Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Oct;16(10):1457-66.
doi: 10.1128/CVI.00172-09. Epub 2009 Aug 26.

Yersinia pestis can bypass protective antibodies to LcrV and activation with gamma interferon to survive and induce apoptosis in murine macrophages

Betty L Noel  1 , Sarit Lilo, Daniel Capurso, Jim Hill, James B Bliska
Affiliations

Yersinia pestis can bypass protective antibodies to LcrV and activation with gamma interferon to survive and induce apoptosis in murine macrophages

Betty L Noel et al. Clin Vaccine Immunol. 2009 Oct.

Abstract

Yersinia pestis, the agent of plague, uses a type III secretion injectisome to deliver Yop proteins into macrophages to counteract phagocytosis and induce apoptosis. Additionally, internalized Y. pestis can survive in the phagosomes of naïve or gamma interferon (IFN-gamma)-activated macrophages by blocking vacuole acidification. The Y. pestis LcrV protein is a target of protective antibodies. The binding of antibodies to LcrV at the injectisome tip results in neutralization of the apoptosis of Y. pestis-infected macrophages and is used as an in vitro correlate of protective immunity. The cytokines IFN-gamma and tumor necrosis factor alpha can cooperate with anti-LcrV to promote protection against lethal Y. pestis infection in mice. It is not known if these phagocyte-activating cytokines cooperate with anti-LcrV to increase the killing of the pathogen and decrease apoptosis in macrophages. We investigated how anti-LcrV and IFN-gamma impact bacterial survival and apoptosis in cultured murine macrophages infected with Y. pestis KIM5. Y. pestis KIM5 opsonized with polyclonal or monoclonal anti-LcrV was used to infect macrophages treated with or without IFN-gamma. The phagocytosis and survival of KIM5 and the apoptosis of macrophages were measured at different time points postinfection. The results show that anti-LcrV reduced apoptosis at an early time point (5 h) but not at a later time point (24 h). Polyclonal anti-LcrV was unable to inhibit apoptosis at either time point in IFN-gamma-activated macrophages. Additionally, anti-LcrV was ineffective at promoting the killing of KIM5 in naïve or activated macrophages. We conclude that Y. pestis can bypass protective antibodies to LcrV and activation with IFN-gamma to survive and induce apoptosis in murine macrophages.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Phagocytosis assay with KIM5-infected naïve or IFN-γ-stimulated RAW 264.7 macrophages. KIM5/GFP was grown under T3SS-inducing conditions and incubated with PBS (KIM5), anti-V (+ anti-V serum), or control serum (+ Serum) prior to infecting naïve macrophages (A) or IFN-γ-stimulated macrophages (B) on coverslips. Twenty-five minutes p.i., the cells were fixed and extracellular bacteria were labeled for detection by immunofluorescence. Extracellular and intracellular bacteria from three fields per experiment were counted by fluorescence microscopy, and the percentage of internalization was determined. Results were taken from three independent experiments and averaged. Error bars show standard deviations. *, P < 0.05 compared to the control serum condition.
FIG. 2.
FIG. 2.
Examination of YopJ-mediated cell death in Y. pestis-infected macrophages through determination of LDH release. Naïve BMDMs (A and B) and IFN-γ-stimulated BMDMs (C and D) were left uninfected or infected with nonopsonized KIM5 or KIM5 yopB or KIM5 opsonized with the indicated serum. LDH levels in the supernatants of the BMDMs at 5 h (A and C) and 24 h (B and D) p.i. were measured. Results were normalized by subtracting background levels of LDH from uninfected macrophages and are shown as percentages of the total LDH. Results were taken from three independent experiments and averaged. Error bars show standard deviations. *, P < 0.05; **, P < 0.01 compared to the control serum.
FIG. 3.
FIG. 3.
Comparison of the intracellular survival of Y. pestis and Y. pseudotuberculosis 32777 in BMDMs. Naïve BMDMs (A, B) and IFN-γ-stimulated BMDMs (C) were infected with the indicated strains, with or without prior opsonization with serum. Intracellular bacterial survival was determined at 25 min, 1.5 h, and 5 h p.i. by CFU assay. Results were taken from three independent experiments and averaged. Error bars show standard deviations. ***, P < 0.001 compared to the level at 25 min.
FIG. 4.
FIG. 4.
Monitoring TNF-α and IL-1β levels in the supernatants of Y. pestis-infected BMDMs. BMDMs were left uninfected or infected with the indicated strains with or without prior opsonization with serum. Supernatants were collected after 24 h of infection, and TNF-α (A) and IL-1β (B) levels were measured by ELISA. Results were taken from three independent experiments and averaged. Error bars show standard deviations. **, P < 0.01; ***, P < 0.001 compared to serum.
FIG. 5.
FIG. 5.
Phagocytosis assay with KIM5-infected naïve or IFN-γ-stimulated RAW 264.7 macrophages. KIM5/GFP was left unopsonized or incubated with anti-LcrV MAb 7.3 or anti-YopD MAb prior to infecting naïve macrophages (A) or IFN-γ-stimulated macrophages (B). Twenty-five minutes p.i., the cells were fixed and the percentage of internalization was determined as described in the legend to Fig. 1. Results were taken from three independent experiments and averaged. Error bars show standard deviations. *, P < 0.05 compared to MAb YopD.
FIG. 6.
FIG. 6.
Effect of MAbs on the intracellular survival of Y. pestis in BMDMs. Naïve BMDMs (A) and IFN-γ-stimulated BMDMs (B) were infected with the indicated strains with or without preincubation with MAbs, and intracellular bacterial survival was determined at 25 min, 1.5 h, and 5 h p.i. as indicated in the legend to Fig. 3. Results were taken from three independent experiments and averaged. Error bars show standard deviations.
FIG. 7.
FIG. 7.
Effect of MAbs on YopJ-mediated cell death through determination of LDH release. Naïve BMDMs (A and B) or IFN-γ-stimulated BMDMs (C and D) were infected with the indicated strains of KIM5 with or without preincubation with MAbs or left uninfected. LDH levels in the supernatants of the BMDMs at 5 h (A and C) and 24 h (B and D) p.i. were measured as indicated in the legend to Fig. 2. Results were taken from three independent experiments and averaged. Error bars show standard deviations. *, P < 0.05; **, P < 0.01 compared to MAb YopD.
FIG. 8.
FIG. 8.
Effect of bacterial growth, temperature, and serum opsonization on phagosome acidification within naïve J774A.1 cells. J774A.1 cells were infected with fixed or live KIM5/GFP pregrown at 28°C or 37°C and left unopsonized or opsonized as indicated. LysoTracker Red DND 99 was used to label acidic compartments, and colocalization with KIM5/GFP was determined at 1.25 h p.i. by fluorescence microscopy. Results were taken from three independent experiments and averaged. Error bars show standard deviations. ***, P < 0.001 compared to the unopsonized KIM5 28°C subculture.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Achtman, M., K. Zurth, G. Morelli, G. Torrea, A. Guiyoule, and E. Carniel. 1999. Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Proc. Natl. Acad. Sci. USA 96:14043-14048. - PMC - PubMed
    1. Aderem, A., and D. M. Underhill. 1999. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17:593-623. - PubMed
    1. Bashaw, J., S. Norris, S. Weeks, S. Trevino, J. J. Adamovicz, and S. Welkos. 2007. Development of in vitro correlate assays of immunity to infection with Yersinia pestis. Clin. Vaccine Immunol. 14:605-616. - PMC - PubMed
    1. Bergsbaken, T., and B. T. Cookson. 2007. Macrophage activation redirects Yersinia-infected host cell death from apoptosis to caspase-1-dependent pyroptosis. PLoS Pathog. 3:e161. - PMC - PubMed
    1. Black, D. S., and J. B. Bliska. 2000. The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence. Mol. Microbiol. 37:515-527. - PubMed

Publication types

MeSH terms

LinkOut - more resources