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 ~  Abstract
 ~  Materials and Me...
 ~  Liquid EMJH cont...
 ~  Liquid EMJH cont...
 ~  Results
 ~  Discussion
 ~  Acknowledgement
 ~  References

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Year : 2004  |  Volume : 22  |  Issue : 2  |  Page : 92-96

Iron-regulated proteins (IRPS) of leptospira biflexa serovar Patoc strain Patoc I

Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad - 500 046, India

Correspondence Address:
Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad - 500 046, India

 ~ Abstract 

BACKGROUND: Iron deficiency has been shown to induce the expression of siderophores and their receptors, the iron-regulated membrane proteins in a number of bacterial systems. In this study, the response of Leptospira biflexa serovar Patoc strain Patoc I to conditions of iron deprivation was assessed and the expression of siderophores and iron-regulated proteins is reported. MATERIALS AND METHODS: Two methods were used for establishing conditions of iron deprivation. One method consisted of addition of the iron chelators ethylenediamine-N, N'-diacetic acid (EDDA) and ethylenediamine di-o-hydroxyphenylacetic acid (EDDHPA) and the second method involved the addition of iron at 0.02 g Fe/mL. Alternatively, iron sufficient conditions were achieved by omitting the chelators in the former method and adding 4 g Fe/mL of the medium in the latter protocol. Triton X-114 extraction of the cells was done to isolate the proteins in the outer membrane (detergent phase), periplasmic space (aqueous phase) and the protoplasmic cylinder (cell pellet). The proteins were subjected to SDS-PAGE for analysis. RESULTS: In the presence of the iron-chelators, four iron-regulated proteins (IRPs) of apparent molecular masses of 82, 64, 60 and 33 kDa were expressed. The 82-kDa protein was seen only in the aqueous phase, while the other three proteins were seen in both the aqueous and detergent fractions. These proteins were not identified in organisms grown in the absence of the iron chelators. The 64, 60 and the 33 kDa proteins were also demonstrated in organisms grown in media with 0.02 g Fe/mL. In addition, a 24 kDa protein was found to be down-regulated at this concentration of iron as compared to the high level of expression in organisms grown with 4 g Fe/mL. The blue CAS agar plates with top agar containing 0.02g Fe/mL showed a colour change to orange-red. CONCLUSION: The expression of siderophores and iron-regulated proteins under conditions of iron deprivation was demonstrated in the non-pathogenic L.biflexa serovar Patoc.

How to cite this article:
Sritharan M, Asuthkar S. Iron-regulated proteins (IRPS) of leptospira biflexa serovar Patoc strain Patoc I. Indian J Med Microbiol 2004;22:92-6

How to cite this URL:
Sritharan M, Asuthkar S. Iron-regulated proteins (IRPS) of leptospira biflexa serovar Patoc strain Patoc I. Indian J Med Microbiol [serial online] 2004 [cited 2020 Nov 26];22:92-6. Available from:

Iron is an essential micronutrient for almost all organisms, playing an important role in electron transport and in intermediary metabolism as a cofactor for many enzymes. Iron containing reductase, catalysing the formation of 2'deoxyribonucleotides needed for DNA synthesis is ubiquitously present in all organisms from  E.coli   to humans, except in lactobacilli that are devoid of haeme and do not need iron for growth.
Iron, by virtue of its chemical nature does not exist in a freely available soluble form. In the inorganic environment, iron exists as the insoluble ferric hydroxide and in the mammalian host it is complexed to proteins and held as protein bound iron both in the circulation as well as in the tissues, where excess iron is stored. Microorganisms have adapted to this low iron environment by the induction of novel pathways for obtaining iron. One of the common and well-studied mechanisms is the production of siderophores and their receptors, the iron-regulated membrane proteins.[1],[2] Another equally important mode of iron withdrawal is the direct removal of the protein bound iron by the elaboration of specific receptors for these protein molecules, as seen in  Neisseria More Detailse spp. which produce transferrin and lactoferrin specific receptors.[3] At the molecular level, iron controls the expression of virulence factor(s) in many microorganisms like Corynebacterium diphtheriae,  Escherichia More Details coli, Pseudomonas aeruginosa and other microorganisms.[4] For example, the tox gene expression in  C.diphtheriae   occurs only upon iron deprivation and is switched off by the dtxR repressor-iron complex when the level of iron increases.
Leptospirosis, a bacterial zoonoses is a re-emerging disease of considerable public concern. This disease is caused by the spirochaetes belonging to the genus Leptospira, which is comprised of the non-pathogenic Leptospira biflexa and the pathogenic Leptospira interrogans, the latter consisting of over 220 serovars. It is well known that many members of the pathogenic  L.interrogans   cause hemolysis. They elaborate the virulence factor hemolysin. The recently sequenced genome of the pathogenic L.interrogans serovar Lai5 is shown to contain five hemolysin genes. Little is known about the acquisition of iron by these microorganisms. Also, the probable link about iron levels and hemolysin expression is not known and remains to be investigated.
In this study, we report the expression of unique proteins under conditions of iron starvation in the non-pathogenic L.biflexa serovar Patoc strain Patoc I.

 ~ Materials and Methods Top

Growth conditions
Leptospira biflexa serovar Patoc strain Patoc I (obtained from the National Leptospiral Repository Facility in Port Blair, Andaman and Nicobar Islands, India) was maintained in the standard liquid EMJH-enrichment medium (Difco) that is employed for the growth of leptospires. They were sub-cultured into EMJH medium with BSA (Sigma, Fraction V) and in this study, they were subjected to growth using the two conditions described below. The bacteria were grown as shake cultures for 7 days. Procedures for preparation of iron-free glassware and medium were done similar to the already established methods for mycobacterial work.[8]

 ~ Liquid EMJH containing 0.1% BSA Top

The bacteria were gradually acclimatised to growth from medium with 1% BSA to growth with 0.1% BSA. Glassware was made iron free and water used for preparation of the medium was autoclaved with alumina. Iron was added at concentration of 4 g Fe/mL for iron sufficient growth and 0.02 g Fe/mL for iron deficient growth.

 ~ Liquid EMJH containing 0.1% BSA plus EDDA and EDDPHA as chelators Top

The medium was prepared as above and iron was added at 4 g Fe/mL. Bacteria were grown in three separate aliquots of the above medium. To two separate aliquots was added 100 M each of ethylenediamine-N, N'-diacetic acid (EDDA, Sigma) and ethylenediamine di-o-hydroxyphenylacetic acid (EDDHPA, ICN BioMedicals) respectively and the third, without any chelator served as medium for growing the cells in iron sufficiently.
Triton X-114 detergent extraction and preparation of proteins for analysis by SDS-PAGE
Triton X-114 extraction was done based on the method of Haake et al.[7] The bacterial cells were harvested by centrifugation at 3000 x g, washed twice with phosphate buffered-saline-5mM MgCl2 and then incubated overnight at 4C in 1% Triton X-114 in 10mM Tris (pH 8.0) and 1 mM phenylmethlsulphonyl fluoride. The insoluble material (cell pellet) was separated by centrifugation at 17,000 x g for 10 min. The TritonX-114 concentration of the supernatant was increased to 2%. Phase separation was performed by warming the supernatant to 37C and subjecting it to centrifugation at 2000 x g. The proteins in the detergent and the aqueous phases were precipitated with acetone.
Electrophoretic separation of the proteins by SDS-PAGE
The proteins in the insoluble cell pellet, detergent phase and the aqueous phase were subjected to discontinuous SDS-PAGE separation.[9] The samples were boiled for 5 min. in the sample buffer (60 mM Tris-HCl pH 6.8-10% glycerol-1% SDS-2% mercaptoethanol-0.1% bromophenol blue). The samples were run on 10% gels and visualised after staining with Coomassie Brilliant blue.
Chrome azurol S (CAS) agar plates
The blue CAS agar plates were prepared by the method of Schwyn and Neilands[6] with some modifications. 60.5 mg CAS (Fluka) was dissolved in 50 mL water and mixed with 10 ml of 1 mM ferric chloride solution (1mM FeCl3.6H2O in 10 mM HCl). This solution was slowly added to 72.9 mg HDTMA (hexadecyltrimethyl ammonium bromide, Sigma) dissolved in 40 ml of water. The resultant dark blue liquid was autoclaved. Simultaneously, EMJH medium containing 2% Difco Bacto agar was autoclaved. After cooling the solutions to about 50C, one volume of the CAS solution was added to 9 volumes of the molten agar, swirled to mix without foaming and immediately poured into sterile petri plates to half-fill the plates. After the agar solidified, another layer of top agar containing only the agar solution (minus CAS) was poured over it. Thus, two layers of the agar can be seen, the lower containing the CAS and the top layer without it. It is necessary to maintain a thin layer of the top agar to appreciate the colour change from blue to orange. In order to achieve growth under iron sufficient and iron deficient conditions, iron was added either at 4 g Fe/mL (for iron sufficient growth) or 0.02 g Fe/mL (for iron deficient growth) to the molten top agar before pouring. The plates were inoculated with the bacteria from the corresponding liquid culture that was maintained as iron sufficient and iron deficient. The plates were incubated for 5 days and then photographed.

 ~ Results Top

Ability of L.biflexa serovar Patoc I to remove iron from CAS agar plates
When plated on CAS agar plates, L.biflexa serovar Patoc I was able to remove the dye bound iron. The [Figure - 1] represents the plates A and B that were photographed after 5 days of growth.
Plate A represents the cells plated on top agar containing 0.02 g Fe/mL (iron deprived) and Plate B represents the cells plated on top agar containing 4 g Fe/mL. Both the plates were initially blue in colour. They began to turn light green to orange and within 2-3 days of growth, cells in the Plate A developed a darker orange red colour. The blue colour disappeared in the plate B also within 3-4 days but the cells remained colourless even after 10 days of incubation.
Iron-regulated expression of proteins in L.biflexa serovar Patoc I
In the presence of the chelators EDDA and EDDHPA, L.biflexa serovar Patoc I expressed four proteins (iron-regulated proteins IRPs) of apparent molecular size 82, 64, 60 and 33 kDa whose synthesis was significantly lower in the control cells [Figure - 2]. The 82 kDa protein was seen in the aqueous extract [Figure - 2], lanes 2,3), while the other three bands were seen in the aqueous extract [Figure - 2], lanes 2,3) and the detergent extract of the EDDA and EDDHPA treated cells [Figure - 2], lane 5,6), with little or no expression in the control cells (lane 1 and 4 respectively).
In the alternate protocol in which the cells were grown in medium with 0.02 g Fe/mL, the 64, 60 and the 33 kDa proteins can be seen in the lanes 1 and 3 [Figure:3], representing the detergent and aqueous phases respectively. These proteins are still seen in the lanes 2 and 4, representing the detergent and aqueous extract in cells grown with 4 g Fe/mL. Also, a prominent 24 kDa band is found in both the detergent and aqueous extracts of cells grown in 4 g Fe/mL (lanes 2 and 4, [Figure:3], whose synthesis is significantly down-regulated when the iron levels are decreased to 0.02 g Fe/mL.

 ~ Discussion Top

This is a report on the influence of iron deprivation in L.biflexa serotype Patoc I. In the CAS agar plate with 0.02 g /mL of iron, the disappearance of the blue colour is seen along with the orange red colouring of the cells, while the cells remained colourless when iron concentration was raised to 4 g Fe/mL. Analysis of the Triton X-114 extracts of the cells grown in the presence of the iron chelators identified four proteins (iron-regulated proteins, IRPs) of apparent molecular size of 82, 64, 60 and 33 kDa under conditions of iron limitation. The 82 kDa was observed in the aqueous extract indicating it might be a periplasmic protein, while the other three proteins were seen in both the aqueous and detergent extract. Further work is needed to confirm the localization of these proteins.
In the organisms grown with iron added at 0.02 g Fe/mL, the expression of the 64, 60 and the 33 kDa proteins were seen [Figure:3]. However, their expression could be seen in cells that were grown with 4 g Fe/mL. This concentration of iron is still probably not sufficient to completely repress the expression of these proteins. At this concentration of iron, these cells expressed a prominent 24 kDa protein that was down-regulated at 0.02 g Fe/mL (lanes 2 and 4, [Figure:3]. This protein band was seen in both the aqueous and detergent extracts. Cullen et al,10 demonstrated the down-regulation of two proteins LipL36 and pL50 upon iron depletion and reported the failure of cleavage of LipL32/Hap-1, a protein associated with hemolysis of host cells. In view of their observations and our findings on the influence of iron concentration on the regulation of several proteins, further work is in progress in our lab to study the expression of some of these proteins in cells grown over a range of varying iron concentrations.
Intracellular iron concentration regulated the synthesis of the iron acquisition machinery in several microorganisms, not only under in vitro conditions but also in vivo.[2],[11],[13] Deprivation of iron has been shown to induce the expression of not only the machinery for iron acquisition but also exert a direct influence on the expression of the virulence factors.[14] Insight into the iron acquisition of pathogenic Leptospira could have important implications in the host-pathogen understanding and help in the development of a strategy for controlling the disease. Work is in progress in our lab with the pathogenic Leptospira to look for iron-regulated proteins. This will be of importance in the light of the virulence factor hemolysin produced by the pathogens, due to a possible link between iron availability and hemolysin expression.

 ~ Acknowledgement Top

The authors thank Andhra Pradesh-Netherlands Biotechnology Programme (Institute for Public Enterprise) for helping to establish the culture facilities for leptospirosis and the UGC-SAP, India for providing maintenance funds to the department. 

 ~ References Top

1.Braun V, Hantke K, Koster W. Bacterial iron transport: mechanisms, genetics and regulation. In: Metal Ions in Biological systems. Iron transport and storage in Microorganisms, Plants and Animals. Sigel, A and Sigel, H. Eds. (Marcel Dekker, New York) 1998: 67.  Back to cited text no. 1    
2.Sritharan M. Iron as a candidate in virulence and pathogenesis in mycobacteria and other microorganisms W J Microbiol Biotechnol 2000;16:769-780.  Back to cited text no. 2    
3.Owen SDG, Schryvers AB. Bacterial transferrin and lactoferrin receptors. Trends Microbiol 1996;4(5):185-190.  Back to cited text no. 3    
4.Griffiths E, Chart H. Iron as a regulatory signal. In: Iron and Infection: Molecular, Physiological and Clinical Aspects. Bullen JJ, Griffiths E. Eds. (John Wiley & Sons) 1999:213.  Back to cited text no. 4    
5.Ren SX, Fu G, Jiang XG, Zeng R, Miao YG, Xu H, et al. Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing. Nature 2003;422:888-893.  Back to cited text no. 5    
6.Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of siderophores. Anal Biochem 1987;160:47-56.  Back to cited text no. 6    
7.Haake DA, Martinich C, Summers TA, Shang ES, Pruetz JD, Mc Coy AM, et al. Characterisation of leptospiral outer membrane lipoprotein LipL36: Down-regulation associated with late-log phase growth and mammalian infection. Infect Immun 1998;66:1579-1587.  Back to cited text no. 7    
8.Hall RM, Sritharan, M, Messenger AJM, Ratledge C. Iron transport in M.smegmatis: occurrence of iron regulated envelope proteins (IREPS) as potential receptors for iron uptake. J Gen Microbiol 1987;133:2107-2114.   Back to cited text no. 8    
9.Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685.  Back to cited text no. 9    
10.Cullen PA, Cordwell SJ, Bulach DM, Haake DA, Adler B. Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai. Infection and Immunity 2002:70(5);2311-2318.   Back to cited text no. 10    
11.Payne SM, Lawlor KM. Molecular studies on iron acquisition by non-Escherichia coli species. In Molecular Basis of Bacterial Pathogenesis Iglewski BH, Clark VL Eds. (California Academic Press) 1990:225.  Back to cited text no. 11    
12.Neilands JB. Molecular biology and regulation of iron acquisition by Escherichia coli K12. In: Molecular Basis of Bacterial Pathogenesis. Iglewski BH, Clark VL Eds. (California Academic Press) 1990:205.  Back to cited text no. 12    
13.Sritharan M, Ratledge C. Iron-regulated envelope proteins of mycobacteria grown in vitro and their occurrence in Mycobacterium leprae grown in vivo. Biol Metals 1990;2:203-208.  Back to cited text no. 13    
14.Salyers AA, Whitt DD. In: Bacterial Pathogenesis. A Molecular Approach (New York ASM Press) 1994:250.  Back to cited text no. 14    
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