Scientific Publications - Work Done by Microbiology Reader Bioscreen C

FEMS Immunology and Medical Microbiology, 1995, vol. 11, pp. 257-264

Iron supplying systems of  Salmonella in diagnostics,  epidemiology and infection

Robert Kingsley, Wolfgang Rabsch, Peter Stephens, Mark Roberts, Rolf Reissbrodt and Peter H. Williams

ABSTRACT

Well-known and newly characterised mechanisms, both endogenous and exogenous, for the uptake of iron by Salmonella are outlined, and their possible roles at various stages in infection are discussed. The contributions of a detailed understanding of iron supplying systems to techniques for diagnosis, epidemiology and disease management are described.
Keywords: Salmonella; Siderophore; Growth supplement; Infection; Vaccine

 ---

1. The role of iron in bacterial infection

Iron is essential for the growth of most microor­ganisms. It functions as a catalyst for electron trans­port and is an essential component of several impor­tant enzymes. The key features of iron are its two stable valencies and its ability to generate a wide range of redox potentials, from +300 mV in cy­tochrome a, for example, to - 490 mV in various iron-sulphur proteins. In anaerobic and mi­croaerophilic conditions and at pH < 7 iron exists in the highly soluble ferrous form which is readily available to microorganisms. Escherichia coli ex­presses a specific mechanism for the uptake of fer­rous iron [1], and it is likely that other bacterial species have analogous systems. In aerobic condi­tions and at pH > 7, however, iron converts to the ferric form in which it exists either as insoluble complexes (K_sol ~ 10³⁸ M) or bound to various pro­teins. Ferric iron is therefore much less readily avail­able to microorganisms.

The role of iron in infection is best understood in terms of a competition between pathogenic microor­ganisms and cells of the host organism. Bacteria require micromolar amounts of iron for growth; the adult human body contains approximately 4 g of iron, mostly inside cells, the actual quantity and form varying from tissue to tissue. However, the majority

of intracellular iron is either in haem or complexed with the iron-storage proteins ferritin and haemosiderin. Body fluids of mammals, birds and reptiles contain only a tiny amount of iron (< 1% of the total), mostly bound to the iron-binding glycopro­teins transferrin in serum and lactoferrin in milk and mucosal secretions. In egg-white, iron is bound to the related protein ovotransferrin (conalbumin). Be­cause the transferrin class of proteins has a very high affinity for iron (binding constant approximately 10²³), levels of free iron in body fluids and within eggs are of the order of 10^-18 M, far too low to support the growth of microorganisms.

2. Siderophores and iron-regulated outer mem­brane proteins

Microorganisms use a variety of strategies to satisfy their demand for iron [2]. Enteric bacteria, including Salmonella, primarily utilise siderophores, low molecular weight (typically < 1000) iron­specific ligands whose biosynthesis and mechanisms for uptake are modulated by internal iron concentra­tions. Uptake mechanisms vary from siderophore to siderophore, but may involve (a) siderophore-specific iron-regulated outer membrane proteins (IROMPs) as surface receptors, (b) the TonB protein, which couples the energised states of the inner and outer membranes, (c) various periplasmic and inner mem­brane transport proteins, and (d) soluble enzymes (reductases, esterases) whose function is to release

iron from ferrisiderophore complexes. The genes encoding such proteins are transcriptionally regu­lated according to the availability of iron by the regulatory protein Fur (ferric iron uptake regulator).

Table 1

Endogenous and exogenous siderophores utilised by Salmonella

Siderophore systems utilised by Salmonella are listed in Table 1. It is an indication of just how important iron is for growth that many bacterial species, including Salmonella, have evolved mecha­nisms for the uptake and utilisation of exogenous siderophores secreted by other microorganisms [3,4]. As far as endogenous production is concerned, it has been known for more than 20 years that the major siderophore of Salmonella is the phenolate molecule enterobactin [5], also known as enterochelin, a cyclic triester of 2,3-dihydroxy-N-benzoyl serine (DHBS). More than 99% of Salmonella isolates make enter­obactin. It has also been known for many years that some strains express the hydroxamate siderophore aerobactin in addition to enterobactin. The aerobactin system is usually encoded by large plasmids in Salmonella [6]. More recently, however, the applica­tion of siderophore-pattern analysis [4] has indicated the existence of several new iron supplying systems among clinical and environmental salmonella iso­lates. For example, a hydroxamate siderophore, whose structure has not yet been determined, has been isolated from a S. stanleyville strain that makes neither enterobactin nor aerobactin [7]. In addition, 2,3-dihydroxybenzoic acid (DHBA), an intermediate in enterobactin biosynthesis, is a growth factor for Salmonella in iron limited conditions, either alone or in the presence of endogenous aerobactin [8,9]. Simi-

larly, Salmonella can utilise breakdown products of enterobactin, notably the linear dimer and trimer forms of DHBS, to supply iron for growth [10]. More interestingly, perhaps, is our recent observation that a-keto- and a-hydroxyacids also have siderophore activity for Salmonella [11], following reports that these primary metabolites of pro- and eukaryotic cells are able to supply iron to the Pro­teus-Providencia-Morganella group [12] and to E. coli [13]. With the exception of DHBA and ferriox­amines B and E, ferrisiderophore uptake by Salmonella is TonB-dependent.

In common with many other bacterial species, Salmonella responds to conditions of iron limitation by changing the composition of proteins in the outer membrane [14-16]. Electrophoretic analysis of membrane proteins shows a cluster of high molecu­lar weight IROMPs, including the enterobactin re­ceptor (FepA, 81 kDa), the aerobactin receptor (IutA, 74 kDa) and the ferrichrome receptor (FhuA, 78 kDa). Unlike E. coli, there is no active ferric-di­citrate uptake system in Salmonella, and homologues of the E. coli Cir and Fiu proteins have also not been demonstrated unequivocally. It is possible that

IROMPs may be effective potential targets for vac­cines.

3. Iron supply and diagnostics

We have recently described a sensitive method for the rapid detection of Salmonella contamination of eggs, based on the observation that siderophores can be used as selective growth factors [17]. Ovotransfer­rin in hens' egg albumen binds available iron, exert­ing a strong bacteriostatic effect under which con­taminating bacteria may remain dormant within eggs for several weeks [18]. Our detection method in­volves supplementation of standard Salmonella pre­enrichment medium with ferrioxamines, particularly E or G. These are excellent growth factors for a range of Salmonella species associated with food poisoning, resulting in a significantly shortened lag phase and a faster growth rate (Fig. 1). Thus, it may be possible to detect contaminating bacteria within a working day, rather than after standard overnight incubation and further enrichment steps. The reason for describing the natural ferrioxamines E and G as selective is that they do not promote the growth of E. coli and the Proteus-Providencia-Morganella group [17]. However, ferrioxamines do su^pport the growth of certain species that may also contaminate egg products, particularly Klebsiella pneumoniae and Citrobacter freundii. Interestingly, the presence of ferrioxamine in pre-enrichment medium appears to cause a characteristic and very striking increase in the swarming behaviour of isolates subsequently in­oculated onto an appropriatesemi-solid medium [19]. The nature of this phenotypic effect is not yet known, but it does provide the potential for a definitive specific test for Salmonella even in the presence of other competitors.

Fig. 1. Effect of ferrioxamine E on the growth in Buffered Peptone Water (BPW, Oxoid CM503) of S. enteritidis PT4 following storage in hens^' egg albumen. Albumen was aseptically removed from a fresh egg, artificially inoculated (< 10 cells/ml), and stored overnight at room temperature. The albumen was split into two samples and diluted ten-fold in BPW; one sample was supplemented with ferrioxamine E (1 µg/ml, n ), the other was unsupplemented (q ). Growth was measured in terms of increasing turbidity at 600 nm using the Bioscreen (Labsystems); each sample of albumen was inoculated across 100 wells (400 µl/well). The experiment shown represents a realistic level of contamination; only 11 of 200 wells were positive, and it is therefore likely that each positive well contained only a single organism at the start of the experiment (Stephens, manuscript in preparation).

Table 2

Qiderophore patternn analysis of various salmonella sn l,cpecies

4. Iron supply and epidemiology

While virtually all Salmonella isolates secrete enterobactin, the application of siderophore pattern analysis to strains of various subspecie [20-22] indicates significant differences in their ability to make aerobactin and occasional differences in the secretion of enterobactin intermediates and break­down products (Table 2). Particularly notable is the relatively high proportion of aerobactin-producing strains among Salmonella subspecies IIIa and IIIb (i.e. monophasic and biphasic S. arizona). Aerobactin production by subspecies I is almost exclusively restricted to isolates from hospital infections, particularly in children's wards, the most frequent serotypes being S. typhimurium, S. wien, S. infantis and S. haifa. Isolates from cases of food-poisoning, by contrast, are generally unable to synthesise aer­obactin.

Table 3 summarises a semi-quantitative analysis of enterobactin and aerobactin production by the most frequently isolated salmonella species. Of par­ticular interest is the observation that host-adapted

Table 3

Semi-quantitative bioassays of enterobactin and aerobactin synthesis by Salmonella.

species (S. typhi for man, S. gallinarum/pullorum for chickens, S. choleraesuis for pigs and S. dublin for cattle, as well as some S. typhimurium strains from pigeons) produce no aerobactin and only low levels of enterobactin. Moreover, IROMPs of S. typhi strains show remarkable consistency [23], and it is likely that iron supplying systems are highly conserved among these species.

Most salmonella isolates give a positive result in bioassays with the indicator strain Aureobacterium flavescens JG-9. This strain is generally used to detect hydroxamate siderophores other than aer­obactin, but it also grows in the presence of various a-keto- and a-hydroxyacids. HPLC analysis of cul­ture supernatants indicated the excretion of these growth-promoting primary metabolites by all salmonella strains tested [11].

5. Iron supply and infection

The mammalian body responds to infection by invasive organisms such as Salmonella in a variety of ways. Of particular relevance here is the signifi­cant reduction in levels of iron in the serum, primar­ily by removal of transferrin-bound iron to storage in ferritin in reticuloendothelial macrophages in the spleen and liver. This reaction, the so-called hypofer­raemia of infection, is regarded as a powerful gener­alised host defence mechanism. In addition, the rise in body temperature characteristic of inflammatory disease may also play a defensive role, since the production and uptake of enterobactin are known to be reduced at elevated temperatures. However, al­though there is agreement that enterobactin produc­tion is necessary for the growth of S. typhimurium in normal mouse serum, the importance of enterobactin in the virulence of Salmonella has been investigated only in laboratory mice and has produced contradic­tory results [24,25]. For a number of reasons, labora­tory mice may not be the most appropriate host for studying the role of siderophore-mediated iron up­take in virulence. For example, mice are unable to develop hyperthermia in response to infection. More­over, the transferrin iron saturation level of labora­tory mice is usually about 60%, about twice that of healthy human adults. Nevertheless, serum iron lev­els do influence the outcome of infection in mice; the severity of infection is reduced in mice fed a low iron diet, while, conversely, excess iron injected with a bacterial inoculum can significantly reduce the LD₅₀ of a challenge strain [26]. Note however that this may not be due solely to increasing bacterial iron supply, because excess iron is detrimental to the normal functioning of polymorphonuclear phago­cytes and monocytes.

S. typhimurium strains with mutations in genes coding for enzymes that catalyse the pre-chorismate pathway (aroA mutants) are unable to produce enter­obactin and other aromatic compounds. Such mu­tants are highly attenuated in a variety of vertebrate hosts, including man. They are also excellent live single dose oral vaccines against salmonellosis in laboratory and domestic animals [27], and are being investigated as next-generation live vaccines against typhoid in humans [28]. Similarly, iron uptake mu­tants of S. typhi are attenuated in mice [29]. When iron is freely available, attenuated strains are in fact able to overcome the immune system of infected individuals. Moreover, strains grown in conditions of iron starvation to derepress iron supplying systems, and then killed by heat or sonication, can be used to elicit cellular (but not humoral) immunity in condi­tions of excess iron [30].

The role of aerobactin in the pathogenesis of Salmonella is also unclear, but by analogy with other enteric pathogens it is likely that the activity of an additional high affinity system may confer a selec­tive advantage at one or more stages in the pathogen­esis of salmonella infection. One such stage is initial adherence to the intestinal epithelium, and prolifera­tion prior to invasion of enterocytes or M-cells. In this case, iron stress is imposed by the presence of lactoferrin in the secretion that bathes the intestinal mucosa. Another environment in which aerobactin may provide an advantage is the bloodstream, which, especially during hypoferraemia, is rich in unsatu­rated transferrin. Support for this comes from the finding that aerobactin-producing strains of certain salmonella serotypes (e.g. S. typhimurium and S. wien) which do not normally cause systemic disease have been isolated from the blood of severely ill patients. This is similar to the situation with pathogenic E. coli; aerobactin production is much more frequently associated with strains that cause extraintestinal infection in man and domestic animals compared with isolates from enteric infections. The importance of aerobactin lies in the fact that it is highly effective at removing ferric iron from trans­ferrin and (presumably) related proteins [31].

Table 4

Minimum inhibitory concentrations of ampicillin- and amoxycillin-derivatives substituted with benzoylhydrazidoglyoxyl groups

It is generally believed that invasive pathogens such as Salmonella do not require high affinity iron uptake systems once they are inside host cells. How­ever, iron levels within vacuoles are of the order of 1

M [32], sufficiently high to repress the expression of genes encoding high affinity siderophore systems, but not high enough to prevent the uptake of iron complexed with a-keto- and a-hydroxyacids. Thus, these compounds, primary metabolites of bacteria and host cells, may be essential iron supplying sys­tems in what might be termed intermediate levels of iron stress, promoting proliferation of the pathogen within the infected cell before transcytosis and systemic spread. Which of several routes of iron uptake contribute to growth in any particular phase of an invasive infection will therefore depend on (a) differ­ential regulation of the different uptake systems in response to iron availability, and (b) the presence of particular iron chelating compounds, originating from either bacterial or host cells.

Fig. 2. Glyoxylic acid benzhydrazone derivatives of aminoacyl penicillins ampicillin (R¹ = H) and amoxycillin (R¹ = OH). R² side chains are indicated in Table 4.

The normal `home' of the intracellular pathogen is the macrophage [33]. There is increasing evidence that the amount of iron available to bacteria resident in macrophages varies according to the activation state of the cells and that this reflects their antimicro­bial capabilities. Macrophages activated with y-inter­feron are able to inhibit intracellular bacterial growth. This correlates with down-regulation of transferrin receptors on the surface of macrophages and can be reversed by adding iron-saturated transferrin. Also, there is a critical balance between depriving bacteria resident in macrophages of iron and providing suffi­cient iron for the generation of iron-mediated antimi­crobial redox reactions [34]. Such experiments have, however, not yet been performed with Salmonella. Interestingly, some in vitro studies suggest that siderophores might play a role in pathogenesis by interfering with T cell function. Deferrated aer­obactin, ferrioxamine and ferrichrome (but not the iron-loaded forms of these siderophores) inhibit mi­togen-induced proliferation of murine T-cells, while enterobactin and ferrienterobactin are directly cyto­toxic to T-cells [35].

An interesting recent development in chemother­apy that stems from research on bacterial iron sup­plying systems concerns the use of antibiotics chemically coupled to siderophore-like molecules. The ef­fectiveness of aminoacyl penicillins, for example, is significantly increased by coupling to a structure that recognises the enterobactin receptor FepA (Fig. 2, Table 4), and which can, on its own, act as a TonB-dependent siderophore for Salmonella [36]. t is hoped that continued detailed analysis of the com­plexity of iron supplying systems will lead to the development of rational approaches to the identifica­tion and control of Salmonella infection.

Acknowledgements

We thank the DAAD (313-ARC-VI-92/149/scu) and the British Council (ARC Project 354) for financial support for some of this work.

References

[1]Hantke, K. (1987) Ferrous iron transport mutants in Es­cherichia coli K12. FEMS Microbiol. Lett. 44, 53-57.

[2]Wooldridge, K.G. and Williams, P.H. (1993) Iron uptake mechanisms of pathogenic bacteria. FEMS Microbiol. Rev. 12, 325-248.

[3]Ernst, J.F., Bennett, R.L. and Rothfield, L.I. (1978) Constitu­tive expression of the iron-enterochelin and ferrichrome up-take systems in a mutant strain of Salmonella typhimurium. J. Bacteriol. 135, 928-934.

[4]Reissbrodt, R. and Rabsch, W. (1988) Further differentiation of enterobacteriaceae by means of siderophore-pattern analy­sis. Zbl. Bakt. Hyg. A 268, 306-317.

[5]Pollock, J.R. and Neilands, J.B. (1970) Enterobactin, an transport compound from Salmonella typhimurium. Biochem. Biophys. Res. Commun. 38, 989-992.

[6]Colonna, B., Nicoletti, M., Visca, P., Casalino, M., Valenti, P. and Maimone, F. (1985) Composite IS1 elements encod­ing hydroxamate-mediated iron uptake in Fl me plasmids from epidemic Salmonella spp. J. Bacteriol. 162, 307-316.

[7]Rabsch, W., Paul, P. and Reissbrodt, R. (1987) A new hydroxamate siderophore for iron supply of Salmonella. Acta Microbiol. Hung. 34, 85-92.

[8]Rabsch, W., Paul, P. and Reissbrodt, R. (1986) DHBA (2,3-Dihydroxybenzoesaure)-Ausscheidung durch einen en­terobactinnegativen multiresistenten Salmonella ty­phimurium-Wildstamm. J. Basic Microbiol. 26, 113-116.

[9]Rabsch, W., Tkacik, J., Lindemann, W., Mikula, I. and Reissbrodt, R. (1991) Different systems for iron supply of Salmonella typhimurium and Escherichia coli-wild strains for typing. Zbl. Bakt. 274, 437-445.

[10]                   Wilkins, T.D. and Lankford, C.E. (1970) Production by Salmonella typhimurium of 2,3-dihydroxybenzoylserine, and its stimulation of growth in human serum. J. Infect. Dis. 121, 129-136.

[11]                   Reissbrodt, R., Kingsley, R., Rabsch, W. and Williams, P.H. (1994) a-keto-/a-hydroxyacids as novel siderophores of Salmonella (poster). VAAM Meeting, Hannover, Germany.

[12]                   Drechsel, H., Thieken, A., Reissbrodt, R., Jung, G. and Winkelmann, G. (1993) a-keto acids are novel siderophores in the genera Proteus, Providencia, and Morganella and are produced by amino acid deaminases. J. Bacteriol. 175, 2727-2733.

[13]                   Reissbrodt, R., Kuhn, S., Beer, W. and Braun, V. (1993) Iron supply of E. coli by a-ketoacids (poster). Conference on Iron and Iron Metabolism, Brugge, Belgium.

[14]                   Braun, V., Hantke, K. and Stauder, W. (1977) Identification of the sid outer membrane receptor protein in Salmonella typhimurium SL1027. Mot. Gen. Genet. 155, 227-229.

[15]                   Braun, V. and Hantke, K. (1991) Genetics of bacterial iron transport. In: CRC Handbook of Microbial Iron Chelates (Winkelmann, G., Ed.) pp. 107-138, CRC Press, Boca Ra­ton.

[16]                   Chart, H. and Rowe, B. (1993) Iron restriction and the growth of Salmonella enteritidis. Epidemiol. Infect. 110, 41-47.

[17]                   Reissbrodt, R. and Rabsch, W. (1993) Selective pre-enrich­ment of Salmonella from eggs by siderophore supplements. Zbl. Bakt. 279, 344-353.

[18]                   Lock, J.L. and Board, R.G. (1992) Persistence of contamina­tion of hens' egg albumen in vitro with Salmonella serop­types. Epidemiol. Infect. 108, 389-396.

[19]                   Pless, P. and Reissbrodt, R. (1995) Improvement of Salmonella detection on motility enrichment media by fer­rioxamine E supplementation of pre-enrichment culture. Int. J. Food Microbiol., in press.

[20]                   Aznar, R., Amaro, C., Alcaide, E. and Lemos, M.L. (1989) Siderophore production by environmental strains of Salmonella species. FEMS Microbiol. Lett. 57, 7-12.

[21]                   Rabsch, W. and Reissbrodt, R. (1988) Investigations of Salmonella strains from different clinical-epidemiological origin with phenolate and hydroxamate (aerobactin)-sidero­phore bioassays. J. Hyg. Epidemiol. Microbiol. Immunol. 32, 353-360.

[22]                   Visca, P., Filetici, E., Anastasio, M.P., Vetriani, C., Fantasia, M. and Orsi, N. (1991) Siderophore production by Salmonella species isolated from different sources. FEMS Microbiol. Lett. 79, 225-232.

[23]                   Faundez, G., Aron, L. and Cabello, F.C. (1990) Chromoso­mal DNA, iron-transport systems, outer membrane proteins, and enterotoxin (heat labile) production in Salmonella typhi strains. J. Clin. Microbiol. 28, 894-897.

[24]                   Benjamin, W.H., Turnbough, C.L., Posey, B.S. and Briles, D.E. (1985) The ability of Salmonella typhimurium to pro-duce the siderophore enterobactin is not a virulence factor in mouse typhoid. Infect. Immun. 50, 392-397.

[25]                   Yancey, R.J., Breeding, S.A.L. and Lankford, C.E. (1979) Enterochelin (enterobactin): virulence factor for Salmonella typhimurium. Infect. Immun. 24, 174-180.

[26]                   Puschmann, M and Ganzoni, A.M. (1977) Increased resis­tance of iron-deficient mice to Salmonella infection. Infect. Immun. 17, 663-664.

[27]                   Hoiseth, S.K. and Stocker, B.A.D. (1981) Aromatic-depen­dent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238-239.

[28]                   Roberts, M., Chatfield, S.K. and Dougan, G. (1994) Salmonella as carriers of heterologous antigens. In: Novel Delivery Systems for Oral Vaccines (O'Hagan, D.T., Ed.) pp. 27-58, CRC Press, Boca Raton.

[29]                   Furman, M., Fica, A., Saxena, M., Di Fabio, J.L. and Ca­bello, F.C. (1994) Salmonella typhi iron uptake mutants are attenuated in mice. Infect. Immun. 62, 4091-4094.

[30]                   Kochan, I., Wagner, S.K. and Wasynczuk, J. (1984) Effect of iron on antibacterial immunity in vaccinated mice. Infect. Immun. 43, 543-548.

[31]                   Ford, S., Cooper, R.A., Evans, R.W., Hider, R.C. and Williams, P.H. (1988) Domain preference in iron removal from human transferrin by the bacterial siderophores aer­obactin and enterochelin. Eur. J. Biochem. 178, 477-481.

[32]                   Garcia-del Portillo, F., Foster, J.W., Maguire, M.E. and Finlay, B.B. (1992) Characterization of the micro-environ­ment of Salmonella ryphimurium-containing vacuoles within MDCK epithelial cells. Molec. Microbiol. 6, 3289-3297.

[33]                   Horwitz, M.A. (1988) Intracellular parasitism. Curr. Opin. Immunol. 1, 41-46.

[34]                   Alford, C.E., King Jr., T.E. and Campbell, P.A. (1991) Role of transferrin, transferrin receptors, and iron in macrophage listericidal activity. J. Exp. Med. 174, 459-466.

[35]                   Autenrieth, I., Hantke, K. and Heesemann, J. (1991) Im­munosuppression of the host and delivery of iron to the pathogen: a possible dual role of siderophores in the patho­genesis of microbial infections? Med. Microbial. Immunol. 180, 135-141.

[36]                   Reissbrodt, R. and Rabsch, W. (1994) Ability of catecholate derivatives to promote growth of Gram-negative bacteria. In: The Development of Iron Chelators for Clinical Use (Bergeron, R. and Brittenham, G.M., Eds.) pp. 209-235. CRC Press, Boca Raton.

(order Full Text from publisher)

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005