Enterococcus faecalis bearing aggregation substance is resistant to killing by human neutrophils despite phagocytosis and neutrophil activation

Abstract

Enterococcus faecalis aggregation substance (AS) mediates efficient bacterium-bacterium contact to facilitate plasmid exchange as part of a bacterial sex pheromone system. We have previously determined that AS promotes direct, opsonin-independent binding of E. faecalis to human neutrophils (PMNs) via complement receptor type 3 and other receptors on the PMN surface. We have now examined the functional consequences of this bacterium-host cell interaction. AS-bearing E. faecalis was phagocytosed and internalized by PMNs, as determined by deconvolution fluorescence microscopy. However, these bacteria were not killed by PMNs, and internalized bacteria excluded propidium iodide, indicating intact bacterial membranes. Resistance to killing occurred despite activation of PMNs, as indicated by an increase in both functional and total surface Mac-1 expression, shedding of L-selectin, and an increase in PMN extracellular superoxide and phagosomal oxidant production. Deconvolution fluorescence microscopy also revealed that phagosomes containing AS-bearing bacteria were markedly larger than phagosomes containing opsonized E. faecalis, suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. PMN phagosomal pH was significantly higher after ingestion of nonopsonized AS-bearing E. faecalis than after that of opsonized bacteria. The novel ability of AS to promote intracellular survival of E. faecalis inside PMNs suggests that AS may be a virulence factor used by strains of E. faecalis.

FIG. 1

Internalization of E. faecalis INY401 (lacking AS) (A) and INY1801 (expressing AS) (B) by PMNs. FITC-labeled bacteria were opsonized with 10% serum (for INY401) or not opsonized (for INY1801) and incubated with DiI-labeled PMNs followed by fixation with formaldehyde and labeling of nucleic acid with DAPI. Cells were examined by deconvolution fluorescence microscopy, as described in Materials and Methods. The rims of the bacteria appear green from the fluorescein, PMN nuclei and bacterial nucleoids appear blue from the DAPI, and PMN plasma membrane and phagosomal membrane appear red from the DiI. Opsonized INY401 organisms are internalized by PMNs in small phagosomes, each containing one bacterium or set of diplococci, while nonopsonized INY1801 organisms are internalized in large phagosomes. The yellow rims of internalized INY401, due to overlap of the green and red fluorescent dyes, suggest close apposition of the phagosomal membrane and bacteria. Bars, 5 μm.

FIG. 2

Bacterial killing by PMNs. E. faecalis INY1801 or INY401 was opsonized with normal serum or not opsonized and then exposed to PMNs or HBSS alone (no PMN); microbial viability was determined. Data are the means (+ standard errors of the means for opsonized enterococci) for 5 to 12 experiments. ∗, P < 0.001 versus opsonized strain INY401.

FIG. 3

Exclusion of propidium iodide by intracellular bacteria. Bacteria were opsonized with normal serum or not opsonized, exposed to PMNs for 60 min, and stained with SYTO 9 and propidium iodide. Representative fluorescence micrographs of opsonized E. faecalis INY401 demonstrating that the majority of intracellular bacteria stained orange-red from the propidium iodide (A) and nonopsonized INY1801 demonstrating that most of the intracellular bacteria were able to exclude the propidium iodide and stained green (B). Original magnification, ×1,000.

FIG. 4

PMN surface protein expression after incubation with bacteria. PMNs were incubated with E. faecalis INY1801 or INY401 or phosphate-buffered saline buffer control and surface expression of Mac-1, an activation epitope of Mac-1, and l-selectin were determined by flow cytometry with MAb MHM23, MAb24-Cy3, and Leu-8, respectively. Data are expressed as the mean channel fluorescence for five or six experiments; error bars represent standard errors of the means. ∗, P < 0.05 versus INY401.

FIG. 5

MPO-mediated killing of enterococci. Bacteria were exposed to an MPO cell-free system, as described in Materials and Methods, and viability was determined. Data are the means for three experiments; error bars represent standard errors of the means. ∗, P < 0.05 versus INY401.