The ferric aerobactin receptor IutA , a protein isolated on agarose column , is not essential for uropathogenic Escherichia coli infection

Corresponding Author: Ademilson Panunto-Castelo Universidade de São Paulo. Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto Departamento de Biologia Av. Bandeirantes, 3900 Bairro: Monte Alegre CEP: 14040-901, Ribeirão Preto, SP, Brasil E-mail: apcastelo@usp.br The ferric aerobactin receptor IutA, a protein isolated on agarose column, is not essential for uropathogenic Escherichia coli infection1


Introduction
The intestinal microbiota of mammals includes several species of bacteria (1) , which generally establish a relationship of mutualism with the host.The first step in the process of colonization of a host is the attachment of the colonizing agent on the epithelium of the colonized organism (2) .In Gram-negative bacteria, a wide variety of molecules or structures can function as adhesins, which promote better adhesion to the epithelial cell receptor (3) .E. coli is the most abundant of all microorganisms that colonize the gut of many animals, and rarely causes disease except in immunocompromised hosts or when the gastrointestinal barriers are broken.The role of adhesins and lectins in the symbiosis of E. coli with the host is not well known, in contrast to pathogenic E. coli, which has some fimbriae associated with virulence.After the pioneer work (4) that obtained the first direct evidence that E. coli contains lectin, some authors demonstrated that proteins with lectin property, such as type 1 and P frimbriae, were determinant virulence factors important to the process of colonization and infection, especially of the bladder epithelium.The type 1 fimbria is one of the Rev. Latino-Am.Enfermagem 2012 Mar.-Apr.;20(2):340-5.
most studied virulence factor of E. coli, present in 80% of UPEC strains (5) .Besides type 1 and P fimbriae, F17 fimbria was described as important to the pathogenesis of enterotoxigenic E. coli in ruminants (6) .
Since lectins have shown to be such important components in the colonization and infection processes by bacteria and because some mechanisms underlying E. coli virulence are not fully understood, we sought to identify other non-described lectins as molecular markers.
Molecular markers from pathogens have been useful to hasten diagnosis, prevention and treatment for many infectious diseases.In urinary tract infection (UTI), studies using molecular methods have shown to be essential to understand the epidemiology, because the background rate of UTI is so high, and many different organisms can be the ethiological agent of these infections.Furthermore, the bacteria that most commonly cause UTI are E. coli strains (7) , which comprise an extremely heterogeneous group (8) .UTI is a public health problem that requires the development of an effective treatment for prevention (9) .So, an understanding of the principles that guide such studies about molecular markers is essential for nurses in all areas of clinical practice, since these professionals are responsible for the planning, assessment and implementation of such strategies, leading to more effective assistance for patients and community.
In this work, we identified an agarose-binding protein of 75-kDa in E. coli as a candidate for a new protein with lectin-activity, with a putative role in E. coli virulence.This protein was identified as the precursor of the ferric aerobactin receptor (IutA), a siderophore receptor encoded by a gene from the plasmid pColV-30 (10) , which is associated with virulence of some E. coli strains (11) .We also evaluated the presence of iutA gene in UPEC strains to correlate with bacterial infection.

Identification of proteins
Coomassie blue-stained proteins were excised and trypsinized in the gel prior to analysis by 1D nLC-MS-MS, using a 4000 QTrap (Applied Biosystems) tandem MS system (Fingerprints Proteomics Facility, University of Dundee).The raw QTrap data were analyzed using Mascot (www.matrixscience.com).

Polymerase chain reaction (PCR) of iutA gene in UPEC strains
UPEC strains, which were isolated from urine of hospitalized patients, were collected in the Emergency Unit of the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, in the period from July to September 2009.These strains were submitted to PCR to detect iutA gene.The plasmid pPOC9-iutA, kindly provided by Dr. Michael O'Connell, Dublin City University, Ireland (13) , was used as positive control.The PCR assays was carried out with 20 μL of

E. coli presents an agarose-binding 75-KDa protein
To identify carbohydrate-binding proteins that could be specific for colonization we first cultured E. coli in minimum medium (DMEM) that mimics the gastrointestinal tract conditions (12) .When the supernatant lysate from E. coli was serially incubated with Sepharose CL-4B and carbohydrate-immobilized resins, we did not detect adsorbed proteins to Sepharose (Figure 1A, lane 1) or carbohydrate-immobilized resins (data not shown).
Based on the work of Kenny et al. (12) , who obtained an increase in the production of virulence factors of EPEC when the minimum medium was supplemented with salts, we cultured bacteria in DMEM, supplemented with 22 mM KH 2 PO 4 , 10 mM CaCl 2 , 0.25 mM Fe(NO 3 ) 3 or 22 mM KH 2 PO 4 plus 0.25 mM Fe(NO 3 ) 3 .The induction of Sepharose-binding proteins was observed only in bacteria grown in DMEM supplemented with CaCl 2 (Figure 1A, lane 3).In the preparation, we obtained a major protein of 75-kDa, followed by minor proteins of >97-kDa, ~ 85-kDa and a duplex of ~40-kDa.Because agarose is a support to immobilize carbohydrate, we first incubated the lisate with Sepharose CL-4B, followed by others resins.Because the 75-kDa protein bound Sepharose CL-4B, we were not able to detect proteins in other resins.

Detection of iutA gene in UPEC strains
Since IutA is expressed in conditions that mimic the site of E. coli colonization and has been associated with virulence (11) , we sought to determine the presence of the iutA gene in E. coli strains and correlated to bacterial infection.To detect the iutA gene in UPEC strains, initially, we performed an amplification reaction in an iutA-subcloned plasmid (pPOC9-iutA).As shown in Figure 2, lane +, amplified product had a size between 2,322 and 2,027 bp, consistent with the expected size of 2,202 bp for iutA gene.The pPOC9-iutA was used as positive control (Figure 2, lane +) in all reactions.
The gene was detected in 14 out of 37 UPEC isolates (data not shown).To confirm the amplifications, positive isolates were submitted to another amplification reaction (Figure 2).Though the samples of lanes 5 and 6 had a weak amplification, we validated that 14 UPEC isolates Landgraf TN, Berlese A, Fernandes FF, Milanezi ML, Martinez R, Panunto-Castelo A.
protein-carbohydrate interaction in this system.Though the glycosylation of proteins in bacteria is limited, other glycoconjugates are abundant (19) .These glycoconjugates could somehow interact with protein, like in organisms of higher complexity.Moreover, glycosylated siderophores have been found, such as salmochelin, although it is not clear why this modification occurs (20) .

Conclusion
The correlation between Sepharose-binding IutA and protein system for iron uptake of E. coli offers an interesting study of the participation of lectins in this process.The identification of novel carbohydratebinding proteins is an open field for many discoveries, as a possible involvement of these proteins in the tissue colonization process.In this study, we demonstrated that, although IutA might be important to the EPEC infection, it did not play an essential role in this process.
4) and sonicated on ice with 5 pulses of 30 seconds each, 60 Hz (Vibra-cell, Sonics & Materials Inc., Danbury, USA).Bacterial lysates were centrifuged at 10,000 × g for 15 minutes at 4°C.The lysate supernatants were submitted to two processes: a. serial incubation with Sepharose CL-4B and carbohydrate-immobilized beads (d-galactose, d -mannose, d -GlcNAc and lactose) for 1 hour at room temperature.After washing the beads with TBS to remove non-absorbed material, the beads were submitted to SDS-PAGE.b.Affinity chromatography on column of Sepharose CL-4B, bed volume of 1 mL, by using ÄKTA Purifier System (GE Healthcare Life Sciences, Uppsala, Sweden).The material adsorbed to the column was eluted with 1 M NaCl or 0.1% acetic acid.Chromatography was monitored by absorbance at 280 nm and the eluted fractions were collected in pools, concentrated and dialyzed against TBS, using Amicon centrifugal filters Ultra-15 (membrane exclusion size of 10-kDa) (Millipore, Billerica, USA), according to manufacturer instructions .Concentrated eluates were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and the gels were stained with Coomassie blue.
Landgraf TN, Berlese A, Fernandes FF, Milanezi ML, Martinez R, Panunto-Castelo A. reaction mixture, containing 1 colony of each tested strain, 1,25 μM of the primers 5'-AAATTCCATATGATG ATAAGCAAAAAG-3' and 5'-TTCAAGCTTTCAGAACAGCA CAGAGTAG-3', 0.2 mM of each deoxynucleoside triphosphate, 10X PCR buffer, 1.5 mM MgCl 2 , 2 U of Taq DNA polymerase (Fermentas, Burlington, Canada), performed in an Eppendorf Master Cycler Gradient Thermocycler (Eppendorf Scientific, Inc., Westbury, USA), as follows: 94°C for 5 min, 30 cycles at 94°C for 1 minute, 51°C for 1 minute and 72°C for 2.5 minutes and, at the end of these 30 cycles, 72°C for 5 minutes.Reactions were kept at 4°C until use.PCR products were analyzed on 1% agarose gel after ethidium bromide staining and the amplicons were identified based on the size of the amplified product.This work was approved by the Ethics Committee of the College of Nursing of Ribeirão Preto, University of São Paulo, protocol 0737/2006.

(
A) Lysate obtained from E. coli, which was grown in minimum medium (1) without additional supplementation with salts or supplemented with (2) KH 2 PO 4 , (3) CaCl 2 , (4) Fe(NO 3 ) 3 , (5) KH 2 PO 4 and Fe(NO 3 ) 3 , was incubated with Sepharose CL-4B.After washing, the beads containing adsorbed proteins were submitted to SDS-PAGE.Lysate from E. coli grown in minimum medium supplemented with CaCl 2 was also submitted to affinity chromatography on Sepharose.Adsorbed material was eluted with (B) 1 M NaCl and (C) 0.1% acetic acid, dialyzed and submitted to SDS-PAGE.Gels were stained with Coomassie Blue.Migration positions of molecular mass markers are shown on the left in kDa.

Figure 1 -
Figure 1 -Protein of 75-kDa from UPEC binds to Sepharose CL-4B in a manner dependent on Ca2 +