Yersinia pestis type III secretion system-dependent inhibition of human polymorphonuclear leukocyte function

Abstract

Human polymorphonuclear leukocytes (PMNs, or neutrophils) are the primary innate host defense against invading bacterial pathogens. Neutrophils are rapidly recruited to sites of infection and ingest microorganisms through a process known as phagocytosis. Following phagocytosis by human PMNs, microorganisms are killed by reactive oxygen species (ROS) and microbicidal products contained within granules. Yersinia pestis, the causative agent of plague, is capable of rapid replication and dissemination from sites of infection in the host. Although Y. pestis survives in macrophages, the bacterial fate following interaction with human PMNs is less clear. The ability of Y. pestis to inhibit phagocytosis by human PMNs was assessed by differential fluorescence microscopy and was shown to be dependent on expression of the type III secretion system (TTSS). Previous studies have demonstrated that TTSS expression in enteropathogenic Yersinia spp. also inhibits the respiratory burst in PMNs and macrophages, and we show here that human PMN ROS production is similarly repressed by Y. pestis. However, exclusion of uningested TTSS-expressing Y. pestis with gentamicin revealed that intracellular bacteria are eliminated by human PMNs, similar to bacteria lacking the TTSS. In summary, our results suggest that the Y. pestis TTSS contributes to extracellular survival following interactions with human PMNs and that the intracellular fate is independent of TTSS inhibition of neutrophil ROS production.

FIG. 1.

Influence of the Y. pestis TTSS on phagocytosis by human neutrophils. Y. pestis isogenic strains with the presence (KIM5) or absence (KIM6) of the pCD1 virulence plasmid were grown at 37°C to induce expression of the TTSS. Phagocytosis was assessed by immunofluorescence microscopy, and the results are expressed as means ± standard deviations (SD) for three experiments. *, significant difference between strains (P < 0.01) at indicated time points.

FIG. 2.

Inhibition of human PMN ROS production by Y. pestis requires expression of the TTSS. Neutrophil ROS production was measured during phagocytosis of Y. pestis strains KIM5 (pCD1 positive) and KIM6 (pCD1 negative). The rates of ROS production are the means for three experiments. ΔFL, change in fluorescence. (A) PMN ROS production following interaction with Y. pestis grown at 28°C. *, KIM5 and KIM6 resulted in increased ROS production (P < 0.001) compared to that of the PMN control. (B) PMN ROS production following interaction with Y. pestis grown at 37°C. Y. pestis strain KIM5 cured of pCD1 was included as a control [KIM5(−)]. *, KIM5 reduced (P < 0.001) neutrophil ROS production compared to that of KIM5(−) and KIM6.

FIG. 3.

Y. pestis survival following interaction with human neutrophils is enhanced by expression of the TTSS. PMNs were incubated with Y. pestis strains KIM5 and KIM6 grown at 37°C. Y. pestis strain KIM5 grown at 28°C was included for comparison. At each time point, PMNs were lysed and bacterial viability was determined by enumeration of CFU. PMN bactericidal activity was calculated as described in Materials and Methods. Results are expressed as means ± SD for three experiments. *, significant difference (P < 0.01) in bactericidal activity between KIM5 at 37°C and either KIM5 at 28°C or KIM6 at 37°C.

FIG. 4.

Y. pestis intracellular survival following phagocytosis by human PMNs. PMNs were incubated with Y. pestis strains KIM5 and KIM6 grown at 37°C. (A) Gentamicin was added to PMNs 15 min following phagocytosis to eliminate noningested (extracellular) Y. pestis. At each time point, PMNs were washed to remove gentamicin, lysed, and plated on growth agar. PMN bactericidal activity was calculated as described in Materials and Methods. Results are expressed as means ± SD for three experiments. No differences (P > 0.05) were detected between KIM5 and KIM6 at any time point. (B) Flow cytometric analysis of early PMN apoptosis following phagocytosis of Y. pestis. PMNs with exposed phosphatidylserine (early apoptosis) bound annexin V-FITC. (C) Late apoptotic/necrotic PMNs dually stained with annexin V-FITC and propidium iodide (PI). Results are expressed as means ± SD for at least 12 experiments. No significant differences (P > 0.05) were detected between KIM5 and KIM6 at any time point. (D) Neutrophil ROS production in the presence of gentamicin. 2′,7′-Dichlorodihydrofluorescein diacetate-treated PMNs were incubated alone or with Y. pestis strains KIM5 and KIM6 (37°C) for 15 min, followed by the addition of gentamicin. The rates of ROS production are means for three experiments. ΔFL, change in fluorescence. PMNs not treated with gentamicin were included as a reference control (none).