Klebsiella pneumoniae manipulates human macrophages to acquire iron
Philipp GrubwieserRichard HilbeClemens M. GehrerManuel GranderNatascha BrigoAlexander HoffmannMarkus SeifertSylvia BergerIgor TheurlManfred NairzGünter Weiß
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Background Klebsiella pneumoniae (KP) is a major cause of hospital-acquired infections, such as pneumonia. Moreover, it is classified as a pathogen of concern due to sprawling anti-microbial resistance. During infection, the gram-negative pathogen is capable of establishing an intracellular niche in macrophages by altering cellular metabolism. One factor critically affecting the host-pathogen interaction is the availability of essential nutrients, like iron, which is required for KP to proliferate but which also modulates anti-microbial immune effector pathways. We hypothesized, that KP manipulates macrophage iron homeostasis to acquire this crucial nutrient for sustained proliferation. Methods We applied an in-vitro infection model, in which human macrophage-like PMA-differentiated THP1 cells were infected with KP (strain ATCC 43816). During a 24-h course of infection, we quantified the number of intracellular bacteria via serial plating of cell lysates and evaluated the effects of different stimuli on intracellular bacterial numbers and iron acquisition. Furthermore, we analyzed host and pathogen specific gene and protein expression of key iron metabolism molecules. Results Viable bacteria are recovered from macrophage cell lysates during the course of infection, indicative of persistence of bacteria within host cells and inefficient pathogen clearing by macrophages. Strikingly, following KP infection macrophages strongly induce the expression of the main cellular iron importer transferrin-receptor-1 (TFR1). Accordingly, intracellular KP proliferation is further augmented by the addition of iron loaded transferrin. The induction of TFR1 is mediated via the STAT-6-IL-10 axis, and pharmacological inhibition of this pathway reduces macrophage iron uptake, elicits bacterial iron starvation, and decreases bacterial survival. Conclusion Our results suggest, that KP manipulates macrophage iron metabolism to acquire iron once confined inside the host cell and enforces intracellular bacterial persistence. This is facilitated by microbial mediated induction of TFR1 via the STAT-6-IL-10 axis. Mechanistic insights into immune metabolism will provide opportunities for the development of novel antimicrobial therapies.Keywords:
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Incubation of human erythroleukaemia K562 cells with Al-transferrin inhibited iron uptake from 59Fe-transferrin by about 80%. The inhibition was greater than that produced by a similar quantity of Fe-transferrin. Preincubation of cells for 6 h with either Al-transferrin or Fe-transferrin diminished the number of surface transferrin receptors by about 40% compared with cells preincubated with apo-transferrin. Al-transferrin did not compete significantly with Fe-transferrin for transferrin receptors and, when cells were preincubated for 15 min instead of 6 h, the inhibitory effect of Al-transferrin on receptor expression was lost. Both forms of transferrin also decreased the level of transferrin receptor mRNA by about 50%, suggesting a common regulatory mechanism. Aluminium citrate had no effect on iron uptake or transferrin-receptor expression. AlCl3 also had no effect on transferrin-receptor expression, but at high concentration it caused an increase in iron uptake by an unknown, possibly non-specific, mechanism. Neither Al-transferrin nor AlCl3 caused a significant change in cell proliferation. It is proposed that aluminium, when bound to transferrin, inhibits iron uptake partly by down-regulating transferrin-receptor expression and partly by interfering with intracellular release of iron from transferrin.
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Cell culture data have demonstrated that transferrin, the major iron (Fe) transport protein, is a necessary requirement for cellular proliferation. Evidence suggests that transferrin supports proliferation by providing Fe for critical cellular processes including DNA synthesis. Lymphocytes, similar to other cell types, respond to an increased Fe requirement during proliferation by increased synthesis and expression of surface transferrin receptors. Moreover, under transferrin-Fe-deplete conditions, certain lymphocyte lines exhibit other specialized adaptations that allow for sufficient Fe uptake to support cellular proliferation. These other adaptations include specialized transferrin synthesis and utilization of a transferrin-independent Fe uptake pathway. Lymphocyte proliferation is inhibited by agents that interfere with cellular Fe metabolism; these agents include Fe chelators, class 3a metals that bind to transferrin, and antibodies directed against the transferrin receptor. The data presented in this paper, demonstrate that differences in sensitivity to the effects of these agents are influenced by the amount of available transferrin-Fe and differences in the mechanisms that individual lymphocyte cell lines utilize to ensure adequate Fe uptake to support proliferation. These data support the hypothesis that these agents, if used appropriately, will be useful in the treatment of different lymphoproliferative disorders.
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The possibilities that the recycling of the transferrin receptor is a rate-limiting step in the efflux of endocytosed transferrin, and that the receptor functions as a trans-membrane Fe transporter were investigated in untransfected Ltk- cells and in cells transfected with different levels of DNA for wild-type, mutant and chimeric human transferrin receptors. The uptake of transferrin-bound Fe and non-transferrin-bound Fe(II), and the surface binding, endocytosis and recycling of transferrin were measured. In cells that expressed increasing numbers of surface transferrin receptors, the rate of Fe uptake increased at a slower rate than the number of receptors. By measurement of the rates of endocytosis and recycling of transferrin it was shown that this effect was not due to a deficiency of endocytosis, but to a slower rate of recycling as the receptor numbers increased. Hence, a restricted recycling rate of the transferrin receptor appeared to be responsible for the slower rate of Fe uptake by cells with high receptor numbers, presumably because one or more cytosolic components required for recycling were in limited supply. The rate of uptake of non-transferrin-bound Fe(II) was not influenced by the number of transferrin receptors present on the surface of the cells even though this varied more than 20-fold between the different cell lines. Hence, this investigation does not support the hypothesis that the receptors play a direct role in the transport of Fe(II) across cell membranes, as has been proposed previously [Singer, S. J. (1989) Biol. Cell 65, 1-5].
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