Indian Journal of Experimental Biology Vol. 42, July 2004, pp.68 1-685

Effect of trace elements on surface hydrophobicity and adherence of Escherichia coli to uroepithelial cells

Vishwas Saralaya K, Gopalkrishna Bhatt, Asha Kamath & P G Shivananda*

Departments of Microbiology & Community Medicine, Kasturba Medical College, Mangalore, 575 001, India *Department of Microbiology, Manipal College of Medical Sciences, Pokhra, Nepal

. Received 27 February 2004

Trace elements have significant effect on the physiology of bacteria. Variation in the concentration of trace elements may affect the expression of virulence by microorganisms. The effect of trace elements on hydrophobicity and adherence of E.coli to uroepithelial cells was studied. Increasing concentrations of Ca2+, Mg2+, Fe3+ and Zn2+ significantly decreased the surface hydrophobicity. Toxic trace elements like C02+, Cu2+, Mn2+. and Ni2+ did not alter surface hydrophobicity. With re­gards to adherence of E.coli to uroepithelial cells, only Mg2+ had significant effect. Toxic trace elements decreased the rate of cell adherence. The pathogenic strains of E.coli showed higher surface hydrophobicity and better cell adherence com­pared to the nonpathogenic strains. There was good correlation between surface hydrophobicity and cell adherence at higher concentrations (0.1 to 0.2mM) of Fe2+ and Zn2+. The results indicated that trace elements can significantly affect surface hy­drophobicity and adherence of E.coli to uroepithelial cells. Such effect may have a significant impact on the initial stages of bacterial infection.

Keywords: Escherichia coli, Trace elements, Surface hydrophobicity, Adherence

Adherence of micro-organisms to host tissues is an essential step in the pathogenesis of infectious dis­eases. The mechanisms by which bacteria adhere to host cells are many and varied, and may include lectin like interactions, hydrogen bonds, van der Waals forces, electrostatic and hydrophobic interactions I . The high hydrophobicity of the bacterial cell surface promotes their adherence to various surfaces like mu­cosal epithelial cells, phagocytes, cell cultures, non wettable plastic sUlfaces and even to hydrophobic surfaces such as liquid hydrocarbons2.

Pathogenic bacteria exhibit higher cell surface hy­drophobicity than commensals and this property is considered to play an important role in their adher­ence to host cell surfaces3

.4. Cell surface · structures which contain hydrophobic domains include fibro­nectin binding proteins, protein A, outer membrane adhesins and more importantly, the fimbriae of gram negative bacteria5

•6

. Certain specialized fimbriae such as the P pili, F fimbriae, colonization factor antigens I, II, and IV impart upon the bacteria exhibiting them with a certain advantage over others in their ability to adhere to specific receptors on the epithelial cells of

tCorresponding author E-mail: gkbhat61@ yahoo.co.in

mucosal surfaces7. This step is essential for coloniza­

tion and infection. Divalent cations have been shown to affect the

composition, envelope properties and production of fimbriae in various bacteria8

.9

. Metal ions are also re­quired for the stabilization of the ce.ll envelope, espe­cially the outer membrane of gram negative bacteria lO

•

Several metal ions can affect cell surface hydropho­bicity and adherence properties in bacterial 1.12.

The present study was conducted to determine the effect of trace metals on surface hydrophobicity and adherence of Escherichia coli to uroepithelial cells.

Materials and Methods Bacteria - E.coli strains MTCC 729, MTCCI687,

VI05 and F23 were used in the study. The strains MTCC 729 and MTCC 1687 were obtained from the "Microbial Type Culture Collection & Gene Bank", Institute of Microbial Technology, Chandigarh, India. E.coli MTCC 729 is an uropathogen with F7 fimbriae and mannose resistant hemagglutination (MRHA). MTCC 1687 is a non-pathogenic strain which was used as a control in the various assays. E.coli strain V 105 was isolated from urine of a case of upper urinary tract infection at the Department of Microbiology, Kasturba Medical College, Mangalore, India. E. coli F

682 INDIAN J EXP BIOL, JULY 2004

23, a non-pathogenic strain isolated from feces, was used as a control in the study.

Media - Chemically defined medium (COM) used in the study contained KH2P04 - 0.78g; K2HP04 -2.3g; (NH4hS04 - 1.0g; Na3C6Hs07 - 0.6g; glucose -1O.0g in 11 of deionised water. MgS04 was added to the medium to obtain a Mg2+ concentration of 0.02mM. The COM was filter sterilized (0.2/lm po­rosity, Millipore) and had a pH of 7.4. Stock solutions of trace metal salts were prepared at a concentration of 10mM, usingCaCb.2H20; CoCb.6H20; CUS04' 5H20; MgS04.7H20; MnChAH20; NH4 Fe(S04h. 12H20; NiS04.6 H20; and 2nS04.7 H2. All chemicals used were of analytical grade, obtained from SO Fine Chemicals, Bombay, India. The stock solutions were filter sterilized (0.2J.1.m porosity, MiIJipore).

Growth conditions - The test strains were grown twice in COM containing 0.02mM Mg2+, each time at 37° C for 24hr. The cells were washed thrice in sterile physiological saline and finally suspended in saline to an 0062S of 0.1 (approximately 1.5 x 108 cells/mI). 100 ml ErlenMeyer flasks with 10 ml COM contain­ing various levels of trace metal ions were inoculated with the test strains to achieve a concentration of ap­proximately 1.5 x 106 cells/ml and incubated at 37° C ina rotary water bath at a speed of 160 rpm for 24hr. The cultures were centrifuged at 3000x g for 15 min to sediment the cells and these cells were used for the determination of surface hydrophobicity and in the adherence assays.

Determination of surface hydrophobicity - The cell surface hydrophobicity of the E.coli strains was assayed by the method of Rosenberg et al 13

, with mi­nor modifications. Cells from the cultures were washed in lOmM phosphate buffered saline (PBS, pH 7.3) and resuspended in PBS to get 00600 of 0.3 (OOlnilial)' Three ml of the bacterial suspension was mixed with 0.3 ml of P - xylene (BDH), vortexed for I min and left for 30 min at room temperature for the separation of water - hydrocarbon phases. The aque­ous phase was aspirated and the 00600 (OOFinal) was measured. The hydrophobicity index was calculated by applying the formula {(OOlnilial - OOFinal)/ OOlnilial } X 100.

Adherence assay - The adherence assay was con­ducted according to the method of Harber et al 14 with minor modifications. Bacterial cells from the cultures were washed thrice in PBS (PH 6.8) and finally sus­pended in PBS to get a concentration of 1 x 108

cells/ml. Uroepithelial cells were obtained from urine

deposits of healthy females on the day of the assay. These cells were washed three times in PBS and fi­nally suspended in PBS to get a concentration of 1 x lOs cells/ml.

One ml each, of bacteria and uroepithelial cells were incubated in duplicate at 37° C in a shaking wa­ter bath for 1 hr with continuous rotation. The suspen­sion was then filtered under vacuum through a 5J.1.m membrane filter (Nucleopore) and washed with 15 ml of PBS. The filter membrane was pressed on a glass slide to give an imprint containing uroepithelial cells. This preparation was air dried, fixed with methanol and stained with dilute carbol fuchsin. The smear was examined by light microscopy (x 1000). Adherence to uroepithelial cells was quantified by determining the mean number of adherent bacteria per epithelial cell after counting 30 cells.

Statistical analysis - The results were statistically analyzed using the Kruskal Wallis test to determine their significance.

Results Effect of metal ions on the surface hydrophobicity

of E.coli - Optimal growth of E.coli in COM re­quired at least 0.02mM of Mg2+. Hence, the surface hydrophobicity of cells grown in COM with 0.02mM Mg2+ was taken as the control. Metal ions such as C02+, Cu2+, Ni2+ and 2n2+ could be studied only in a small range of concentrations as they were toxic for E.coli at higher concentrations. Increasing concentra­tions of Ca2+, Mg2+ and 2n2+ had significant effect on the cell surface hydrophobicity of E.coli (Table I) . Whereas higher concentrations (0.2 - 1.6mM) Ca2+and Mg2+ ions decreased the expression of sur­face hydrophobicity, Fe3+ and 2n2+ ions decreased hydrophobicity at lower levels (0.1 - 0.2 mM). Other metal ions such as C02+, Cu2+, Mri2+ and Ni2+ did not show any significant effect on the expression of cell surface hydrophobicity by E.coli.

Effect of metal ions on adherence of E.coli to uro­epithelial cells - Mg2+ had a profound effect on the adherence properties of E.coli (Table 2). When Mg2+ ions was supplied at a lesser than optimal level , (0.01 mM), there was a significant decrease in the number of adherent bacteria per uroepithelial cell and with increasing concentrations of the same ion, there was a similar, significant increase in the number of adherent bacteria. Toxic metal ions like as C02+, Cu2+, Ni2+, 2n2+ and even Fe3+ at higher concentrations, signifi­cantly decreased the adherence properties of E.coli.

VISHW AS et at.: EFFECT OF TRACE ELEMENTS ON E. COLI 683

It is obvious from the results (Table 3), that there exists a vast difference between pathogenic and com­mensal E.coli in the level of expression of surface hydrophobicity and cell adherence. Cell surface hy­drophobicity of the commensal strains MTCC 1687 and F 23 was significantly lower than that of the uropathogenic strains MTCC 729 and U 105. The pathogenic E.coli adhered more effectively to uro­epithelial cells.

Correlation of the effect of metal ions on the ex­pression of surface hydrophobicity and adherence

properties - At higher concentrations (0.1 - 0.2mM), Fe3+ and Zn2+ significantly decreased both cell surface hydrophobicity and adherence properties of E.coli MTCC 729 (Fig. 1 A, B). Although metal ions such as Ca2+, C02+, Cu2+, Mg2+ and Ni2+ did have significant effect on the individual virulence factors, no correla­tion could be observed on their effect on both the factors. Metal ions had a similar, comparable effect on the virulence properties of U 105 also (data not shown), indicating that there was not much difference in their action on different strains of E.coli.

Trace element

0.01

Ca2+ 42.0 ± 1.6

C02+ 42.5 ± 1.6

Cu2+ 41.6 ± 1.5

Fe3+ 40.7 ± 1.7

Mg2+ 42.4 ± 1.6

Mn2+ 41.4 ± 1.9

Ni2+ 40.8 ± 1.4

Zn2+ 41.5 ± 2.5

Table I-Effect of trace elements on the surface hydrophobicity of E. coli MTCC 729 [Values are mean ± SO for six experiments]

Hydrophobicity Index

Trace element concentration (mM)

0.02 0.05 0.1 0.2 0.4 0.8

NO 41.7 ± 0.8 37.2* ± 1.3 35.9* ± 2.6 35.7* ± 1.4

41.3 ± 1.3 40.5 ± 2.5 40.9 ± 1.1

41.5 ± 2.2 41.6 ± 0.9 42.1 ± 1.4 41.2±2.4

41.3 ± 0.8 40.6 ± 1.8 33.9* ± 1.4 32.3* ± 2.6

42.6 ± 2.l[C] 41.5 ± 2.1 39.7 ± 1.4 38.5* ± 0.8 37.8* ± 1.2

41.4 ± 0.6 41.1 ± 1.1 41.4 ± 1.6 40.4 ± 1.4

41.2 ± 0.9 41.0 ± 2.4 41.4 ± 0.5

40.6 ± 1.7 38.0* ± 0.9 37.7* ± 1.1 33.5* ± 2.3

* Statistically significant at P< 0.05. [C]- Control.

Trace element

Ca2+

C02+

Cu2+

Fe3+

Mg2+

Mn2+

Ni2+

Zn2+

Table 2~ffect of trace elements on the adherence of E.coli MTCC 729 to uroepithelial cells [Values are ± SO of six experiments]

q .

Adherent bacteria per uroepithelial cell

Trace element concentration (mM)

om 0.02 0.05 0.1 0.2 0.8

13.3 ± 1.3 11.9 ± 1.4 12.5 ± 1.5 11.6 ± 2.3

11.9 ± 2.1 8.9* ± 1.1 4.3* ± 1.2

13.1 ± 2.2 10.3 ± 1.7 6.8* ± 1.2 3.9* ± 1.5

13.6 ± 0.7 13.2 ± 1.9 7.8* ± 1.4 5.9 *± 1.3

4.8* ± 1.9 12.1 ± 1.6 [C] 15.3* ± 0.9 15.0* ± 2.3 15.9* ± 1.7

12.9 ± 1.7 12.7 ± 2.1 10.8 ± 1.3 10.3 ± 1.1

11.9 ± 2.5 6.6* ± 1.2 3.1*±0.7

13.2 ±0.9 12.8 ± 1.3 10.5 ± 1.6 4.7* ± 1.1

* Statistically significant at P< 0.05. [C] - control

1.6

35.5* ± 3.1

36.4* ± 1.6

41.0 ± 1.3

1.6

11.8 ± 2.7

14.6* ± 1.3

8.5* ± 2.4

684 INDIAN J EXP BIOL, JULY 2004

Discussion The present study was designed to investigate the

effect of physiological, limiting and excess concen­trations of various metal ions on the expression sur­face hydrophobicity and adherence of uropathogenic and non-pathogenic strains of E.coli: Higher concen­trations of Mg2+, Ca2+, Fe3+ and Zn2+ could signifi­cantly lower the expression of cell surface hydropho­bicity in E.coli. These results are consistent with re­ports of a previous study which have made similar

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Fig 1-Correlation between effect of-(A) Fe3+; and (B) Zn2+ on hydrophobicity(-. - ) & adherence ( - • -) of E.coli to uroepithelial cells.

Table 3-Surface hydrophobicity and adherence properties of E.coli strains MTCC 729, MTCC 1687, U 105 and F 23 grown in

CDM containing 0.02 mM Mg2+ [Values are ± SD of six experiments]

Virulence MTCC 729 MTCC 1687 U 105 F23 factor

Surface 42.6 ± 2.1 8.9 ± 1.3 38.7 ± 1.6 11.6 ± 2.4 Hydro-phobicity Index

Adherent 12.1 ± 1.6 2.4 ± 1.1 14.3 ± 1.2 4.6 ± 2.1 bacteria per Uro-epithelial cell

observations with Staphylococcus aureus ll• Higher

surface hydrophobicity and a net anionic charge have been considered to be important for the interaction between the bacterial cell surface and the host cell membrane15

. The capacity of trace metals to reduce surface hydrophobicity may be due to either an inhi­bition of synthesis of surface structures which are rich in hydrophobic domains or, the binding of these ca­tions by the outer membrane and the lipopolysaccha­ride (LPS) reduced the net negative charge of the cell envelope, resulting in a change in the physico­chemica] properties of the cell surface. Not surpris­ingly, the ions which have lowered the surface hydro­phobicity, namely, Ca2+, Mg2+ Fe3+ and Zn2+ have been shown to be either native to the cell envelope or are more avidly bound by the outer membrane than others 10. This might have led to the reduction in the partitioning of the bacterial cells into the hydrocarbon phase in our assay. It is possible that both the mecha­nisms mentioned above may be involved in decreas­ing the surface hydrophobicity of bacteria.

Adherence to the uroepithelium by uropathogenic bacteria is the initial step in the process of ascending infection of the urinary tract. Various factors such as adhesins on the bacterial surface, presence of appro­priate receptors . on the host cells, the ionic charge of the two surfaces, all play an important role in this step. We investigated the role that certain cations play, in the process of adherence of certain uropatho­genic E.coli strains to uroepithelial cells. Results of the present study indicate that metal ions do play an important role in this adherence process. Under Mg2+ limited conditions, adherence to uroepithelial cells by E.coli was significantly lower than in physiological concentrations or more. Mg2+ has been shown to be absolutely essential for normal metabolism, synthesis of various proteins and production of fimbrial struc­tures of the bacterial cell 16.9. In Mg2+ limited condi­tions, adhesins such as outer membrane proteins and fimbriae involved in adherence are probably not ex­pressed and this might have led to the decrease in ad­herence to uroepithelial cells. It is interesting to note that adherence was maximum at physiological con­centrations (0.8 - 1.2mM) of Mg2+ ions.

The toxic metal ions such as C02+, Cu2+, Ne+, Zn2+ significantly lowered the adherence of E.coli to uro­epithelial cells. Even Fe3+ at higher concentrations had a similar effect. This is in contrast to previous studies, which had demonstrated a higher degree of adherence of E.coli to buccal epithelial cells 12 and

VISHWAS et al.: EFFECT OF TRACE ELEMENTS ON E. COLI 685

HeLa cells'7 in the presence of Fe3+ and Zn2

+ at a con­centration range between 5 x 10.5 to 10-3 M. This dif­ference in the results may be due to the reason that Fe3

+ and Zn2+ were added during the adherence assays

and not into the medium which was used to grow the bacteria. The toxic effect of higher concentrations of these metal ions may block the formation of outer membrane proteins or pili involved in adherence, at the transcriptional or translational level of protein synthesis and decrease the rate of adherence.

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