vitro growth is inhibited by iron chelators. Moreover, in vivo administration of DFO in mice infected with Leishmania leads to a slight delay in the development of cutaneous lesions. Unexpectedly however, systemic iron delivery at early time points of infection, decreased parasite load at the site of parasite inoculation, the regional lymph node, the liver and spleen. The protective effect of iron correlated with lower IL-4 and IL-10, but higher type-1 cytokine transcripts (IFN-γ and inducible ΝΟ synthase) at the site of inoculation, as well as by increased serum levels of IgG2a.143 An iron-dependent superoxide dismutase from *Leishmania Chagas* is expressed at low levels in the early logarithmic stage of development and increases at later stages of growth. The parasite demonstrates significant growth reduction when endogenous superoxide levels are increased, following the addition of paraquat in culture. There is a protective gene, LcFeSODB, which plays an important role in the parasite growth and survival by protecting the glycosomes from superoxide toxicity.144

Malnutrition alters the innate immune response against *L.donovani*. Thus, diets deficient in calories, protein, and in the metal elements zinc and iron represent a risk factor for the development of visceral leishmaniasis, and in malnourished mice, a greater parasite burden is found in the spleen and liver, which is attributed to a failure of lymph node barrier function. Lymph node cells from the malnourished group produced increased levels of PGE2 and decreased levels of IL-10 and inducible NO synthase activity.145 Iron deficiency may finally favor the host and impair *L.donovani* growth. When iron availability is restricted the parasite's growth may be reduced and the infection attenuated.146

Another *Leishmania spp*, *Leishmania amazonensis,* elaborates an inducible ferrous iron transport system through LIT1, a novel parasitic membrane protein. LIT1 is only detectable upon intracellular invasion of the parasite and its expression is accelerated under irondeprived conditions. *L. amazonensis* lacking LIT1 protein abolishes its virulence and its replicating capacity within macrophages.147

*Trichomonas vaginalis* is the most common non viral pathogen, transmitted sexually and is highly-dependent on iron. *T. vaginalis* is adhered to vaginal epithelial cells, through specific surface proteins (AP65, AP51, AP33 and AP23) named adhesins. Free iron, heme and hemoglobin induce AP65 mRNA and protein expression on the parasistic membrane, thus favoring virulence. Heme-induced AP65 expression was about 10-fold higher in a low-iron culture medium, indicating that *T. vaginalis* can use heme as an alternative source of iron, important to its growth and regulation of expression of the adhesin genes.148 An ironresponsive promoter and other iron regulatory elements (IRE) in the 5'-UTR of the ap65-1 gene, as well as two IRE-like hairpin-loop structures in mRNAs of TVCP4 and TVCP12 cysteine proteinases, have been identified in *T.vaginalis,* suggesting the existence of a posttranscriptional iron regulatory mechanism of critical genes by an IRE/IRP-like system in this protozoon.149 DFO killed all *T.vaginalis* isolates with a minimum lethal concentration of 30 μM after 48 h of exposure, and a potent and persistent inhibitory effect of DFO on the parasite viability and growth was observed, with lower drug concentration and shorter time of exposure.150

*Tritrichomonas foetus* is a protozoan pathogen of cattle, and its growth and virulence is greatly influenced by the iron concentration of the culture medium. In iron-restricted media both, Lf and Tf support *T.foetus* growth. However, a specific binding to the outer parasitic membrane has been demonstrated only for Lf, whose uptake at 370 C is about 3.5-fold higher, a finding indicating a mechanism of receptor-mediated endocytosis. In contrast, Tf sources.154

310 Insight and Control of Infectious Disease in Global Scenario

vitro growth is inhibited by iron chelators. Moreover, in vivo administration of DFO in mice infected with Leishmania leads to a slight delay in the development of cutaneous lesions. Unexpectedly however, systemic iron delivery at early time points of infection, decreased parasite load at the site of parasite inoculation, the regional lymph node, the liver and spleen. The protective effect of iron correlated with lower IL-4 and IL-10, but higher type-1 cytokine transcripts (IFN-γ and inducible ΝΟ synthase) at the site of inoculation, as well as by increased serum levels of IgG2a.143 An iron-dependent superoxide dismutase from *Leishmania Chagas* is expressed at low levels in the early logarithmic stage of development and increases at later stages of growth. The parasite demonstrates significant growth reduction when endogenous superoxide levels are increased, following the addition of paraquat in culture. There is a protective gene, LcFeSODB, which plays an important role in the parasite growth and survival by protecting the glycosomes from superoxide toxicity.144 Malnutrition alters the innate immune response against *L.donovani*. Thus, diets deficient in calories, protein, and in the metal elements zinc and iron represent a risk factor for the development of visceral leishmaniasis, and in malnourished mice, a greater parasite burden is found in the spleen and liver, which is attributed to a failure of lymph node barrier function. Lymph node cells from the malnourished group produced increased levels of PGE2 and decreased levels of IL-10 and inducible NO synthase activity.145 Iron deficiency may finally favor the host and impair *L.donovani* growth. When iron availability is restricted

Another *Leishmania spp*, *Leishmania amazonensis,* elaborates an inducible ferrous iron transport system through LIT1, a novel parasitic membrane protein. LIT1 is only detectable upon intracellular invasion of the parasite and its expression is accelerated under irondeprived conditions. *L. amazonensis* lacking LIT1 protein abolishes its virulence and its

*Trichomonas vaginalis* is the most common non viral pathogen, transmitted sexually and is highly-dependent on iron. *T. vaginalis* is adhered to vaginal epithelial cells, through specific surface proteins (AP65, AP51, AP33 and AP23) named adhesins. Free iron, heme and hemoglobin induce AP65 mRNA and protein expression on the parasistic membrane, thus favoring virulence. Heme-induced AP65 expression was about 10-fold higher in a low-iron culture medium, indicating that *T. vaginalis* can use heme as an alternative source of iron, important to its growth and regulation of expression of the adhesin genes.148 An ironresponsive promoter and other iron regulatory elements (IRE) in the 5'-UTR of the ap65-1 gene, as well as two IRE-like hairpin-loop structures in mRNAs of TVCP4 and TVCP12 cysteine proteinases, have been identified in *T.vaginalis,* suggesting the existence of a posttranscriptional iron regulatory mechanism of critical genes by an IRE/IRP-like system in this protozoon.149 DFO killed all *T.vaginalis* isolates with a minimum lethal concentration of 30 μM after 48 h of exposure, and a potent and persistent inhibitory effect of DFO on the parasite viability and growth was observed, with lower drug concentration and shorter time

*Tritrichomonas foetus* is a protozoan pathogen of cattle, and its growth and virulence is greatly influenced by the iron concentration of the culture medium. In iron-restricted media both, Lf and Tf support *T.foetus* growth. However, a specific binding to the outer parasitic membrane has been demonstrated only for Lf, whose uptake at 370 C is about 3.5-fold higher, a finding indicating a mechanism of receptor-mediated endocytosis. In contrast, Tf

the parasite's growth may be reduced and the infection attenuated.146

replicating capacity within macrophages.147

of exposure.150

binding is nonspecific, and iron retrieval is achieved via extracellular release and siderophore assistance.151 Many microbial siderophores can also support *T. foetus* growth under iron-limited conditions, providing iron to ferredoxin, the major siderophore of the parasite. Iron uptake is not mediated by previous extracellular reduction, although *T.foetus* possesses some ferrireductase activity. Siderophores are pinocytosed by the parasites in small vesicles, exhibiting a very acidic environment. Hemin also supports *T. foetus* growth, probably with the involvement of heme oxygenase.152 Parasites grown in iron-depleted media exhibit reduced capability to destroy epithelial cell monolayers and reduced activity of several cysteine proteases, indicating that iron is an extracellular signal, modulating T.foetus' ability to interact with host epithelial cells.153 In one study, mice inoculated intraperitoneally with a moderately- or a highly-virulent strain of *T.foetus* and treated with ferric citrate exhibited high mortality rate by the moderately-virulent strain up to the level of the highly-virulent strain. Peritoneal cultures showed that iron overload was associated with stimulation of parasite replication, which was strongly suppressed in untreated mice, and the less virulent strains showed lower efficiency for iron acquisition from Tf and other

The greatest experience about the influence of iron metabolism on parasite growth has emerged from the study of malaria infection. *Plasmodium* grows up fluently in the intraerythrocyte environment, where plenty of iron, contained in hemoglobin, can be easily accessed and uptaken. Since about 3 decades ago there has been emerging evidence that, iron deprivation might represent an important mechanism in the battle of man against malaria.155 Asymptomatic parasitemia has been associated with the existence of hypochromic anemia, in the absence of a prominent acute phase reaction. These patients exhibit higher serum hepcidin concentration, higher ferritin, lower iron and transferrin levels, and lower transferrin saturation, and consequently have impaired intestinal iron absorption and dietary iron utilization. On the other hand malaria commonly coexists with a background of frank iron deficiency. Antimalarial treatment partly restores low-grade inflammation and decreases serum hepcidin, ferritin, and other indeces of inflammation, and should be preceded of any effort for anemia correction with iron. Clearance of parasitemia increases dietary iron absorption but did not affect systemic iron utilization. Therefore, in areas of high prevalence of malaria, since asymptomatic parasitemia has a protracted course, careful clinical evaluation of anemic patients is mandatory, because the unjustified or mistimed iron supplementation will be ineffective and may even be hazardous and render malaria symptomatic.156, 157 Among pregnant women in areas with high malaria prevalence, malaria parasitemia, hookworm infection, gravidity and advanced gestational age were associated with lower hemoglobin and iron deficiency. Malaria parasitemia, *Ascaris lumbricodes* and *Trichuris trichiura* infections and older age were associated with lower serum ferritin levels.158

Intraerythrocytic malaria parasites digest hemoglobin to obtain the amino acids needed for their own protein synthesis. Hemoglobin degradation and total parasite protein content increase in parallel with parasite maturation, but the rate of hemoglobin degradation is higher, than the utilized amount of amino acids.159 Hemoglobin degradation yields also large quantities of ferriprotoporphyrin IX and iron, which create a highly oxidative erythrocyte environment and high requirements for detoxification. Redox-active iron released inside the erythrocyte, mediate the conversion of H2O2 to hydroxyl radical [HO] which is more reactive. Superoxide dismutase (SOD) and nitroxide SOD detoxifies the

Iron and Microbial Growth 313

Entamoeba histolytica trophozoites can grow in vitro within culture media, containing ferrous or ferric iron, and they can use hemoglobin, holotransferrin, hololactoferrin and ferritin as iron sources. Iron-binding proteins are specifically bound to the amoeba surface, are uptaken by endocytosis, traffick through the endosomal/lysosomal route and are degraded by neutral and acidic cysteine-proteases. Tf and ferritin are mainly uptaken as clathrin-coated vesicles. However, apolactoferrin bound to membrane lipids and cholesterol, induces cell death. In vivo trophozoites secrete products capable to destroy enterocytes, erythrocytes and hepatocytes, releasing Tf, hemoglobin, ferritin and other iron-containing proteins, which, together with Lf derived from neutrophils and acinar cells, can be used as iron supplies by amoebas.168 Many biological functions and pathogenicity of the free-living amoeba *Naegleria fowleri* are dependent on the composition of the culture medium. The ironcontaining porphyrins hemin or hematin or the iron-free protoporphyrin IX, can support *N.fowleri* growth in serum free media, whereas iron-binding proteins, including hemoglobin cannot.169 Some growth-promoting factors for *Entamoeba* species are low molecular weight substances, found in cellular fractions of various cells, and are probably siderophores, such

Hookworm infection has been associated with growth delay and iron deficiency anemia. In a mouse model of this disease, infected animals, fed with a standard diet exhibited significant growth delay and reduced hemoglobin levels, compared to uninfected controls, whereas no significant difference in weight or hemoglobin concentration was observed between infected and uninfected animals, fed with an iron-restricted diet. Moreover, ironrestricted animals exhibited reduced intestinal worm burden, compared to animals fed with the standard diet. Finally, infected animals fed with intermediate-iron containing diet exhibited greater weight loss and anemia, than animals fed with iron-restricted- or high-iron diets. Mortality was also higher in the intermediate-iron containing diet. Therefore, severe dietary iron restriction impairs hookworm development, but moderate iron restriction

The human blood fluke *Schistosoma japonicum* is responsible for significant morbidity and mortality in tropical areas. For this fluke and some other invertebrates, an additional role for iron has been postulated, and this concerns the stabilization of the extracellular matrix. *Schistosoma* requires iron for its development and stores abundant iron in the vitelline (eggshell-forming) cells of the female system, in the form of yolk ferritin that is upregulated in females and is also expressed at low levels in egg-stages and adult males. Iron concentrations have been found higher in the female- than the male adult parasite, but also in the parasite eggs and purified eggshell, whose matrix is composed of heavily cross-linked

As previously noted, iron is crucial for the growth and proliferation of all microorganisms, due to its role in mitochondrial respiration and DNA synthesis. Iron starvation and oxidative stress are the hurdles that bacteria must overcome to establish an infection. In some cases there is excess iron available and specific infections are more common. Iron overload may be secondary to lysis of red cells from free heme compounds, as a result of trauma and due to altered metabolism (hemochromatosis, hepatic disease or post

**9. Clinical considerations - infections in iron overloaded patients**

as ferredoxins and rubredoxin.170

eggshell precursor proteins.172

chemotherapy).

enhances host susceptibility to severe disease.171

erythrocyte and acts similarly to the antimalarial drug 4-OH,2,2,6,6,tetramethyl piperidine-N-oxyl (Tempol) in *P.falciparum* growth. Tempol inhibits parasite growth, and induces accelerated mortality in a SOD-overexpressing mouse model of malaria.160 SOD has therefore a protective role for the erythrocytes, and transgenic copper/zinc superoxide dismutase- - (CuZnSOD) mouse strains show higher sensitivity to infection by *Plasmodium berghei*. Moreover, treatment of infected erythrocytes, either SOD transgenic or normal, with oxidative stress inducers, reduces parasite viability. Therefore, CuZnSOD does not support plasmodium development, and impairment of its activity results in higher oxidative stress, favoring malaria growth.161

Iron deficiency modulates *Plasmodium yoelii* development in hepatocytes, by inactivating hepatic xanthine-oxidase. Iron-deficient mice infected with *Pl. yoelii* sporozoites, exhibited enhanced development of hepatic stage, resulting in the earlier appearance of blood parasites. An iron-starving diet increased penetration of sporozoites into liver cells, whereas inactivation of hepatic xanthine-oxidase inhibited both, sporozoite penetration and schizont maturation. Moreover, inhibition of heme synthesis also results in inhibition of parasite development.162 Another mechanism, favorably influencing the clinical course of *Pl.falciparum* infection in iron deficient subjects, is the faster clearance of infected erythrocytes. Iron deficiency accelerates unifected erythrocyte death and enhances death and removal of infected erythrocytes by phagocytosis, which is evident from phosphatidylserine exposure. Indeed, parasitized iron deficient erythrocytes are more susceptible to phagocytosis in vitro, than normal erythrocytes.163,164 The importance of iron in plasmodium growth has shifted antimalarial treatment strategies and research towards the identification and application of new drugs intervening with the parasite iron metabolism. More details on the topic are mentioned in the following paragraphs.

Trypanosomiasis or Chagas' disease has been associated with iron overload. *Trypanosoma* possesses a unique mechanism of adaptation and iron acquisition from the host environment. *Trypanosoma brucei* escapes destruction by the host immune system, by regularly replacing its *Variant Surface Glycoprotein* (VSG) coat. The VSG is expressed together with expression site associated genes, encoding the heterodimeric Tf receptor. There are about 20 VSG expression sites and trypanosomes can change the active site, according to environmental conditions. Since the various Tf receptor genes, localized in different expression sites, differ somewhat in sequence, expression site switching results in the production of a slightly different Tf receptor. Trypanosomes can adapt the expression site of its Tf receptor to achieve the highest affinity for the host Tf molecule.165

Hypochromic anemia is a dominant characteristic of this disease and its severity is correlated with the severity of trypanosomiasis. The parasite induces a strong type-I immune response, activating bone marrow and tissue macrophages and establishing an imbalance between erythropoiesis and erythrophagocytosis or erythroblastic apoptosis, which is the typical pathogenetic mechanism of anemia of chronic disease.166 In a murine model of trypanosomiasis, erythrophagocytosis by cytokine-activated M1 macrophages was the main initial cause of aggressive anemia during the acute phase of infection. Persistence of type I cytokine production in the chronic phase of infection perpetuates and deteriorates anemia. Meanwhile, iron homeostasis is perturbed and there is increased iron sequestration by macrophages, resulting after upregulation of Fp, Tf and ceruloplasmin genes, indicating that iron export is reduced. In the chronic phase of trypanosomiasis, iron sequestration worsens, while the enhanced uptake of iron-containing molecules is maintained.167

erythrocyte and acts similarly to the antimalarial drug 4-OH,2,2,6,6,tetramethyl piperidine-N-oxyl (Tempol) in *P.falciparum* growth. Tempol inhibits parasite growth, and induces accelerated mortality in a SOD-overexpressing mouse model of malaria.160 SOD has therefore a protective role for the erythrocytes, and transgenic copper/zinc superoxide dismutase- - (CuZnSOD) mouse strains show higher sensitivity to infection by *Plasmodium berghei*. Moreover, treatment of infected erythrocytes, either SOD transgenic or normal, with oxidative stress inducers, reduces parasite viability. Therefore, CuZnSOD does not support plasmodium development, and impairment of its activity results in higher oxidative stress,

Iron deficiency modulates *Plasmodium yoelii* development in hepatocytes, by inactivating hepatic xanthine-oxidase. Iron-deficient mice infected with *Pl. yoelii* sporozoites, exhibited enhanced development of hepatic stage, resulting in the earlier appearance of blood parasites. An iron-starving diet increased penetration of sporozoites into liver cells, whereas inactivation of hepatic xanthine-oxidase inhibited both, sporozoite penetration and schizont maturation. Moreover, inhibition of heme synthesis also results in inhibition of parasite development.162 Another mechanism, favorably influencing the clinical course of *Pl.falciparum* infection in iron deficient subjects, is the faster clearance of infected erythrocytes. Iron deficiency accelerates unifected erythrocyte death and enhances death and removal of infected erythrocytes by phagocytosis, which is evident from phosphatidylserine exposure. Indeed, parasitized iron deficient erythrocytes are more susceptible to phagocytosis in vitro, than normal erythrocytes.163,164 The importance of iron in plasmodium growth has shifted antimalarial treatment strategies and research towards the identification and application of new drugs intervening with the parasite iron

metabolism. More details on the topic are mentioned in the following paragraphs.

its Tf receptor to achieve the highest affinity for the host Tf molecule.165

Trypanosomiasis or Chagas' disease has been associated with iron overload. *Trypanosoma* possesses a unique mechanism of adaptation and iron acquisition from the host environment. *Trypanosoma brucei* escapes destruction by the host immune system, by regularly replacing its *Variant Surface Glycoprotein* (VSG) coat. The VSG is expressed together with expression site associated genes, encoding the heterodimeric Tf receptor. There are about 20 VSG expression sites and trypanosomes can change the active site, according to environmental conditions. Since the various Tf receptor genes, localized in different expression sites, differ somewhat in sequence, expression site switching results in the production of a slightly different Tf receptor. Trypanosomes can adapt the expression site of

Hypochromic anemia is a dominant characteristic of this disease and its severity is correlated with the severity of trypanosomiasis. The parasite induces a strong type-I immune response, activating bone marrow and tissue macrophages and establishing an imbalance between erythropoiesis and erythrophagocytosis or erythroblastic apoptosis, which is the typical pathogenetic mechanism of anemia of chronic disease.166 In a murine model of trypanosomiasis, erythrophagocytosis by cytokine-activated M1 macrophages was the main initial cause of aggressive anemia during the acute phase of infection. Persistence of type I cytokine production in the chronic phase of infection perpetuates and deteriorates anemia. Meanwhile, iron homeostasis is perturbed and there is increased iron sequestration by macrophages, resulting after upregulation of Fp, Tf and ceruloplasmin genes, indicating that iron export is reduced. In the chronic phase of trypanosomiasis, iron sequestration

worsens, while the enhanced uptake of iron-containing molecules is maintained.167

favoring malaria growth.161

Entamoeba histolytica trophozoites can grow in vitro within culture media, containing ferrous or ferric iron, and they can use hemoglobin, holotransferrin, hololactoferrin and ferritin as iron sources. Iron-binding proteins are specifically bound to the amoeba surface, are uptaken by endocytosis, traffick through the endosomal/lysosomal route and are degraded by neutral and acidic cysteine-proteases. Tf and ferritin are mainly uptaken as clathrin-coated vesicles. However, apolactoferrin bound to membrane lipids and cholesterol, induces cell death. In vivo trophozoites secrete products capable to destroy enterocytes, erythrocytes and hepatocytes, releasing Tf, hemoglobin, ferritin and other iron-containing proteins, which, together with Lf derived from neutrophils and acinar cells, can be used as iron supplies by amoebas.168 Many biological functions and pathogenicity of the free-living amoeba *Naegleria fowleri* are dependent on the composition of the culture medium. The ironcontaining porphyrins hemin or hematin or the iron-free protoporphyrin IX, can support *N.fowleri* growth in serum free media, whereas iron-binding proteins, including hemoglobin cannot.169 Some growth-promoting factors for *Entamoeba* species are low molecular weight substances, found in cellular fractions of various cells, and are probably siderophores, such as ferredoxins and rubredoxin.170

Hookworm infection has been associated with growth delay and iron deficiency anemia. In a mouse model of this disease, infected animals, fed with a standard diet exhibited significant growth delay and reduced hemoglobin levels, compared to uninfected controls, whereas no significant difference in weight or hemoglobin concentration was observed between infected and uninfected animals, fed with an iron-restricted diet. Moreover, ironrestricted animals exhibited reduced intestinal worm burden, compared to animals fed with the standard diet. Finally, infected animals fed with intermediate-iron containing diet exhibited greater weight loss and anemia, than animals fed with iron-restricted- or high-iron diets. Mortality was also higher in the intermediate-iron containing diet. Therefore, severe dietary iron restriction impairs hookworm development, but moderate iron restriction enhances host susceptibility to severe disease.171

The human blood fluke *Schistosoma japonicum* is responsible for significant morbidity and mortality in tropical areas. For this fluke and some other invertebrates, an additional role for iron has been postulated, and this concerns the stabilization of the extracellular matrix. *Schistosoma* requires iron for its development and stores abundant iron in the vitelline (eggshell-forming) cells of the female system, in the form of yolk ferritin that is upregulated in females and is also expressed at low levels in egg-stages and adult males. Iron concentrations have been found higher in the female- than the male adult parasite, but also in the parasite eggs and purified eggshell, whose matrix is composed of heavily cross-linked eggshell precursor proteins.172

#### **9. Clinical considerations - infections in iron overloaded patients**

As previously noted, iron is crucial for the growth and proliferation of all microorganisms, due to its role in mitochondrial respiration and DNA synthesis. Iron starvation and oxidative stress are the hurdles that bacteria must overcome to establish an infection. In some cases there is excess iron available and specific infections are more common. Iron overload may be secondary to lysis of red cells from free heme compounds, as a result of trauma and due to altered metabolism (hemochromatosis, hepatic disease or post chemotherapy).

Iron and Microbial Growth 315

more often when ferritin levels were >500 ng/ml.182 *Y. enterocolitica* has also been identified as a causative agent of posttransfusion septic shock. *Yersinia* bacteremias complicate the transfusions of blood that has been stored for more than 3 weeks.183,184 The highpathogenicity island (HPI), present in pathogenic *Yersinia* and encoding the siderophore yersiniabactin, has been found in *E. coli* pathotypes, responsible for bacteremias, neonatal

The spleen, an important part of the reticuloendothelial system, acts as a filter for circulating debris, including bacteria and as an important source of lymphoid cells and antibody production. Splenectomy may alter the ability to prevent or suppress some infections. There appears to be a high risk of severe bacterial infections when splenectomy is performed in patients with thalassaemia major, hepatitis, cirrhosis, histocytosis or inborn errors of metabolism.186,187 Seventy three patients with β-thalassemia/HbE were studied 1-28 years after splenectomy. Serum ferritin levels in both, HbH and β-thalassemia/Hb E patients were higher than normal. They were higher in β-thalassemia/HbE than HbH disease. Most striking was the significantly higher serum ferritin levels in splenectomized patients with βthalassemia/HbE disease than in the nonsplenectomized ones. After splenectomy, in patients with β-thalassemia/HbE disease, there was an increase of the Tf saturation in

Levels of serum iron are elevated in patients undergoing hematopoietic stem cell transplantation (HSCT), as a result of disturbed iron metabolism, pre-transplantation blood transfusions, or cytotoxic therapy, for conditioning before HSCT. The complications of iron overload in HSCT patients include bacterial and fungal infections, mucositis, chronic liver disease (fibrosis progression), sinusoidal obstruction syndrome, and other regimen-related toxicities. Iron overload can be considered as an independent adverse prognostic factor in allogeneic HSCT. Screeneing for iron overload at various time points before and after transplantation may be beneficial especially in patients with thalassemia and

Singh et al, assessed the role of hepatic explant iron overload as a risk factor for *Staph. aureus* bacteremia in liver transplant recipients. Noncarriers (patients without *S. aureus* nasal carriage) who developed *S. aureus* bacteremia were more likely to have hepatic iron overload. A quantifiable assessment of hepatic iron in patients without carriage at the time of transplantation can potentially identify those who may be at risk for early *S. aureus* bacteremia.190 In healthy humans the lower respiratory tract as well as all mucosa, contains a very low free iron concentration (10-18 M), while in cystic fibrosis (CF) patients, sputum iron concentration is very high, showing a median value of 63x10-6 M. Accumulation of catalytic reactive iron contributes to subsequent clinical complications in the lung disorders by the production of ROS and increases bacterial growth and virulence. The iron-overload of the sputum of CF patients induces nonmotile forms, aggregation and biofilm formation both in *Pseudomonas aeruginosa* and *Burkholderia cenocepacia* which are the main pathogens in these patients, facilitating the penetration of host epithelial barriers and contributing to the establishment of infection, colonization, persistence and systemic spread of these pathogens.191 In human plasma, a fall in Eh (oxidation-reduction potential) or pH results in the abolition or marked reduction of its bactericidal properties. This is highly relevant to infection after trauma, where a fall in Eh and pH frequently accompanies tissue damage. Hypoxia interferes with the oxidative killing of many bacteria by polymorphonuclear leukocytes. In

addition to increased circulating non-trasferrin bound iron.188

meningitis and urosepsis.185

myelodysplastic syndromes.189

In the presence of hemolytic disorders, caused by malaria or *Bartonella bacilliformis* (in cases of Oroya fever) *salmonella* infections are noted.173 The presence of free hemoglobin or heme may effectively impair or completely destroy the mechanism of natural resistance. Bullen et al. showed that ferric citrate, hematin hydrochloride, lysed guinea-pig red cells and crystalline human hemoglobin greatly enhanced *E. coli* virulence, when injected intraperitoneally into normal guinea-pigs.174

Blood transfusions may increase the free hemoglobin. Red blood cell transfusions should be used sparingly, keeping in mind the potential risks of infection and poor outcomes in critically ill patients. In a prospective, observational cohort study by Taylor et al. the posttransfusion nosocomial infection rate was 14.3% in 428 evaluable patients, significantly higher than that observed in nontransfused patients (5.8%; p <0.0001).175 In a multivariate analysis controlling for patient age, maximum storage age of red blood cells, and number of red blood cell transfusions, only the number of transfusions was independently associated with nosocomial infection (odds ratio 1.097; p=0.005). In addition mortality and length of stay (in intensive care unit and hospital) were significantly higher in transfused patients, even when corrected for illness severity.176 Secondary analysis of a multicentered, prospective observational study of transfusion practice in intensive care units in the United States showed that transfusion of packed red blood cells increases the risk of developing VAP (ventilator associated pneumonia). The effect of transfusion on late-onset VAP was more pronounced (odds ratio 2.16; 95% CI, 1.27-3.66) and demonstrated a positive doseresponse relationship.177 To determine whether blood transfusion influences infection after trauma, Agarwal et al. analyzed data on 5366 consecutive patients, hospitalized for more than 2 days. Even when patients were stratified by Injury Severity Score, the infection rate increased significantly with the higher numbers of transfused blood units. Blood transfusion in the injured patients is an important independent statistical predictor of infection. Its contribution cannot be attributed to age, sex, or the underlying mechanism of severity of injury.178 Both, modified and native human hemoglobin may promote infection.179 They showed that pyridoxalated polymerised human hemoglobin promotes fulminant *E. coli* septicemia in mice, which draws attention to the potential danger of such products in the clinic.

Hereditary hemochromatosis is the prototype disease for primary iron overload. *Vibrio vulnificus* has been linked to primary sepsis, which usually occurs in patients with underlying liver disease (cirrhosis or hemochromatosis).180 Although this pathogen can be destroyed by human plasma, it multiplies rapidly when free iron is available. After eating raw sea food, like oysters, the patient develops high fever, prostration, hypotension and in most cases characteristic cutaneous manifestations (initially erythematous patches followed by ecchymoses, vesicles and bullae) with a mortality rate up to 50% without the prompt therapy. Primary hemochromatosis was the commonest underlying disease in patients with liver abscesses caused by *Yersinia enterocolitica*.181 Some *Yersinia* strains are unable to synthesize siderophores but they can exploit host-chelated iron stores and the drug DFO. As a result, iron overload appears to be independent risk factor for *Y. enterocolitica* bacteremia, mainly by the serotypes O:3 and O:9. *Yersinia* bacteremia must be considered as an indicator of possible iron overload and *Yersinia* infection must be suspected in febrile hemochromatosic patients. In the past, patients with chronic renal failure, undergoing dialysis received multiple transfusions and frequent parenteral iron preparations. In a study by Boelaer *Yersinia* bacteremias (*Y. enterocolitica* and *Y. pseudotuberculosis*) were detected

In the presence of hemolytic disorders, caused by malaria or *Bartonella bacilliformis* (in cases of Oroya fever) *salmonella* infections are noted.173 The presence of free hemoglobin or heme may effectively impair or completely destroy the mechanism of natural resistance. Bullen et al. showed that ferric citrate, hematin hydrochloride, lysed guinea-pig red cells and crystalline human hemoglobin greatly enhanced *E. coli* virulence, when injected

Blood transfusions may increase the free hemoglobin. Red blood cell transfusions should be used sparingly, keeping in mind the potential risks of infection and poor outcomes in critically ill patients. In a prospective, observational cohort study by Taylor et al. the posttransfusion nosocomial infection rate was 14.3% in 428 evaluable patients, significantly higher than that observed in nontransfused patients (5.8%; p <0.0001).175 In a multivariate analysis controlling for patient age, maximum storage age of red blood cells, and number of red blood cell transfusions, only the number of transfusions was independently associated with nosocomial infection (odds ratio 1.097; p=0.005). In addition mortality and length of stay (in intensive care unit and hospital) were significantly higher in transfused patients, even when corrected for illness severity.176 Secondary analysis of a multicentered, prospective observational study of transfusion practice in intensive care units in the United States showed that transfusion of packed red blood cells increases the risk of developing VAP (ventilator associated pneumonia). The effect of transfusion on late-onset VAP was more pronounced (odds ratio 2.16; 95% CI, 1.27-3.66) and demonstrated a positive doseresponse relationship.177 To determine whether blood transfusion influences infection after trauma, Agarwal et al. analyzed data on 5366 consecutive patients, hospitalized for more than 2 days. Even when patients were stratified by Injury Severity Score, the infection rate increased significantly with the higher numbers of transfused blood units. Blood transfusion in the injured patients is an important independent statistical predictor of infection. Its contribution cannot be attributed to age, sex, or the underlying mechanism of severity of injury.178 Both, modified and native human hemoglobin may promote infection.179 They showed that pyridoxalated polymerised human hemoglobin promotes fulminant *E. coli* septicemia in mice, which draws attention to the potential danger of such products in the

Hereditary hemochromatosis is the prototype disease for primary iron overload. *Vibrio vulnificus* has been linked to primary sepsis, which usually occurs in patients with underlying liver disease (cirrhosis or hemochromatosis).180 Although this pathogen can be destroyed by human plasma, it multiplies rapidly when free iron is available. After eating raw sea food, like oysters, the patient develops high fever, prostration, hypotension and in most cases characteristic cutaneous manifestations (initially erythematous patches followed by ecchymoses, vesicles and bullae) with a mortality rate up to 50% without the prompt therapy. Primary hemochromatosis was the commonest underlying disease in patients with liver abscesses caused by *Yersinia enterocolitica*.181 Some *Yersinia* strains are unable to synthesize siderophores but they can exploit host-chelated iron stores and the drug DFO. As a result, iron overload appears to be independent risk factor for *Y. enterocolitica* bacteremia, mainly by the serotypes O:3 and O:9. *Yersinia* bacteremia must be considered as an indicator of possible iron overload and *Yersinia* infection must be suspected in febrile hemochromatosic patients. In the past, patients with chronic renal failure, undergoing dialysis received multiple transfusions and frequent parenteral iron preparations. In a study by Boelaer *Yersinia* bacteremias (*Y. enterocolitica* and *Y. pseudotuberculosis*) were detected

intraperitoneally into normal guinea-pigs.174

clinic.

more often when ferritin levels were >500 ng/ml.182 *Y. enterocolitica* has also been identified as a causative agent of posttransfusion septic shock. *Yersinia* bacteremias complicate the transfusions of blood that has been stored for more than 3 weeks.183,184 The highpathogenicity island (HPI), present in pathogenic *Yersinia* and encoding the siderophore yersiniabactin, has been found in *E. coli* pathotypes, responsible for bacteremias, neonatal meningitis and urosepsis.185

The spleen, an important part of the reticuloendothelial system, acts as a filter for circulating debris, including bacteria and as an important source of lymphoid cells and antibody production. Splenectomy may alter the ability to prevent or suppress some infections. There appears to be a high risk of severe bacterial infections when splenectomy is performed in patients with thalassaemia major, hepatitis, cirrhosis, histocytosis or inborn errors of metabolism.186,187 Seventy three patients with β-thalassemia/HbE were studied 1-28 years after splenectomy. Serum ferritin levels in both, HbH and β-thalassemia/Hb E patients were higher than normal. They were higher in β-thalassemia/HbE than HbH disease. Most striking was the significantly higher serum ferritin levels in splenectomized patients with βthalassemia/HbE disease than in the nonsplenectomized ones. After splenectomy, in patients with β-thalassemia/HbE disease, there was an increase of the Tf saturation in addition to increased circulating non-trasferrin bound iron.188

Levels of serum iron are elevated in patients undergoing hematopoietic stem cell transplantation (HSCT), as a result of disturbed iron metabolism, pre-transplantation blood transfusions, or cytotoxic therapy, for conditioning before HSCT. The complications of iron overload in HSCT patients include bacterial and fungal infections, mucositis, chronic liver disease (fibrosis progression), sinusoidal obstruction syndrome, and other regimen-related toxicities. Iron overload can be considered as an independent adverse prognostic factor in allogeneic HSCT. Screeneing for iron overload at various time points before and after transplantation may be beneficial especially in patients with thalassemia and myelodysplastic syndromes.189

Singh et al, assessed the role of hepatic explant iron overload as a risk factor for *Staph. aureus* bacteremia in liver transplant recipients. Noncarriers (patients without *S. aureus* nasal carriage) who developed *S. aureus* bacteremia were more likely to have hepatic iron overload. A quantifiable assessment of hepatic iron in patients without carriage at the time of transplantation can potentially identify those who may be at risk for early *S. aureus* bacteremia.190 In healthy humans the lower respiratory tract as well as all mucosa, contains a very low free iron concentration (10-18 M), while in cystic fibrosis (CF) patients, sputum iron concentration is very high, showing a median value of 63x10-6 M. Accumulation of catalytic reactive iron contributes to subsequent clinical complications in the lung disorders by the production of ROS and increases bacterial growth and virulence. The iron-overload of the sputum of CF patients induces nonmotile forms, aggregation and biofilm formation both in *Pseudomonas aeruginosa* and *Burkholderia cenocepacia* which are the main pathogens in these patients, facilitating the penetration of host epithelial barriers and contributing to the establishment of infection, colonization, persistence and systemic spread of these pathogens.191

In human plasma, a fall in Eh (oxidation-reduction potential) or pH results in the abolition or marked reduction of its bactericidal properties. This is highly relevant to infection after trauma, where a fall in Eh and pH frequently accompanies tissue damage. Hypoxia interferes with the oxidative killing of many bacteria by polymorphonuclear leukocytes. In

Iron and Microbial Growth 317

The use of iron chelators as adjuvant treatment in systemic zygomycosis and other mycoses appears to be rational, and has been shown to be effective in sporadic cases. Reed et al. reported a case of a 40-year-old diabetic patient with aggressive rhinocerebral zygomycosis and progressive central nervous system involvement, despite combination treatment with high-dose LAmB plus caspofungin and surgical debridement. As brain magnetic resonance imaging (MRI) showed new parenchymal lesions and left cavernous sinus thrombosis, he was given a 7-day salvage treatment with DFX 1000 mg daily. A new brain MRI scan showed significant improvement, and treatment with LAmB was discontinued. The patient, 4 months later, remained in good condition without any neurological deficit. This is the first reported case of zygomycosis being successfully treated with a combination of classical antifungal treatment and an iron chelator.194 We have recently treated two patients with acute lymphoblastic leukemia in remission (one of them following allogeneic transplantation) with zygomycosis (one with concurrent rhinocerebral and pulmonary form, the second with classical rhinocerebral form) with a combination of LAmB 10 mg/kg, posaconazole and DFX 20 mg/kg daily. Restricted intranasal and intrasinus surgical debridement was also applied repeatedly. Both patients responded very well, with rapid defervescence, resolution of pain and chymosis, and disappearance of the dense pulmonary and sinonasal infiltrates (unpublished data). Some more published cases have also shown encouraging results195 however in other cases, iron chelation treatment was unsuccessful.196 Therefore, the possible benefit of iron chelation as adjuvant treatment in systemic mycoses, and particularly in zygomycosis, had to be tested in a prospective randomized trial. Such a clinical trial, the DEFEAT mucor study, investigated the existence of synergy between the classical treatment plus or minus DFX. Twenty patients with proven or probable zygomycosis were randomized to receive LAmB plus DFX (20 mg/kg/day for 14 days) or LAmB plus placebo. Surprisingly, death was more frequent in the DFX than in the placebo arm and global success was worse for the DFX arm, since patients of this arm had higher mortality rate at 90 days. This was attributed to population imbalances between the two

arms, and therefore, make generalizable conclusions cannot be drawn.197

as a curative treatment.198

(P=0.001).200

DFO, although is a xenosiderophore for *Zygomycetes*, may have direct and irreversible toxic effects on *P.carinii,* independently of iron chelation. This direct and irreversible damage of *P.carinii* by DFO was confirmed in vivo, in an animal model, in which a once-a-week aerosol treatment of PCP with DFO was effective in 100% of the animals, both as a prophylactic and

All the available iron chelators can inhibit the growth of malaria parasites. Using a flow cytometric method for testing in vitro drug susceptibility of *Pl.falciparum* to hydroxypyridinone derivatives and to DFO, it has been found that both classes of chelators exhibited dose-dependent inhibition of parasite growth, but DFO demonstrated a stronger inhibitory effect. The MIC required for the parasite growth, correlated with observed abnormal microscopic morphology, and sensitivity to iron chelators was shown for both, chloroquine- and pyrimethamine-resistant parasites.199 In another study, comparing the efficacy of DFO and DFX at 30 μM/l or 60 μΜ/l, added in cultures of *Pl. falciparum* in human erythrocytes, it was observed that DFX had marked antimalarial activity by 6 h after exposure, and over 48 h of culture, and although the IC50s were similar for DFX and DFO, malarial growth was significantly lower with DFX than with DFO at both concentrations

addition it produces a fall in tissue Eh and as a result free ferrous iron is produced, leading to overwhelming growth of bacteria. If the Eh is lowered, the ferric iron is reduced to the ferrous form, no longer bound to Tf.

The bactericidal power of fresh human plasma against *Klebsiella pneumoniae* and *E. coli* is extremely sensitive to changes in Eh and pH. At a high Eh (approx. +200 mV) the bacteria were destroyed, but rapid regrowth was observed when the Eh was lowered to -400 mV. Abolition of the bactericidal effect was also produced by adding ferric iron at a high Eh (approx. +200 mV). Lowering the pH to 6.5 reduced or prevented the bactericidal effect. Rising the Eh from –400 to +200 mV restored the bactericidal effect.192 Some bacteria like *Cl. Perfringens* or *E. coli* have developed reducing systems. They may take advantage of a reduction in skin Eh and they are capable of lowering the Eh of tissue fluids to a level where Fe2+ is freely available. These results are probably related to the availability of iron for bacterial growth, and could be important for understanding the development of infection in injured or diseased tissue.Iron supplementation to treat anemia is controversial, since it may promote the progression of the underlying infectious disease but existing data are insufficient to support this hypothesis.193

#### **10. Iron chelators as adjuvant treatment in systemic fungal and protozoan infections**

It is self-evident if we take into consideration all the above, that an important weapon in the war against the various infectious microorganisms might be iron deprivation. Many efforts have been performed for this task, in targeting the appropriate microbial pathway, identifying the ideal compound for each microorganism, evaluating its efficacy, confirming its safety and testing its clinical usefulness. The usually acute clinical course of bacterial infections, the abundance of antibiotics and the relative satisfactory handling has restricted research programs testing iron chelators for fungal and protozoan infections and infestations.

Among fungal infections the most challenging is zygomycosis, for which effective treatment is still unavailable. Many studies, elaborating animal models for this disease have tried to address the efficacy of iron chelating agents, against this mycosis. In a mouse model of zygomycosis, animals were infected with *R. oryzae* spores, and 24 h later were treated with DFP at dose levels of 50, 100 or 200 mg/kg every day or every other day. The dose of 100 mg/kg every other day resulted in a significant survival advantage of DFP-treated mice, as compared with placebo-treated animals. The other dose schedules were either ineffective or toxic. The survival advantage was comparable to, although lower than, that of Liposomal Amphotericin-B (LAmB)-treated mice. Both drugs significantly reduced the brain fungal burden as compared with placebo. The beneficial effect of DFP was abrogated when animals were given ferric chloride.133 In a similar mouse model of established zygomycosis, the administration of DFX was associated with comparable efficacy to that of LAmB. DFX has shown efficacy in neutropenic and diabetic ketoacidotic mice with zygomycosis. In these experiments, DFX at a daily dose of 20 mg/kg, starting 24 h after infection was synergistic with LAmB at a high-dose schedule of 15 mg/kg daily, in the reduction of fungal burden from the brain and the kidney. Moreover, the combination of the two drugs significantly improved survival time as compared with placebo or each drug separately.134 Similar results have been obtained with the combination treatment in a mouse model of aspergillosis.135

addition it produces a fall in tissue Eh and as a result free ferrous iron is produced, leading to overwhelming growth of bacteria. If the Eh is lowered, the ferric iron is reduced to the

The bactericidal power of fresh human plasma against *Klebsiella pneumoniae* and *E. coli* is extremely sensitive to changes in Eh and pH. At a high Eh (approx. +200 mV) the bacteria were destroyed, but rapid regrowth was observed when the Eh was lowered to -400 mV. Abolition of the bactericidal effect was also produced by adding ferric iron at a high Eh (approx. +200 mV). Lowering the pH to 6.5 reduced or prevented the bactericidal effect. Rising the Eh from –400 to +200 mV restored the bactericidal effect.192 Some bacteria like *Cl. Perfringens* or *E. coli* have developed reducing systems. They may take advantage of a reduction in skin Eh and they are capable of lowering the Eh of tissue fluids to a level where Fe2+ is freely available. These results are probably related to the availability of iron for bacterial growth, and could be important for understanding the development of infection in injured or diseased tissue.Iron supplementation to treat anemia is controversial, since it may promote the progression of the underlying infectious disease but existing data are

**10. Iron chelators as adjuvant treatment in systemic fungal and protozoan**

It is self-evident if we take into consideration all the above, that an important weapon in the war against the various infectious microorganisms might be iron deprivation. Many efforts have been performed for this task, in targeting the appropriate microbial pathway, identifying the ideal compound for each microorganism, evaluating its efficacy, confirming its safety and testing its clinical usefulness. The usually acute clinical course of bacterial infections, the abundance of antibiotics and the relative satisfactory handling has restricted research programs testing iron chelators for fungal and protozoan infections and

Among fungal infections the most challenging is zygomycosis, for which effective treatment is still unavailable. Many studies, elaborating animal models for this disease have tried to address the efficacy of iron chelating agents, against this mycosis. In a mouse model of zygomycosis, animals were infected with *R. oryzae* spores, and 24 h later were treated with DFP at dose levels of 50, 100 or 200 mg/kg every day or every other day. The dose of 100 mg/kg every other day resulted in a significant survival advantage of DFP-treated mice, as compared with placebo-treated animals. The other dose schedules were either ineffective or toxic. The survival advantage was comparable to, although lower than, that of Liposomal Amphotericin-B (LAmB)-treated mice. Both drugs significantly reduced the brain fungal burden as compared with placebo. The beneficial effect of DFP was abrogated when animals were given ferric chloride.133 In a similar mouse model of established zygomycosis, the administration of DFX was associated with comparable efficacy to that of LAmB. DFX has shown efficacy in neutropenic and diabetic ketoacidotic mice with zygomycosis. In these experiments, DFX at a daily dose of 20 mg/kg, starting 24 h after infection was synergistic with LAmB at a high-dose schedule of 15 mg/kg daily, in the reduction of fungal burden from the brain and the kidney. Moreover, the combination of the two drugs significantly improved survival time as compared with placebo or each drug separately.134 Similar results have been obtained with the combination treatment in a mouse model of aspergillosis.135

ferrous form, no longer bound to Tf.

insufficient to support this hypothesis.193

**infections**

infestations.

The use of iron chelators as adjuvant treatment in systemic zygomycosis and other mycoses appears to be rational, and has been shown to be effective in sporadic cases. Reed et al. reported a case of a 40-year-old diabetic patient with aggressive rhinocerebral zygomycosis and progressive central nervous system involvement, despite combination treatment with high-dose LAmB plus caspofungin and surgical debridement. As brain magnetic resonance imaging (MRI) showed new parenchymal lesions and left cavernous sinus thrombosis, he was given a 7-day salvage treatment with DFX 1000 mg daily. A new brain MRI scan showed significant improvement, and treatment with LAmB was discontinued. The patient, 4 months later, remained in good condition without any neurological deficit. This is the first reported case of zygomycosis being successfully treated with a combination of classical antifungal treatment and an iron chelator.194 We have recently treated two patients with acute lymphoblastic leukemia in remission (one of them following allogeneic transplantation) with zygomycosis (one with concurrent rhinocerebral and pulmonary form, the second with classical rhinocerebral form) with a combination of LAmB 10 mg/kg, posaconazole and DFX 20 mg/kg daily. Restricted intranasal and intrasinus surgical debridement was also applied repeatedly. Both patients responded very well, with rapid defervescence, resolution of pain and chymosis, and disappearance of the dense pulmonary and sinonasal infiltrates (unpublished data). Some more published cases have also shown encouraging results195 however in other cases, iron chelation treatment was unsuccessful.196

Therefore, the possible benefit of iron chelation as adjuvant treatment in systemic mycoses, and particularly in zygomycosis, had to be tested in a prospective randomized trial. Such a clinical trial, the DEFEAT mucor study, investigated the existence of synergy between the classical treatment plus or minus DFX. Twenty patients with proven or probable zygomycosis were randomized to receive LAmB plus DFX (20 mg/kg/day for 14 days) or LAmB plus placebo. Surprisingly, death was more frequent in the DFX than in the placebo arm and global success was worse for the DFX arm, since patients of this arm had higher mortality rate at 90 days. This was attributed to population imbalances between the two arms, and therefore, make generalizable conclusions cannot be drawn.197

DFO, although is a xenosiderophore for *Zygomycetes*, may have direct and irreversible toxic effects on *P.carinii,* independently of iron chelation. This direct and irreversible damage of *P.carinii* by DFO was confirmed in vivo, in an animal model, in which a once-a-week aerosol treatment of PCP with DFO was effective in 100% of the animals, both as a prophylactic and as a curative treatment.198

All the available iron chelators can inhibit the growth of malaria parasites. Using a flow cytometric method for testing in vitro drug susceptibility of *Pl.falciparum* to hydroxypyridinone derivatives and to DFO, it has been found that both classes of chelators exhibited dose-dependent inhibition of parasite growth, but DFO demonstrated a stronger inhibitory effect. The MIC required for the parasite growth, correlated with observed abnormal microscopic morphology, and sensitivity to iron chelators was shown for both, chloroquine- and pyrimethamine-resistant parasites.199 In another study, comparing the efficacy of DFO and DFX at 30 μM/l or 60 μΜ/l, added in cultures of *Pl. falciparum* in human erythrocytes, it was observed that DFX had marked antimalarial activity by 6 h after exposure, and over 48 h of culture, and although the IC50s were similar for DFX and DFO, malarial growth was significantly lower with DFX than with DFO at both concentrations (P=0.001).200

Iron and Microbial Growth 319

[2] Weiss G. Modification of iron regulation by the inflammatory response. Best Pract Res

[3] Aisen P, Enns C, Wessling-Resnick M. (2001) Chemistry and biology of eukaryotic iron

[4] Imlay JA, Chin SM, Linn S. Toxic DNA damage by hydrogen peroxide through the

[5] Gazitt Y, Reddy SV, Alcantara O, Yang J, Boldt DH. A new molecular role for iron in

[6] Alcantara O, Kalidas M, Baltathakis I, Boldt DH. Expression of multiple genes regulating

[7] Dong Fu, Richardson DR. Iron chelation and regulation of the cell cycle: 2 mechanisms of

[8] Kehrer JP. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology 2000;

[9] Shaw GC, Cope JJ, Li L, Corson K, Hersey C, Ackermann GE et al. Mitoferrin is essential

[10] Hentze MW, Muckenthaler MU, Andrews NC. Balancing acts: Molecular control of

[11] Hentze MW, Kuhn LC. Molecular control of vertebrate iron metabolism: mRNA-based

[12] Napier I, Ponka P, Richardson DR. Iron trafficking in the mitochondrion: Novel

[13] Renton FJ, Jeitner TM. Cell cycle-dependent inhibition of the proliferation of human neural tumor cell lines by iron chelators. Biochem Pharmacol 1996; 51:1553-61. [14] Gao J, Richardson DR. The potential of iron chelators of the pyridoxal isonicotinoyl

[15] Darnell G, Richardson DR. The potential of analogues of the pyridoxal isonicotinoyl

[18] Wang G, Miskimins R, Miskimins WK. Regulation of p27(Kip1) by intracellular iron

[19] Lee SH, Pyo CW, Hahm DH, Kim J, Choi SY. Iron-saturated lactoferrin stimulates cell cycle progression through PI3K/Akt pathway. Mol Cells. 2009; 28(1):37-42. [20] Le NT, Richardson DR. The role of iron in cell cycle progression and the proliferation

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regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl

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149(1):43-50.

Dexrazoxane is an iron chelating prodrug, used for the protection of anthracyclin-induced cardiotoxicity, which must undergo intracellular hydrolysis to bind iron. Investigating the antimalarial properties of dexrazoxane on *Pl. falciparum* cultured in human erythrocytes, and on *P.yoelii* cultured in mouse hepatocytes, it was found that dexrazoxane inhibited *P. falciparum* growth, only at suprapharmacologic concentrations. In contrast, pharmacologic concentrations of dexrazoxane inhibited *P.yoelli* growth by 45-69%, implying the presence of a dexrazoxane-hydrolyzing enzyme in hepatocytes but not in erythrocytes or malaria parasites.201 Novel aroylhydrazone and thiosemicarbazone iron chelators exhibit strong inhibitory activity on cultured tumor cells. These compounds were tested as antimalarials on chloroquine-sensitive- and -resistant strains of *Pl. falciparum*, and were significantly more active in both strains than DFO. The anti-malarial activity correlated with anti-proliferative activity against neoplastic cells. This class of lipophilic chelators may be potentially useful agents as anti-malarials.202 Among various other synthetic siderophores the most promising profile (low MIC for plasmodia and minimum toxicity to mammalian cells) was demonstrated by an acylated monocatecholate or a triscatecholate as substituent.203 To examine the site of action of antimalarial iron chelators, Loyevsky et al. have shown that specific fluorescence indicating the presence of iron chelators was observed within the parasites, implying that iron chelators bind labile iron within the plasmodium.204

The antimalarial activity of zinc-desferrioxamine (Zn-DFO) was found to be superior to that of DFO in vitro. A possible explanation is that the complex Zn-DFO might be more easily permeable into parasitized erythrocytes, exchange zinc for ferric ions due to higher affinity and deprive iron from the parasite. Parasites treated with Zn-DFO were less likely to recover at a later stage, in comparison to parasites treated with DFO, therefore, the complex Zn-DFO, which is more effective in vitro, should be examined for its in vivo activity.205

Many iron chelators are very effective in the treatment of trypanosomiasis and almost as effective as benznidazole, the classical drug used for the treatment of this disease. Some of them inhibit *T.Cruzi* growth at very low concentrations, thanks to their ability to interfere with and disrupt essential steps of epimastigote iron, copper or zinc metabolism at intracellular sites.206 Eleven out of 13 other iron chelators inhibited trypanosoma growth in vitro, but many of these chelators were also cytotoxic for human HL-60 cells and therefore were not further tested. Newer, more specific, lipophilic iron-chelators may serve as lead compounds for novel anti-trypanosomal drug development.207 Bloodstream forms of *T.brucei* are 10 times more sensitive than mammalian cells to iron depletion, and treatment with DFO inhibits parasite proliferation, inducing inhibition of DNA synthesis and decrease in oxygen consumption, findings implying that DFO impairs ribonucleotide reductase and alternative oxidase activity, apparently by chelating cellular iron and preventing its incorporation into the newly synthesized apoproteins. DFO treatment for 24 h has no effect on superoxide dismutase activity.208 Three compounds of an aminothiol family of iron chelators were tested against *Trypanosoma Cruzi*. BAT-TE completely arrested the growth of trypomastigote forms in mouse blood, while BAT-TM arrested growth in *T.cruzi*-infected mice. These results render BAT derivatives potential candidates for the clearing of donated blood from trypomastigotes in endemic areas.209

#### **11. References**

[1] Thelander L, Graslund A, Thelander M. Continual presence of oxygen and iron required for mammalian ribonucleotide reduction: Possible regulation mechanism. Biochem Biophys Res Commun 1983; 110:859-865.

Dexrazoxane is an iron chelating prodrug, used for the protection of anthracyclin-induced cardiotoxicity, which must undergo intracellular hydrolysis to bind iron. Investigating the antimalarial properties of dexrazoxane on *Pl. falciparum* cultured in human erythrocytes, and on *P.yoelii* cultured in mouse hepatocytes, it was found that dexrazoxane inhibited *P. falciparum* growth, only at suprapharmacologic concentrations. In contrast, pharmacologic concentrations of dexrazoxane inhibited *P.yoelli* growth by 45-69%, implying the presence of a dexrazoxane-hydrolyzing enzyme in hepatocytes but not in erythrocytes or malaria parasites.201 Novel aroylhydrazone and thiosemicarbazone iron chelators exhibit strong inhibitory activity on cultured tumor cells. These compounds were tested as antimalarials on chloroquine-sensitive- and -resistant strains of *Pl. falciparum*, and were significantly more active in both strains than DFO. The anti-malarial activity correlated with anti-proliferative activity against neoplastic cells. This class of lipophilic chelators may be potentially useful agents as anti-malarials.202 Among various other synthetic siderophores the most promising profile (low MIC for plasmodia and minimum toxicity to mammalian cells) was demonstrated by an acylated monocatecholate or a triscatecholate as substituent.203 To examine the site of action of antimalarial iron chelators, Loyevsky et al. have shown that specific fluorescence indicating the presence of iron chelators was observed within the

parasites, implying that iron chelators bind labile iron within the plasmodium.204

blood from trypomastigotes in endemic areas.209

Biophys Res Commun 1983; 110:859-865.

**11. References**

The antimalarial activity of zinc-desferrioxamine (Zn-DFO) was found to be superior to that of DFO in vitro. A possible explanation is that the complex Zn-DFO might be more easily permeable into parasitized erythrocytes, exchange zinc for ferric ions due to higher affinity and deprive iron from the parasite. Parasites treated with Zn-DFO were less likely to recover at a later stage, in comparison to parasites treated with DFO, therefore, the complex Zn-DFO, which is more effective in vitro, should be examined for its in vivo activity.205

Many iron chelators are very effective in the treatment of trypanosomiasis and almost as effective as benznidazole, the classical drug used for the treatment of this disease. Some of them inhibit *T.Cruzi* growth at very low concentrations, thanks to their ability to interfere with and disrupt essential steps of epimastigote iron, copper or zinc metabolism at intracellular sites.206 Eleven out of 13 other iron chelators inhibited trypanosoma growth in vitro, but many of these chelators were also cytotoxic for human HL-60 cells and therefore were not further tested. Newer, more specific, lipophilic iron-chelators may serve as lead compounds for novel anti-trypanosomal drug development.207 Bloodstream forms of *T.brucei* are 10 times more sensitive than mammalian cells to iron depletion, and treatment with DFO inhibits parasite proliferation, inducing inhibition of DNA synthesis and decrease in oxygen consumption, findings implying that DFO impairs ribonucleotide reductase and alternative oxidase activity, apparently by chelating cellular iron and preventing its incorporation into the newly synthesized apoproteins. DFO treatment for 24 h has no effect on superoxide dismutase activity.208 Three compounds of an aminothiol family of iron chelators were tested against *Trypanosoma Cruzi*. BAT-TE completely arrested the growth of trypomastigote forms in mouse blood, while BAT-TM arrested growth in *T.cruzi*-infected mice. These results render BAT derivatives potential candidates for the clearing of donated

[1] Thelander L, Graslund A, Thelander M. Continual presence of oxygen and iron required

for mammalian ribonucleotide reduction: Possible regulation mechanism. Biochem

Iron and Microbial Growth 321

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**Part 4**

**Molecular Epidemiology and Mitigation Strategy**

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## **Part 4**

## **Molecular Epidemiology and Mitigation Strategy**

330 Insight and Control of Infectious Disease in Global Scenario

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bloodstream forms of Trypanosoma brucei by the iron chelator deferoxamine. Int J

**17**

*1UK 2USA 3Scotland 4Canada 5Germany*

**Diphtheria Disease and Genes Involved in**

Shanow Uthman1, Shihui Liu2, Flaviano Giorgini1, Michael J. R. Stark3,

Within shared ecological niches, secretion of lethal protein toxins by microorganisms is a common strategy to ensure positive selection and survival of the toxin producing killer strains amongst other microbial competitors including bacteria, yeast and fungi (Schmitt & Schaffrath, 2005). Frequently, toxin production and secretion are traits that are genetically associated with extrachromosomal elements such as linear DNA killer plasmids and doublestranded RNA mycoviruses from yeast and fungi or episomal and prophage DNA integrated into the chromosome of the microbial toxin producer (reviewed by Meinhardt et al., 1997; Schaffrath & Meinhardt, 2005; Leis et al., 2005). The latter scenario is predominantly found in bacterial species and accounts for lysogenic conversions, phenomena in which phenotypes of the microbial host including pathogenicity, growth performance and production of virulence factors and toxins can be significantly affected by expression of prophage encoded genes. Prominent examples for phage-dependent toxin expression and disease formation include exotoxin-associated scarlet fever by *Streptocoocus pyogenes* (Johnson et al., 1986; Broudy et al., 2001), dysentery causing Shiga toxins Stx1 and Stx2 from *Shigella dysenteriae* (Newland et al., 1985; Willshaw et al., 1985; Huang, et al., 1986), phage CTXφ encoded cholera toxin from *Vibrio cholera* (Waldor & Mekalanos, 1996; Faruque & Nair, 2002) and diphtheria toxin encoded on a beta prophage from lysogens of *Corynebacterium diphtheriae* (Holmes & Barksdale, 1969; Bishai & Murphy 1988)*.* Together

**1. Introduction**

**Formation of Diphthamide, Key Effector**

**of the Diphtheria Toxin**

*University of Dundee,*

 *Universität Kassel, Kassel,*

*5Institut für Biologie, Mikrobiologie,*

Michael Costanzo4 and Raffael Schaffrath1,5

*1Department of Genetics, University of Leicester, Leicester, 2Laboratory of Bacterial Diseases, NIAID, NIH, Bethesda, MD, 3Wellcome Trust Centre for Gene Regulation & Expression,*

*4The Donnelly Centre, University of Toronto, Toronto, Ontario,*

## **Diphtheria Disease and Genes Involved in Formation of Diphthamide, Key Effector of the Diphtheria Toxin**

Shanow Uthman1, Shihui Liu2, Flaviano Giorgini1, Michael J. R. Stark3, Michael Costanzo4 and Raffael Schaffrath1,5 *1Department of Genetics, University of Leicester, Leicester, 2Laboratory of Bacterial Diseases, NIAID, NIH, Bethesda, MD, 3Wellcome Trust Centre for Gene Regulation & Expression, University of Dundee, 4The Donnelly Centre, University of Toronto, Toronto, Ontario, 5Institut für Biologie, Mikrobiologie, Universität Kassel, Kassel, 1UK 2USA 3Scotland 4Canada 5Germany*

#### **1. Introduction**

Within shared ecological niches, secretion of lethal protein toxins by microorganisms is a common strategy to ensure positive selection and survival of the toxin producing killer strains amongst other microbial competitors including bacteria, yeast and fungi (Schmitt & Schaffrath, 2005). Frequently, toxin production and secretion are traits that are genetically associated with extrachromosomal elements such as linear DNA killer plasmids and doublestranded RNA mycoviruses from yeast and fungi or episomal and prophage DNA integrated into the chromosome of the microbial toxin producer (reviewed by Meinhardt et al., 1997; Schaffrath & Meinhardt, 2005; Leis et al., 2005). The latter scenario is predominantly found in bacterial species and accounts for lysogenic conversions, phenomena in which phenotypes of the microbial host including pathogenicity, growth performance and production of virulence factors and toxins can be significantly affected by expression of prophage encoded genes. Prominent examples for phage-dependent toxin expression and disease formation include exotoxin-associated scarlet fever by *Streptocoocus pyogenes* (Johnson et al., 1986; Broudy et al., 2001), dysentery causing Shiga toxins Stx1 and Stx2 from *Shigella dysenteriae* (Newland et al., 1985; Willshaw et al., 1985; Huang, et al., 1986), phage CTXφ encoded cholera toxin from *Vibrio cholera* (Waldor & Mekalanos, 1996; Faruque & Nair, 2002) and diphtheria toxin encoded on a beta prophage from lysogens of *Corynebacterium diphtheriae* (Holmes & Barksdale, 1969; Bishai & Murphy 1988)*.* Together

Diphtheria Disease and Genes Involved

the disease symptoms seen in the above laboratory animals.

in Formation of Diphthamide, Key Effector of the Diphtheria Toxin 335

toxin, French Pierre-Paul-Émile Roux (1853-1933) and Swiss-French Alexandre Émile Jean Yersin (1863-1943) showed elegantly at the Pasteur Institute that the major bacterial virulence factor was indeed a potent exotoxin (Roux & Yersin, 1888). Upon sterile-filtration of *C. diphtheriae* cultures, they injected toxin-containing, cell free supernatants into laboratory animals and found that disease symptoms (including the eventual death of the animals) were developed in a manner indistinguishable from animals infected with the bacterium alone or even from infected humans. In addition, they proved that toxincontaining urine obtained from children infected with *C. diphtheriae* was sufficient to induce

Fig. 1. *Corynebacterium diphtheriae*, the causative agent of the diphtheria disease*.* Formation of *C. diphtheriae* colonies is shown on blood agar (left panel) according to the CDC (Centers for Disease Control and Prevention, USA). Stained cells of *C. diphtheriae* (right panel). Their barred appearance is due to metachromatic granules which contain polyphosphate. Permission by Professor Kenneth Todar, University of Wisconsin, USA, to show the

Löffler was the first to show that the pathogen could selectively be cultured from nasopharyngeal infections, indicating that diphtheria spreads within the upper respiratory tract. The disease causes a sore throat, low fever and an adherent green-grey membrane on the tonsils, pharynx, and nasal cavities. This thick and fibrinous pseudomembrane, which can severely obstruct airways and suffocate patients, is the result of a combination of bacterial and host effects in response to pathogen cell growth and toxin production as well as the host's immune response and necrosis of the underlying host cell tissue. In 1890, German scientists Emil Adolf von Behring (1854–1917) and Paul Ehrlich (1854–1915) began to study the immunization of horses against diphtheria in order to generate a serum for medical use in humans. Considered to be Ehrlich's first bacteriological achievement attracting world-wide renown, the transformation of diphtheria antitoxin into an effective protective preparation was successfully used during an epidemic in Germany. Rather controversially, however, only von Behring was awarded the first Nobel Prize in Medicine

photographs (Todar, 2004) is gratefully acknowledged by the authors.

in 1901 for developing a serum therapy against diphtheria.

with tRNase ribotoxins and anticodon nucleases from prokaryal and eukaryal microbes that have been shown to be encoded by transposable elements as well as circular and nonconventional linear DNA plasmids (Tokunaga et al., 1990; Kaufmann, 2000; Schaffrath & Meinhardt, 2005; Schaffrath et al., 1999), all of these genetic constellations implicate scenarios in which killer phenotypes have been evolved and spread by way of viral DNA transduction pathways or other forms of horizontal gene transfer. In support of this notion, certain cytoplasmic killer plasmids and their associated toxin phenotypes can be transferred between distinct yeast genera by means of cytoduction (Gunge 1983; Sugisaki et al., 1985) and horizontal transfer of the diphtheria toxin encoding *tox* gene from phages has been assigned to *in situ* lysogenic conversion of non-toxigenic to virulent corynebacteria (Freeman, 1951).

With regards to individual toxin response pathways in sensitive target cells, the specific cellular components being targeted by individual microbial protein toxins, not surprisingly, vary significantly depending on the nature of the essential cellular process that is targeted by the toxin in question (Schmitt & Schaffrath, 2005). For instance, microbial toxins capable of inhibiting the process of protein biosynthesis not only have been shown to target individual steps of mRNA translation (e.g. initiation, elongation and termination) but also to attack different components of the ribosomal machinery or associated factors required for mRNA translation (e.g. proteins, mRNAs, tRNAs and rRNAs). In this review, we will focus on one such microbial protein toxin that targets the essential process of mRNA translation and protein biosynthesis: diphtheria toxin (DT) from the Gram-positive bacterium *Corynebacterium diphtheriae* (for previous reviews, see Pappenheimer, 1977, 1984; Murphy, 1996). For DT to unfold its lethal action, the toxin needs to hijack a post-translationally modified residue known as diphthamide in its target protein EF2 (eukaryotic translation elongation factor 2). Next and by virtue of its enzymatic activity, DT ADP-ribosylates its target protein, an irreversible modification that inactivates the essential function of EF2 in mRNA translation. Eventually, EF2 inactivation by DT causes depletion of *de novo* protein biosynthesis and results in the death of the target cell including the model eukaryote and budding yeast *Saccharomyces cerevisiae*  (reviewed by Collier, 2001; Todar 2004; Ratts & Murphy, 2005)*.* Here, we review recent advances in the molecular biology of DT and present new insights into DT mode of action and DT response pathway components. An attractive idea emerging from research into DT mode of action is to take its basic molecular biology and apply it to biomedical intervention schemes against tumour cells, microbial pathogens or other biomedically and biotechnically relevant cell systems whose proliferation heavily relies on mRNA translation and protein biosynthesis (White-Gilbertson et al., 2009; Uthman et al., 2011). Such strategies are particularly informed by the use of chimeric DT fusion proteins that combine the lethal ADP-ribosylation activity of DT with a specific cell surface receptor domain for target cell or tissue specificity (Kreitman, 2006, 2009).

#### **2. History and discovery of the diphtheria pathogen**

Since the discovery in 1884 by German bacteriologists and physicists Edwin Klebs (1834– 1912) and Friedrich Löffler (1852–1915), that *Corynebacterium diphtheriae* (also known as the *Klebs-Löffler* bacillus) is the causative agent of diphtheria (Fig. 1), diphtheria has arguably developed into one of the prototypic, toxigenic and infectious human diseases. Soon after Löffler speculated that organ damage during diphtheria was the consequence of a bacterial

with tRNase ribotoxins and anticodon nucleases from prokaryal and eukaryal microbes that have been shown to be encoded by transposable elements as well as circular and nonconventional linear DNA plasmids (Tokunaga et al., 1990; Kaufmann, 2000; Schaffrath & Meinhardt, 2005; Schaffrath et al., 1999), all of these genetic constellations implicate scenarios in which killer phenotypes have been evolved and spread by way of viral DNA transduction pathways or other forms of horizontal gene transfer. In support of this notion, certain cytoplasmic killer plasmids and their associated toxin phenotypes can be transferred between distinct yeast genera by means of cytoduction (Gunge 1983; Sugisaki et al., 1985) and horizontal transfer of the diphtheria toxin encoding *tox* gene from phages has been assigned to *in situ* lysogenic conversion of non-toxigenic to virulent corynebacteria

With regards to individual toxin response pathways in sensitive target cells, the specific cellular components being targeted by individual microbial protein toxins, not surprisingly, vary significantly depending on the nature of the essential cellular process that is targeted by the toxin in question (Schmitt & Schaffrath, 2005). For instance, microbial toxins capable of inhibiting the process of protein biosynthesis not only have been shown to target individual steps of mRNA translation (e.g. initiation, elongation and termination) but also to attack different components of the ribosomal machinery or associated factors required for mRNA translation (e.g. proteins, mRNAs, tRNAs and rRNAs). In this review, we will focus on one such microbial protein toxin that targets the essential process of mRNA translation and protein biosynthesis: diphtheria toxin (DT) from the Gram-positive bacterium *Corynebacterium diphtheriae* (for previous reviews, see Pappenheimer, 1977, 1984; Murphy, 1996). For DT to unfold its lethal action, the toxin needs to hijack a post-translationally modified residue known as diphthamide in its target protein EF2 (eukaryotic translation elongation factor 2). Next and by virtue of its enzymatic activity, DT ADP-ribosylates its target protein, an irreversible modification that inactivates the essential function of EF2 in mRNA translation. Eventually, EF2 inactivation by DT causes depletion of *de novo* protein biosynthesis and results in the death of the target cell including the model eukaryote and budding yeast *Saccharomyces cerevisiae*  (reviewed by Collier, 2001; Todar 2004; Ratts & Murphy, 2005)*.* Here, we review recent advances in the molecular biology of DT and present new insights into DT mode of action and DT response pathway components. An attractive idea emerging from research into DT mode of action is to take its basic molecular biology and apply it to biomedical intervention schemes against tumour cells, microbial pathogens or other biomedically and biotechnically relevant cell systems whose proliferation heavily relies on mRNA translation and protein biosynthesis (White-Gilbertson et al., 2009; Uthman et al., 2011). Such strategies are particularly informed by the use of chimeric DT fusion proteins that combine the lethal ADP-ribosylation activity of DT with a specific cell surface receptor

domain for target cell or tissue specificity (Kreitman, 2006, 2009).

Since the discovery in 1884 by German bacteriologists and physicists Edwin Klebs (1834– 1912) and Friedrich Löffler (1852–1915), that *Corynebacterium diphtheriae* (also known as the *Klebs-Löffler* bacillus) is the causative agent of diphtheria (Fig. 1), diphtheria has arguably developed into one of the prototypic, toxigenic and infectious human diseases. Soon after Löffler speculated that organ damage during diphtheria was the consequence of a bacterial

**2. History and discovery of the diphtheria pathogen**

(Freeman, 1951).

toxin, French Pierre-Paul-Émile Roux (1853-1933) and Swiss-French Alexandre Émile Jean Yersin (1863-1943) showed elegantly at the Pasteur Institute that the major bacterial virulence factor was indeed a potent exotoxin (Roux & Yersin, 1888). Upon sterile-filtration of *C. diphtheriae* cultures, they injected toxin-containing, cell free supernatants into laboratory animals and found that disease symptoms (including the eventual death of the animals) were developed in a manner indistinguishable from animals infected with the bacterium alone or even from infected humans. In addition, they proved that toxincontaining urine obtained from children infected with *C. diphtheriae* was sufficient to induce the disease symptoms seen in the above laboratory animals.

Fig. 1. *Corynebacterium diphtheriae*, the causative agent of the diphtheria disease*.* Formation of *C. diphtheriae* colonies is shown on blood agar (left panel) according to the CDC (Centers for Disease Control and Prevention, USA). Stained cells of *C. diphtheriae* (right panel). Their barred appearance is due to metachromatic granules which contain polyphosphate. Permission by Professor Kenneth Todar, University of Wisconsin, USA, to show the photographs (Todar, 2004) is gratefully acknowledged by the authors.

Löffler was the first to show that the pathogen could selectively be cultured from nasopharyngeal infections, indicating that diphtheria spreads within the upper respiratory tract. The disease causes a sore throat, low fever and an adherent green-grey membrane on the tonsils, pharynx, and nasal cavities. This thick and fibrinous pseudomembrane, which can severely obstruct airways and suffocate patients, is the result of a combination of bacterial and host effects in response to pathogen cell growth and toxin production as well as the host's immune response and necrosis of the underlying host cell tissue. In 1890, German scientists Emil Adolf von Behring (1854–1917) and Paul Ehrlich (1854–1915) began to study the immunization of horses against diphtheria in order to generate a serum for medical use in humans. Considered to be Ehrlich's first bacteriological achievement attracting world-wide renown, the transformation of diphtheria antitoxin into an effective protective preparation was successfully used during an epidemic in Germany. Rather controversially, however, only von Behring was awarded the first Nobel Prize in Medicine in 1901 for developing a serum therapy against diphtheria.

Diphtheria Disease and Genes Involved

in Formation of Diphthamide, Key Effector of the Diphtheria Toxin 337

toxigenic strains may acquire virulence following lysogenic conversion *in situ* (see below). Although it is clear that events other than production and secretion of DT promote host tissue colonization, detailed knowledge about *C. diphtheriae* factors involved in virulence is scarce. Putative roles in the colonization process have been discussed for a sialic acid splitting neuraminidase from the pathogen and for a corynebacterial cell surface component known as cord factor, i.e. 6,6'-di-O-mycoloyl-,-D-trehalose (reviewed by Murphy, 1996). The emergence of continuously changing lysotypes in the pathogen's population is likely due to their ability to compete more efficiently in segments of the nasopharyngeal ecologic niche. Thus, a given lysotype may persist for a while only to be replaced during later stages

Diphtheria is a contagious disease; although toxigenic strains have been isolated from horses, the pathogen *C. diphtheriae* is usually spread by direct physical contact among humans, namely by droplets or through inhaling aerosols of infected individuals. In addition, *in situ* conversion of avirulent strains to pathogenic ones may involve lateral gene transfer following bacterial lysis and release of the DT encoding phage gene (Freeman, 1951; Holmes & Barksdale, 1969). In regions of active immunization programs, isolated focal outbreaks can be associated with carriers who returned from visits to regions still endemic for diphtheria. In the US, Europe and elsewhere, diphtheria was a disease typical of children before mass immunization with diphtheria toxoid. Today, virulent biotypes of *C. diphtheriae* are rarely isolated and clinical diphtheria has largely been eradicated from industrialized nations through global vaccination schemes. Due to effective Diphtheria–Pertussis–Tetanus (DPT) vaccines, the number of diphtheria cases among school-aged children in the US has significantly dropped from 52 in 1980-2000 to 3 in 2000-2007 (Atkinson et al., 2007). For adults, however, vaccine boosts, are strongly advocated since immunity wears off with age and 30-60% of adults are estimated to be at risk, in particular, persons travelling to countries in which diphtheria has not been fully eradicated but poses a constant endemic healththreat. In the 1980s and 1994, public health breakdown in Sweden and Russia caused epidemic clinical diphtheria (Rappuoli et al., 1988) with officials recording more than 80,000 cases including 2,000 deaths. Focal outbreaks reported thereafter were almost certainly

associated with diphtheria carriers who returned from Russia to Europe and the US.

Recognition that systemic organ damage associated with diphtheria is due to the action of the lethal DT rather than dissemination of blood-borne pathogens led to the development of a highly successful toxoid vaccine in which inactivated DT that remains antigenic is able to raise an immune response. The toxoid is prepared by incubating DT with formaldehyde at 37° C under alkaline conditions. Although immunization with the toxoid has made diphtheria a rare disease, diphtheria outbreaks do still occur in non-immunized and immune-compromised groups. Control of diphtheria, therefore, depends upon adequate immunization with antigenically intact yet inactivated diphtheria toxoid. Immunization against diphtheria should begin in the second month of life with a series of three primary doses spaced 4 to 8 weeks apart, followed by a fourth dose approximately 1 year after the last primary inoculation. Diphtheria toxoid is widely used as a component in the DPT vaccine (see above). Epidemiologic surveys have shown that immunization against diphtheria is approximately 97% effective. Although mass immunization against diphtheria

**4. Host defence, diagnosis and control**

in the infection by another one that is more adapted to its niche.

#### **3. Manifestations, pathogenesis and epidemiology of diphtheria**