Iron is important factor for bacterial survival and many biological functions. Iron presents in one of two states: ferric [Fe(III)] or ferrous [Fe(II)] iron. In aerobic environment, iron usually present in an oxidized and relatively insoluble Fe(III) form. Less is understood that the facts ferrous iron should be predominant in niches within the host that is oxygen limited. The study of ferrous iron acquisition system should provide knowledge and insight on bacterial strategy in competing limited iron source within host cell. Sequence analysis of K. pneumoniae CG43 genome revealed three major ferrous iron uptake transporter-encoding gene clusters: feoABC, sitABCD and efeUOB. In this study, we analyzed the preferential expression of individual ferrous iron acquisition systems in response to different environment stimuli and their possible effects on K. pneumoniae growth and virulence-related properties such as oxidative stress response, biofilm formation and type 3 fimbriae expression.
Generally, individual or cumulative loss of Feo, Sit and Efe transporter proteins do not affect the growth of CG43S3. DIP, 2, 2’-dipyridy, is a high-affinity chelating agent for ferrous iron while DFX, deferoxamine is a siderophore produced from Stretomyces pilosus that function to chelate ferric iron [130-132]. The expression of feo is induced by DFX but not DIP suggesting Fe(II) transport system could be stimulated by the scarcity of Fe(III). As described in H. pylori, when Fe(III) supplied in the environment, uptake of Fe(II) by FeoB involved a Fe(III) reductase. This was further verified by a study in Leptospira spp, feoB mutant unable to transport Fe when iron sources were Fe(III)-dicitrate and iron sulfate. Thus, FeoB is involved in the uptake of Fe(III) as well as Fe(II) [133, 134].
In the promoter activity analysis, FeoC displayed a negative regulation on the feo expression under iron-depleted and static cultured condition. This suggests that FeoC
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represses feo expression when the environmental oxygen contents and iron concentration are low. However, the regulatory role on feo expression was not observed in S. enterica or V.
cholerae [53, 61]. The activity of Pfeo1 is much lower than Pfeo2 implies a negative regulatory element is located in the region. Sequence analysis of the region reveals a possibility of NagC repressor binding element. NagC which participates in regulating the E. coli phosphotransferase system is N-acetylglucosamine regulator. The regulatory role of NagC on feo expression is pending for verification.
In either of Pfeo, Psit or Pefe, a relatively conserved Fur box could be found. The promoter activity analysis further supports a negative role of Fur in the expression of feo, sit and efe. A recent report in CG43 suggested an involvement of small RNA, RyhB in SitA transcription. If RyhB plays a negative role in sit expression remained to be investigated for Psit activity in ∆ryhB or ∆ryhB∆fur background. The level of promoter activity is ranked Psit>
Pefe> Pfeo2>Pfeo1. An ArcA box located in Psit might explain the microaeribic induction of sit expression. However, the study in S. enterica showed transcriptional activity of sitABCD was not affected by the deletion of arcA or fnr. Highest expression of feoABC, sitABCD and efeUOB occurred in stationary phase, whether the involvement of growth-regulated elements (e.g. sigma S factor, rpoS) requires further study.
Fe(II) is known to predominant under acidic and reducing environment. It’s reasonable to speculate these Fe(II) transporter would have a potential role under acidic media. EfeUOB system is an acid inducible and CpxR-regulated Fe(II) transporter identified in E. coli O157:H7 [46]. A single-base-pair deletion in efeU caused efeUOB cryptic in E. coli K12. In another word, EfeUOB was particularly function in E. coli O157:H7. To investigate whether EfeUOB is cryptic in KPCG43, sequence alignment as shown in Appendix 3
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revealing that the amino acid similarities of EfeU, EfeO and EfeB between CG43 and O157:H7 are 82.9 %, 86.9 % and 82.9 % respectively. The gene organization analysis showed the flanking genes are the phosphate starvation gene phoH (56.8% amino acid similarity) and pgaABCD in E. coli [135]. However, pgaABCD is located somewhere in CG43 genome. Consistent with E. coli, Pefe of CG43S3 is induced by weak acid (pH 5) and cpxR deletion. The microaerobic-repressed Pefe expression implies EfeUOB plays an important role in the presence of oxygen. Unlike result from promoter activity of this study, contrasted observation was found in a comparative transcriptome analysis of CG43S3 which using RNA-sequence approach to identify the genes responded to acid stress (pH 3) [136].
Among the significantly down-regulated genes, the expression of D364_05435 (EfeO) was 6.26-fold decreased. This could be resulted from different pH treatment (pH 5 versus pH 3) or different roles of EfeO between the two bacteria.
In summary, all three systems are induced by iron-depletion (low Fe) but negatively regulated by Fur-Fe2+. FeoC possibly represses expression of feo via the iron-sulfur cluster when both iron and oxygen are depleted (low Fe and low O2). CpxAR represses efe expression when CG43S3 cultured in a rather alkaline environment (OH-), i.e., when the environment switches to acidic, CpxAR repression on efe expression is released, leading to the uptake of ferrous iron. Increased expression of feo, sit and efe were observed by addition of Mn2+. The de-repression of Fur is controlled by a manganese-dependent regulator, PerR [137]. When Mn2+ becomes excess, Mn2+-PerR represses the expression of fur, subsequently releases the repressing effect of Fur on these iron-acquisition systems (Fig.21).
Accumulation of excess Fe(II) is hazardous to bacteria since it lead to formation of ROS when participating in Fenton reaction. Mn is required for some enzymes involved in
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oxidative stress response proteins, such as superoxide dismutase SodA and the non-heme catalase KatN. [138, 139]. The potential of Mn/Fe transporter SitABCD in protection against oxidative stress was studied by H2O2 stress response. From the oxidative stress response study, SitABCD may be more important in protecting from H2O2 stress, especially in the depletion of divalent transition metal cations. As reported in Salmonella [12], the loss of sit and feo resulted in increased-sensitivity to oxidative stress. The interruption of Mn and Fe hemostasis triggered by loss of multiple Fe(II) transporter proteins might be the major reason in causing an oxidative-sensitive phenotype. On the other hand, aerobically grown ∆feoC loaded with DIP showed decreased bacterial oxidative stress resistance further supports a negative role of FeoC for Feo system, which is induced by iron-depletion and oxygen-depletion. In this study, the inhibition zone formed in disk assay did not show significant difference from each other. A quantitative assay such as measurement of survival rate between treated bacteria versus untreated bacteria might provide more convincing evidence of their role in oxidative resistance.
Biofilm formation in bacteria was found very much dependent on iron availability.
Our study showed deletion of these iron acquisition genes decrease CG43S3 biofilm formation ability. Yet, the combined deletion of these genes did not show gradient decrement of biofilm formation. Nevertheless, deletion of sitCD and efeUOB rendered an increase of biofilm formation. FeoB may also be the sole Fe(II) uptake system under iron depletion condition. Increasing intracellular Fe levels by FeoB permease leads to escalate biofilm formation. Under iron- and oxygen-depleted condition, ∆feoC exhibits increasing feoAB expression which may in turn increase Fe(II) uptake by feoB and consequently increase biofilm formation.
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The availability of iron could activate expression of type 3 fimbriae and also the biofilm formation in CG43S3 [26]. However, none of the Fe(II) transporter systems had significant effect on the MrkA expression. It is interesting that ∆feoB in iron-depleted condition enhanced MrkA expression. The loss of feoB may induce iron-acquisition related element(s) to enhance MrkA expression. It is possible that the regulation of these iron acquisition system-mediated biofilm formation is independent to the iron-regulated MrkA expression. Moreover, manganese may also play an important role in regulating type 3 fimbriae since the loss sit (∆sitCD and ∆ESB) increased MrkA expression.
From the results above, a proposed functional model of these systems is suggested (Fig. 22). The ferrous iron-acquisition is mediated by FeoB which contains a cytoplasmic GTPase domain to hydrolysis GTP to GDP; cytoplasmic FeoC senses environmental oxygen and iron to regulate feo system; the role of FeoA is unclear yet (this model is based on a model proposed by Cartron et al. [49]). Transport of ferrous iron or/ and manganese by permease SitC and SitD is activated by the periplasmic SitA which in turn facilitated by SitB that hydrolyze ATP to ADP (this model is based on their structural analysis). The acid-inducible EfeU uptakes ferrous iron with the assistance of EfeO and EfeB (the real mechanism is little known in Gram-negative bacteria). All three systems affect the formation of biofilm in CG43S3 while SitABCD protects CG43S3 against H2O2. In a Western blot analysis, FeoB and Sit system negatively regulate the production of major pilin of type 3 fimbriae in iron-depleted (low Fe) or / and microaerobic condition (low O2)
In most of the assessments performed, the addition of DIP caused slower growth rate in compared to general cultured condition. Hence, the toxic effect of DIP toward bacterial growth has to be taken into consideration. The divalent-cations chelator EDTA or
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chelating agent ferrozine may be better agent to create an iron-depleted condition. Besides, an alternative reporter system to determine ferrous iron level in cell could provide a better understand of these system. The quinone antibiotic, streptonigrin (SNG) binds specifically to iron and promotes formation of hydroxyl radicals [140]. Hence, it has been used to select mutant of defective in iron-uptake in E. coli [141]. SNG could serve as an indicator of Fe level in deletion mutants, where deletion mutants will grow better than wild-type strain when treated with SNG. However, the thick mucoid capsule formed by CG43 protects it from harsh oxidative stress, a higher concentration of SNG might be needed.
Iron availability affects bacterial biological function in many ways. Environmental iron availability apparently affects the expression of bacterial iron-acquisition system. On the other hand, the intracellular Fe(II) level in bacteria may affecting their biological functions as well. The homeostasis of Fe in bacteria is tightly regulated and hence the intracellular Fe deficiency caused by deletion of the ferrous iron transport system may very likely be compensated by other ferric iron acquisition systems.
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