Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 24, Num. 3, 2006, pp. 163-164

Indian Journal of Medical Microbiology, Vol. 24, No. 3, July-September, 2006, pp. 163-164

Guest Editorial

Iron and bacterial virulence

Sritharan M

School of Life Sciences, University of Hyderabad, Hyderabad - 500 046, AP
Correspondence Address:School of Life Sciences, University of Hyderabad, Hyderabad - 500 046, AP, srimanju@yahoo.com

Code Number: mb06051

An invading pathogen must have the ability to multiply successfully within the hostile environment of the mammalian host to establish an infection. Bacterial and other pathogens produce a range of virulence determinants required for pathogenicity, many of which are regulated by environmental factors; level of iron is one of the important factors intimately connected to the synthesis of some of the virulence determinants.[1] There is direct evidence to show that iron, in association with the iron-regulator Fur (in many gram negative organisms)/DtxR (in several gram positive organisms) operates at the molecular level and acts as a regulatory molecule, controlling not only the iron acquisition machinery but the expression of toxins and other bacterial virulence determinants, that are not related to iron metabolism.[2]

So, how do iron levels play an important role in infection? Low iron levels have been shown to induce the expression of a number of bacterial toxins and virulence factors. Do pathogenic bacteria encounter conditions of iron limitation within the mammalian host? Iron is an essential nutrient, which by virtue of its low solubility at biological pH is not easily available. This was perhaps nature′s way of restricting the level of this element, whose toxicity is well known due to its role in the generation of free radicals. The mammalian host maintains low levels of circulating free iron by means of its iron-binding proteins, the extracellular transferrin and lactoferrin and the intracellular ferritin, haemosiderin and haeme. Normal human transferrin is about 30% saturated with iron and has a high association constant for Fe ³⁺ which ensures that the amount of free Fe ³⁺ in plasma is about 10 ^-18 M. Lactoferrin, predominantly found in polymorphonuclear leukocytes and macrophages, the first lines of defense against an invading pathogen, has a greater affinity for iron and possesses the additional property of holding the iron at the low pH prevailing in the immediate environment of the inflammatory sites. These two molecules account significantly for the bactericidal and bacteriostatic effect of plasma, lymph or cell-free exudates. Kochan[3] referred this phenomenon of limiting the iron availability to an invading pathogen as "nutritional immunity". There is no involvement of the immune system in this process. Circulating iron levels are lowered by increased synthesis of transferrin and ferritin with simultaneous suppression of the assimilation of dietary iron by decreasing its absorption by the intestine.

The question now arises as to how pathogens multiply successfully in vivo despite this severe restriction of freely available iron? To obtain host iron, successful pathogens employ one of the following strategies: (1) production of low molecular weight Fe ³⁺ specific ligands called siderophores that chelates iron from host iron-binding proteins, followed by uptake of the ferric siderophore via specific cell surface receptors, the iron-regulated membrane/envelope proteins (IRMPs/IREPs), (2) direct uptake of iron from host iron-containing molecules via specific receptors that include receptors for hemin, hemoglobin, transferrin, lactoferrin. Escherichia coli provided an ideal experimental model for the understanding of adaptation of bacteria to iron-restriction and today there is a vast amount of information from E. coli about iron acquisition systems and molecular mechanisms of iron acquisition machinery.[4] About 6 iron acquisition systems have been identified in E. coli and one among them is the enterobactin-mediated high affinity transport system seen not only in E. coli but also in Klebsiella pneumoniae , Salmonella typhimurium and some species of Shigella . The secreted siderophore enterobactin (also called enterochelin) is taken up by a TonB-dependant outer membrane receptor, the ferric-enterobactin receptor FepA. A wide range of bacterial siderophores and their receptors have been extensively reviewed.[5],[6] The second important mechanism of iron acquisition is seen in a number of human and animal pathogens and in particular the members of the Pasteurellaceae and Neisseriaceae[7] that exploit a siderophore-independent mechanism for acquiring iron from host iron-binding proteins. These pathogens express specific cell surface receptors and there is direct contact between these receptors and the host iron-binding proteins followed by removal of the iron directly from the latter. These receptors are highly specific as exemplified by the high degree of specificity of human transferrin as compared to that from other species. Bacterial receptors for transferrin, lactoferrin, haemin, haemoglobin are well understood, both in chemistry and in uptake mechanisms. All the iron-controlled genes, irrespective of the nature of the iron-acquisition system, are regulated by level of intracellular iron in association with the iron regulator Fur/DtxR, the details of which are well deciphered in E. coli . The Fur protein is a repressor molecule, which on complexing with Fe ²⁺ blocks the transcription of iron-regulated genes by binding to specific operator sequences called Fur box/ Iron box within their promoter regions, whose consensus sequence was determined as 5′- GATAATGATAATCATTATC.

Overwhelming evidence has accumulated over the past decade[2] that shows that iron-restricted conditions favoured the expression of a number of toxins and other potential virulence determinants: few examples include diphtheria toxin by Corynebacterium diphtheriae , a-hemolysin by E. coli , Shiga toxin by Shigella dysenteriae , verocytotoxin by E. coli and exotoxin A by Pseudomonas aeruginosa . The association of iron and virulence is obvious. It is clear that pathogens employ these molecules for gaining access to host nutrients, by effecting host cell lysis. Haemolysins, for example cause the lysis of not only erythrocytes but all cells resulting in the release of haeme and iron, along with other cellular nutrients for utilization by the infecting bacteria. Molecular mechanisms of regulation of the expression of tox gene in C. diphtheriae showed that in the presence of sufficient intracellular iron, the DtxR-Fe ²⁺sub complex bound to the iron box upstream of the tox gene and inhibited its transcription, while upon iron deprivation, there was induction of its expression as the DtxR cannot bind to the iron box in the absence of Fe ²⁺ .

The importance of iron in tuberculosis has been well described by Ratledge,[8] with emphasis on understanding the basic mechanisms of the pathogenesis of this disease, that has assumed to be one of the worst bacterial disease in terms of the number of deaths per year. This is particularly important in our country, with reports of tuberculosis being the leading killer among the infectious diseases. It has been aptly brought out by the author that iron level is very crucial in the outcome of an infection. While the host tries to limit infection by lowering iron, there is adaptation by the pathogen with increased expression of virulence factors, causing damage to the host. At the same time, administration of iron is detrimental as the increased availability of iron increases multiplication of bacterial growth, again contributing to increased virulence, as demonstrated experimentally. While iron is important in the establishment of an infection, it appears that iron may also play a role in the effect of anti-bacterial agents. Our observations on the effect of iron deprivation on the anti-tubercular drug INH on Mycobacterium tuberculosis grown in vitro showed that the peroxidase activity of the catalase-peroxidase KatG is abolished upon iron limitation, resulting in the failure of activation of the prodrug INH to active form.[9] The potentiating effect of iron on another anti-mycobacterial drug pyrazinamide was also shown by Somoskovi et al .[10]

While it is clear that iron levels are important in infection, it is not an easy task to control their levels in the host. The pros and cons of low and high iron levels, as explained above needs to be considered by the physician in treating a patient with chronic infection. In tuberculosis, for example, administering iron to a patient presenting with anemia with a low blood cell count needs to be done with caution. The iron-withholding capacity of the host serves to control the infection ad if this is compromised by iron supplements, this favors the pathogen rather than benefiting the host. This, of course needs to be weighed against the consequences of severe anemia, if left untreated. There should a slow influx of iron with monitoring of the levels at regular intervals to effectively control the infection.

References

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