Effects of iron limitation on adherence and cell surface carbohydrates of Corynebacterium diphtheriae strains

Abstract

Iron limitation may cause bacterial pathogens to grow more slowly; however, it may also stimulate these microorganisms to produce greater tissue damage, given that many virulence factors are controlled by the iron supply in the environment. The present study investigated the influence of low iron availability on the expression of proteins and surface sugar residues of two toxigenic strains of Corynebacterium diphtheriae subsp. mitis and evaluated their adherence to human group B erythrocytes and HEp-2 cells. A comparison was made between bacteria grown in (i) Trypticase soy broth (TSB), (ii) TSB treated with dipyridyl to deplete free iron, and (iii) TSB enriched with FeCl(3). The effects of iron concentration on adhesive properties were different for strains 241 and CDC-E8392, of the sucrose-fermenting and non-sucrose-fermenting biotypes, respectively. Iron-limited conditions enhanced interaction of strain 241 with erythrocytes and HEp-2 cells. Inhibition assays suggested the involvement of nonfimbrial protein combination 67-72p on hemagglutination of diphtheria bacilli grown under iron-limited conditions. Conversely, iron limitation inhibited adherence to glass and expression of electron-dense material on the bacterial surface. Lectin binding assays demonstrated a reduction in the number of sialic acid residues and an increase in D-mannose and D-galactose residues on the surfaces of both strains. Thus, iron exerts a regulatory role on adhesive properties of diphtheria bacilli, and low iron availability modulates the expression of C. diphtheriae surface carbohydrate moieties. The significant changes in the degree of lectin binding specific for D-mannose, D-galactose and sialic acid residues may have an effect on binding of host cells. The expression of dissimilar microbial virulence determinants may be coordinately controlled by common regulatory systems. For C. diphtheriae, the present results imply regulation of adherence and slime production as part of a global response to iron-limited environmental conditions that includes derepression of genes for the synthesis of cytotoxin and siderophores and for transport of the Fe(III)-siderophore complexes.

FIG. 1.

Protein profiles of strains CDC-E8392 (A) and 241 (B) of C. diphtheriae grown under standard and iron-limited conditions. Total protein was analyzed by SDS-10% PAGE. Lane 1, molecular mass markers; lanes 2 and 4, strains CDC-E8392 and 241, respectively, from standard cultures; lanes 3 and 5, strains CDC-E8392 and 241, respectively, from iron-limited cultures. Arrows indicate proteins bands expressed at high levels, and dots indicate proteins bands that were absent or expressed at low levels in iron-limited cultures.

FIG. 2.

(A) Binding of iodinated lectins SNA (•, ○) and ConA (▴, ▵) to C. diphtheriae strain 241 grown under standard (•, ▵) and iron-limited (○, ▴) conditions. (B) For each lectin, the binding data for high-affinity receptors (filled symbols from panel A have been plotted by the method of Steck and Wallach) (23) according to the equation C/[lectin] bound = 1/Kn × 1/[lectin] free + 1/n, where C is the concentration of bacteria, n is the number of lectin molecules bound per cell, K is the lectin association constant, and [lectin] is the concentration of lectin in molar. All points represent means from triplicate experiments; the standard deviations is less than 10%.

FIG. 3.

Electron micrographs showing structural differences on the surface of the sucrose-fermenting C. diphtheriae strain 241 grown under standard (A) and iron-limited (B) conditions. Magnifications, ×40,000 and ×70,500, respectively.