanisms to take effect. The mechanism of nutritional immunity relies mainly
on the invading organisms being dependent on the nutritionally-rich fluids of the host to provide
them with the nutrients they need. Iron plays a particularly important role here, and it is this
which is the nutritionally-limiting factor for bacterial multiplication in serum, saliva, tears, milk
and eggs. Thus when mammals are invaded by bacteria they limit the amount of available iron by
decreasing the concentration in their sera. This is termed hypoferraemia and is accomplished by
decreasing the amount of iron taken up from the intestine and by increasing the amount stored
in the liver. Fever might also develop as a result of bacterial invasion and this could further serve
to restrict the uptake of iron into bacterial cells, because a slight increase in the host's body
temperature above 37°C could significantly decrease the production of bacterial iron-chelators
(Table 2). Such a temperature effect could even conceivably prevent some bacteria from infect-
ing animals, such as birds, which have relatively high body temperatures (40 ° to 44°C).

Table 2 Production of iron-chelators by Salmonella typhimurium Tm-1 at different temperatures

Amount of phenolate iron

transport compoundst

°C 0.03 ,ug Fe/ml 0.3 /ag Fe/ml

31.0 0.2 0.52

34.7 0.3 0.34
36.9 0.18 0.20
40.3 < 0.005 < 0.005

~fExpressed as microequivalents of 2,3-dihydroxybenzoic acid per ml of cell-free supernatant fluid. Data

from Garibaldi) s

Although the amount of iron in serum declines in response to bacterial invasion, theoretically
there still remains enough to support bacterial growth. Depending on the circumstances, human
serum contains between 1 and 3/ag iron/ml whilst bacteria typically require between 0.02 and
0.5 ~g/ml. Yet, despite the presence of what would seem to be enough iron in the host fluids
these materials cannot support the growth of bacteria unless more iron is added to them. At
first sight this seems contradictory, and opposes the general view that the addition of these fluids
to laboratory culture media enhances the growth of bacteria. However, the prevention of bac-
terial growth can be explained by the presence in host fluids of compounds such as transferrin,
ferritin, lactoferrin and conalbumin which have a high affinity for iron, and which can therefore
withhold the iron from invading bacteria.
Transferrin, which has a molecular weight of 76 000, can strongly bind two atoms of iron
(each with a stability constant of about 1036). Under normal circumstances human serum, for
example, contains 2 to 3 mg of transferrin per ml. Therefore, in relation to the amount of iron
present in serum (see above) transferrin is in such an excess that only about one third of it is
saturated. The net result is that the amount of iron which is available to the bacteria is limited
to less than 0.01 /zg/ml, which is insufficient to support bacterial growth. The need for such an
excess of transferrin can also be explained by the fact that an uptake of more iron into the blood-
stream could easily over-saturate a low concentration of transferrin in the serum. Furthermore,
some bacteria produce their own iron-binding compounds which have higher affinities for iron
than their host equivalents. With tubercle bacilli, for example, the more the serum transferrin is
saturated with iron then the greater the growth of the bacteria. However, even virulent myco-
bacteria find it difficult to withdraw iron from transferrin whose iron saturation is less than 70%

BIOCHEMICAL EDUCATION 11(2) 1983

 61

unless a large quantity of mycobactin is present within the cells. Bacterial iron-chelators are
therefore important and advantageous contributors to the growth of pathogenic organisms.
However, even though a bacterium is capable of producing a siderophore this does not necess-
arily mean that the siderophore is actually produced in vitro or is used to the advantage of the
invading organism. For example, the virulence of E coli has been related to the production of
enterochelin, but some serotypes o r E coli cannot multiply in undiluted sera or milk even though
they can produce enterochelin. This is probably because of other factors, such as the production
of specific antibodies by the host, which could affect the uptake or secretion of the chelating
agent) 4 Care must be taken with such studies because there is also the possibility that the in-
vading organisms can produce additional siderophores. As mentioned above, the Colicin V
plasmid of E coli codes not only for a specific antibacterial compound but also for the iron-
binding aerohactin. These two compounds can individually enhance the virulence of E coli
when it is grown iron-deficiently. This has been shown by using mutants which are deficient in
the appropriate parts of the plasmid. 14 In contrast to enterochelin, (see above) aerobactin can-
not be transported across the membranes of those strains which do not carry a Col V plasmid.
Clearly this could give Col V strains a further advantage when they are invading hosts, since aero-
bactin also helps these strains overcome serum bacteriostasis. 14 When bacterial siderophores are
looked at from an evolutionary viewpoint one would have difficulty in deciding whether the pro-
duction and use of these bacterial siderophores to overcome bacteriostasis evolved in response to
nutritional immunity or was instead a fortuitous development which arose from their need to
solubflize iron in the inanimate environment.

T h e acquisition o f iron
without siderophores
The production of specific iron-binding compounds is not the only way in which bacteria can
acquire iron from their hosts. Other methods include the binding of iron to less specific cell-
wall components, the release of iron from the host by tissue destruction, or the acquisition of
iron by the infection of sites that have a low pH. For E coli and other Gram-negative bacteria the
most important non-specific cation-binding compound is probably lipopolysaccharide (LPS),
a major cell-wall component which is also responsible for the endotoxicity and antigenicity of
the cell. Virulent strains of E coli contain more LPS than avirulent strains (3.7 mg and 1.6 mg
LPS per 100 mg dry cell weight, respectively) but this enhancement of virulence might reflect
the toxic or the antigenic properties of LPS rather than its ability to bind iron. In addition, the
ability of LPS to bind iron may be offset by its tendency to induce hypoferraemia (ie to decrease
serum iron levels) and fever (thus decreasing the production of enterochelin) as host responses
to infection. In Gram-positive bacteria teichoic acid acts as the non-specific cation-binding com-
pound. This has no fever-inducing properties. An alternative method by which iron can be
acquired in vivo by some bacteria relies on the fact that iron need not bind directly to the cell
wall, but may instead be acquired via a non-bacterial mediator. For example, a mechanism of
iron uptake, which involves the possible binding of iron-saturated transferrin to the bacterial
cell wall, has been described for Neisseria. 2 s This binding of transferrin, probably is to specific
receptors in the cell wall.
Tissue destruction may result either from the production of toxins and hydrolytic enzymes by
the infecting organism, or by the host's own response to infection. Thus, the bacteria might
acquire iron by (a) the utilization of haem, in which case the production of haemolysins would
be an advantage, or (b) by the marked increase in the solubility of iron associated with an in-
crease in acidity as might follow, say, the release of the lysosomal contents of phagocytic cells
after these cells had ingested some invading bacteria. Although iron is more soluble at low pH
and transferdn is a poor iron-chelator below pH 7, this need not, however, mean that more iron
will def'mitely be made available to the invading bacteria. This is because the iron released from
transferrin may instead bind to lactoferrin, which is present in low concentrations (0.01/aM) in
serum and can hold Fe 3+ at pH 2. There will, therefore, be some interplay between transferrin
and lactoferrin for iron-binding at sites of infection.

Iron and virulence Although the addition of iron to an infected animal appears to enhance the extent of infection
by a variety of bacteria, some of which are listed in Table 3, it is experimentally difficult to show
how iron or its lack of availability affects the growth of bacteria in vivo. There is also the
problem of relating the production of the various siderophores with virulence since saprophytes
also produce iron-chelators, often in greater quantities than a corresponding pathogen. When the
effects of iron on the pathogenicity of a given bacterium are studied, the way in which the iron

B I O C H E M I C A L E D U C A T I O N 11(2) 1983
62

Table 3 Infections enhanced by iron

(a ) Mycobacterium tuberculosis (h ) Klebsiella pneumonia

(b ) Escherichia coli (i) Neisseria gonorrhoea
(c ) Clostridium perfringens q) Vibrio spp
(d) Yersinia pestis, Y septica (k ) Staphylococcus aureus
(e) Listeria monocytogenes (l) Pseudomonas aeruginosa
(0 Legionella spp (m ) Brucella abortis
(g) Salmonella spp

is administered to the host is important. The ability of an iron compound to promote a bacterial
infection is improved when the compound is administered during the early incubation period, or
when administered with the infecting inoculum, rather than when the bacteria have been trying
for some time to grow in the host. After administration, the iron must also be able to diffuse
to the site of invasion. As might be expected, the administration of iron usually decreases the
number of bacteria needed to cause disease or death. For example, the 50% lethal dose of Vibrio
vulnificus for mice was lowered dramatically from 6 x 10 6 to 1.1 x 10 ° cells by the intra-
peritoneal injection of 4/ag of ferric ammonium citrate per g of body weight into the mice. 26
Extreme iron overload in a host, such as can happen during a blood transfusion, can lead not
only to bacterial growth but also to degenerative changes, especially in the liver, myocardium and
the endocrine organs, because high iron-concentrations can be toxic to animal ceils. These
degenerative changes would be expected to weaken the animal and thus make it more susceptible
to bacterial infection. Bacteriostasis, however, can be overcome with lower concentrations of
iron than is needed to cause the disorders observed above, but even these might degeneratively
affect other host defence mechanisms. Bullen and Wailis 27 suggested that the iron-binding pro-
teins in polymorphonuclear leukocytes may play an essential role in the bactericidal activity of
these cells. Kochan et al 2 a showed that immune mice, when infected with virulent Salmonella
typhimurium and injected with iron, developed lethal infections as rapidly and nearly as fre-
quently as similarly-treated normal mice. Siderophores did not promote lethal infections in
immune mice, which implied that iron exerted a deleterious effect on acquired immunity.
The influence of iron on the virulence of bacteria is not restricted to it promoting the growth
of these organisms. Iron can also affect the production of toxins and extracellular enzymes that
act as virulence factors (Table 1). A low iron concentration could be advantageous here because
the production of many of these proteins is repressed by high iron concentrations. Thus, the
virulence of Pseudomonas aeruginosa may be increased by it being exposed to iron limiting con-
ditions because an increase in the iron concentration decreases the production of toxin A,
elastase and alkaline protease) 9 Iron at concentrations between 5 x 10 -6 and 10-4 M can depress
the synthesis of Clostridium perfringens a-toxin, Shigella shigae neurotoxin and Corynebacteria
diphtheria toxin, whilst tetanus toxin requires an optimum Fe: + concentration of 10-~ M, and is
suppressed if the iron concentration is trebled.

Disease and The intricate checks and balances between the host and pathogen for iron can be readily and
e x t e r n a l factors markedly upset either by external factors, or other diseases. Predictably, persons whose serum
transferrin has been saturated with iron (ie those with hyperferraemia) show increased suscep-
tibilities to bacterial infection. Hyperferraemia can occur because of liver damage, haemolytic
anaemia or an overload of iron. In the acute phases of the conditions listed in Table 4 the plasma
iron concentration may be double the normal value and, because the amount of transferrin does
not appreciably increase or may even decrease due to malnutrition (as in kwashiorkor), then the
saturation of transferrin is often greater than 60%. However, the risk of infection also depends on
other factors such as stress, population overcrowding and genetic predisposition, as well as the
virulence and prevalence of the invading organism.

Table 4 Diseases which affect the balance of iron in the host

(1) Sickle cell anaemia (4) Leukaemia (7) Haemochromatosis

(2) Viral hepatitis (5) Lymphonia (8) Kwashiorkor
(3) Malaria (6) Thalassemia (9) Alcoholism

BIOCHEMICAL EDUCATION 11(2) 1983

 63

The treatment of hyperferraemia is usually made by lowering the level of iron saturation with
desferrioxamine B (see Fig ld), or its methane sulphonate derivative (Desferal). s° Derivatives of
2,3-dihydroxybenzoic acid, such as enterochelin, have also been used. Desferrioxamine B has, in
a similar way, been used successfully to treat Listeria monocytogenes infections by reducing the
amount of iron available to it during the initial stages of infection. However, treatments which
involve the use of microbial products (desferrioxarnine B comes from Streptomyces species)
carry the risk that this compound might stimulate the growth of other invading micro-organisms.
For example, desferfioxamine B has been shown to stimulate the growth of Vibrio vulnificus in
serum, 26 and enterochelin, if this were to be given as an alternative, can stimulate the growth of
most enterobacteda in serum. Because enterochelin can bind metal ligands other than iron, these
enterochelin-metal complexes can compete with iron-enterochelin complexes to act as specific
antibacterial inhibitors. Rogers et al s ~ have shown that complexes of enteroehelin with scan-
dium or indium are particularly effective against enterobacteria such as E coli and KIebsiella
pneumonia.
The treatment to reverse a patient's iron-deficiency may be another way by which his serum
can end up being overloaded with iron, mainly because of previously diminished serum trans-
ferrin levels and the use of relatively large doses of iron. Barry and Reeve s ~ observed that newly
born Polynesian infants who had been treated for iron-deficiencies by intramuscular injections of
iron-dextran were about eight times more susceptible to bacterial infections than before, and that
the incidence of infection declined when the treatment was stopped. This was partly due to the
administration of iron coinciding with a period of high transferrin saturation which is normal at
this age. McFarlane et al s s found that when children suffering from kwashiorkor were fed on a
high protein-plus-iron diet a large proportion of them died soon after. This was assumed to be
due to the diminished transferrin levels which are associated with kwashiorkor, and which by the
absence of transferrin effectively allowed the concentration of free iron to reach an overload.
Although iron-deficiency is generally considered to be detrimental to the health of an indi-
vidual, it may have its advantages. For example, Somali nomads have a high incidence of iron-
deficiency because they live on an all-dairy diet, which contains large amounts of lactoferrin.
When they were treated with iron, 36 out of 71 of them became infected by micro-organisms,
whilst only 7 out of 67 untreated persons became infected, s4 The infections included the
activation of pre-existing malaria, brucellosis and tuberculosis, and it was therefore speculated
that the iron-deficiency was an ecological compromise which allowed normally susceptible hosts
to survive in a highly infective environment.

Acknowledgements Ann Messenger and Raymond Barclay are postdoctoral research assistants whose work is sup-
ported by the SERC and ARC respectively. We wish to thank Dr Colin Ratledge for his help in
the preparation of this review.

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B I O C H E M I C A L E D U C A T I O N 11(2) 1983