Doctoral Dissertations

Date of Award

12-1982

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

T. C. Montie

Committee Members

J. M. Becker, D. Doyle, C. J. Wust, B. T. Rouse

Abstract

The adherence of Bordetella bronchiseptica smooth, intermediate, and rough phase isolates to Hamster Lung Fibroblasts (HLF), as well as to a variety of mammalian cells, was investigated and the mechanism of adherence to HLF was studied. Isolates representing the three phases adhered to the HLF in a manner that was rapid and reproducible. Adherence occurred within 5 min, and increased to a stationary level of adherence by 30 min. Adherence was not appreciably affected by pH or growth substrate. Carbon source did not affect piliation or flagellation of the isolates. Piliation and adherence of the rough and intermediate phase isolates were, however, affected by stage of growth; the level of adherence and percent of bacterial cells with pili increased with age of culture. Piliation and adherence of the smooth phase isolate were similar throughout its growth cycle. The level of adherence increased with bacterial concentration and temperature. Adherence of the isolates to other cell lines and freshly isolated epithelial cells was similar to that observed for the HLF except that the level of adherence to freshly isolated cells was more variable than that observed for cell lines. Although the three phases were similar with respect to the number of adherent bacteria per cell, attachment of all rough and intermediate phase isolates tested (n=13) regardless of animal species from which the isolates were obtained was inhibited and reversed by citrate. EGTA also inhibited adherence of these two phases, except when an excess of the divalent cations Ca+2, Sr+2, Cd+2, or Mn+2 were present. Adherence of smooth phase isolates (n=4) was inhibited and reversed by N-acetylglucosamine (GlcNAc). The entire configuration of this molecule appeared to be important in blocking attachment since N-acetylgalactosamine, N-acetymannosamine, N-actylcysteine, and glucosamine blocked adherence to a lesser extent than N-acetylglucosamine. Also, mannoseamine and galactosamine had no blocking effects. The GlcNAc appeared to act by binding to the bacterial adhesin and not to the HLF since it was demonstrated that smooth phase bacteria bound [3H]-GlcNAc in a manner that was specific and saturable, and that bacteria preincubated with GlcNAc then washed free of the unbound ami no sugar were nonadherent whereas HLF similarly treated still bound bacteria to their surface. The bacterial adhesin(s) of all three phases of B. bronchiseptica were heat labile proteins which could be removed from the bacterial surface by relatively gentle agitation in a homogenizer. The pili of B. bronchiseptica were also heat labile and were removed by brief agitation in a homogenizer. The results suggested that B. bronchiseptica adhered to eukaryotic cells by at least two mechanisms which may in part have been mediated by pili.

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