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Transferrin blocks growth of B. anthracis by iron deprivation. A , growth of B. anthracis Sterne 34F2 after 5 h of incubation with human apo-transferrin (apo-hTF, iron-free) or holo-transferrin (holo-hTF, iron-saturated). B , growth of B. anthracis Sterne 34F2 after 5 h of incubation in the presence of recombinant apo-transferrin ( apo-rhTF ), recombinant holo-transferrin ( holo-rhTF ), or apo-rhTF mutated in its iron-binding sites ( apo-rhTF ⌬ Fe ). C , growth of B. anthracis Sterne 34F2 after 5 h of incubation in the presence of human serum (0 or 10%) in the presence of iron(III) citrate. D , relative growth of different Gram-positive pathogens grown for 5 h in the presence of human apo-transferrin (apo-hTF). Growth in buffer alone was set at 100%. Data represent mean Ϯ S.E. of three independent experiments. Growth indices indicate ratio of bacterial cfu surviving after incubation versus the initial inoculum. **, p Ͻ 0.001 (apo-hTF compared with buffer).
Source publication
The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the ide...
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Context 1
... in buffer alone (RPMI) was set at 100%. Gel Filtration Chromatography and Functional Screening — Heat-inactivated serum (250 l) was separated on a Superdex- 200 GL 10/300 (GE Healthcare, Piscataway, NJ) equilibrated with RPMI 1640 (flow 1 ml/min), and 1-ml fractions were collected. The column was calibrated with thyroglobulin (670 kDa), gamma globulin (158 kDa), ovalbumin (44 kDa), myoglo- bin (17 kDa), and vitamin B12 (1.4 kDa). Fractions were incubated with 500 cfu of B. anthracis Sterne 7702 and grown over- night at 37 °C. Overnight cultures were subcultured in THB and grown for 7 h after which A 600 was assessed. Immunoblotting —Gel filtration fractions were separated by SDS-PAGE and transferred to an Immobilon PVDF membrane (Millipore, Billerica, MA). Transferrin was detected using per- oxidase-labeled goat-anti-transferrin antibodies (Bethyl Labo- ratories, Montgomery, TX) and enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, IL). Hypotransferrinemic (Trf hpx/hpx ) Mice —The Trf hpx/hpx mice, maintained on a BALBc/J background, were produced and housed as described previously (16). Trf hpx/hpx mice were treated with weekly intraperitoneal injections of 6 mg of human Trf (Roche Applied Science, Indianapolis, IN) until weaning. Serum was collected from female mice at 8 weeks of age, when all injected transferrin is degraded. Statistical Analysis —Student’s t test analysis was performed using the Microsoft Excel Software. Human Serum Inhibits Growth of B. anthracis —While studying the role of humoral blood components in the defense against Gram-positive pathogens, we observed that growth of B. anthracis Sterne is strongly impaired in the presence of normal human serum (Fig. 1 A ). In contrast, serum did not affect growth of S. aureus nor S. pneumoniae . The two Sterne strains showed similar growth inhibition at 1% serum but B. anthracis Sterne 7702 was more sensitive at 5% serum. This difference may be due to the fact that these two strains have been cultured under different conditions for many years. Growth inhibition of B. anthracis was dose-dependent and complete at serum concentrations of 1% or higher (Fig. 1 B ). Serum was bacteriostatic rather than bactericidal to B. anthracis , since the initial bacterial inoculum did not decrease in its presence. Although Gram- positive bacteria have a thick peptidoglycan layer that makes them resistant to complement lysis, we wondered whether bacteriostasis could be complement-dependent. Heat-inactivated serum could still inhibit growth of B. anthracis Sterne, although the activity was somewhat lower than in normal serum (Fig. 1 C ). This finding suggests that complement contributes to the growth inhibitory effect, but that serum contains another component that blocks growth of anthrax bacilli. Factor —To identify the serum component that inhibits growth of B. anthracis , we fractionated heat-inactivated human serum by gel filtration chromatography and analyzed collected fractions for their ability to block growth of B. anthracis (Fig. 2 A ). We found that the bacteriostatic activity eluted from the column in a single wide peak, indicative of a single protein. The active protein was estimated to have a molecular weight between ϳ 15–150 kDa (Fig. 2 A ). (Moderate growth inhibition seen in fractions smaller than 1 kDa can most likely be attrib- uted to high salt). Because the iron regulator human transferrin has a molecular mass of 80 kDa and is relatively abundant in serum (2– 4 mg/ml) (17), we tested for the presence of trans- tion —In normal persons, only 30% of transferrin in serum is in complex with iron (holo-transferrin), leaving a pool of transferrin with no iron, ϳ 40% (apo-transferrin) (17). To study whether iron binding by transferrin is important for B. anthracis growth inhibition, we compared growth in the presence of these naturally occurring forms of transferrin. We observed a dose-dependent growth inhibition by apo-transferrin, whereas growth was not affected by holo-transferrin (Fig. 3 A ). These data strongly suggest that transferrin blocks growth by iron sequestration. However, in the case of Gram-negative bacteria, transferrin has also been suggested to directly injure the bacterial outer membrane (18, 19). Because holo-transferrin has a different conformation than apo-transferrin (20, 21), we could not exclude that apo-transferrin has direct membrane damag- ing properties. However, we studied the anti-bacterial mechanism in more detail by analyzing growth in the presence of a recombinant apo-transferrin mutant that does not bind iron, but has the same conformation as wild-type apo-transferrin (15). Fig. 3 B shows that, in contrast to recombinant apo-trans- ferrin, the iron-binding mutant did not block growth of B. anthracis Sterne. Also, recombinant holo-transferrin showed no bacteriostatic activity. Furthermore we showed that growth of B. anthracis in human serum could be restored by supplemen- tation with additional iron (Fig. 3 C ). Collectively these data show that transferrin effectively blocks the growth of B. anthracis by depriving the bacilli from acquiring the iron that they need to grow. Because our initial experiments in human serum showed that S. aureus and S. pneumoniae could resist the bacteriostatic effects of serum, we re-tested the growth of these and other Gram-positive bacteria in the presence of purified apo- transferrin. Growth of all tested pathogens was unaffected by purified apo-transferrin (Fig. 3 D ), indicating that they have evolved mechanisms to circumvent the iron scavenging effects of human transferrin. The human innate immune system provides an essential first-line of defense against bacterial infections and is endowed with both cellular and humoral components that eradicate such as heme or transferrin (36). For instance, bacteria produce iron chelators that compete with transferrin (37, 38). Among Gram-positive species, only S. aureus is known to express such a molecule: the staphylococcal transferrin-binding protein A (39). Indeed, we observe that S. aureus grows even better in the presence of purified transferrin than in its absence. Our studies indicate that S. pneumoniae is also resistant to the antibacterial effects of transferrin in serum. Whereas the normal ecology of S. aureus and S. pneumoniae involves asymptomatic coloniza- tion of human mucosal surfaces, B. anthracis exists in the soil causing a zoonotic infection and only accidentally infects humans, perhaps explaining why it has not evolved transferrin resistance mechanism. In addition, many of the described bacterial iron-uptake systems are highly host-specific. Even though B. anthracis is known to express siderophores, small molecules that chelate iron (40), we observe that under the relevant phys- iological conditions (human serum at 37 °C) that we used these molecules are not effective in competing with transferrin in human serum. The sensitivity of B. anthracis to human transferrin could provide an explanation for the observed differences in disease severity between B. anthracis introduced via the skin or the lungs. Anthrax spores that enter the body via the skin have been shown to germinate extracellularly, which allows serum proteins in interstitial tissue fluids to interact with the bacteria and control the infection (41, 42). In contrast, inhaled spores are quickly phagocytosed by macrophages in which they germinate intracellularly. In this case, bacilli are protected from serum resistance factors allowing them to initially grow unimpeded, multiply rapidly and potentially cause an overwhelming sys- temic infection. In conclusion, although the antibacterial properties of transferrin are not generally appreciated, our data show that transferrin is a critical host defense molecule that controls growth of B. anthracis in human ...
Context 2
... and grown over- night at 37 °C. Overnight cultures were subcultured in THB and grown for 7 h after which A 600 was assessed. Immunoblotting —Gel filtration fractions were separated by SDS-PAGE and transferred to an Immobilon PVDF membrane (Millipore, Billerica, MA). Transferrin was detected using per- oxidase-labeled goat-anti-transferrin antibodies (Bethyl Labo- ratories, Montgomery, TX) and enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, IL). Hypotransferrinemic (Trf hpx/hpx ) Mice —The Trf hpx/hpx mice, maintained on a BALBc/J background, were produced and housed as described previously (16). Trf hpx/hpx mice were treated with weekly intraperitoneal injections of 6 mg of human Trf (Roche Applied Science, Indianapolis, IN) until weaning. Serum was collected from female mice at 8 weeks of age, when all injected transferrin is degraded. Statistical Analysis —Student’s t test analysis was performed using the Microsoft Excel Software. Human Serum Inhibits Growth of B. anthracis —While studying the role of humoral blood components in the defense against Gram-positive pathogens, we observed that growth of B. anthracis Sterne is strongly impaired in the presence of normal human serum (Fig. 1 A ). In contrast, serum did not affect growth of S. aureus nor S. pneumoniae . The two Sterne strains showed similar growth inhibition at 1% serum but B. anthracis Sterne 7702 was more sensitive at 5% serum. This difference may be due to the fact that these two strains have been cultured under different conditions for many years. Growth inhibition of B. anthracis was dose-dependent and complete at serum concentrations of 1% or higher (Fig. 1 B ). Serum was bacteriostatic rather than bactericidal to B. anthracis , since the initial bacterial inoculum did not decrease in its presence. Although Gram- positive bacteria have a thick peptidoglycan layer that makes them resistant to complement lysis, we wondered whether bacteriostasis could be complement-dependent. Heat-inactivated serum could still inhibit growth of B. anthracis Sterne, although the activity was somewhat lower than in normal serum (Fig. 1 C ). This finding suggests that complement contributes to the growth inhibitory effect, but that serum contains another component that blocks growth of anthrax bacilli. Factor —To identify the serum component that inhibits growth of B. anthracis , we fractionated heat-inactivated human serum by gel filtration chromatography and analyzed collected fractions for their ability to block growth of B. anthracis (Fig. 2 A ). We found that the bacteriostatic activity eluted from the column in a single wide peak, indicative of a single protein. The active protein was estimated to have a molecular weight between ϳ 15–150 kDa (Fig. 2 A ). (Moderate growth inhibition seen in fractions smaller than 1 kDa can most likely be attrib- uted to high salt). Because the iron regulator human transferrin has a molecular mass of 80 kDa and is relatively abundant in serum (2– 4 mg/ml) (17), we tested for the presence of trans- tion —In normal persons, only 30% of transferrin in serum is in complex with iron (holo-transferrin), leaving a pool of transferrin with no iron, ϳ 40% (apo-transferrin) (17). To study whether iron binding by transferrin is important for B. anthracis growth inhibition, we compared growth in the presence of these naturally occurring forms of transferrin. We observed a dose-dependent growth inhibition by apo-transferrin, whereas growth was not affected by holo-transferrin (Fig. 3 A ). These data strongly suggest that transferrin blocks growth by iron sequestration. However, in the case of Gram-negative bacteria, transferrin has also been suggested to directly injure the bacterial outer membrane (18, 19). Because holo-transferrin has a different conformation than apo-transferrin (20, 21), we could not exclude that apo-transferrin has direct membrane damag- ing properties. However, we studied the anti-bacterial mechanism in more detail by analyzing growth in the presence of a recombinant apo-transferrin mutant that does not bind iron, but has the same conformation as wild-type apo-transferrin (15). Fig. 3 B shows that, in contrast to recombinant apo-trans- ferrin, the iron-binding mutant did not block growth of B. anthracis Sterne. Also, recombinant holo-transferrin showed no bacteriostatic activity. Furthermore we showed that growth of B. anthracis in human serum could be restored by supplemen- tation with additional iron (Fig. 3 C ). Collectively these data show that transferrin effectively blocks the growth of B. anthracis by depriving the bacilli from acquiring the iron that they need to grow. Because our initial experiments in human serum showed that S. aureus and S. pneumoniae could resist the bacteriostatic effects of serum, we re-tested the growth of these and other Gram-positive bacteria in the presence of purified apo- transferrin. Growth of all tested pathogens was unaffected by purified apo-transferrin (Fig. 3 D ), indicating that they have evolved mechanisms to circumvent the iron scavenging effects of human transferrin. The human innate immune system provides an essential first-line of defense against bacterial infections and is endowed with both cellular and humoral components that eradicate such as heme or transferrin (36). For instance, bacteria produce iron chelators that compete with transferrin (37, 38). Among Gram-positive species, only S. aureus is known to express such a molecule: the staphylococcal transferrin-binding protein A (39). Indeed, we observe that S. aureus grows even better in the presence of purified transferrin than in its absence. Our studies indicate that S. pneumoniae is also resistant to the antibacterial effects of transferrin in serum. Whereas the normal ecology of S. aureus and S. pneumoniae involves asymptomatic coloniza- tion of human mucosal surfaces, B. anthracis exists in the soil causing a zoonotic infection and only accidentally infects humans, perhaps explaining why it has not evolved transferrin resistance mechanism. In addition, many of the described bacterial iron-uptake systems are highly host-specific. Even though B. anthracis is known to express siderophores, small molecules that chelate iron (40), we observe that under the relevant phys- iological conditions (human serum at 37 °C) that we used these molecules are not effective in competing with transferrin in human serum. The sensitivity of B. anthracis to human transferrin could provide an explanation for the observed differences in disease severity between B. anthracis introduced via the skin or the lungs. Anthrax spores that enter the body via the skin have been shown to germinate extracellularly, which allows serum proteins in interstitial tissue fluids to interact with the bacteria and control the infection (41, 42). In contrast, inhaled spores are quickly phagocytosed by macrophages in which they germinate intracellularly. In this case, bacilli are protected from serum resistance factors allowing them to initially grow unimpeded, multiply rapidly and potentially cause an overwhelming sys- temic infection. In conclusion, although the antibacterial properties of transferrin are not generally appreciated, our data show that transferrin is a critical host defense molecule that controls growth of B. anthracis in human ...
Context 3
... by plating serial dilutions on THA. Growth index was calculated as the ratio of bacterial colony forming units (cfu) surviving after incubation versus the initial inoculum. Relative growth was used when growth of B. anthracis was compared with different bacteria ( S. aureus , S. pneumonia , GAS, and GBS); in this case growth in buffer alone (RPMI) was set at 100%. Gel Filtration Chromatography and Functional Screening — Heat-inactivated serum (250 l) was separated on a Superdex- 200 GL 10/300 (GE Healthcare, Piscataway, NJ) equilibrated with RPMI 1640 (flow 1 ml/min), and 1-ml fractions were collected. The column was calibrated with thyroglobulin (670 kDa), gamma globulin (158 kDa), ovalbumin (44 kDa), myoglo- bin (17 kDa), and vitamin B12 (1.4 kDa). Fractions were incubated with 500 cfu of B. anthracis Sterne 7702 and grown over- night at 37 °C. Overnight cultures were subcultured in THB and grown for 7 h after which A 600 was assessed. Immunoblotting —Gel filtration fractions were separated by SDS-PAGE and transferred to an Immobilon PVDF membrane (Millipore, Billerica, MA). Transferrin was detected using per- oxidase-labeled goat-anti-transferrin antibodies (Bethyl Labo- ratories, Montgomery, TX) and enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, IL). Hypotransferrinemic (Trf hpx/hpx ) Mice —The Trf hpx/hpx mice, maintained on a BALBc/J background, were produced and housed as described previously (16). Trf hpx/hpx mice were treated with weekly intraperitoneal injections of 6 mg of human Trf (Roche Applied Science, Indianapolis, IN) until weaning. Serum was collected from female mice at 8 weeks of age, when all injected transferrin is degraded. Statistical Analysis —Student’s t test analysis was performed using the Microsoft Excel Software. Human Serum Inhibits Growth of B. anthracis —While studying the role of humoral blood components in the defense against Gram-positive pathogens, we observed that growth of B. anthracis Sterne is strongly impaired in the presence of normal human serum (Fig. 1 A ). In contrast, serum did not affect growth of S. aureus nor S. pneumoniae . The two Sterne strains showed similar growth inhibition at 1% serum but B. anthracis Sterne 7702 was more sensitive at 5% serum. This difference may be due to the fact that these two strains have been cultured under different conditions for many years. Growth inhibition of B. anthracis was dose-dependent and complete at serum concentrations of 1% or higher (Fig. 1 B ). Serum was bacteriostatic rather than bactericidal to B. anthracis , since the initial bacterial inoculum did not decrease in its presence. Although Gram- positive bacteria have a thick peptidoglycan layer that makes them resistant to complement lysis, we wondered whether bacteriostasis could be complement-dependent. Heat-inactivated serum could still inhibit growth of B. anthracis Sterne, although the activity was somewhat lower than in normal serum (Fig. 1 C ). This finding suggests that complement contributes to the growth inhibitory effect, but that serum contains another component that blocks growth of anthrax bacilli. Factor —To identify the serum component that inhibits growth of B. anthracis , we fractionated heat-inactivated human serum by gel filtration chromatography and analyzed collected fractions for their ability to block growth of B. anthracis (Fig. 2 A ). We found that the bacteriostatic activity eluted from the column in a single wide peak, indicative of a single protein. The active protein was estimated to have a molecular weight between ϳ 15–150 kDa (Fig. 2 A ). (Moderate growth inhibition seen in fractions smaller than 1 kDa can most likely be attrib- uted to high salt). Because the iron regulator human transferrin has a molecular mass of 80 kDa and is relatively abundant in serum (2– 4 mg/ml) (17), we tested for the presence of trans- tion —In normal persons, only 30% of transferrin in serum is in complex with iron (holo-transferrin), leaving a pool of transferrin with no iron, ϳ 40% (apo-transferrin) (17). To study whether iron binding by transferrin is important for B. anthracis growth inhibition, we compared growth in the presence of these naturally occurring forms of transferrin. We observed a dose-dependent growth inhibition by apo-transferrin, whereas growth was not affected by holo-transferrin (Fig. 3 A ). These data strongly suggest that transferrin blocks growth by iron sequestration. However, in the case of Gram-negative bacteria, transferrin has also been suggested to directly injure the bacterial outer membrane (18, 19). Because holo-transferrin has a different conformation than apo-transferrin (20, 21), we could not exclude that apo-transferrin has direct membrane damag- ing properties. However, we studied the anti-bacterial mechanism in more detail by analyzing growth in the presence of a recombinant apo-transferrin mutant that does not bind iron, but has the same conformation as wild-type apo-transferrin (15). Fig. 3 B shows that, in contrast to recombinant apo-trans- ferrin, the iron-binding mutant did not block growth of B. anthracis Sterne. Also, recombinant holo-transferrin showed no bacteriostatic activity. Furthermore we showed that growth of B. anthracis in human serum could be restored by supplemen- tation with additional iron (Fig. 3 C ). Collectively these data show that transferrin effectively blocks the growth of B. anthracis by depriving the bacilli from acquiring the iron that they need to grow. Because our initial experiments in human serum showed that S. aureus and S. pneumoniae could resist the bacteriostatic effects of serum, we re-tested the growth of these and other Gram-positive bacteria in the presence of purified apo- transferrin. Growth of all tested pathogens was unaffected by purified apo-transferrin (Fig. 3 D ), indicating that they have evolved mechanisms to circumvent the iron scavenging effects of human transferrin. The human innate immune system provides an essential first-line of defense against bacterial infections and is endowed with both cellular and humoral components that eradicate such as heme or transferrin (36). For instance, bacteria produce iron chelators that compete with transferrin (37, 38). Among Gram-positive species, only S. aureus is known to express such a molecule: the staphylococcal transferrin-binding protein A (39). Indeed, we observe that S. aureus grows even better in the presence of purified transferrin than in its absence. Our studies indicate that S. pneumoniae is also resistant to the antibacterial effects of transferrin in serum. Whereas the normal ecology of S. aureus and S. pneumoniae involves asymptomatic coloniza- tion of human mucosal surfaces, B. anthracis exists in the soil causing a zoonotic infection and only accidentally infects humans, perhaps explaining why it has not evolved transferrin resistance mechanism. In addition, many of the described bacterial iron-uptake systems are highly host-specific. Even though B. anthracis is known to express siderophores, small molecules that chelate iron (40), we observe that under the relevant phys- iological conditions (human serum at 37 °C) that we used these molecules are not effective in competing with transferrin in human serum. The sensitivity of B. anthracis to human transferrin could provide an explanation for the observed differences in disease severity between B. anthracis introduced via the skin or the lungs. Anthrax spores that enter the body via the skin have been shown to germinate extracellularly, which allows serum proteins in interstitial tissue fluids to interact with the bacteria and control the infection (41, 42). In contrast, inhaled spores are quickly phagocytosed by macrophages in which they germinate intracellularly. In this case, bacilli are protected from serum resistance factors allowing them to initially grow unimpeded, multiply rapidly and potentially cause an overwhelming sys- temic infection. In conclusion, although the antibacterial properties of transferrin are not generally appreciated, our data show that transferrin is a critical host defense molecule that controls growth of B. anthracis in human ...
Context 4
... All recombinant transferrins have a hexa-His tag at the N terminus and are nonglycosylated. Nei- ther the lack of carbohydrates nor the presence of the His tag affect their function (15). Bacterial Growth Assays —Bacterial cultures were grown to post-log phase in THB media and diluted 1:10,000 in RPMI 1640 tissue culture medium (Invitrogen, Carlsbad, CA). 50 l of bacteria were mixed with 50 l RPMI (untreated controls), serum or transferrin (both diluted in RPMI) in 96-well plates. Iron(III) citrate was obtained from Sigma. Plates were incubated at 37 °C under continuous shaking and surviving bacteria were enumerated by plating serial dilutions on THA. Growth index was calculated as the ratio of bacterial colony forming units (cfu) surviving after incubation versus the initial inoculum. Relative growth was used when growth of B. anthracis was compared with different bacteria ( S. aureus , S. pneumonia , GAS, and GBS); in this case growth in buffer alone (RPMI) was set at 100%. Gel Filtration Chromatography and Functional Screening — Heat-inactivated serum (250 l) was separated on a Superdex- 200 GL 10/300 (GE Healthcare, Piscataway, NJ) equilibrated with RPMI 1640 (flow 1 ml/min), and 1-ml fractions were collected. The column was calibrated with thyroglobulin (670 kDa), gamma globulin (158 kDa), ovalbumin (44 kDa), myoglo- bin (17 kDa), and vitamin B12 (1.4 kDa). Fractions were incubated with 500 cfu of B. anthracis Sterne 7702 and grown over- night at 37 °C. Overnight cultures were subcultured in THB and grown for 7 h after which A 600 was assessed. Immunoblotting —Gel filtration fractions were separated by SDS-PAGE and transferred to an Immobilon PVDF membrane (Millipore, Billerica, MA). Transferrin was detected using per- oxidase-labeled goat-anti-transferrin antibodies (Bethyl Labo- ratories, Montgomery, TX) and enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, IL). Hypotransferrinemic (Trf hpx/hpx ) Mice —The Trf hpx/hpx mice, maintained on a BALBc/J background, were produced and housed as described previously (16). Trf hpx/hpx mice were treated with weekly intraperitoneal injections of 6 mg of human Trf (Roche Applied Science, Indianapolis, IN) until weaning. Serum was collected from female mice at 8 weeks of age, when all injected transferrin is degraded. Statistical Analysis —Student’s t test analysis was performed using the Microsoft Excel Software. Human Serum Inhibits Growth of B. anthracis —While studying the role of humoral blood components in the defense against Gram-positive pathogens, we observed that growth of B. anthracis Sterne is strongly impaired in the presence of normal human serum (Fig. 1 A ). In contrast, serum did not affect growth of S. aureus nor S. pneumoniae . The two Sterne strains showed similar growth inhibition at 1% serum but B. anthracis Sterne 7702 was more sensitive at 5% serum. This difference may be due to the fact that these two strains have been cultured under different conditions for many years. Growth inhibition of B. anthracis was dose-dependent and complete at serum concentrations of 1% or higher (Fig. 1 B ). Serum was bacteriostatic rather than bactericidal to B. anthracis , since the initial bacterial inoculum did not decrease in its presence. Although Gram- positive bacteria have a thick peptidoglycan layer that makes them resistant to complement lysis, we wondered whether bacteriostasis could be complement-dependent. Heat-inactivated serum could still inhibit growth of B. anthracis Sterne, although the activity was somewhat lower than in normal serum (Fig. 1 C ). This finding suggests that complement contributes to the growth inhibitory effect, but that serum contains another component that blocks growth of anthrax bacilli. Factor —To identify the serum component that inhibits growth of B. anthracis , we fractionated heat-inactivated human serum by gel filtration chromatography and analyzed collected fractions for their ability to block growth of B. anthracis (Fig. 2 A ). We found that the bacteriostatic activity eluted from the column in a single wide peak, indicative of a single protein. The active protein was estimated to have a molecular weight between ϳ 15–150 kDa (Fig. 2 A ). (Moderate growth inhibition seen in fractions smaller than 1 kDa can most likely be attrib- uted to high salt). Because the iron regulator human transferrin has a molecular mass of 80 kDa and is relatively abundant in serum (2– 4 mg/ml) (17), we tested for the presence of trans- tion —In normal persons, only 30% of transferrin in serum is in complex with iron (holo-transferrin), leaving a pool of transferrin with no iron, ϳ 40% (apo-transferrin) (17). To study whether iron binding by transferrin is important for B. anthracis growth inhibition, we compared growth in the presence of these naturally occurring forms of transferrin. We observed a dose-dependent growth inhibition by apo-transferrin, whereas growth was not affected by holo-transferrin (Fig. 3 A ). These data strongly suggest that transferrin blocks growth by iron sequestration. However, in the case of Gram-negative bacteria, transferrin has also been suggested to directly injure the bacterial outer membrane (18, 19). Because holo-transferrin has a different conformation than apo-transferrin (20, 21), we could not exclude that apo-transferrin has direct membrane damag- ing properties. However, we studied the anti-bacterial mechanism in more detail by analyzing growth in the presence of a recombinant apo-transferrin mutant that does not bind iron, but has the same conformation as wild-type apo-transferrin (15). Fig. 3 B shows that, in contrast to recombinant apo-trans- ferrin, the iron-binding mutant did not block growth of B. anthracis Sterne. Also, recombinant holo-transferrin showed no bacteriostatic activity. Furthermore we showed that growth of B. anthracis in human serum could be restored by supplemen- tation with additional iron (Fig. 3 C ). Collectively these data show that transferrin effectively blocks the growth of B. anthracis by depriving the bacilli from acquiring the iron that they need to grow. Because our initial experiments in human serum showed that S. aureus and S. pneumoniae could resist the bacteriostatic effects of serum, we re-tested the growth of these and other Gram-positive bacteria in the presence of purified apo- transferrin. Growth of all tested pathogens was unaffected by purified apo-transferrin (Fig. 3 D ), indicating that they have evolved mechanisms to circumvent the iron scavenging effects of human transferrin. The human innate immune system provides an essential first-line of defense against bacterial infections and is endowed with both cellular and humoral components that eradicate such as heme or transferrin (36). For instance, bacteria produce iron chelators that compete with transferrin (37, 38). Among Gram-positive species, only S. aureus is known to express such a molecule: the staphylococcal transferrin-binding protein A (39). Indeed, we observe that S. aureus grows even better in the presence of purified transferrin than in its absence. Our studies indicate that S. pneumoniae is also resistant to the antibacterial effects of transferrin in serum. Whereas the normal ecology of S. aureus and S. pneumoniae involves asymptomatic coloniza- tion of human mucosal surfaces, B. anthracis exists in the soil causing a zoonotic infection and only accidentally infects humans, perhaps explaining why it has not evolved transferrin resistance mechanism. In addition, many of the described bacterial iron-uptake systems are highly host-specific. Even though B. anthracis is known to express siderophores, small molecules that chelate iron (40), we observe that under the relevant phys- iological conditions (human serum at 37 °C) that we used these molecules are not effective in competing with transferrin in human serum. The sensitivity of B. anthracis to human transferrin could provide an explanation for the observed differences in disease severity between B. anthracis introduced via the skin or the lungs. Anthrax spores that enter the body via the skin have been shown to germinate extracellularly, which allows serum proteins in interstitial tissue fluids to interact with the bacteria and control the infection (41, 42). In contrast, inhaled spores are quickly phagocytosed by macrophages in which they germinate intracellularly. In this case, bacilli are protected from serum resistance factors allowing them to initially grow unimpeded, multiply rapidly and potentially cause an overwhelming sys- temic infection. In conclusion, although the antibacterial properties of transferrin are not generally appreciated, our data show that transferrin is a critical host defense molecule that controls growth of B. anthracis in human ...
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Citations
... Specifically, transferrin in its apo (empty), but not holo (full), state inhibits in vitro growth of B. cereus [39] and B. anthracis [40] in a dose-dependent manner. When iron-binding proteins such as transferrin are saturated (full), as was demonstrated by the low total iron-binding capacity of case patient 1, the antibacterial activity, including phagocytic activity, of plasma may be significantly diminished [41]. ...
Bacillus anthracis has traditionally been considered the etiologic agent of anthrax. However, anthrax-like illness has been documented in welders and other metal workers infected with Bacillus cereus group spp. harboring pXO1 virulence genes that produce anthrax toxins. We present 2 recent cases of severe pneumonia in welders with B. cereus group infections and discuss potential risk factors for infection and treatment options, including antitoxin.
... Due to their high affinity to iron, transferrins have been shown to inhibit the growth of certain microbes (17). While numerous studies reported the potent antimicrobial activity of purified transferrins in vitro, in vivo studies addressing transferrin function are rather limited (10,15,(18)(19)(20)(21)(22)(23)(24). Although hypotransferrinemic (hpx) mice devoid of serum transferrin exist, how they respond to microbial infection has yet to be examined (25). ...
Significance
Hosts sequester iron as a strategy to limit pathogen acquisition of this essential nutrient in a process termed nutritional immunity. Due to their in vitro antimicrobial activity, iron-binding proteins transferrins are suspected to play a role in iron sequestration. However, little is known about the in vivo role of transferrins. Here, we found that Drosophila melanogaster exhibits infection-induced hypoferremia mediated by Transferrin 1. Due to excessive iron in hemolymph, Transferrin 1 ( Tsf1 )-deficient flies are hypersusceptible to certain infections. Our study reveals that nutritional immunity is an important, previously unrecognized arm of immune defense in Drosophila . Using fly and bacterial genetics, we show that Tsf1 mediates nutritional immunity by sequestering iron from the pathogens in vivo on the whole-organism level.
... Hp and Hx can accelerate the clearance of free hemoglobin and heme, limiting the availability of the iron necessary for bacterial growth [12]. The ability of EV76 to induce the rapid expression of Hx and Hp may explain the early antibacterial effect induced by immunization with EV76, which is consistent with the suggestion that exogenously elevating the levels of the host iron-binding proteins represents a therapeutic approach to preventing iron acquisition by pathogens [12][13][14][15]. ...
Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing contagious disease. In the plague mouse model, a single immunization with the EV76 live attenuated Y. pestis strain rapidly induced the expression of hemopexin and haptoglobin in the lung and serum, both of which are important in iron sequestration. Immunization against a concomitant lethal Y. pestis respiratory challenge was correlated with temporary inhibition of disease progression. Combining EV76-immunization and second-line antibiotic treatment, which are individually insufficient, led to a synergistic protective effect that represents a proof of concept for efficient combinational therapy in cases of infection with antibiotic-resistant strains.
... Thus far, nonpathogenic Gram positives appear to be resistant to killing by serum (21). Only B. anthracis was previously shown to be sensitive to normal serum; however, this was fully dependent on growth inhibition by transferrin-mediated iron deprivation (48). hGIIA is an acute-phase reactant protein, whose concentration in serum upon bacterial infection can increase up to 100-fold, which is high enough to kill some Gram-positive pathogens (31). ...
Human innate immunity employs cellular and humoral mechanisms to facilitate rapid killing of invading bacteria. The direct killing of bacteria by human serum is mainly attributed to the activity of the complement system that forms pores in Gram-negative bacteria. Although Gram-positive bacteria are considered resistant to serum killing, we here uncover that normal human serum effectively kills Enterococcus faecium. Comparison of a well-characterized collection of commensal and clinical E. faecium isolates revealed that human serum specifically kills commensal E. faecium strains isolated from normal gut microbiota, but not clinical isolates. Inhibitor studies show that the human group IIA secreted phospholipase A2 (hGIIA), but not complement, is responsible for killing of commensal E. faecium strains in human normal serum. This is remarkable since the hGIIA concentration in ‘non-inflamed' serum was considered too low to be bactericidal against Gram-positive bacteria. Mechanistic studies showed that serum hGIIA specifically causes permeabilization of commensal E. faecium membranes. Altogether, we find that a normal serum concentration of hGIIA effectively kills commensal E. faecium and that hGIIA resistance of clinical E. faecium could have contributed to the ability of these strains to become opportunistic pathogens in hospitalized patients.
... This iron-binding blood plasma glycoprotein controls the level of free iron in biological fluids. Transferrin has a bacteriostatic activity, a property assigned to its iron-binding function (10), thus chelating the available iron necessary for bacterial growth. Transferrin is also considered an acute phase protein (APP) which acts in the inflammatory response to remove iron from the bloodstream (11). ...
Although several efforts have been made to describe the immunoendocrine interaction in fish, there are no studies to date focusing on the characterization of the immune response and glucocorticoid synthesis using the host–pathogen interaction on larval stage as an early developmental stage model of study. Therefore, the aim of this study was to evaluate the glucocorticoid synthesis and the modulation of stress- and innate immune-related genes in European sea bass (Dicentrarchus labrax) larvae challenged with Vibrio anguillarum. For this purpose, we challenged by bath full-sibling gnotobiotic sea bass larvae with 10⁷ CFU mL⁻¹ of V. anguillarum strain HI 610 on day 5 post-hatching (dph). The mortality was monitored up to the end of the experiment [120 hours post-challenge (hpc)]. While no variations were registered in non-challenged larvae maintained under gnotobiotic conditions (93.20% survival at 120 hpc), in the challenged group a constant and sustained mortality was observed from 36 hpc onward, dropping to 18.31% survival at 120 hpc. Glucocorticoid quantification and expression analysis of stress- and innate immunity-related genes were carried out in single larvae. The increase of cortisol, cortisone and 20β-dihydrocortisone was observed at 120 hpc, although did not influence upon the modulation of stress-related genes (glucocorticoid receptor 1 [gr1], gr2, and heat shock protein 70 [hsp70]). On the other hand, the expression of lysozyme, transferrin, and il-10 differentially increased at 120 hpc together with a marked upregulation of the pro-inflammatory cytokines (il-1β and il-8) and hepcidin, suggesting a late activation of defense mechanisms against V. anguillarum. Importantly, this response coincided with the lowest survival observed in challenged groups. Therefore, the increase in markers associated with glucocorticoid synthesis together with the upregulation of genes associated with the anti-inflammatory response suggests that in larvae infected with V. anguillarum a pro-inflammatory response at systemic level takes place, which then leads to the participation of other physiological mechanisms at systemic level to counteract the effect and the consequences of such response. However, this late systemic response could be related to the previous high mortality observed in sea bass larvae challenged with V. anguillarum.
... Presumably, these immune-mediated regulations of Fpn1, Tf, Tfr1 and Dtm1 have evolved during evolution to limit the availability of iron for extracellular microbes. The ability of highly virulent bacteria such as Yersinia pestis, Bacillus anthracis or Klebsiella pneumoniae to cause bacteraemia may have provided some of the evolutionary force to drive hypoferraemia as adaptive response [163][164][165][166][167]. Also, Gram-positive cocci including Streptococcus pneumoniae are able to use multiple iron sources and hypoferraemia impairs their growth [168]. ...
The acute-phase response is triggered by the presence of infectious agents and danger signals which indicate hazards for the integrity of the mammalian body. One central feature of this response is the sequestration of iron into storage compartments including macrophages. This limits the availability of this essential nutrient for circulating pathogens, a host defence strategy known as ‘nutritional immunity’.
Iron metabolism and the immune response are intimately linked. In infections, the availability of iron affects both the efficacy of antimicrobial immune pathways and pathogen proliferation. However, host strategies to withhold iron from microbes vary according to the localization of pathogens: Infections with extracellular bacteria such as Staphylococcus aureus, Streptococcus, Klebsiella or Yersinia stimulate the expression of the iron-regulatory hormone hepcidin which targets the cellular iron-exporter ferroportin-1 causing its internalization and blockade of iron egress from absorptive enterocytes in the duodenum and iron-recycling macrophages. This mechanism disrupts both routes of iron delivery to the circulation, contributes to iron sequestration in the mononuclear phagocyte system and mediates the hypoferraemia of the acute phase response subsequently resulting in the development of anaemia of inflammation. When intracellular microbes are present, other strategies of microbial iron withdrawal are needed. For instance, in macrophages harbouring intracellular pathogens such as Chlamydia, Mycobacterium tuberculosis, Listeria monocytogenes or Salmonella Typhimurium, ferroportin-1-mediated iron export is turned on for the removal of iron from infected cells. This also leads to reduced iron availability for intra-macrophage pathogens which inhibits their growth and in parallel strengthens anti-microbial effector pathways of macrophages including the formation of inducible nitric oxide synthase and tumour necrosis factor.
Iron plays a key role in infectious diseases both as modulator of the innate immune response and as nutrient for microbes. We need to gain a more comprehensive understanding of how the body can differentially respond to infection by extra- or intracellular pathogens. This knowledge may allow us to modulate mammalian iron homeostasis pharmaceutically and to target iron-acquisition systems of pathogens, thus enabling us to treat infections with novel strategies that act independent of established antimicrobials.
... Antimicrobial mechanisms of transferrins and their selective inhibition of H þ -ATPase have been reported in Pseudomonas aeruginosa and Lactococcus lactis [34]. Human transferrin also possesses the antimicrobial activity against anthrax [35]. Moreover, goldfish [19] and carp transferrin [36] serve as the primary activating agents of macrophage antimicrobial responses. ...
... The sequestration of iron promoted by hemopexin and transferrin could explain the early antibacterial effect induced by immunization with EV76. In fact, exogenously elevating the level of the serum iron-binding protein transferrin was suggested to represent a therapeutic approach to preventing iron acquisition by pathogens, such as Staphylococcus aureus, Acinetobacter, Candida and B. anthracis, consequently improving the survival of the infected host (Rooijakkers et al., 2010;Lin et al., 2014;Bruhn and Spellberg, 2015). ...
Prompt and effective elicitation of protective immunity is highly relevant for cases of rapidly deteriorating fatal diseases, such as plague, which is caused by Yersinia pestis. Here, we assessed the potential of a live vaccine to induce rapid protection against this infection. We demonstrated that the Y. pestis EV76 live vaccine protected mice against an immediate lethal challenge, limiting the multiplication of the virulent pathogen and its dissemination into circulation. Ex vivo analysis of Y. pestis growth in serum derived from EV76-immunized mice revealed that an antibacterial activity was produced rapidly. This activity was mediated by the host heme- and iron-binding proteins hemopexin and transferrin, and it occurred in strong correlation with the kinetics of hemopexin induction in vivo. We suggest a new concept in which a live vaccine is capable of rapidly inducing iron nutritional immunity, thus limiting the propagation of pathogens. This concept could be exploited to design novel therapeutic interventions.
... Moreover, infections associated with MBL deficiency, such as S. pneumoniae and Mycoplasma infection, are also those that are commonly associated with pulmonary infection in CVID (Litzman et al., 2008). As Gram-positive bacteria, such as S. pneumoniae, are intrinsically resistant to complement mediated lysis (Rooijakkers et al., 2010), MBL deficiency may increase susceptibility to such infection via reduced complement mediated opsonization and subsequent phagocytosis. Cross talk between complement and tolllike receptors (TLRs), another crucial innate immune component, has been shown to drive the differentiation of Th-17 cells suggesting that complement deficiency may have a negative impact on both innate and adaptive immune responses (Song, 2012). ...
One of the most common and most severe forms of primary antibody deficiency encountered in the clinical setting is a heterogeneous group of syndromes termed common variable immune deficiency (CVID). This disorder is characterized by reduced immunoglobulin production and increased susceptibility to infection, particularly of the respiratory tract. Infection and subsequent immunological/inflammatory processes may contribute to the development of pulmonary complications such as bronchiectasis and interstitial lung disease. Immunoglobulin replacement and/or antibiotic therapy, to prevent infection, are routinely prescribed treatments. However, chronic lung disease, the major cause of morbidity and mortality in this patient cohort, may still progress. This clinical progression suggests that pathogens recalcitrant to currently prescribed treatments and other immunological defects may be contributing to the development of pulmonary disease. This review describes the potential role of microbiological and non-B cell immunological factors, including T-cells, neutrophils, complement, toll like receptors, and antimicrobial peptides, in the pathogenicity of chronic lung disease in patients with CVID.
... Apo-Tf is the denomination of its iron-depleted form; holo-Tf is the iron-loaded form; both forms are collectively named Tfs. Tf has been shown to exert both a bacteriostatic and bactericidal effect in vitro on a variety of microbial pathogens [53,54]. The antimicrobial activity of transferrin is conventionally related to the iron-depleted form but some studies demonstrated that the mechanism of its antibacterial activity could not be referred only to iron deprivation [55,56]. ...
Biofilm infections represent a new medical challenge that drives towards the discovery of new diagnostics and new drugs specifically designed for this purpose. All living organisms offer a huge source of compounds which represent the biochemical substrate of the biological competition on the Earth and can be used to this aim. We describe an innovative diagnostic tool to early diagnose medical device infections sustained by Staphylococci; then we list new compounds that modulate bacterial phenotype and reduce virulence without affecting bacterial viability so as to avoid the emergence of genetic resistances. These compounds are all derived from natural sources: prokaryotes, plants, and human body. From prokaryotes we studied new compounds extracted from different environmental bacterial species, including Antarctic species growing in extreme environments. We describe also the anti-biofilm properties of extracts obtained from plants well known since centuries in folk medicine. The humoral immune response is the source of the last anti-biofilm compound: transferrin (Tf), a protein derived from human plasma involved in inflammation and natural immunity. All these compounds can be used as scaffolds for the design of new drugs active on the sessile form of pathogens prevalent in human biofilm infections.
