Transferrins and heme-compounds as iron sources for pathogenic bacteria.

Critical Reviews in Microbiology, 18(3):217-233 ( 1 992)

Transferrins and Heme-Compounds as Iron Sources for Pathogenic Bacteria B. R. Otto,A. M. J. J. Verweij-van Vught, and D. M. MacLaren

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Department of Medical Microbiology, Faculty of Medicine, Vrije Universiteit, Van der Boechorststraat 7,1081 BT Amsterdam, The Netherlands

ABSTRACT: The low concentration of free iron in body fluids creates bacteriostatic conditions for many microorganisms and is therefore an important defense factor of the body against invading bacteria. Pathogenic bacteria have developed several mechanisms for acquiring iron from the host. Siderophore-mediated iron uptake involves the synthesis of low molecular weight iron chelators called siderophores which compete with the host iron-binding glycoproteins lactofemn (LF) and transfemn (TF) for iron. Other ways to induce iron uptake, without the mediation of siderophores, are the possession of outer membrane protein receptors that actually recognize the complex of TF or LF with iron, resulting in the internalization of this metal, and the use of hemecompounds released into the circulation after lysis of erythrocytes. In this review, the nonsiderophore-mediated iron-uptake systems used by certain pathogenic bacteria are emphasized. The possible contribution of these ironuptake systems to the virulence of pathogens is also discussed.

KEY WORDS: iron-uptake systems, heme uptake, virulence, iron-repressible outer membrane proteins

1. INTRODUCTION Iron is an essential growth factor of virtually all bacteria. The low concentration of free iron on the mucous membranes and in tissues is one of the first lines of host defense against bacterial infection. Bacteria have several mechanisms to overcome the ability of the host to withhold iron. The best-studied system is that of iron chelators, called siderophores, which compete with lactofenin (LF) and transfemn (TF) for iron. Ironrepressible outer membrane proteins (ROMPS) usually serve as receptors for iron-siderophore complexes and are essential for iron uptake. Such high affinity iron transport systems have been detected in many bacterial pathogen^.'-^ Little is known about bacterial iron-uptake systems which do not depend on the synthesis of siderophores. Bacterial species such as Neisseria gomrrhoeae and Haemophilus influenzae are able to use TF- and LF-iron directly without mediation of siderophores. Listeria monocytogenes appears

to remove iron from TF by production of a soluble reductant which dissociates the iron-TF complex.6 There are some reports of iron acquisition from heme-containing serum The mechanisms of these nonsiderophore-mediated iron-uptake systems are still not clear. This review presents first a brief overview of the iron-withholding capacity of the human body and the siderophore-mediated iron-uptake systems of bacteria. Thereafter, the nonsiderophore-mediated iron uptake of pathogenic bacteria is extensively discussed.

II. IRON-WITHHOLDING CAPACITY OF THE HUMAN BODY In humans, most of the iron is located intracellularly as femtin or as heme-compounds (Table 1). This iron is normally not available to invading microorganisms. The small amount of extracellular iron that appears in body fluids is

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217

TABLE 1 Iron Distribution in Human Adults"


lntracellular Hemoglobin (2300mg) Myoglobin (320mg) Ferritin (700mg) Hemosiderin (300 mg) Heme-type enzymes (80 mg) Nonheme type enzymes (100 mg)

attached to the host iron-binding and transport proteins, i.e., TF in serum and lymph and LF in phagocytic cells and mucosal secretions, thereby effectively creating an iron-restricted environment for extracellular microorganisms. Femtins are iron-storage proteins present in most animal tissues. These proteins are water soluble, have a high and variable content of iron, and are present in the extramitochondrial spaces of the cell. The fenitin molecule consists of two components: an iron hydroxide-phosphate micelle (Fe00H)8(Fe00P03H2)and a multi-subunit protein shell (apofemtin) which encompasses the former. This apofemtin has a number of channels connecting the interior of the molecule with the outside. Apofemtin is composed of 24 subunits and can accommodate approximately 4500 iron atoms. The average molecular weight of each subunit is close to 20,000.Hemosiderin is present as insoluble granules within secondary lysosomes. It is thought to be derived from ferritin by degradation.Is Hemoglobin, another intracellular iron-protein, is the most abundant heme-containing protein in the body. Other heme-containing proteins are myoglobin, enzymes such as catalases, peroxidases and some oxygenases, and electron carriers such as the various cytochromes. Hemoglobin is the oxygen-binding protein of red blood cells that consists of two pairs of polypeptide chains, which are termed a- and P-chains. The heme residue of hemoglobin is located in a hydrophobic pocket of each polypeptide chain. The complete molecule has a molecular weight of 64,450. Hemoglobin, if released by lysis of erythrocytes, is bound by haptoglobin ( M W 100,OOO). One molecule of this plasma glycoprotein binds one molecule hemoglobin. The hemoglobin-haptoglobin complex is rapidly cleared by the hepatocytes (Figure 1).l6 Free 218

Extracellular Transferrins (3mg)

hemoglobin appears when haptoglobin is saturated but is oxidized and dissociates into globin and heme. Free heme is bound by a specific hemebinding protein called hemopexin. This glycoprotein, with a molecular weight of 57,000, binds one heme molecule per molecule of protein. The complex is transported to the liver where the heme may be degraded to bilirubin or incorporated into cytochrome P-450. TFs are extracellular iron-binding glycoproteins with molecular weights in the range of 75,000 to 80,000.They are present in human body fluids in two types: LF and TF. These proteins have a homology of 49.8% in their amino acid sequence. TF is present mainly in blood, whereas LF occurs both intracellularly in neutrophils and in various secretions (Table 2). The TFs function as bacteriostatic agents and, in the case of TF, as a mean of delivering iron to various tissues. TF and LF have the capacity to bind two atoms of femc iron in association with the binding of an anion, usually bicarbonate. Both ironbinding proteins are usually only partially saturated with iron (30%). The amount of free iron in equilibrium with the proteins is about lo-'* M. In the case of TF, the iron-binding ability is diminished below pH 6 , whereas LF retains its iron-binding properties in more acidic conditions (stable until pH 4). Thus, although there is an abundance of iron present in the body, the amount of free iron is far too small to sustain bacterial g r ~ w t h . ~ Dur~*'~ ing infection, the host further reduces the total amount of extracellular iron. This phenomenon is called the hypoferremia of infection. Two mechanisms are thought to be responsible for the hypoferremic response: one is an increase in the synthesis of femtin in the liver, the other is the release of LF from neutrophils. The released LF, in its iron-poor state, captures the iron from TF.

LYSlS ERYTHROCYTES

1 HAPTOGLOBIN GNSFERRIN

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FERRlTlN

FIGURE 1. Schematic representationof the clearance of hemoglobin from the blood circulation after lysis of erythrocytes. Hemoglobin released by erythrocytes is bound in blood by haptoglobin and transported to the liver. Free hemoglobin appears when haptoglobin is saturated. The free hemoglobin is oxidized and dissociates into globin and heme. Hemopexin binds and transports heme to the liver. A small part of the heme-molecules disintegrates into protoporphyrin and iron (FE). This iron is bound by transferrin and transported to the iron-storage protein ferritin.

TABLE 2

Distribution of Transferrins in Body Fluids2 Lactoferrin

Transferrin

Saliva, tears, nasal secretions, intestinal and seminal fluids, cervical mucus, colostrum, milk, neutrophils Plasma, lymph

The LF-iron complex is then picked up by fixed or circulating macrophages and the latter are removed rapidly from the circulation by the reticuloendothelial system. l8 All this makes it clear that the possession of specialized iron-uptake systems is crucial fdr bacteria to override the iron limitation imposed by the host.

111. SIDEROPHORE-MEDIATED IRONUPTAKE One efficient system of acquiring iron involves the synthesis of low molecular weight, high-affinity iron chelators, termed siderophores. Siderophores are secreted in response to iron deprivation and, once complexed to iron, they are transported back into the cell via specific receptors on the outer membrane. 1920 These iron chelators are able to remove iron from TF or LF (Figure 2). Several comprehensive reviews on siderophores and iron transport systems in microorganisms have been published.2.21*22

Microbial siderophores can be divided into two dominant chemical types: phenolates and hydroxamates. Many enteric bacteria synthesize the phenolate siderophore enterobactin and the hydroxamate siderophore aerobactin (Table 3). Enterobactin has a very high affinity for Fe3+ (Ks = at neutral pH). The formation constant of aerobactin is 1023.14,23 Escherichia coli, Salmonella, Klebsiella, and Campylobacter also have the capability of obtaining iron via a variety of hydroxamate-type siderophores which are not synthesized by the organisms themselves but are produced by other microorganisms.2*20.2L26 Ferrichrome and ferrioxamine are the best known exogenous chelators. They are produced by certain fungi and Actinomyces, respectively. It is not known if such chelators normally operate in vivo. Exogenously supplied desferrioxamine B can enhance infections of Klebsiella and Salmonella.2 IROMPs usually serve as receptors for ironsiderophore complexes and are essential for iron uptake. The IROMPs of the enteric bacteria, involved in siderophore-mediated iron uptake, have apparent molecular masses in the range 74 to 84 kDa and are expressed under iron-limited cond i t i o n ~ . *In * ~E. ~ coli, a 81-kDa protein is the receptor for Fe3+-enterobactin and is the product of the fep gene. This gene is a part of the ent, fep, fes gene cluster, which is responsible for the biosynthesis, transport, and hydrolysis of enterobactin. This iron transport system is controlled 219

by the product of thefur gene, a universal regulator of all iron transport systems in E. cofi and possibly other bacteria (Figure 3). The aerobactin iron-assimilation system mediated by a plasmid consists of five genes organized in one operon. The operon includes the genes responsible for the biosynthesis of aerobactin and the gene iutA, which encodes a 74-kDa polypeptide that acts as

the receptor for femc aerobactin. This operon is also regulated by the product of thefur gene, The Fur protein (17 m a ) acts as a repressor, using Fez+ as a cofactor by binding to the operator sites of the enterobactin and aerobactin iron-uptake The expression of certain toxins in . E . coli and Corynebacterium diphtheriae is also under the control of the fur and fur-like


Bacterial cell Specific ~ ~ 3 ' 0

4

J

0 I

Siderophore

Transferrin or Lactoferrin

Bacterial cell Specific

Transferrin or Lactoferrin

b)

Bacterial cell

Hemoglobin (heme) Haptoglobin-hemogl obin Hemopexin-heme

FIGURE 2. Schematic representation of different ways by which pathogenic bacteriaobtain iron from iron-binding or heme-containingproteins: (a) siderophore-mediated iron uptake; (b) iron uptake by direct interaction with transferrin or tactoferrin; (c) iron uptake by direct interaction with heme-containing proteins. Symbols: (El)the , whole heme-molecule has been internalized; (&I), only the iron of the heme-molecule has been removed.

220

needed before we can fully understand the importance of these high-affinity iron-uptake systems in promoting the growth of pathogenic bacteria in vivo.

TABLE 3 Synthesis of Siderophores in Clinical Isolates of Enteric Bacteria4*"

Genus

Enterobactin production

Percent aerobactin producers

+ + + +

46 18 4 67 71


Escherichia Klebsiella Salmonella Shigella Enterobacier

+a

IV. NONSIDEROPHORE-MEDIATED IRON UPTAKE A. Iron-Glycoproteins as Iron Sources 1. Neisseria Species

Shigella boydii and S. flexneri only secrete aerobactin. Other Shigella species produce the siderophore enterobactin and a few produce both chelators.

l o ~ i . ~ &The ~ Oexpression of these toxin genes are repressed by thefir system during growth in high iron. Although a great deal is known about the regulation and the mechanism of siderophore ironuptake systems in v i m , much more work is

The human pathogen N . gonorrhoeae usually causes localized infections on the mucosal surfaces of the urogenital tract, the oropharynx, and the rectum. Occasionally, it may invade the bloodstream and the synovial membranes of joints. N . rneningitidis, also an obligate human pathogen, is normally restricted to the nasopharynx, but occasionally it invades the bloodstream from where it reaches the meninges and sometimes the synovial membranes of joints.

E n t e r o b a c t i n system fes

entF

fepE

entC entE e n t B e n t A

f e p C fepG f e p D f e p

f

R e r o b a c t i n system

iucA 63 kD

iucB

iucC

iucD

iutA

32 kD

62 kD

53 kD

74 kD

aerobactin

biosynthesis

transport

FIGURE 3. Organization of the enterobactin and aerobactin systems and sites of interaction of the Fur-Fez+complexes. Symbol: iron boxes. The sites of Fur action on the enterobactin system are based on sequence analysis only. (From Crosa, J. H., Microbiol. Rev., 53, 517, 1989. With permission.)

4,

221


a. Transferrin and Lactoferrin as Iron Sources TF and LF are probably the two major iron sources which commonly support these pathogens during human infection because the virulence of meningococci in mice is enhanced by injecting the animals with iron-saturated TF or LF prior to i n f e ~ t i o n . ~In ’ -N~ .~gonorrhoeae and N . meningitidis, an ability to use TF-iron for their g r ~ w t hand ~ ~a. TF-binding ~~ activity have been demonstrated.36 Siderophores are not involved in this kind of iron acquisition since Neisseria species do not secrete siderophores (Figure 2) .35.37.38 Most commensal species of Neisseria are unable to use TF-iron,”~~~ in spite of being able to bind TF.36Therefore, it seems that it is not simply the binding of TF to the cell surface but the release and uptake of iron from TF that determines in part pathogenicity or nonpathogenicity. The contribution of LF-iron to the pathogenicity of either the gonococcus or the meningococcus is not clear; whereas all meningococci can obtain iron from LF, only 53% of gonococci and 24% of commensal Neisseria species can do Schryvers and Lee36detected LF-binding activity in all isolates of meningococci, gonococci, and in all commensal Neisseria species. It seems that the ability to use iron in LF is not a prerequisite for the survival of Neisseria on mucosal surfaces since most commensal Neisseria species which normally colonize the nasopharynx cannot obtain iron from LF. However, all of these species are able to use hemin as an iron source, and it is possible that heme released by dying cells sloughed from the epithelium is an alternative source of iron on mucosal surfaces.39The relative importance of LF-iron for virulence is not known. Griffiths2 suggested that pathogenic species may need to multiply more rapidly on the mucosa than the commensals and that an ability to use LFiron might help. The suggestion that the ability to take iron from LF contributes to the pathogenicity of Neisseria in general is not plausible because 50% of the gonococci cannot use LF as an iron source. Schryvers and Lee36 suggested that the discrepancy between the LF-binding activity of these gonococcal strains and the inability to obtain iron from LF can be explained by the different experimental conditions used by differ-

222

ent research groups. The in virro growth conditions applied by Mickelsen et al.39 may not adequately mimic the in vivo circumstances so that adequate expression of the LF-iron-uptake system may not always be achieved.36Therefore, the possibility has not been satisfactorily ex.eluded that some of these gonococcal strains are able to acquire iron directly from LF in vivo. b. Iron-Repressible Outer Membrane Proteins N. meningitidis and N . gonorrhoeae synthesize new outer membrane proteins during iron restriction. The ROMPS may play specific roles in the binding of TF or LF, in the release of iron from these proteins, and in the transport of iron across the membrane. Mietzner et aL40and West and confirmed the earlier work of Norqvist et aL4’ in which they demonstrated that under conditions of iron limitation N . gonorrhoeue produced several new outer membrane proteins (OMPs) with apparent molecular masses of 70 to 100 m a . They also detected the expression of two more proteins with molecular masses of about 37 and 19.5 m a . At low pH (6.6) and under iron-limited growth conditions, N . menExpresingitidis also expresses extra OMPs sion of the IRPs in gonococci was highly variable, not only with the strain used but also with the source of iron. A specific subset of proteins was expressed when the bacteria were grown in the presence of TF or LF. In media containing either hemin or hemoglobin, expression of all the IRPs characteristic of the strain studied was obThe 37-kDa protein was unique in that it was expressed in all strains examined and in the presence of all iron-containing molecules tested.37This protein appears to be also present in gonococci grown under other nutrient limitations like cystine or glucose lirnitati~n.~~ An explanation for this phenomenon could be a depletion of the energy generation necessary for the energy-dependent transport of iron during ironreplete, but glucose- or cystine-limited growth. This resulted in a low intracellular iron concentration and induction of proteins normally associated with iron transport during iron stress.43 Mietzner et al.,er who detected the appearance of a 37-kDa protein in all pathogenic Neisseria

species, suggested that the conservation of this IRP in these species points to an essential role of this protein in the pathogenesis of neisserial


infection. To study the functional and pathogenic roles of this protein, Mietzner et al.45isolated it. The purified protein displayed an unusually basic isoelectric point of >9.35. The amino acid composition of the meningococcal and gonococcal protein was nearly identical, and iron was associated with the 37-kDa protein. This may imply that this protein plays a direct role in neisserial iron assimilation.

c. Mechanism of Iron Uptake N . gonorrhoeae and N. meningitidis possess a mechanism of iron acquisition in which iron is removed directly from TF or LF. High-affinity iron uptake from TF by N. meningitidis needs the contact of TF with the cell surface, but TF is not taken up by the cells during iron uptake.35 The binding of TF to the cell surface seems to be energy independent since the amount of bound TF to cyanide-treated meningococci was similar to that bound by metabolically active cells. McKenna et al.38 showed that gonococci and meningococci removed iron very efficiently from TF and LF in an energy-dependent manner. This mechanism was repressed by prior growth in iron. Growth with iron-loaded TF or LF was dependent on the amount of the iron-protein complex available rather than the saturation level of the prot e i n ~ . They ~ * suggested a cycle consisting of TF binding to the cell surface followed by iron transport, release of iron-unloaded TF, and binding of another molecule of TF (Figure 2). The identity of the TF receptor is still unknown. The results of several TF-binding studies show a variation in size of the TF-binding proteins, depending on the method used and/or on the strains examined (Table 4). Schryvers and Morris46first reported the identification of a 70kDa IRP on the cell surface of N. meningitidis by means of SDS-PAGE and Western blot analysis. This protein was shown to be highly specific for human-TF binding. Recently, Banerjee-Bhatnagar and Frasch4’ confmed these findings. However, Schryvers and Lee,36 using affinity chromatography, failed to confirm with certainty the interaction of this protein with TF. Further-

more, studies with mutants lacking the common 70-kDa protein showed TF-binding activity equal to that of the wild type.48 In these studies, the amount of cell-associated *251-TFwas determined. Competitive binding experiments with antibodies against the 70-kDa protein demonstrated that it could not be the TF receptor.49 By the use of mutants, Tsai et al.48demonstrated that the 65-, 85-, or 95-kDa IRPs also cannot be the meningococcal TF receptor. Other possible candidates for the TF receptor in N . meningitidis, detected by Schryvers and Lee,36 are a 68- or a 98-kDa TF-binding protein. Electrophoretic analysis showed that the 68-kDa protein is not the same protein as the IRP with a molecular weight of about 70,000.50A 98-kDa TF-binding protein has also been detected in N. gonorrhoeae by means of affinity chromatography. 51 Griffiths et al.’O detected a TF-binding protein of 68 kDa following SDS-PAGE and electroblotting in only 4 of the 35 Neisseria strains tested. Most of the TF-binding proteins had a molecular mass between 78 and 83 kDa. A TF-binding protein of 98 kDa could not have been detected by using this method. Evidence to date shows that in N. meningitidis, by using affinity isolation procedures, two transfemn-binding proteins can be found; one with a higher molecular weight of about 98,000, called TF-binding protein 1 , and another with a lower but variable molecular weight in the range of 68,000 to 85,000, called TFbinding protein 2. 36.52 Only TF-binding protein 2 is able to bind TF after SDS-PAGE and electroblotting . The human LF-binding protein was identified Schryas a 105-kDa IROMP in N . rneningitidi~.~~ vers and Lee36also demonstrated the presence of this LF-binding protein in other species of the genus Neisseria and in Moraxella catarrhalis (formerly Branhamella catarrhalis), whereas in the gonococcus a 101-kDa protein was identified as the LF receptor.” The mechanism of binding of TF or LF to the receptors is still unknown. The mechanism proposed by McKenna et al.38 implies a higher affinity of the receptor for iron-saturated TF or LF than for apo-TF or apo-LF. In competitive binding experiments comparing iron-saturated TF with apo-TF, Schryvers and clearly showed that iron-saturated TF is more effective

223

TABLE 4 Survey of Several Studies of Microbial Species Whose Outer Membrane Proteins' Were Shown To Bind or Not To Bind TF or LF TF-binding protein ' Species


Neisseria meningitidis N. gonorrboeae Haemophilus influenzae Trichomonas vaginalis a

Wa) 7P.47 60,98= 60,70-03m 9W1 50, 98" 72"

7oW.4L3.40 9 P 65, 05, 95"

LF-binding protein (kD4 1053e-u

10151 105"

170, 7V7

SDS-PAGE and electroblotting were used in References 46, 47, 40, 49, 50, and 60 to detect TF-binding activity. Affinity isolation procedures for detecting TF or LF binding activity were used in References 36, 51, 53, 64, and 67.

in blocking the receptor than apo-TF. On the other hand, Simonson et al.35and Tsai et al.48 failed to demonstrate differences in recognition of iron-loaded or iron-free TF by N . meningitidis. This discrepancy can be strain dependent or can be attributed to the methods used. The formeP used horseradish peroxidase (HRP)-labeled TF, while the latter investigators4"used radioactively labeled TF (lZsI-TF)in competitive binding experiments. TF may be modified by HRP in such a way that binding is less sensitive to competition by apo-TF, or iron could be removed from the '251-labeledTF by cold TF, thereby reducing its affinity for the receptor. No difference was detected in the binding of iron-loaded LF and apoLF in competitive binding experiment^.^^ The biochemical mechanisms of release of iron from TF or LF and of its transport across the cell membranes in Neisseriu species are likewise unknown. Evans et reported that Streptococcus mutuns made use of a membranebound iron reductase to obtain iron. In L. monocytogenes, an extracellular reductase was It is possible that in Neisseriu too the release of iron from TF is mediated by a reductase. Keevil et al.43 found that the 37-kDa protein is present in the outer membrane and also in high concentrations in the soluble cell fractions. They suggest that this protein may serve to interact with the LF and TF receptors on the outer mem224

Not the TF-binding protein (kDa)

brane, abstracting their bound iron and providing a shuttle mechanism to convey the iron across the periplasmic space to the inner membrane. This hypothesis is supported by the findings of Lee and Bryan,5' who purified TF complexes by affinity chromatography. Several proteins were copurified by this process. A 98- and a 37-kDa protein were present in all gonococcal strains examined, whereas a third copurified protein exhibited interstrain molecular mass heterogeneity, varying from 66.5 to 74.5 m a . They suggest that the TF-receptor consists of a functional complex of three proteins. The 37-kDa protein could be the shuttle vector which transports the iron from the outer membrane to the cytoplasmic membrane, while the other two proteins are involved in the binding of TF and the subsequent release of iron from this protein. Recently, the structural gene for the 37-kDa protein expressed by N. gonorrhoeue was cloned and sequenced.56 The data from this and the other studies will be used to uncover the role of this protein in the iron uptake of Neisseriu. 2. Haemophilus influenzae

Various Huernophilus species colonize the mucosa of the upper respiratory tract of humans. Of these species, H . influenzae type b is by far


the most important human pathogen. Infection by this pathogen may lead to sepsis or meningitis in children. Other invasive infections due to H. influenzae include pneumonia, epiglottitis, and arthritis.57Other species such as H . parainfluenzae, H. paraphrophilus, and H. haemolyticus are only rarely pathogenic. H. influenzae type b does not produce side r ~ p h o r e s , ~nor . ~ ~does . ~ ~it use exogenous siderophores.60-62It can acquire iron from TF, but not from LF.9,58.M).62 None of the H. paruinfluenzae isolates could use TF as a source of iron in ~ i n - 0This . ~ ~might be responsible for their lower ability to invade the bloodstream. This microorganism also cannot obtain iron from LF. Since both pathogenic and nonpathogenic Haemophilus strains are able to grow on human mucosal surfaces, they must be able to acquire iron in vivo. A potential iron source could be heme released by dying cells sloughed from the epithelium. The specificity by which Huemophilus species acquired iron from TF was determined by Morton and Williams.6o In contrast to the Neisseria species, H. influenzae used iron bound to human, bovine, and rabbit TFs. During substantial iron restriction, H. influenzue type b expresses three minor IRPs of about 94 to 98 ma.’’ An IROMP of 43 kDa, described by Coulton and Pang,63has not been detected by other workers.M) In addition to these minor IRPs, major alterations in the pattern of OMP were detected at suboptimal iron concentrations by Pidcock et aL9 They observed the expression of a 77-, 6 5 , and 24kDa OMP under these conditions. Williams and Brown,61however, showed that iron restriction had little effect on the outer membrane protein profile of their isolate of H. influenzue type b. It is not clear whether the differences in membrane proteins under iron stress observed by the different groups were due to differences in experimental conditions or to the use of different strains. Although H. influenzae has been shown to use human TF-bound iron as the sole source of iron for growth, the mechanism of iron acquisition from TF and its importance in pathogenesis are still unknown. Schryvers64suggested the involvement of three proteins (58,67, and 98 kDa) in iron acquisition from human TF in this bacterium. The 58-kDa protein was capable of binding TF even after SDS-PAGE and electroblot-

ting. Some variability in the size of the higher molecular weight protein (94 to 106 kDa) was demonstrated in the TF-binding proteins from type b and nontypable H . influenzae strains. Morton and Williams,M)however, demonstrated a TFbinding protein of 72 kDa after SDS-PAGE and electroblotting in H . influenzae (Table 4). Mutants of H . influenzue, lacking a 72-kDa protein, showed a reduced binding or were unable to bind TF.65These results suggest that this protein probably functions as the receptor for TF. SchryversM also isolated LF-binding proteins with molecular masses of 105 and 106 kDa from an H. influenzue strain. The similarity in size and in binding specificity of the LF-receptor in this strain with the LF-receptor in meningococcal strains suggests a structural and functional conservation. However, as stated above, H . influenzue cannot acquire its iron from LF in vitro. The presence of a LFreceptor in this microorganism implies that the in vitro growth studies may not adequately mimic the in vivo situation. Clearly, more work is needed to clarify the response of different isolates of H. influenzae to iron restriction. Also, the precise contribution of the IROMPs to TF-receptor function and iron acquisition in H . influenzae remains to be established. 3. Other Pathogenic Microorganisms Various other pathogens are able to obtain iron in vivo via a mechanism involving direct contact between the pathogen and an iron-binding protein. Mycoplasma pneumoniaeMand the pathogenic protozoan Trichomonus vaginaW7 have specific receptors for human LF (Table 4). The binding of LF in preference to TF may be a significant factor in the noninvasive nature of these parasites. Peterson and Alderete67 detected no internalization of bound lactofemn by T. vaginalis. Putative candidates as LF-receptors in this organism are two proteins with molecular masses of 178 and 75 kDa. Bordetella pertussis, the causative agent of whooping-cough, is a noninvasive pathogen of the human upper respiratory tract. This microorganism synthesizes hydroxamate siderophores,68but also uses TF and LF as iron sources by binding these proteins to its outer membrane. In competitive binding experiments,

225


Redhead et al.69showed that LF and TF bound to a same bacterial cell surface component in B. pertussis. This in contrast to the Neisseria species, H . influenzae, M . pneumoniae, and T. vaginalis, which have separate receptors for LF and TF. Under iron-restricted growth conditions, the appearance of a new 77-kDa protein was demonstrated in B. pertussis.69 Recently, Redhead and Hill’O showed that a direct contact between B. pertussis and TF provides far more effective iron uptake than uptake via siderophores. As stated previously, B . pertussis is a noninvasive pathogen and it seems unlikely that this microorganism can use TF as an iron source in vivo. Therefore, the possession of a TF-receptor in B. pertussis is highly questionable. , Some anaerobic bacteria are also capable of obtaining iron from TF. The growth of Clostridium p e e i n g e n s in vivo leads to a decrease in the Eh of the infected tissues (Eh, -400 mV at pH 6.5). These highly reduced conditions, as well as the acid pH, promote the release of iron from TF and facilitate the growth of this pathogen. l8 Black-pigmented Bacteroides species are capable of degrading TF, hemopexin, albumin, and haptoglobin by proteolytic e n ~ y m e s The .~ ability of these organisms to degrade these ironand heme-binding proteins could be a mechanism to obtain iron from the host. Recently, in one of the members of the black-pigmented Bacteroides group, Phorphyromonas gingivalis (formerly Bacteroides gingivalis), a heme-repressible outer membrane protein of 26 kDa was dete~ted.~’

6. Heme-Compounds as Iron Sources Heme-compounds are normally not available for infecting microorganisms because of their intracellular localization. Therefore, pathogenic bacteria can only utilize the iron in heme-compounds after it is made available by some form of tissue damage which releases intracellular material. The lysis of erythrocytes is a good example (Figure 1). Hemolysis spontaneously occurs at low levels, but is much more enhanced in sickle cell anemia, bartonellosis, malaria, and trauma. These patients are unusually susceptible to infections.l 8 Also bacterial toxins like hemolysins can cause the lysis of erythrocyte^.^^,^^ 226

Heme-compounds are known to be an iron source for bacteria in vivo. The presence of free hemoglobin markedly enhances the lethality of E. coli for rats. However, when bacteria were injected with hemoglobin bound to haptoglobin, the majority of the animals Thus, the organisms were unable to use heme-iron from the hemoglobin-haptoglobin complex. Yersinia pestis needed very low concentrations of heme (0.5 pmol) to allow growth in an otherwise inhibitory medium.75It is not known if this organism can utilize heme bound to hemopexin. B ~ l l e nsug~~ gested that sufficient heme to initiate an infection might be injected along with Y. pestis by the rat flea, Xenopsylla cheopis, during the transmission of plague. 7. Neisseria Species

Several studies have demonstrated the ability of N . meningitidis and N . gonorrhoeae to use heme-compounds for their growth. Yancey and F i n k e l ~ t e i nand ~ ~ Mickelsen and S ~ a r l i n g ~ ~ showed that all meningococci and gonococci tested could use free heme as a source of iron in vitro. Furthermore, almost all meningococcal strains tested and 60% of the gonococcal strains were capable of using hemoglobin for growth,34 although TF and LF were more readily utilized as iron sources than hemoglobin and hemin.37*78 Provision of exogenous hemoglobin resulted in increased meningococcal lethality for mice .78*79 This increase was not as great as that observed with amounts of TF with equivalent iron content, which parallels the more effective utilization of TF and LF in in vitro growth experiment^.^^ Nevertheless, these observations suggested that hemoglobin may act as an iron source in vivo. This hypothesis was supported by the findings of Dyer et al. ,8 who found that hemoglobin bound to haptoglobin was used as an iron source by some meningococci and gonococci strains in vitro. Heme complexed to hemopexin could not be used by these two pathogenic species. Although these pathogens can utilize released hemoglobin, they are not known to produce specific hemolysins that would facilitate hemoglobin release. The role of IROMPs in the acquisition of iron from hemoglobin is unknown. West and


Spading3' observed a variation in the expression of the IROMPs of N . gonorrhoeue depending upon the iron source used. Fewer IRPs were seen when cells were supplied with TF or LF than when the cultures were grown with either hemin or hemoglobin. As described earlier, heme and hemoglobin are poorly utilized iron sources. This implies that when these compounds are the only iron sources growth is more iron-limited for these bacteria. The expression of the additional IRPs under these circumstances could be an indication of the induction in these bacteria of an extra iron uptake system, designed to obtain iron from hemoglobin (Figure 2).

2. Haemophilus influenzae

fluenzue remove heme-iron efficiently from those complexes. How utilization of various heme sources contributes to the pathogenesis of invasive infections by this microorganism is not known. It is possible the heme-uptake systems in H. influenzue are repressed during infection because anaerobic conditions would prevail. Under these circumstances, H . influenzue would totally depend on TF for its iron sources. Heme-repressible OMPs of 43 and 38 m a g o were detected in this bacterium. Coulton and Pang63suggested that the 43-kDa protein may play a role in the transport of heme across the cell envelope of this microorganism. Recently, Hanson and H a n s e P demonstrated a 51-kDa hemin-binding protein in this microorganism. This hemin-binding protein is associated with the inner, and possibly outer, membrane of H. influenzue. It is clear that H . influenzue acquires iron from a variety of heme and nonheme sources (Table 5).

Only under aerobic conditions does H. influenzue have an absolute requirement for heme. Coulton and Pang63demonstrated that both iron and the porphyrin ring were taken up at the same rate, suggesting that H. influenzae could utilize heme as an iron source. In vitro studies showed 3. Shigella Species that H. influenzue can acquire heme-iron from heme bound to hemopexin and to serum albumin, Members of the genus Shigella are enteroinand from hemoglobin bound to h a p t o g l ~ b i n . ~ . ~ ~vasive pathogens that have the ability to penetrate Both invasive and noninvasive isolates of H. inand multiply within intestinal epithelial cells. This

TABLE 5 Utilization of Heme and Nonheme Iron Proteins by Human Pathogens Utilization of ~

Species Neisseria meningitidis N. gonorrhoeae Haemophilus influenzae Shigella Bacteroides fragilis Vibrio vulnificus V. cholerae Bordetella pertussis

TF

LF

+ + +

+ + ' + - +

-

-

-

?

? +

? +

? +

+ ?

-

-

HM

+

HB

HP-HB

HX-HM

+

+ + +

-

? ?

? ? ? ? ?

2

+ ?

+ + + ?

+ ? ?

-

+

Symbols: +, -, able or unable to utilize as an iron source: 2 , 50 to 60% of the gonococcal strains are capable of using these iron sources; ?, not determined; TF, transferrin; LF,lactoferrin; HM, heme; HB, hemoglobin; HP-HB, haptoglobin-hemoglobincomplex; HX-HM, hemopexinheme complex.

227


causes epithelial cell death and tissue destruction, leading to ulceration and bloody diarrhea. All the species of this genus, Shigellu dysenteriue, S. flexneri, S . sonnei, and S . boydii, synthesize siderophores.4 Recently, Payne4 reviewed studies about iron-uptake systems of Shigellu species, the genetics and regulation of these systems, and their role in the production of disease by these organisms. These studies suggest that in the intracellular environment iron may be acquired from heme-compounds. The components of the hemeiron transport system have not been elucidated. A relationship between Congo red binding (Crb’ phenotype) and adsorption of hemin has been noted.82The ability to bind these compounds in v i m correlates with virulence. An enhancement in the ability to invade HeLa cell monolayers was seen when Congo red or hemin were prebound to either the bacterial or target cells.82These compounds appeared to be bound to a plasmid-encoded cell-surface protein of 101 kDa in shigellae.83No correlation was seen between the binding of hemin and the utilization of heme-iron. Crbmutants grew as well as wild-type Crb’ strains with hemin as the sole iron source.82Therefore, this heme-binding protein is probably not involved in a heme-uptake system in these bacteria. Daskaleros and Payne8zclaimed that this protein plays a crucial role in the invasion of the host cells by shigellae. The bacterial cell coated with heme could be a desirable molecule for the intestinal epithelial cell. The heme receptors of the host bind the bacterium-heme complex and then actively endocytose the bacterial cells, the socalled Trojan horse effect. These studies clearly show how shigellae use heme to invade host cells; however, it still remains unknown whether these bacteria can use heme as an iron source. 4. Bacteroides fragilis

B. frugilis is a clinically important anaerobic bacterium. This opportunistic pathogen is often found in infections in which trauma, surgery, vascular constriction, and necrosis are predisposing factors. Infections with B. fragilis mostly originate from the gut and are usually present as generalized peritonitis or intraabdominal abscesses which may spread to other organs and

228

systems.8’86 In animal infection models, the greater virulence of B. fragilis in comparison to the more common members of the intestinal flora, such as B. vulgutus, has been established by several author^.^'-^^ B. fragilis did not produce iron chelators.90Several in vitro studies demonstrated ,that B. frugilis has an efficient system of iron ~ p t a k e . ~This l . ~ microorganism ~ was better able to grow in the presence of the iron chelator bipyridyl than the other less virulent members of ~ ~ , ~ ~ Verweijthe B. frugilis g r o ~ p .Furthermore, van Vught et a1.91*93 showed that only B. frugilis strains could grow in serum and plasma. The growth inhibition of the other members of the fragilis group in these media can be abolished by adding iron sulfate or heme-compounds. B. fragilis cannot utilize TF-iron for its These results suggest the presence of a more efficient system in B. frugiZis for the sequestration of iron from the minute quantities of heme-compounds present in normal serum. IROMPs with the apparent molecular masses of 89,49, 44,and 23.5 kDa were found in B. fragilis.* the 44-kDa protein appears to be one of the major OMPs in B. fragilis under iron stress. In B. vulgutus cells, this protein and the 23.5-kDa protein were absent, whereas the expression of the 49-kDa protein stayed at a low level under comparable ironlimited growth conditions. The 44-kDa protein appears to be a lipopolysaccharide-associated protein with an isoelectric point of approximately pH 5.5.% Recently, Otto et al.95 showed that the 44-kDa IROMP is expressed in vivo and induces an antibody response in patients and animals infected with B . frugilis. They also demonstrated a conservation of this protein within B. frugilis strains. These results may imply an essential function of this protein in the fight for survival of this microorganism. Otto et al.92reported that the 44-kDa outer membrane protein plays an important role in the uptake of heme by B. frugilis. They observed that antiserum specific to the 44kDa protein inhibited the growth of B. fragilis in a medium where heme was the only iron source. It would be of interest to determine whether this protein is a receptor for heme or whether it is involved in the passage of heme through the envelope. Also the role of the other IROMPs in the heme uptake of this bacterium remains to be elucidated.


5. Vibrio Species

Vibrio species are known to obtain iron from heme-compounds. Vibrio vulnificus is an opportunistic marine pathogen capable of causing fatal septicemias and wound infections in humans. It causes disease via skin lesions or after the ingestion of raw shellfish. Although the fatality rate after wound infections is not great, ingestion of this bacterium results in primary septicemia, with 46% of these infections proving fatal.%In cases with life-threatening septicemia, most patients had underlying diseases which result in iron overload, like hemochromatosis.2.96.97 In vivo studies by Wright et aL9*directly correlate the pathogenicity of V. vulnificus with the availability of iron; iron overload in mice resulted in a dramatic reduction of the 50% lethal dose of V . vufnijicus. So, it seems that the organism is an opportunistic pathogen which relies on the iron which is often freely available in the serum of individuals who are suffering from iron-overload diseases.97The mechanism of wound infection by V. vufnzjicus in persons with apparently normal serum iron levels is unknown. The bacterium cannot use TFor LF-iron for its However, it has been shown to produce hemolysins.99Therefore, localized infections may be accompanied by an increase in free hemoglobin. Helms et al.99 and Chart et al.97 indeed demonstrated in vivo and in vitro that this bacterium is able to utilize various heme-compounds . Moreover, V. vufnzjicusovercame the bacteriostatic effect of haptoglobin and probably acquired iron from the haptoglobinhemoglobin complex. The ability to use this complex as an iron source was confirmed by the work of Zakaria-Meehan et al.'O0 The findings that V . v u f n i j h s can use various heme-compounds as a source of iron suggest that the availability of heme at the site of infection might be an important factor in p a t h o g e n e s i ~ . ~ ~ . ~ ~ Stoebner and Payne'O reported that in vitro both hemin and hemoglobin could serve as the sole sources of iron for V. choferae. This study also indicated that V. choferae hemolysin production is iron regulated, i.e., there is a derepression of the synthesis under iron stress. The bacterium produces a siderophore vibriobactin,'O' although it has been claimed that the ferricvibriobactin transport system is not required for

the virulence of V. cholerue.*02Therefore, the cytotoxic hemolysin of this bacterium could effect release of intracellular heme-compounds from damaged epithelial cells in the gut, thus providing an iron source.

V. CONCLUDING REMARKS The contribution of the siderophore-dependent iron-uptake systems to the virulence of some pathogenic bacteria is still unclear. The production of the siderophore aerobactin appears to be a more important virulence factor for enteric bacteria than the synthesis of the siderophore enterobactin since the results of epidemiological studies showed that the incidence of aerobactin is remarkably high in strains causing invasive infectious However, it must be noted that there is a large group of enteropathogenic and enterotoxigenic E. cofi causing severe diarrheal disease that produces only enterobactin as an iron chelator.2*23 Moreover, Payne4 reported that siderophores are not essential for shigellae to either survive within the intestinal lumen or for invasion of intestinal epithelial cells, intracellular survival, and multiplication. The functional iron-uptake system of aerobactin-negative strains in vivo has not been identified; it could be the enterobactin system andlor the iron-uptake systems as described in this review. It must be noted that only bacterial species that are able to survive outside the human body possess siderophore-dependent iron-uptake systems. It is tempting to speculate that these systems play an important role in the acquisition of iron by these bacteria under aerobic conditions in the environment. Studies of siderophore-independent iron uptake from TF have suggested that at least three proteins may be involved. One protein could be the shuttle vector which transports the iron from the outer membrane to the cytoplasmic membrane, while the other two proteins are involved in the binding of TF and the subsequent release of iron from this protein. A possible candidate responsible for the transport of iron in Neisseria could be a 37-kDa protein. The TF-receptor may consist of two proteins. In N . meningitidis, two TF-binding proteins have been found: TF-bind-

229


ing protein 1, with a molecular mass about 98 kDa, and TF-binding protein 2, with a variable molecular mass ranging from 68 to 85 kDa. TFbinding protein 1 could only bind TF under nondenaturing conditions, while TF-binding protein 2 is able to bind TF under both denaturing and nondenaturing circumstances. The release of iron from TF is probably mediated by a reductase. It is tempting to speculate that one of the TF-binding proteins is also involved in the release of iron. A possible candidate for this function could be TF-binding protein 1. The nonspecific binding of TF to bacterial cells in addition to a specific interaction with TF could be a problem in the interpretation of the results of TF-binding studies. Ellison et aL6 reported that TF and LF, as part of the host defense, bind to the outer membrane of Gram-negative bacteria. Thus, it is possible that this mechanism masks the more specific binding of TF to a receptor involved in the iron uptake from this ironcontaining glycoprotein. It is not surprising that in Neisseriu, T. vuginulis, and H. infzuenzue LF-receptors are found since all these species have an intimate association with mucosal tissue. The similarity in size and in binding specificity of the LF-receptor in H. influenme and the LF-receptor in Neisseria suggests a structural and functional conservation. These two species are also capable of utilizing heme-compounds as iron sources. The ability of H . infzuenzue and Neisseriu to acquire iron from a variety of heme and nonheme sources may make an important contribution to the virulence of these bacteria. Many in vitro and in vivo studies have demonstrated that bacterial pathogens are able to obtain their iron from a variety of heme-compounds (Table 5). However, the mechanism of iron uptake from these compounds is still unknown. Proteins with molecular weights of approximately 43,OOO, involved in the uptake andlor binding of heme, were found in Shigellu, E. coli. H. injluenzue, and B. fragilis. It would be of interest to determine whether these proteins are antigenically related. Clearly, there are still many gaps in our knowledge of the nonsiderophore-dependent ironuptake systems in bacteria. Nevertheless, the last 10 years have seen a marked progression in our 230

understanding of these iron-uptake systems. We are coming out the Stone Age into the Iron Age!

REFERENCES 1 . Carniel, E., Mazigh, D., and Mollaret, H. H., Expression of iron-regulatedproteins in Yersinia species and their relation to virulence, Infect. Immun., 55, 277, 1987. 2. Grifllths, E., The iron-uptake systems of pathogenic bacteria, in Iron and Infection: Molecular, Physiological and Clinical Aspects, 1st ed., Bullen, J. J. and Griffiths, E., Eds., John Wiley & Sons, Chichester, U.K., 1987, chap. 3. 3. Neilands, J. B., Bindereif, A., and Montgornerie, J. Z., Genetic basis of iron assimilation in pathogenic Escherichia coli, Curr. Top. Microbiol. Immunol., 118, 179, 1985. 4. Payne, S. M., Iron and virulence in Shigella, Mol. Microbiol., 3, 1301, 1989. 5. Sigel, S. P. and Payne, S. M., Effect of iron limitation on growth, siderophore production, and expression of outer membrane proteins of Vibrio cholerae, J . Bacteriol., 150, 148, 1982. 6. Ellison, R. T., Giehl, T. J., and LaForce, M., Damage of the outer membrane of enteric gram-negative bacteria by lactofemn and transfemn, Infect. Immun., 56, 2774, 1988. 7. Carlsson, J., Hotling, J. F., and Sundqvist, G. K., Degradation of albumin, hemopexin, haptoglobin and transfemn by black-pigmented Bacreroides species, J . Med. Microbiol., 18, 39, 1984. 8. Dyer, D. W., West, E. P., and Sparling, P. F., Effects of serum carrier proteins on the growth of pathogenic Neisseriae with heme-bound iron, Infect. Immun., 55, 2171, 1987. 9. Pidcock, K. A., Wooten, J. A., Daley, B. A., and Stull, T. L., Iron acquisition by Haemophilus influenzae, Infect. Immun., 56, 721, 1988. 10. Stwbner, J. A. and Payne, S. M., Iron-regulated hemolysin production and utilization of heme and hemoglobin by Vibrio cholerae, Infect. Immun., 56, 2891, 1988. 1 1 . Weinberg, E. D., Cellular iron metabolism in health and disease, Drug Metab. Health Dis., 22,53 1 , 1 9 9 0 . 12. Finkelstein, R. A., Sciortino, V., and McIntosh, M. A., Role of iron in microbe-host interactions, Rev. Infect. Dis.,5 , S759, 1983. 13. Barclay, R., The role of iron in infection, Med. Lab. Sci., 42, 166, 1985. 14. Chrichton, R. R. and Charloteaux-Wauters,M., Iron transport and storage, Eur. J . Biochem., 164, 485, 1987. 15. Crichton, R. R., Iron uptake and utilization by mammalian cells. II. Intracellulariron utilization, Trends Biochem. Sci.. 6 , 283, 1984.


16. Hershko, C., The fate of circulating hemoglobin, Br. J . Haematol.. 29, 199, 1975. 17. Muller-Eberhard, U. and Morgan, W. T., Porpbyrin-binding proteins in serum, Ann. N.Y. Acad. Sci., 244, 624, 1975. 18. Kluger, M. J. and Bullen, J. J., Clinical and physiological aspects, in Iron and Infection: Molecular, Physiological and Clinical Aspects, 1st ed., Bullen, J. J. and Griffiths, E., Eds., John Wiley & Sons, Chichester, U.K., 1987, chap. 7. 19. Neilands, J. B., Microbial envelope proteins related to iron, Annu. Rev. Microbiol.. 36, 285, 1982. 20. Neilands, J. B., Microbial iron compounds, Annu. Rev. Biochem., 50, 715, 1981. 21. Neilands, J. B., Molecular biology and regulation of iron acquisition by Escherichia coli K12, in The Bacteria, Vol. 11, Iglewski, B. H. and Clark, V. L., Eds., Academic Press, New York, 1990, chap. 10. 22 Payne, S. M. and Lawlor, K. M., Molecular studies on iron acquisition by non-Escherichia coli species, in The Bacteria, Vol. 11, Iglewski, B. H. and Clark, V. L., Eds., Academic Press, New York, 1990, chap. 11. 23. de Lorenzo, V. and Martinez, J. L., Aerobactin production as a virulence factor: a reevaluation, Eur. J . Clin. Microbiol. Infect. Dis., 7, 621, 1988. 24. Baig, B. H., Wachsmuth, I. K., and Morris, G. K., Utilization of exogenous siderophores by Campylobarter species, J . Clin. Microbiol., 23, 431, 1986. 25. Field, L. H., Headley, V. L., Payne, S. M., and Berry, L. J., Influence of iron on growth, morphology, outer membrane protein composition and synthesis of siderophores in Campylobacter jejuni, Infect. Immun., 54, 126, 1986. 26. Luckey, M., Pollack, J. R., Wayne, R., Ames, B. N., and Neilands, J. B., Iron uptake in Salmonella typhimurium: utilization of exogenous siderochromes as iron carriers, J . Bacteriol., 111, 73 1, 1972. 27. Crosa, J. H., Genetics and molecular biology of siderophore-mediated iron transport in bacteria, Microbiol. Rev., 53, 517, 1989. 28. DiRita, V. J. and Mekalanos, J. J., Genetic regulation of bacterial virulence, Annu. Rev. Genet., 23, 455, 1989. 29. Calderwood, S. B. and Mekalanos, J. J., Iron regulation of Shiga-like toxin expression in Escherichia coli is mediated by the fur locus, J . Bacteriol., 169, 4759, 1987. 30. Tai, S. P. S. and Holmes, R. K., Iron regulation of the cloned diphtheria toxin promoter in Escherichia coli, Infect. Immun., 56, 2430, 1988. 31. Holbein, B. E., Enhancement of Neisseria meningitidis infection in mice by addition of iron bound to transfenin, Infect. Immun., 34, 120, 1981. 32. Holbein, B. E., Iron-con.trolled infection with Neisseria meningitidis in mice, Infect. Immun., 29, 886, 1980. 33. Schryvers, A. B. and Gonzalez, G. C., Comparison I

of the abilities of different protein sources of iron to enhance Neisseria meningitidis infection in mice, Infect. Immun., 57, 2425, 1989. 34. Mickelsen, A. and Sparling, P. F., Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species to obtain iron from transfemn and iron compounds, Infect. Immun., 33, 555, 1981. 35. Simonson, C., Brener, D., and Devoe, I. W., Expression of a high-affinity mechanism for acquisition of transfenin iron by Neisseria meningitidis. Infect. Immun., 36, 107, 1982. 36. Schryvers, A. B. and Lee, B. C., Comparative analysis of the transfenin and lactofemn binding proteins in the family Neisseriaceae, Can. J . Microbiol., 35, 409, 1988. 37. West, S. E. H. and Sparling, P. F., Response of Neisseria gonorrhoeae to iron limitation: alterations in expression of membrane proteins without apparent siderophore production, Infect. Immun., 47, 388, 1985. 38. McKenna, W. R., Mickelsen, P. A,, Sparling, P. F., and Dyer, W., Iron uptake from lactofenin and transfenin by Neisseria gonorrhoeae, Infect. Immun., 56, 785, 1988. 39. Mickelsen, P. A., Blackman, E., and Sparling, P. F., Ability of Neisseria gonorrhoeae, Neisseria meningitidis and commensal Neisseria species to obtain iron from lactofenin, Znfecr. Immun., 35, 915, 1982. 40. Mietzner, T. A., Luginbuhl, G. H., Sandstrom, E., and Morse, S. A,, Identification of an ironregulated 37,000-dalton protein in the cell envelope of Neisseria gonorrhoeae, Infect. Immun., 45, 410, 1984. 41 Norqvist, A., Davies, J., Norlander, L., and Normark, S., The effect of iron starvation on the outer membrane protein composition of Neisseria gonorrhoeae. FEMS Microbiol. Lett., 4, 71, 1978. 42. Brener, D., DeVoe, I. W., and Holbein, B. E., Increased virulence of Neisseria meningitidis after in vitro iron-limited growth at low pH, Infect. Immun., 33, 59, 1981. 43. Keevil, W., Davies, D. B., Spillane, B. J., and Mahenthiralingam, E., Influence of iron-limited and replete continuous culture on physiology and virulence of Neisseria gonorrhoeae, J . Gen. Microbiol., 135, 851, 1989. 44. Mietzner, T. A., Barnes, R. C., Jeanlouis, Y. A., Shafer, W. M., and Morse, S. A., Distribution of an antigenically related iron-regulated protein among the Neisseria spp, Infect. Immun., 51, 60,1986. 45. Mietzner, T. A., Bolan, G., Schoolnik, G. K., and Morse, S. A., Purification and characterization of the major iron-regulated protein expressed by pathogenic Neisseria, J . Exp. Med., 165, 1041, 1987. 46. Schryvers, A. B. and Morris, L. J., Identification and characterization of the transfenin receptor from Neisseria meningitidis. Mol. Microbiol.. 2,281, 1988.

231


47. Banerjee-Bhatnagar, N. and Frasch, C. E.,

H.parainjluenzae, FEMS Microbiol. Len. 33, 153,

Expression of Neisseria meningitidis iron-regulated outer membrane proteins, including a 70-kilodalton transfemn receptor, and their potential for use as vaccines, Infect. Immun., 58, 2875, 1990. 48. Tsai, J., Dyer, D. W., and Sparling, P. F., Loss of transfemn receptor activity in Neisseria meningitidis correlates with inability to use transfemn as an iron source, Infect. Immun.. 56, 3132. 1988. 49. Ala’Aldeen, D. A., Davies, H.,Wall, R. A., and Boriello, S. P., The 70-kilodalton regulated protein of Neisseria meningitidis is not the human transfemn receptor, FEMS Microbiol. Lett., 69, 37, 1990. 50. Griffiths, E., Stevenson, P., and Ray, A., Antigenic and molecular heterogeneity of the transfemnbinding protein of Neisseria meningitidis, FEMS Microbiol. Lett., 69, 31, 1990. 51. Lee, B. C. and Bryan, L. E., Identification and comparative analysis of the lactofemn and transfemn receptors among clinical isolates of gonococci, J . Med. Microbiol., 28, 199, 1989. 52. Padda, J. S. and Schryvers, A. B., N-linked oligosaccharides of human transfemn are not required for binding to bacterial transfemn receptors, Infect.

1986.

I

62. Williams, P., Morton, D. J., Towner, K. J.,

,

Stevenson, P., and G M t h s , E., Utilization of enterobactin and other exogenous iron sources by Haemophilus injluenzae, H.parainjluenzae and H.paraphrophilus, J . Gen. Microbiol., 136, 2343, 1990. 63. Coulton, J. W. and Pang, J. C. S., Transport of hemin by Haemophilus infuenzae type b, Curr. Microbiol., 9. 93, 1983. 64. Schryvers, A. B., Identification of the transfemn and lactofemn binding proteins in Haemophilus injluenzae, J . Med. Microbiol.. 29, 121, 1989. 65. Holland, J., Towner, K. J., and Williams, P., Isolation and characterisation of Haemophilus influenzae type b mutants defective in transfemn-binding and iron assimilation, FEMS Microbiol. Lett., 77, 283, 1991. 66. Tryon, V. V. and Baseman, J. B., The acquisition

L., Byers, B. R., Martin, M. E., and Aranha, H., Ferrous iron transport in Streptococcus mutans, J . Bacteriol., 168, 1096,

of human lactofemn by Mycoplasma pneurnoniae, Microb. Pathogenesis, 3, 437, 1987. 67. Peterson, K. M. and Alderete, J. F., Iron uptake and increased inhacellular enzyme activity follow host lactofemn binding by Trichomonas vaginalis receptors, J . Exp. Med., 160, 398, 1984. 68. Gorringe, A. R., Woods, G., and Robinson, A., Growth and siderophore production by Bordetella pertussis under iron-restricted conditions, FEMS Microbiol. Len., 66. 101, 1990. 69. Redhead, K., Hill, T., and Chart, H., Interaction of lactofemn and transfenin with the outer membrane of Bordetella pertussis, J . Gen. Microbiol., 133, 891,

1986. 55. Adams, T. J., Vartivarian, S., and Cowart, R. E.,

1987. 70. Redhead, K. and Hill, T., Acquisition of iron from

Iron acquisition systems of Listeria monocytogenes, Infect. Immun.. 58, 2715, 1990. 56. Berish, S. A,, Mietzner, T. A., Mayer, L. W., Genco, C. A., Holloway, B. P., and Morse, S. A., Molecular cloning and characterization of the structural gene for the major iron-regulated protein expressed by Neisseria gonorrhoeae, J. Exp. Med.,

trans femn by Bordetella pertussis. FEMS Microbiol. Lett., 77, 303, 1991. 71. Bramanti, T. E. and Holt, S. C., Iron-regulated outer membrane proteins in the periodontopathic bacterium Bacteroides gingivalis. Biachem. Biophys. Res.

Immun., 58, 2972, 1990. 53. Schryvers, A. B. and Morris, L. J., Identification

and characterization of the human lactofemn-binding protein from Neisseria meningitidis, Infect. Irnmun., 56, 1144, 1988. 54. Evans, S. L., Arceneaux, J. E.

171, 1535, 1990. 57. Turk, D. C., The pathogenicity of Haemophilus injluenzae, J . Med. Microbiol., 18, 1, 1984. 58. Herrington, D. A. and Sparling, P. F., Haemo-

philus injluenzae can use human transfemn as a sole source for required iron, Infect. Immun., 48, 248, 1985. 59. Morton, D. J. and Williams, P., Utilization of

transfemn-bound iron by Haemophilus species of human and porcine origins, FEMS Microbiol. Lett., 65, 123, 1989.

60. Morton, D. J. and Williams, P., Siderophore-independent acquisition of transfemn-bound iron by Haemophilus injluenzae type-B, J . Gen. Microbiol., . 136. 927, 1990. 61. Williams, P. and Brown, M. R. W., Influence of

iron restriction on growth and the expression of outer membrane proteins by Haemophilus injluenzae and

232

Commun., 166, 1146, 1990. 72. Cavalieri, S. J., Bohach, G. A., and Snyder, I. S.,

Escherichia coli-haemolysin: characteristics and probable role in pathogenicity, Microbiol. Rev., 48, 326, 1984. 73. Wadwijk, C., MacLaren, D. M., and de Graaf,

J., In vivo function of hemolysin in the nephropathogenicity of Escherichia coli, Infect. Immun., 42, 245, 1983. 74. Eaton, J. W., Brandt, P., and Mahoney, J. R., Haptoglobin: a natural bacteriostat, Science, 215, 691, 1982. 75. Perry, R. D. and Brubaker, R. R., Accumulation of iron by Yersiniae, J . Bacteriol., 137, 1290, 1979. 76. Bullen, J. J., The significance of iron in infection, Rev. Infect. Dis., 3, 1127, 1981. 77. Yancey, R. and Finkelstein, R. A., Assimilation

of iron by pathogenic Neisseria spp, Infect. Immun., 32, 592, 1981. 78. Schryvers, A. B. and Gonzalez, G. C., Comparison

of the abilities of different protein sources of iron to enhance Neisseria meningitidis infection in mice, Infect. Immun., 57, 2425, 1989. 79. Brodeur, B. R., Larose, Y., Tsang, P., Hamel, J., Ashton, F., and Ryan, A., Protection against infection with Neisseria meningitidis group B serotype 2b by passive immunization with serotype-specific monoclonal antibody, Infect. Immun., 50, 510,


1985.

80. Stull, T. L., Protein sources of heme for Haemophilus influenzae, Infect. Immun., 55, 148, 1987. 81. Hanson, M. S. and Hansen, E. J., Molecular cloning, partial purification, and characterization of a haemin-binding lipoprotein from Haemophilus influenzae type b, Mol. Microbiol., 5, 267, 1991. 82. Daskaleros, P. A. and Payne, S. M., Congo red binding phenotype is associated with hemin binding and increased infectivity of Shigella ji’exneri in the HeLa cell model, Infect. Immun., 55, 1393, 1987. 83. Stugard, C. E., Daskaleros, P. A., and Payne, S. M., A 101-kilodalton heme-binding protein associated with congo red binding and virulence of Shigella flexneri and enteroinvasive Escherichia coli strains, Infect. Immun., 57, 3534, 1989. 84. Bourn, E., Reig, M., Garcia de la Torre, M., Rodriguez-Creixems, M., Romero, J., Cercenado, E., and Baquero, F., Retrospective analysis of two hundred and twelve cases of bacteremia due to anaerobic microorganisms, Eur. J . Clin. Microbiol., 4, 262. 1985. 85. Heseltine, P. N. R., Appleman, M. D., and

Leedom, J. M., Epidemiology and susceptibility of resistant Bacteroidesfragilis group organisms to new beta-lactam antibiotics, Rev. Infect. Dis., 6, S254, 1984. 86. Brook, I., A 12 year study of aerobic and anaerobic

bacteria in intra-abdominal and postsurgical abdominal wound infections, Surg. Gynecol. Obstet., 169, 387, 1989. 87. Maskel, J. P., The pathogenicity of B. fragilis and

related species estimated by intra-cutaneous infection in the guinea-pig, J. Med. Microbiol., 14, 131, 1981.

88. VerweU-van Vught, A. M. J. J., Namavar, F., Vel, W. A. C., Sparrius, M., and MacLaren, D. M., Pathogenic synergy between Escherichia coli and Bacteroides fiagilis or Bacteroides vulgatus in experimental infections: a non-specific phenomenon, J. Med. Microbiol., 21,43, 1986. 89. Rotstein, 0. D., h e t t , T. L., Wells, C. L., and Simmons, R. L., The role of Bacteroides encapsulation in the lethal synergy between Escherichia coli and Bacteroides species studied in a rat fibrin clot peritonitis model, J . Infection, 15, 135, 1987.

90.

Otto, B. R.,Verweij-van Vught, A. M. J. J., Doon van, J., and MacLaren, D. M., Outer membrane proteins of Bacteroides fragilis and Bacteroides vulgatus in relation to iron uptake and virulence, Microb.

Pathogenesis, 4, 279, 1988. 91. Venveij-van Vught, A. M. J. J., Otto, B.

R.,

Namavar, F., Sparrius,M., and MacLaren, D. M., Ability of Bacteroides species to obtain iron from , iron salts, haem-compounds and transfemn, FEMS Microbiol. Len., 49, 223, 1988. 92. Otto, B. R., Sparrius, M., Verweij-van Vught, A. M. J. J., and MacLaren, D. M., Iron regulated outer membrane protein of Bacteroides fragilis involved in heme uptake, Infect. Immun., 58, 3954, 1990. 93. Verweij-van Vught, A. M. J. J., Sparrius, M.,

Namavar, F., Vel, W. A. C., and MacLaren, D. M., Nutritional requirements of Bacteroidesfragilis and Bacteroides vulgarus with reference to iron and virulence, FEMSMicrobiol. Lett., 36,47, 1986. 94. Otto, B. R., Verweij-van Vught, A. M. J. J., and MacLaren, D. M., Purification and partial characterization of an iron regulated outer membrane protein of B. fragilis under nondenaturing conditions, FEMS Microbiol. Lett., 66, 285, 1990. 95. Otto, B. R., Verweij, W. R., Sparrius, M., Venveij-van Vught, A. M. J. J., Nord, C. E., and MacLaren, D. M., Human immune response to an iron-repressible outer membrane protein of Bacteroides fragilis, Infect. Immun., 59, 2999, 1991. 96. Blake, P. A., Merson, M. H., Weaver, R. E., Hollis, D. G., and Heublein, P. C., Disease caused by a marine vibrio, N . Engl. J. Med., 300, 1, 1979. 97. Chart, H.and Grifiths, E., The availability of iron and the growth of Vibrio vulnijicus in sera from patients with haemochromatosis, FEMS Microbiol. Lett., 26, 227, 1985. 98. Wright, A. C., Simpson, L. M., and Oliver, J. D., Role of iron in the pathogenesis of Vibrio vulnijicus infections, Infect. Immun., 34, 503, 1981. 99. Helms, S. D., Oliver, J. D., and Travis, J. C., Role of heme compounds and haptoglobin in Vibrio vulnijicus pathogenicity, Infect. Immun.. 45, 345, 1984. 100. Zakaria-Meehan, Z., Massad, G., S i p s o n , L. M., Travis, J. C., and Oliver, J. D., Ability of Vibrio vulnificus to obtain iron from hemoglobin-haptoglobin complexes, Infect. Immun., 56, 275, 1988. 101. Griffths, G. L., Sigel, S. P., Payne, S. M., and

Neilands, J. B., Vibriobactin, a siderophore from Vibrio cholerae, J. Biol. Chem., 259, 383, 1984. 102. Sigel, S. P., Stoebner, J. A., and Payne, S. M., Iron vibriobactin transport system is not required for virulence of Vibrio cholerae, Infect. Immun., 47, 360, 1985.

233

Iron sources for Haemophilus ducreyi.

Sociomicrobiology and Pathogenic Bacteria.

Iron deficiency and sources of iron.

RNA-Seq for Plant Pathogenic Bacteria.

Chemical and nutritional evaluation of yeasts and bacteria as dietary protein sources for rats and pigs.

Marine Sponges and Bacteria as Challenging Sources of Enzyme Inhibitors for Pharmacological Applications.

In-vitro screening of Malaysian honey from different floral sources for antibacterial activity on human pathogenic bacteria.

Genetic variation in pathogenic bacteria.

Pathogenic bacteria and timing of laying.

Threats and opportunities of plant pathogenic bacteria.

Shigella IpaH Family Effectors as a Versatile Model for Studying Pathogenic Bacteria.

X-ray solution scattering reveals conformational changes upon iron uptake in lactoferrin, serum and ovo-transferrins.

Binding of mouse and rabbit iron-59 transferrins to lactating mouse mammary epithelial cells.

Host epithelial glycoconjugates and pathogenic bacteria.

Manganese homeostasis and utilization in pathogenic bacteria.

Pathogenic Mechanisms Underlying Iron Deficiency and Iron Overload: New Insights for Clinical Application.

FISHing for bacteria in food--a promising tool for the reliable detection of pathogenic bacteria?

Use of transferrin-iron enterobactin complexes as the source of iron by serum-exposed bacteria.

Examination of slurry from cattle for pathogenic bacteria.

Ferric iron reduction by sulfur- and iron-oxidizing bacteria.

Pathogenic vibrios in environmental, seafood and clinical sources in Germany.

Selective adherence as a determinant of the host tropisms of certain indigenous and pathogenic bacteria.

When pathogenic bacteria meet the intestinal microbiota.

Insights into cross-kingdom plant pathogenic bacteria.