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.1999 Aug;67(8):3757-62.
doi: 10.1128/IAI.67.8.3757-3762.1999.

Loss of resistance to ingestion and phagocytic killing by O(-) and K(-) mutants of a uropathogenic Escherichia coli O75:K5 strain

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Loss of resistance to ingestion and phagocytic killing by O(-) and K(-) mutants of a uropathogenic Escherichia coli O75:K5 strain

S M Burns et al. Infect Immun.1999 Aug.

Abstract

To determine the importance of the O75 O antigen and the K5 capsular antigen in resistance to phagocytosis and phagocytic killing, we used previously described O75(-) and K5(-) mutants from an O75(+) K5(+) wild-type uropathogenic Escherichia coli strain in phagocytosis assays with polymorphonuclear leukocytes (PMNs) and monocytes. At a 10-to-1 ratio of bacteria to phagocytes and in the presence of 10% serum, the parental strain GR-12 was resistant to both PMNs and monocytes over a 2-h incubation period. The O75(-) and K5(-) mutants were similar in sensitivity to killing by both PMNs and monocytes, decreasing in viability by 80% in the first hour. Yet, a significant difference in killing between the O75(-) and K5(-) mutants was observed in the first 15 min of incubation. The K5(-) mutant decreased in numbers by almost 60%, while the O75(-) mutant increased in numbers similarly to GR-12 in the first 15 min. The difference in killing was found not to be due to the rate of opsonization. To further determine the mechanism of resistance, a fluorescence assay was used to differentiate attached and internalized bacteria. The K5 capsule hindered the association of both the wild-type strain and the O75(-) mutant in the initial incubation time with PMNs. In conclusion, both the K5 capsule and O75 O antigen play crucial roles in resistance to phagocytosis over time.

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Figures

FIG. 1
FIG. 1
PMN phagocytosis killing assays. Wild-type strain GR-12 (squares) and O75− (triangles) and K5− (circles) mutants were mixed with PMNs at a 10-to-1 ratio of bacteria to PMNs. Assays were performed in 10% serum with (closed symbols) and without (open symbols) PMNs (as a control) for 2 h; samples were taken in duplicate at the start and at 15, 30, 60, and 120 min. The graph represents results of seven different assays for each strain, although the 15-min time was taken for only five of the seven assays. Error bars indicate standard deviations.
FIG. 2
FIG. 2
Preopsonization PMN phagocytosis assays of wild-type strain GR-12 (squares) and O75− (triangles) and K5− (circles) mutants at a 10-to-1 ratio of bacteria to PMNs. GR-12 and mutants incubated with PMNs (closed symbols) were preopsonized in 10% serum for 30 min prior to addition to PMNs. Controls were done with 10% serum and no PMNs added (open symbols). Assays were performed for 1 h; samples were taken at the start and at 15, 30, and 60 min. The graph represents results of three different assays of each strain. Error bars indicate standard deviations.
FIG. 3
FIG. 3
Bacterial viability in supernatant fraction following separation from PMNs. PMN phagocytosis assays were performed with wild-type strain GR-12 and mutants; bacteria associated with PMNs were separated from bacteria in supernatant at 0, 15, and 30 min; bacterial counts were compared to the initial total number of bacteria. The bar graph represents results of three different assays of each strain.
FIG. 4
FIG. 4
Fluorescence assay to determine the number of attached and internalized bacteria per PMN for the wild-type strain GR-12 and mutants with 10% serum (A) or 10% heat-inactivated serum (B). FITC-labeled bacteria were added to PMNs as described in the text, samples were taken at 0, 5, 15, and 30 min (A) or 60 min (B), and nonassociated bacteria were separated. Ethidium bromide was added to quench the fluorescence of extracellular bacteria. Samples were viewed under a microscope, and attached and internalized bacteria were counted. Solid bar denote mean bacteria internalized, and hatched bars denote attached bacteria. The graph depicts results of four different assays of each strain.
FIG. 5
FIG. 5
Monocyte phagocytosis killing assays of wild-type strain GR-12 (squares) and O75− (triangles) and K5− (circles) mutants at a 10-to-1 ratio of bacteria to monocytes. Assays were performed in 10% serum with (closed symbols) and without (open symbols) monocytes (as a control) for 2 h; samples were taken in duplicate at the start and at 15, 30, 60, and 120 min. The graph represents results of six different assays for each strain. Error bars indicate standard deviations.
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References

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