Survival and Trafficking of Yersinia pestis in Non-acidified Phagosomes in Murine Macrophages

ABSTRACT Yersinia pestis survives and replicates in phagosomes of murine macrophages. Previous studies demonstrated that Y. pestis-containing vacuoles (YCVs) acquire markers of late endosomes or lysosomes in naïve macrophages and that this bacterium can survive in macrophages activated with the cytokine gamma interferon. An autophagic process known as xenophagy, which destroys pathogens in acidic autophagolysosomes, can occur in naïve macrophages and is upregulated in activated macrophages. Studies were undertaken here to investigate the mechanism of Y. pestis survival in phagosomes of naïve and activated macrophages and to determine if the pathogen avoids or co-opts autophagy. Colocalization of the YCV with markers of autophagosomes or acidic lysosomes and the pH of the YCV were determined by microscopic imaging of infected macrophages. Some YCVs contained double membranes characteristic of autophagosomes, as determined by electron microscopy. Fluorescence microscopy showed that ∼40% of YCVs colocalized with green fluorescent protein (GFP)-LC3, a marker of autophagic membranes, and that YCVs failed to acidify below pH 7 in naïve macrophages. Replication of Y. pestis in naïve macrophages caused accumulation of LC3-II, as determined by immunoblotting. While activation of infected macrophages increased LC3-II accumulation, it decreased the percentage of GFP-LC3-positive YCVs (∼30%). A viable count assay showed that Y. pestis survived equally well in macrophages proficient for autophagy and macrophages rendered deficient for this process by Cre-mediated deletion of ATG5, revealing that this pathogen does not require autophagy for intracellular replication. We conclude that although YCVs can acquire an autophagic membrane and accumulate LC3-II, the pathogen avoids xenophagy by preventing vacuole acidification.

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The Ability To Replicate in Macrophages Is Conserved between Yersinia pestis and Yersinia pseudotuberculosis

Infection and Immunity ◽

10.1128/iai.71.10.5892-5899.2003 ◽

2003 ◽

Vol 71 (10) ◽

pp. 5892-5899 ◽

Cited By ~ 121

Author(s):

Céline Pujol ◽

James B. Bliska

Keyword(s):

Yersinia Pestis ◽

Type Iii Secretion ◽

Yersinia Pseudotuberculosis ◽

Type Iii Secretion System ◽

Evolutionary Process ◽

Secretion System ◽

Intracellular Replication ◽

Murine Macrophages ◽

Type Iii ◽

Chromosomal Type

ABSTRACT Yersinia pestis, the agent of plague, has arisen from a less virulent pathogen, Yersinia pseudotuberculosis, by a rapid evolutionary process. Although Y. pestis displays a large number of virulence phenotypes, it is not yet clear which of these phenotypes descended from Y. pseudotuberculosis and which were acquired independently. Y. pestis is known to replicate in macrophages, but there is no consensus in the literature on whether Y. pseudotuberculosis shares this property. We investigated whether the ability to replicate in macrophages is common to Y. pestis and Y. pseudotuberculosis or is a unique phenotype of Y. pestis. We also examined whether a chromosomal type III secretion system (TTSS) found in Y. pestis is present in Y. pseudotuberculosis and whether this system is important for replication of Yersinia in macrophages. A number of Y. pestis and Y. pseudotuberculosis strains of different biovars and serogroups, respectively, were tested for the ability to replicate in primary murine macrophages. Two Y. pestis strains (EV766 and KIM10+) and three Y. pseudotuberculosis strains (IP2790c, IP2515c, and IP2666c) were able to replicate in macrophages with similar efficiencies. Only one of six strains tested, the Y. pseudotuberculosis YPIII(p−) strain, was defective for intracellular replication. Thus, the ability to replicate in macrophages is conserved in Y. pestis and Y. pseudotuberculosis. Our results also indicate that a homologous TTSS is present on the chromosomes of Y. pestis and Y. pseudotuberculosis and that this secretion system is not required for replication of these bacteria in macrophages.

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A Transposon Site Hybridization Screen Identifies galU and wecBC as Important for Survival of Yersinia pestis in Murine Macrophages

Journal of Bacteriology ◽

10.1128/jb.06237-11 ◽

2011 ◽

Vol 194 (3) ◽

pp. 653-662 ◽

Cited By ~ 23

Author(s):

K. A. Klein ◽

H. S. Fukuto ◽

M. Pelletier ◽

G. Romanov ◽

J. P. Grabenstein ◽

...

Keyword(s):

Yersinia Pestis ◽

Murine Macrophages

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Yersinia pestis Can Bypass Protective Antibodies to LcrV and Activation with Gamma Interferon To Survive and Induce Apoptosis in Murine Macrophages

Clinical and Vaccine Immunology ◽

10.1128/cvi.00172-09 ◽

2009 ◽

Vol 16 (10) ◽

pp. 1457-1466 ◽

Cited By ~ 18

Author(s):

Betty L. Noel ◽

Sarit Lilo ◽

Daniel Capurso ◽

Jim Hill ◽

James B. Bliska

Keyword(s):

Yersinia Pestis ◽

Gamma Interferon ◽

Bacterial Survival ◽

Activated Macrophages ◽

Necrosis Factor Alpha ◽

Murine Macrophages ◽

Protective Antibodies ◽

Ifn Γ ◽

Time Point

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-γ)-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-γ 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-γ 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-γ. 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-γ-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-γ to survive and induce apoptosis in murine macrophages.

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