Alteration of the Iron Level in Iron-overloaded Mice by Magnetospirillum Gryphiswaldense

Magnetospirillum gryphiswaldense (MSR-1), a type of magnetotactic bacteria are capable of forming magnetosomes, which are nanometer-sized particles and membrane-enclosed organelles containing crystals of magnetite (Fe 3 O 4 ). For the synthesis of magnetosomes, bacteria require a signicant amount of iron. Iron is an essential element for most life on earth; however, the elevation of iron level leads to hemochromatosis.This research aimed to investigate whether MSR-1 has an impact on the alteration of iron levels in mammalian cells in vivo.Female BALB/c were received intraperitonealinjections of iron-dextran (100mg/kg b.w.) 2 times per week for four consecutive weeks, and the iron was allowed to equilibrate for 15 days. The MSR-1 (10 9 in 200 µl PBS)were injected into healthy (C+MSR-1) and iron overloaded (IO+MSR-1) mice. The control healthy (C+ no MSR-1) and control iron overloaded (IO+ no MSR-1) mice received PBS. After the administration of bacteria, the viable bacterial numbers were evaluated in mice. Iron levels in serum and liver, as well as urinary iron exertion, were detected. Our MSR-1


Introduction
Magnetotactic bacteria (MTB), such as the α-proteobacterium Magnetospirillum gryphiswaldense (MSR-1), are motile and aquatic microorganisms that have a highly controlled bio-mineralization process to synthesize well-ordered nano-sized particles with perfect magnetic (Fe 3 O 4 ) and crystalline properties which allow the cells to align and to navigate along the geomagnetic eld [1,2]. MSR-1 accumulates iron (up to 4% of the dry weight) from the environment. It uses to biomineralize up to 60 magnetites (Fe 3 O 4 ) cuboctahedral crystals with a diameter of approximately 30 to 50 nanometers per cell [1,3,4]. These amounts indicate that MSR-1 has very e cient biological systems for uptake, transport, and precipitation of iron [3,5]. Based on Mössbauer spectroscopy and biochemical analyses, it was suggested the magnetite bio-mineralization pathway begins with iron uptake either as Fe 2+ or Fe 3+ from the environments, followed by a biochemical pool of iron composed of ferritin and Fe 2+ within the cell [3,6,7]. In MSR-1, the iron uptake rate for Fe 3+ is approximately 7-fold higher than that for Fe 2+ at the same extracellular concentration [8].
Iron is a biologically important trace element which required for many metabolic pathways (i.e., DNA synthesis, cell growth, respiration) in living organisms [2,9]; however, an increase of iron level in the body lead to iron-overloaded disorders such as hemochromatosis, is a disease in which too much iron builds up in the body [10]. Iron overload is an inevitable problem in patients because the body has no active mechanism to excrete excess iron [11]. Extra iron can cause damage in several organs such as spleen, liver, heart, and lymph nodes [10]. However, studies have demonstrated that iron-chelating drugs reduce organ damage and improve life expectancy in these patients [12]. In this study, the potential uptake of iron in mice by MSR-1 was investigated.

Animals
Female BALB/c mice (18-22 g, 4-6 weeks) were purchased from the laboratory animal center of Pasteur Institute of Iran. The Institutional Animal Care and Use Committee of Pasteur Institute of Iran (authorization no. 92/0201/3498) approved experiments involving mice. Mice were placed in polypropylene cages with stainless steel lids at room temperature with a 12-hour light/dark cycle. The animals had free access to standard pellet chow and drinking water, and body weights were measured weekly throughout the study period. The mice (n = 120) were divided into four groups, i. normal mice without MSR-1 ii. normal mice with MSR-1 iii. iron-overloaded mice with MSR-1 and iv. Iron-overloaded mice without MSR-1. For iron overloading, the mice received an intraperitoneal injection of 100 mg/kg iron dextran (Sigma-Aldrich) twice a week for 4-consecutive weeks, whereas the control group received PBS buffer. Two weeks after the last injection, iron was allowed to equilibrate in the mice [13][14][15].

MSR-1 injection and bacterial clearance
For bacterial injection, MSR-1 cells were washed twice in PBS, then resuspended in PBS to give a nal concentration of 1 × 10 9 MSR-1 cells in 200 µL PBS buffer, which was then injected into the tail vein of the mice and as a control, PBS buffer was injected [19,20].
To detect the bacterial clearance, animals were sacri ced 24, 48, 72, and 96 hours after the rst MSR-1 injection. The spleen, liver, and lymph nodes were harvested, rinsed with sterile PBS, weighed, and homogenized. The samples were centrifuged (1,000 rpm, 5 minutes), and supernatants from each sample were diluted and plated in 5 ml of MSGM with 0.7% agar and incubated at 28 °C for ten days [20,21].

Detection of the iron level in plasma and liver
The blood samples were collected by cardiac puncture into a centrifuge tube (3 iron-overloaded mice per each time point) [22]. Blood collection was performed between 8 a.m. to 10 a.m, and serum was obtained following centrifugation at 2,000 rpm for 20 minutes. The serum iron level was determined by the standard kit (Pars Azmoon, Tehran, Iran) with 5 µg/dl sensitivity.
The total iron-level was detected by using a wet washing procedure in the liver. The wet weight of the harvested liver was obtained (~ 100 mg), and each one was placed in 2 ml glass vials. The livers were desiccated at 65 °C for 24 hours, then 200 µL of an acid mixture (chloric acid: nitric acid 2:1) was added.
After drying for 5 to 6 hours at 182 °C, 1 ml of 3 M HCl was added and mixed. A 50 µl aliquot of the acidi ed sample was incubated for 30 minutes with 200 µl of chromogen (1.6 mM bathophenanthroline, 2 M sodium acetate, and 11.5 mM thioglycolic acid). The absorbance of the samples and iron standards was performed at 600 nm, measured in triplicate, and the mean values were used for calculating the total amounts of iron in the liver [23].

Analyze of urinary iron extraction
The animals were placed in individual metabolic cages 24 hours before starting the experiment, and during this time, urine was collected and used as an initial reference point for each mouse. After injecting MSR-1 into the iron-overloaded mice, their urine was collected every 24 hours for ten days and then analyzed by using a ame atomic absorption spectrophotometer (Perkin-Elmer Model A Analyst 300) [24].

Statistical analyses
All of the results were measured in a minimum of 3 mice per experimental point. Comparisons between groups were analyzed by t-student test. For all comparisons, results with p values < 0.05 were considered signi cant. Statistical analyses were performed using Graphpad Prism 5.0 (Graphpad Software, San Diego, CA).

Results
For the iron-overloaded mice, no death occurred during the administration of iron dextran, and no weight gain was observed in all groups (2.5 ± 0.5 g). Figure 1 illustrates the alteration of iron levels in serum and liver after administration of iron dextran. Serum iron and total liver iron levels increased during the four weeks of iron dextran administration compared with control animals that received PBS (non-iron overloaded mice). The total liver iron levels remained constant for two weeks after the nal injection of iron dextran in the IO group.
The number of colony-forming units (CFUs) recovered in the liver and spleen peaked 24 hours after the rst injection and then decreased to nearly zero by 72 hours, and after by 96 hours, there were no CFUs of MSR-1. The lymph nodes had virtually no CFUs during the entire experiment (Fig. 2). Figure 3a shows that serum iron levels were decreased in Cont.+MSR-1 compare to Cont.+ no MSR-1 (by 42.1%), and a similar pattern was also observed in IO + MSR-1 versus IO + no MSR-1 after 24 hours postinjection. The serum iron levels in IO + MSR-1continued to be signi cantly less than IO + no MSR-1 during the ten days. However, the serum iron level in Cont. +MSR-1 group returned to the normal level after 96 hours post-injection.
The same pattern was observed in the total liver iron concentrations after biopsy during the rst three days post-MSR-1 injection compare to no MSR-1 group (Fig. 3b). Moreover, the total iron levels in IO + MSR-1 were continued to be lower until the 10th-day post-injection, although it did not reach statistical signi cance.
After MSR-1 injection, the urinary iron excretion was analyzed. The iron levels were slightly increased (between 0 to 24 hours) after MSR-1 injection; however, iron excretion level was signi cantly increased after 24 hours and continued until 96 hours after post-injection compared to groups with no MSR-1. The iron excretion levels in IO and control mice that received MSR-1 were similar ( Figure. 3c).

Discussion
In this preliminary study, we try to understand whether MTB can uptake and accumulate iron from the animal body. This investigation was essential to understand the behavior of bacteria toward iron in in vivo environment. MTB have a remarkable potential to accumulate a large amount of iron in different environments and may play a key role in the regulation of the iron cycle in sedimentary environments, in which this ability is based on magnetosomes [25]. Bacteria require large amounts of iron to synthesis their magnetosomes; they may accumulate up to 100 times more iron than non-magnetotactic heterotrophic bacteria [26,27].
In this study, to obtain the iron overloaded animal model, iron dextran was administrated twice a week for four consecutive weeks, and the iron level was measured as it was described earlier in mice [23] and rats [33]. As it is known, initially, iron dextran is taken up by the reticuloendothelial system, but then equilibrates with the parenchymal system [28]. In an earlier study by Moon et al., the author suggested a chronic iron overloaded model by administration of iron dextran for 2-8 weeks and showed that mice were not dead and adjusted well up to the cumulative iron dose of 300 mg and long term survival was con rmed [29].
To investigate whether bacteria can uptake the iron in in vivo, MSR-1 was injected to iron overloaded mice, and normal mice and alteration of the iron level in plasma and liver were measured and compared with iron overloaded mice and normal mice which received only PBS. In a study by Benoit et al., Magnetospirillum magneticum (AMB-1) were injected intrathecal (i.t.) and intravascular (i.v.) to mice in order to visualize implanted tumors with magnetic resonance imaging by bacteria [30]. The authors showed that bacteria were cleared completely from other organs after six days of post-injection and remained only in tumors [30]. Setayesh et al. injected MSR-1, either i.v., peritoneal (i.p.) and subcutaneous (s.c.), and the clearance of MSR-1 in healthy mice was investigated [20]; the authors showed that mice clear MSR-1 from their body with different routes of injection after 96 hours. In the same study, the serum iron level was measured to understand by which routs of injection; bacteria effect on iron level [20].
In this experiment, after i.v. injection of MSR-1 to mice, serum iron, and total liver iron levels were decreased. The reduction of iron levels were highly signi cant during the rst three days (p < 0.05).
Notably, the percentages of serum and total liver iron reduction were similar in IO + MSR-1 and in Cont.+MSR-1, which may explain the amount of iron taken by the same number of bacteria (10 9 in 200 µl PBS) are a consistent amount. Moreover, the CFUs of MSR-1 after 96 hours were zero in the liver, spleen, and lymph nodes that may explain the sharp reduction of iron levels during the rst three days after the entrance of bacteria into the animal body. Therefore, it may conclude that as long as there are alive MSR-1 in mice (during the rst three days), iron levels in plasma and liver were reduced. The urinary iron excretion was higher between 24 to 96 hours after MSR-1 injection, and in parallel, the number of bacteria in mice was at the highest level in 24 hours post-injection, and it gradually reduced until 96 hours postinjection which we could not detect bacteria in liver, spleen and lymph nodes (see Fig. 3).
In this study, we reported the ability of iron uptake by MSR-1 in in vivo. MSR-1 has the potential to help to design a better biological tool that can multiply the effectiveness of the treatment of iron overloaded disorders. Therefore, our preliminary study opens the question of whether the bacteria can be a better replacement for chemical iron chelation drugs. However, our knowledge concerning the physiological characteristics and mechanism of iron uptake by MSR-1 in in vivo remains fragmentary. There are more studies warranted to understand the mechanism of bacterial clearance and cytotoxicity and genotoxicity effects of bacteria as well as an immune response in in vivo model. We also recommend future studies on optimizing the biological magnetic systems.

Conclusion
We found that MSR-1 signi cantly reduce the iron level in iron overloaded mice and increase the urinary iron excretion.

Declarations
Availability of data and materials All data generated or analysed during this study are included in this published article The authors declare that there is no con ict of interest regarding the publication of this article. Changes in the serum iron level (a) and the liver total iron concentration (b) in mice during the iron overloading process. In iron overloaded mice, serum iron increased, and total liver iron increased during the 4 weeks of iron dextran administration and remained constant for two weeks after the nal injection.

Figure 2
CFU per tissue after MSR-1 injection, the number of CFUs recovered in the liver and spleen peaked 24 hours after the rst injection, then decreased to nearly zero by 72 hours, and by 96 hours, there were no CFUs. The lymph nodes had virtually no CFUs during the entire experiment.