yersinia pestis can reside in autophagosomes and avoid

INFECTION AND IMMUNITY, June 2009, p. 2251–2261 Vol. 77, No. 60019-9567/09/$08.00�0 doi:10.1128/IAI.00068-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Yersinia pestis Can Reside in Autophagosomes and Avoid Xenophagyin Murine Macrophages by Preventing Vacuole Acidification�

Celine Pujol,1†‡ Kathryn A. Klein,1† Galina A. Romanov,1 Lance E. Palmer,1§ Carol Cirota,1Zijiang Zhao,2 and James B. Bliska1*

Center for Infectious Diseases and Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook,New York 11794,1 and Department of Pathology and Immunology and Department of Molecular Microbiology,

Washington University School of Medicine, St. Louis, Missouri 631102

Received 19 January 2009/Returned for modification 21 February 2009/Accepted 10 March 2009

Yersinia pestis survives and replicates in phagosomes of murine macrophages. Previous studies demonstratedthat Y. pestis-containing vacuoles (YCVs) acquire markers of late endosomes or lysosomes in naïve macro-phages and that this bacterium can survive in macrophages activated with the cytokine gamma interferon. Anautophagic process known as xenophagy, which destroys pathogens in acidic autophagolysosomes, can occurin naïve macrophages and is upregulated in activated macrophages. Studies were undertaken here to inves-tigate the mechanism of Y. pestis survival in phagosomes of naïve and activated macrophages and to determineif the pathogen avoids or co-opts autophagy. Colocalization of the YCV with markers of autophagosomes oracidic 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 mi-croscopy. 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 naïvemacrophages. Replication of Y. pestis in naïve macrophages caused accumulation of LC3-II, as determined byimmunoblotting. While activation of infected macrophages increased LC3-II accumulation, it decreased thepercentage of GFP-LC3-positive YCVs (�30%). A viable count assay showed that Y. pestis survived equally wellin macrophages proficient for autophagy and macrophages rendered deficient for this process by Cre-mediateddeletion of ATG5, revealing that this pathogen does not require autophagy for intracellular replication. Weconclude that although YCVs can acquire an autophagic membrane and accumulate LC3-II, the pathogenavoids xenophagy by preventing vacuole acidification.

Yersinia pestis is a gram-negative bacterium and the cause ofplague (32, 34). Zoonotic foci of plague exist in many parts ofthe world, including North America. Y. pestis infections aremost commonly transmitted to humans by infected fleas andtypically develop into bubonic plague or, less frequently, intosepticemic plague (32). Plague infections in humans can alsoresult from contact with body fluids or from inhalation ofrespiratory droplets from infected animals or humans. Inhala-tion of Y. pestis into the lungs can initiate primary pneumonicplague.

Y. pestis is able to survive and replicate in murine macro-phages in vitro (5, 6, 18, 35, 36, 44) and in vivo (23) and istherefore classified as a facultative intracellular pathogen. Mi-croscopic examination of tissues of animals experimentally in-fected with Y. pestis has shown the presence of plague bacilliinside macrophages (11, 23, 25, 50). More often, however, Y,pestis is detected as large numbers of extracellular bacteria intissues (20, 39, 51). Y. pestis produces several antiphagocyticfactors that are upregulated during growth at 37°C. These

factors include several Yop proteins and the LcrV protein andtheir designated type III secretion system encoded on pCD1(48). In addition, a capsule composed of the F1 protein ismaximally expressed after extended growth at 37°C and pro-motes resistance to phagocytosis (8). When grown at ambienttemperatures (e.g., 28°C), Y. pestis is efficiently phagocytosedby macrophages (5). It has been hypothesized that when Y.pestis growing at 28°C is introduced into a mammalian host, itinitially survives and replicates within macrophages that inter-nalize the bacteria (5). Subsequently, the bacteria escape orare released from dying macrophages and replicate in an ex-tracellular niche (5).

After a macrophage engulfs a bacterium, the phagosomechanges rapidly into a less habitable compartment, termed aphagolysosome, through a series of fusion events with endo-cytic compartments (49). Within 2 to 5 min after their forma-tion, phagosomes transiently acquire characteristics of earlyendosomes (49). After 10 to 30 min phagosomes begin to fusewith late endosomes and lysosomes. Late endosomes and ly-sosomes can be characterized by the presence of componentssuch as antimicrobial peptides, lysosome-associated membraneproteins (LAMPs), and lysosomal proteases (cathepsins B, D,and L). In contrast to late endosomes, only lysosomes andphagolysosomes contain significant amounts of mature pro-teases. In addition, the pH of lysosomes and phagolysosomes issignificantly lower (�pH 4.5) than the pH in late endosomes(pH 5.5 to 6). The decrease in pH is due to the action of thevacuolar proton ATPase (vATPase).

* Corresponding author. Mailing address: Center for Infectious Dis-eases, Stony Brook University, Stony Brook, NY 11794-5120. Phone:(631) 632-8782. Fax: (631) 632-4294. E-mail: [email protected].

† C.P. and K.A.K. contributed equally to this work.‡ Present address: UR1282, Infectiologie Animale, Sante Publique

(IASP-311), INRA Centre de Tours, F-37380 Nouzilly, France.§ Present address: Siemens Corporate Research, 755 College Rd.

East, Princeton, NJ 08540.� Published ahead of print on 16 March 2009.

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Early work on the trafficking of Y. pestis-containing vacuoles(YCVs) in primary murine macrophages yielded evidence thatYCVs fuse with lysosomes (6, 45). It was therefore suggestedthat Y. pestis survives in a phagolysosome (45). More recentstudies have confirmed that the YCV colocalizes with markersof late endosomes or lysosomes in murine J774A.1 macro-phage-like cells (13). Results of experiments that utilized lyso-somal tracers or antibodies to the LAMP1 or cathepsin Dprotein in conjunction with immunofluorescence and thin-sec-tion electron microscopy (EM) showed that the YCV colocal-izes with these markers between 1.5 and 8 h postinfection (13).In addition, results of ultrastructural analysis by thin-sectionEM showed that the YCV has a spacious morphology begin-ning around 8 h postinfection, at which time bacterial replica-tion begins (13). How Y. pestis controls phagosome traffickingand expansion of its vacuole in macrophages is not understood.

Activation by the cytokine gamma interferon (IFN-�) isknown to dramatically alter the trafficking and environment ofphagosomes in macrophages (37, 41). Y. pestis is able to survivein primary murine macrophages that are activated with IFN-�(36). A chromosomally encoded operon termed ripCBA thatpromotes survival of Y. pestis in activated macrophages hasbeen identified. The ripCBA genes appear to encode novelmetabolic enzymes (36). However, it is not known how the Ripproteins promote intracellular survival, and it is unclear if themorphology or trafficking of the YCV is modified in IFN-�-activated macrophages.

Autophagy is a membrane trafficking process in eukaryoticcells that sequesters cytoplasmic material (e.g., defective mi-tochondria in the case of macroautophagy) in a vacuole (theautophagosome) and routes the cargo for destruction in anautophagolysosome (19, 21, 28). The autophagy pathway en-compasses several different membrane compartments, begin-ning with the phagophore, which functions to sequester cyto-plasmic material. Following trapping of cytoplasmic materialby the phagophore, the autophagosome is formed, which ma-tures through interactions with late endosomes and lysosomesinto an autophagolysosome, where degradation of luminalcomponents takes place. Autophagy is regulated at multiplesteps by several signaling molecules, including the mTOR ki-nase and class I and class III phosphatidylinositol 3-kinases(19, 21, 28). Recent studies have revealed that autophagy canplay an important role in the protective innate immune re-sponse to cytoplasmic or vacuolar bacterial pathogens (3, 21,28). The process that results in breakdown of microorganismswithin autophagosomes has been referred to as xenophagy(21). IFN-� can upregulate autophagy in macrophages (21). Inmacrophages exposed to IFN-�, upregulation of autophagyappears to override the phagosome maturation block imposedby Mycobacterium tuberculosis, allowing routing of the patho-gen to a microbicidal autophagolysosome-like compartment(21, 28). Alternatively, some intracellular pathogens appear toco-opt autophagy for survival within host cells (26, 28).

In this work we examined the possibility that the replicationof Y. pestis in macrophages exposed to IFN-� resulted from anincrease in autophagy and that the pathogen might exploit thisprocess for survival within phagosomes. Our results show that,although the YCV can interact with an autophagy pathway inmacrophages, this interaction does not appear to require acti-vation by IFN-� nor is it required for intracellular survival of Y.

pestis. Instead, we find that Y. pestis prevents phagosome acid-ification, and it is suggested that this process allows Y. pestis toavoid destruction in phagolysosomes or autophagolysosomesof either naïve or activated macrophages.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The Y. pestis strains used (Table 1)were derived from Y. pestis KIM (molecular group 2.MED) (1). Ampicillinresistance (Apr), chloramphenicol resistance (Cmr), and kanamycin resistance(aph-3� or Knr) cassettes were introduced into Y. pestis strains in compliance withCDC guidelines. Yersinia pseudotuberculosis strain 32777 (previously referred toas strain IP2777) is a serogroup I isolate. Y. pestis strains were routinely grown onheart infusion agar plates or in heart infusion broth at 28°C. Y. pseudotuberculosiswas grown in Luria-Bertani broth or on Luria-Bertani agar plates at 28°C. Y.pestis was killed by fixation for 30 min with 2.5% paraformaldehyde, followed byextensive washing with phosphate-buffered saline (PBS).

Strain construction. KIM6� was converted to the fully virulent background byintroducing pCD1 marked with Apr (pCD1Ap) (12) via electroporation as de-scribed previously (13). The resulting strain was designated KIM5� and ishandled according to select agent guidelines and using biosafety level 3 condi-tions. Screening for spontaneous loss of the pgm locus from KIM6� (13) resultedin KIM6. Introduction of pCD1Ap into KIM6 resulted in KIM5, a strain that isconditionally virulent and exempt from select agent guidelines. PlasmidpMMB207gfp3.1 (Cmr), used to express green fluorescent protein (GFP), wasintroduced into KIM5 or KIM6� by conjugation (13). A plasmid expressingmCherry under control of the tac promoter was generated using pRSET-mCherry (40). The mCherry gene was amplified from pRSET-mCherry by PCRusing oligonucleotides mCherry-EcoRI-F1 (5�-CCGGAATTCATGGTGAGCAAGGGCGAG-3�) and mCherry-HindIII-R1 (5�-CCCAAGCTTTTACTTGTACAGCTCGTCCATGCC-3�). The mCherry coding region was inserted intopMMB207 (27a) downstream of the tac promoter and between the EcoRI andHindIII sites. The resulting plasmid, p207mCherry, was introduced into KIM6�by conjugation. GFP or mCherry expression in Y. pestis was obtained by incu-bation in the presence of 500 �M isopropyl-�-D-thiogalactopyranoside (IPTG)for 1 to 2 h.

To construct a Knr plasmid expressing inducible GFP, pMMB207gfp3.1 wasmodified to inactivate the Cmr gene and to introduce a Knr cassette (aph-3�). Tothis end, the Knr cassette was isolated from pBSL86 (2) by restriction digestionwith SmaI and inserted into the SnaBI site of pMMB207gfp3.1, resulting ininactivation of the Cmr cassette. The resulting plasmid, p207gfp3.1::kan, wasconjugated into KIM5�, resulting in KIM5�/GFP.

Deletion of the ripCBA operon in KIM6� was performed by allelic recombi-nation. To this end, the ripCBA operon and flanking sequence were obtained byPCR amplification using the KIM6� chromosome as the template and primersY2382-F2-PvuII (5�-CAGCTGTTGACAACATCCAGAAATGCAATACCT-3�) and Y2385-R5-XmaI (5�-TCCCCCCGGGTCAGATTAGATGCATTTTTTTGGC-3�). The amplified DNA fragment was cloned into the pGEM-T Easyvector (Promega). The resulting plasmid was digested with AfeI, which recog-nizes and cleaves sites within ripC and ripA. Upon ligation of the ends of the

TABLE 1. Strains used in this study

Strain Relevant characteristics Reference

KIM6� pCD1� pPCP1�, pgm� 12KIM6�/GFP pCD1�, pPCP1�, pgm�,

pMMB207gfp3.1, Cmr36

KIM6�/mCherry pCD1�, pPCP1�, pgm�,p207mCherry, Cmr

This study

KIM6�phoP pCD1�, pPCP1�, pgm� �phoP 13KIM6�ripCBA pCD1�, pPCP1�, pgm� �ripCBA This studyKIM6 pCD1�, pPCP1�, �pgm 22KIM5� pCD1Ap, pPCP1�, pgm�, Apr This studyKIM5�/GFP pCD1Ap, pPCP1�, pgm�,

p207gfp3.1::kan, Knr AprThis study

KIM5 pCD1Ap, pPCP1�, �pgm, Apr 22KIM5/GFP pCD1Ap, pPCP1�, �pgm,

pMMB207gfp3.1, Apr Cmr13

KIM10 pCD1�, pPCP1�, �pgm 3332777 pYV� 42

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plasmid, a deletion was obtained that removed the 3� end of ripC, all of the ripBgene, and the 5� end of ripA. A DNA fragment with the deletion (�ripCBA) wasliberated from pGEM-T Easy and introduced into the suicide vector pSB890using NotI restriction sites (30). The resulting plasmid, pSB890�ripCBA, wasconjugated into KIM6�, and allelic exchange was performed as described pre-viously (14), resulting in KIM6�ripCBA.

Cell culture. Bone marrow-derived macrophages (BMDMs) were isolated andcultured as described previously (35, 36). BMDMs were prepared from femurs ofC57BL/6 mice (Jackson Laboratory), ATG5flox/flox-Lyz-Cre mice, or controlATG5flox/flox mice (52). ATG5flox/flox mice were backcrossed five generationsto a C57BL/6 line prior to use. 293T cells were cultivated at 37°C in the presenceof 5% CO2 in Dulbecco modified Eagle medium (DMEM) with Glutamax-I(Invitrogen) supplemented with 1 mM sodium pyruvate (Invitrogen) and 10%fetal bovine serum (FBS) (HyClone). J774A.1 murine macrophage-like cellswere cultured as described previously (13).

Assays for determination of bacterial survival in macrophages. The proce-dures used for infecting macrophages with Y. pestis or Y. pseudotuberculosis andfor measuring intracellular survival by a CFU or GFP induction assay have beendescribed previously (13, 35, 36).

EM. BMDMs (1.5 105 cells) seeded on plastic coverslips (diameter, 12 mm;Thermanox) were infected with KIM5� at a multiplicity of infection (MOI) of 10for 30 min in 200 �l of infection medium. Activation of BMDMs with IFN-� wasperformed as described previously (36). At the appropriate time, the infectedcells were fixed for 1 h with a mixture of 2% paraformaldehyde and 2.5%glutaraldehyde in 100 mM cacodylate buffer (pH 7.5) supplemented with 2.5 mMCaCl2. Fixative buffer baths were renewed after 30 min of incubation. Fixedsamples were washed three times for 5 min with 100 mM cacodylate buffer (pH7.5) and processed by the Central Microscopy Image Center at Stony BrookUniversity. Digital images of thin sections were acquired with an FEI BioTwinG2

transmission electron microscope.Detection of LC3 by immunoblotting. BMDMs (6.5 105 cells) seeded in

six-well plates were infected with Y. pestis strains as previously described (35),with the following modifications. An MOI of 10 to 15 was used, and BMDMswere infected for 30 min in a small volume (1 ml) of infection medium. BMDMswere activated by adding 150 U/ml IFN-� to the infected wells as previouslydescribed (36). Twenty-four hours postinfection, the infected cells were washedthree times with PBS and lysed with 100 �l RIPA buffer (150 mM NaCl, 1%NP-40, 0.5% sodium deoxycholate, 50 mM Tris [pH 8.0], 0.1% sodium dodecylsulfate [SDS]) containing protease inhibitors (Complete Mini, EDTA-free;Roche). Cell lysate from each well was collected, passed through a 22-gaugeneedle, and centrifuged for 10 min at 16,000 g at 4°C. Supernatants werecollected, protein concentrations were determined (bincinchoninic acid or Brad-ford protein assay), and 25 �g of protein from each sample was separated bySDS-polyacrylamide gel electrophoresis (15% polyacrylamide) and transferredonto a 0.2-�m polyvinylidene difluoride membrane for 2 h at 100 V using aMiniBiorad electrophoresis apparatus. The blots were probed overnight using apolyclonal anti-LC3 antibody (catalog no. NB100-2331; Novus) diluted 1:300 inTris-buffered saline–0.05% Tween containing 5% dry skim milk. To control forloading, blots were probed with a polyclonal anti-actin antibody (catalog no.4967; Cell Signaling). Secondary rabbit immunoglobulin G antibody conjugatedto horseradish peroxidase (catalog no. 111-035-144; Jackson Immunoresearch)was used at a dilution of 1:5,000. Immunoblots were developed using an en-hanced chemiluminescent reagent (Perkin-Elmer Life Science) and Kodak X-MAT film. Band intensities for LC3-II, LC3-I, and �-actin were quantified usingQuantityOne software (Bio-Rad), and ratios of LC3-II to LC3-I or LC3-II toactin were calculated.

Transfection of 293T cells and production of retroviral particles. 293T pack-aging cells (4 106 cells) were seeded in 100-mm cell culture-treated dishes 16 hbefore transfection. Six micrograms of pCL-Eco (Imgenex) and 6 �g of pJC110expressing GFP-LC3 (a gift from Jean Celli) (7) were added to the cell culturemedium using the HEPES-buffered saline calcium phosphate method (31).Seven hours later, the supernatant of transfected 293T cells was replaced withfresh cell culture medium and the preparation was incubated overnight. Thesupernatant containing retroviral particles was collected, centrifuged for 10 minat 834 g, and passed through a 0.45-�m filter. A second supernatant wasobtained after 8 h of incubation with fresh cell culture medium and processed asdescribed above.

Retroviral transduction of BMDMs. BMDMs seeded on coverslips in 24-wellplates at a concentration of 6 104 cells/well and grown for 2 days weretransduced with a 2:1 mixture of retrovirus-containing supernatant (first collec-tion) and BMDM culture medium (DMEM with Glutamax-I supplemented with20% FBS, 1 mM sodium pyruvate, and 45% L-cell supernatant). After 8 h, thesupernatant was replaced with fresh BMDM culture medium containing the sec-

ond retroviral harvest (ratio, 2:1). Twenty-four hours posttransduction, the su-pernatant of transduced BMDMs was removed and replaced with fresh BMDMinfection medium (DMEM with Glutamax-I supplemented with 10% FBS, 15%L-cell-conditioned supernatant, and 1 mM sodium pyruvate), and the prepara-tion was incubated for a minimum of 30 min before infection.

Determination of colocalization of GFP-LC3 with YCVs by fluorescence mi-croscopy. BMDMs were infected with KIM6�/mCherry and treated or nottreated with IFN-� as described previously (36). IPTG was added to the cellculture media 2 h before the end of a time point to induce mCherry expression.At the time indicated below, the infected cells were fixed with 2.5% paraformal-dehyde and processed for immunofluorescence labeling (35). Bacteria werestained with anti-Yersinia primary antibody (35) and a goat anti-rabbit secondaryantibody conjugated to Alexa Fluor 633 (Molecular Probes) for confocal micros-copy analysis or to Alexa Fluor 594 (Molecular Probes) for epifluorescenceanalysis. GFP-LC3 was immunolabeled using an anti-GFP monoclonal antibodydiluted 1:250 (catalog no. A11120; Invitrogen) and a goat anti-mouse antibodyconjugated to Alexa Fluor 488 to enhance detection. Confocal images wereacquired using a Zeiss inverted Axiovert 200 M microscope. GFP-LC3-positiveYCVs were quantified by epifluorescence microscopy using a Zeiss Axioplan2microscope equipped with a 40 objective. Images were captured using a Spotcamera (Diagnostic Instruments, Inc.) and assembled in Adobe Photoshop.GFP-LC3-positive YCVs were enumerated using merged images of green fluo-rescence (combined GFP and Alexa Fluor 488) and red fluorescence (combinedmCherry and Alexa Fluor 594), and the results were expressed as a percentageobtained by dividing the number of YCVs positive for LC3-GFP by the totalnumber of YCVs. Approximately 300 YCVs were enumerated for each conditionin a single experiment.

Phagosomal pH assays. Determination of Lysotracker Red DND-99 colocal-ization with YCVs containing Y. pestis was performed using J774A.1 macro-phage-like cells as described previously for Texas Red ovalbumin (13), exceptthat Lysotracker was added at a concentration of 50 nM 1 h prior to fixation andanalysis of samples. In some experiments macrophages were incubated in me-dium containing 30 �g/ml chloramphenicol to inhibit bacterial protein synthesisduring infection. Coinfection experiments were performed as follows. KIM5/GFP bacteria were induced with IPTG (500 �M) to express GFP and then fixed.Macrophages were infected at an MOI of 5 with fixed KIM5/GFP and unlabeledlive KIM5 (total MOI, 10). At 1.25 h postinfection cells were fixed, permeabilizedwith saponin, and stained with anti-Yersinia primary antibody (1:1,000) and AlexaFluor 350 (Molecular Probes) secondary antibody (1:500). Macrophages werelabeled with Lysotracker as described above.

The YCV pH was measured by ratiometic imaging as described previously(43), with the following modifications. Y. pestis was labeled with fluoresceinisothiocyanate (FITC) at a final concentration of 1 mg/ml in PBS (pH 8) for 30min. After three washes in PBS, the bacteria were used to infect J774A.1 cellsseeded in 35-mm glass coverslip dishes as described previously (13). A Zeiss LSM510 META microscope was used for live cell analysis. Images were obtainedusing excitation wavelengths of 488 nm and 458 nm. The pH of YCVs wasextrapolated from a calibration curve (pH 4 to 7) generated using 10 �Mmonensin and 10 �M nigericin (43). Signal intensities of at least 50 phagosomeswere determined in three different fields per experiment.

RESULTS

Fully virulent Y. pestis replicates in activated macrophages.We previously demonstrated that Y. pestis cured of pCD1(KIM6�) (Table 1) replicates in murine BMDMs activatedwith IFN-� (36). To determine if fully virulent, pCD1-contain-ing Y. pestis can survive in activated BMDMs, a GFP inductionassay was performed (35). KIM5�/GFP (Table 1) pregrown at28°C was used to infect BMDMs, and survival of extracellularbacteria was prevented by use of gentamicin (see Materials andMethods). At different time points postinfection de novo syn-thesis of GFP was induced by addition of IPTG, and live cellswere examined using phase-contrast and fluorescence micros-copy. As shown in Fig. 1, KIM5�/GFP replicated in activatedin BMDMs between 6 and 24 h postinfection. At a highermagnification, GFP-positive bacteria were clearly visible withinspacious YCVs of activated BMDMs at 24 h postinfection (Fig.

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2), with elongated bacteria typically apposed to the phago-somal membrane.

Ultrastructural analysis of YCV morphology in activatedmacrophages. We next looked for evidence that YCVs interactwith an autophagy pathway in activated macrophages. Evi-dence for the interaction of intracellular bacteria with an au-tophagy pathway can be obtained by analysis of phagosomemorphology at the ultrastructural level (19). Nascent autopha-gosomes typically have a double membrane. Fusion of lyso-somes with an autophagosome results in maturation of thevacuole to an autophagolysosome, which has a single delimit-ing membrane. Therefore, the presence of a double membranesurrounding an intracellular bacterium can be taken as evi-dence that the pathogen was residing in a nascent autophago-some-like vacuole at the time of cell fixation. Thin-section EMwas used to examine the possibility that Y. pestis resides inautophagosome-like vacuoles in activated macrophages.BMDMs were infected with KIM5� (Table 1) pregrown at28°C and then exposed to IFN-�. Analysis of the infected

macrophages at 4 h postinfection by thin-section EM (seeMaterials and Methods) showed that some intracellular Y.pestis bacteria were present within a tight-fitting double mem-brane (Fig. 3A and B). However, more commonly, a tight-fitting single membrane surrounded intracellular Y. pestis andother vesicular material (Fig. 3C). At a later time point (22 hpostinfection) Y. pestis bacteria were found to be present inspacious phagosomes with partial double membranes (Fig. 3Dand E and Fig. 3F, inset). These results indicated that, inactivated macrophages, Y. pestis resided in autophagosome-like vacuoles, as well as in vacuoles with morphologies similarto those of standard phagosomes or multivesicular bodies.

IFN-� and replication of Y. pestis increase LC3-II levels inmacrophages. The progress of autophagy is executed by ATGproteins, which comprise two protein conjugation systems. TheATG8 protein (more commonly known as LC3-I in its uncon-jugated form) becomes conjugated to phosphatidylethano-lamine during autophagy, resulting in the formation of LC3-II,which specifically associates with autophagic membranes (19).

FIG. 1. Analysis of Y. pestis replication in activated macrophages by microscopy. BMDMs infected with KIM5�/GFP were activated with IFN-�and incubated for 6 h (A and B) or 24 h (C and D) before microscopic examination. One hour before examination, IPTG was added to inducede novo expression of GFP in viable intracellular bacteria. (A and C) Representative phase-contrast microscopy images. (B and D) Representativefluorescence microscopy images.

FIG. 2. Examination of Y. pestis-containing spacious phagosomes in activated macrophages by microscopy. (A and B) Enlargements of regionsof panels C and D of Fig. 1, respectively. (C) Overlay of panels A and B.



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The conversion of LC3-I to LC3-II and the turnover or deg-radation of LC3-II within autophagolysosomes can be followedby immunoblotting, and this procedure has become a usefultool for measuring rates of autophagy within eukaryotic cells(19, 27). The steady-state level of LC3-II, as detected by im-munoblotting of cell lysates, was used to determine if autoph-agy was altered in macrophages after 24 h of treatment withIFN-� and/or infection by Y. pestis KIM6�. The LC3-II signalobtained by immunoblotting was normalized by using two dif-ferent approaches (19), calculating the ratio of LC3-II to LC3-Iand calculating the ratio of LC3-II to a loading control (actin).Treatment of uninfected BMDMs with IFN-� resulted in anincreased ratio of LC3-II to LC3-I compared to that for un-treated macrophages (Fig. 4A, compare lanes 3 and 4; Fig. 4B).Interestingly, after infection of BMDMs with Y. pestis KIM6�in the absence of IFN-�, there were large increases in thesteady-state levels of both LC3-I and LC3-II (Fig. 4A, comparelanes 1 and 3) and there was a slight increase in the LC3-II/LC3-I ratio compared to the data for uninfected macrophages(Fig. 4B). Infection of BMDMs with KIM6� in the presence ofIFN-� resulted in a further increase in the LC3-II/LC3-I ratio(Fig. 4A, lane 2; Fig. 4B). A time course analysis indicated thatthe increased LC3-II level was observed beginning at 3 hpostinfection (data not shown). To determine if the increase inthe LC3-II level required survival of Y. pestis in macrophages,BMDMs were infected with a KIM6� phoP mutant (Table 1),which is defective for intracellular survival, or with killed form-aldehyde-fixed KIM6�. Compared to the data for macro-phages infected with live KIM6�, the LC3-II/actin ratios werelower after challenge with the phoP mutant or fixed KIM6�

(Fig. 4C and D). Moreover, the LC3-II/actin ratio was higherin BMDMs infected with a Y. pestis strain containing pCD1 butlacking the pgm locus (KIM5) (Table 1) than in uninfectedBMDMs (Fig. 4E and F), although in this case the ratios werehigher in nonactivated macrophages than in IFN-�-treatedBMDMs (Fig. 4E and F). We have observed that KIM5 doesnot replicate in activated BMDMs as well as KIM6� repli-cates, in part because it lacks the ripCBA operon in the pgmlocus (36). A KIM6� ripCBA mutant (Table 1) had a pheno-type similar to that of KIM5 with respect to LC3-II levels (Fig.4E and F). These results indicated that the levels of LC3-II inmacrophages could be increased by treatment with IFN-� or byinfection with replicating Y. pestis.

Analysis of GFP-LC3 colocalization with YCVs in macro-phages. A GFP-LC3 fusion protein can be used as a specificcytological marker for autophagic membranes in eukaryoticcells (19). Fluorescence microscopy was used to quantify theGFP-LC3 in YCVs in macrophages exposed or not exposed toIFN-�. For this purpose, BMDMs were transduced with aretrovirus producing GFP-LC3 (see Materials and Methods).One day after transduction, BMDMs were infected withKIM6�/mCherry (Table 1) in the presence or absence ofIFN-�. IPTG was added to the infected wells for 2 h to inducede novo mCherry expression in viable intracellular Y. pestis.Eight hours postinfection, the infected wells were fixed and thesamples were processed for immunofluorescence microscopy(see Materials and Methods). The colocalization of GFP-LC3and bacterial signals was determined by confocal fluorescencemicroscopy. As shown in Fig. 5B, GFP-LC3 could be detectedon YCVs in macrophages that were treated with IFN-�, con-

FIG. 3. YCV morphology in activated macrophages as determined by thin-section EM. BMDMs were infected with KIM5� and then treatedwith IFN-�. At 4 h (A to C) or 22 h (D to F) the cells were fixed and processed for thin-section EM. (A) BMDM infected with KIM5� at 4 hpostinfection. (B) Magnification of area containing bacteria in panel A. The arrow indicates a double membrane surrounding a single bacterium.(C) BMDM infected with KIM5� at 4 h postinfection, showing two bacteria and other vesicles in a single-membrane compartment. (D) BMDMinfected with KIM5� for 22 h. The arrow indicates a region with a double membrane. (E) Magnification of a region in panel D. (F) BMDMinfected with KIM5� for 22 h. A region containing a double membrane is magnified in the inset. (A) Bar 2 �m. (B to F) Bars 500 nm.



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firming that an autophagic membrane can be recruited tophagosomes containing viable Y. pestis, as suggested by trans-mission EM results (Fig. 3B). However, quantification by epi-fluorescence microscopy showed that only 30.4% of YCVs inactivated macrophages were positive for GFP-LC3 (Fig. 5B). Inaddition, a slightly higher percentage of YCVs colocalized withGFP-LC3 in naïve macrophages (42.5%) (Fig. 5A). Thus, al-though Y. pestis infection modulated autophagy, based on in-creased steady-state levels of LC3-II (Fig. 4), the majority ofYCVs did not colocalize with the GFP-LC3 marker of autophagicmembranes in BMDMs. It was anticipated that the percentage ofGFP-LC3-positive YCVs would be highest in activated macro-phages, conditions under which maximal LC3-II levels were seen(Fig. 4), but this was not the case (see Discussion).

Y. pestis inhibits phagosome acidification. The results de-scribed above suggested that the localization of an autophagicmembrane to YCVs and the increase in LC3-II levels duringinfection may not be critical for intracellular survival, butrather a side effect or downstream consequence of the strategythat Y. pestis uses to survive in macrophages. Turnover ofLC3-II trapped in autophagosomes is mediated by lysosomal

FIG. 5. Analysis of GFP-LC3 colocalization with YCVs. BMDMswere transduced with retrovirus producing GFP-LC3. Twenty-fourhours posttransduction, the macrophages were infected with KIM6�/mCherry. De novo expression of mCherry was induced in viable intra-cellular bacteria by addition of IPTG. After 8 h of infection with Y.pestis in the absence (A) or presence (B) of IFN-�, cells were fixed andprocessed for examination by fluorescence microscopy. Images wereobtained by using confocal fluorescence microscopy. The panels showoverlays of GFP-LC3 (green) and mCherry (red) signals from repre-sentative images. The percentages of colocalization of signals as de-termined by epifluorescence microscopy are shown for each condition,and the numbers of phagosomes scored are indicated in parentheses.

FIG. 4. Immunoblot analysis of LC3 levels in BMDMs exposed to IFN-� and infected with Y. pestis. BMDMs were not infected or were infectedwith KIM6� in the presence or absence of IFN-�. At 24 h postinfection cell lysates were prepared and subjected to SDS-polyacrylamide gelelectrophoresis, followed by immunoblotting with anti-LC3 antibody (A, C, and E) and anti-actin antibody (C and E). For panels C and E,macrophages were infected with the KIM6� phoP mutant, with KIM6� fixed with paraformaldehyde, with KIM5, or with the KIM6� ripCBAmutant. Densitometry was used to quantify band signals for LC3-I and LC3-II (A) or for LC3-II and actin (C and E), and calculated ratios areshown in panels B, D, and F, respectively. For panel B the ratio of LC3-II to LC3-I in uninfected and untreated cells was defined as 1, and otherratios were normalized to this value.



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proteases which are activated in autophagolysosomes. Prevent-ing lysosomal degradation by use of protease inhibitors ordrugs (such as bafilomycin A1) that inhibit vacuole acidifica-tion leads to an increase in the steady-state level of LC3-II(19). Y. pseudotuberculosis, which is closely related to Y. pestis(1), has been shown to inhibit acidification of phagosomes inprimary murine macrophages (46). To determine if Y. pestisprevents acidification of phagosomes, colocalization of YCVswith a probe for acidic lysosomes (Lysotracker DND-99) inmacrophages was assayed by fluorescence microscopy. Whennaïve J774A.1 murine macrophage-like cells were infected withKIM5/GFP (Table 1) in the presence of Lysotracker, very fewGFP-positive YCVs colocalized with Lysotracker up to 5 hpostinfection (Fig. 6A to C and G). In contrast, phagosomescontaining killed fixed KIM5/GFP showed increased colocal-ization with Lysotracker with time, and the value reached 90%by 2.5 h postinfection (Fig. 6D to F and G). This result sug-gested that live Y. pestis could prevent acidification of theYCVs to values below the value required for detection ofLysotracker (pH �5.5).

We next used live cell ratiometric imaging and Y. pestislabeled with the pH-sensitive dye FITC to measure the pH ofthe YCV over a time course (43). As shown in Fig. 7, the pH

FIG. 6. Measurement of YCV acidification using Lysotracker and fluorescence microscopy. J774A.1 macrophages on coverslips were infected withlive KIM5/GFP (A to C) or fixed KIM5/GFP (D to F) in the presence of Lysotracker Red DND-99. At 1.25 h postinfection the samples were fixed, andrepresentative images were obtained by using a fluorescence microscope equipped with a digital camera. The images show the GFP signal (A and D),the Lysotracker signal (B and E), or an overlay of the two signals (C and F). (G) Percent colocalization of GFP and Lysotracker signals at the indicatedtime points, as quantified using three independent experiments. The symbols indicate averages, and the error bars indicate standard deviations.

FIG. 7. Measurement of YCV pH using live cell ratiometric imag-ing. Live KIM5 cells, fixed KIM5 cells, or live 32777 cells labeled withFITC were used to infect J774A.1 macrophages. At the indicated timepoints live cell imaging was performed using a confocal microscope.Images were obtained by using excitation wavelengths of 488 nm and458 nm. A calibration curve was generated using the ionophores mon-ensin and nigericin and media buffered at pHs between pH 4 and 7.Comparison of the 488 nm/458 nm ratio to the pH calibration curvewas used to estimate the pH of YCVs. The symbols indicate theaverages of three experiments (for 32777) or of five experiments (forKIM5 and fixed KIM5), and the error bars indicate standard devia-tions.



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of phagosomes containing live KIM5 was �6.5 at 30 minpostinfection. Over the next several hours the pH of the YCVwas determined to be �8, confirming the finding of the Lyso-tracker assay that Y. pestis inhibits phagosome acidification.Similar results were obtained for Y. pseudotuberculosis 32777(Table 1) (Fig. 7), confirming that this property is shared by thetwo Yersinia species. In contrast, the pH of YCVs containingfixed KIM5 decreased to �4.5 within 2.5 h (Fig. 7). Theseresults showed that Y. pestis inhibits acidification of its phago-somes, regardless of whether it acquires an autophagic mem-brane, and prevents maturation of these vacuoles. Thus, in theYCVs that incorporate an autophagic membrane, the normalturnover of LC3-II may be prevented, resulting in accumula-tion of this autophagy marker.

Autophagy is dispensable for survival of Y. pestis in macro-phages. Based on the results described above (Fig. 5 to 7), wepredicted that autophagy is dispensable for survival of Y. pestisin macrophages, and to test this hypothesis, we utilizedBMDMs deficient for this process. In many studies, 3-methyl-adenine, an inhibitor of class I and class III phosphatidylino-sitol 3-kinases, is used to pharmacologically block autophagy incells. In preliminary control experiments we found that growthof Y. pestis in bacterial culture media was inhibited in thepresence of 5 mM 3-methyladenine, a concentration that istypically used to block autophagy in cells. We therefore soughtan alternative approach to block autophagy that did not in-volve the potential nonspecific effects of chemical inhibitors.The ATG5 protein is essential for autophagy, as it is requiredfor the formation of the phagophore (19, 21). Mice lackingATG5 in myelomonocytic cells have been generated by breed-ing ATG5flox/flox mice with mice in which Cre recombinase isexpressed from the lysozyme M locus (52). BMDMs derivedfrom the ATG5flox/flox-Lyz-Cre mice are defective for auto-phagy and have been used previously to determine the role ofautophagy in coronavirus replication in host cells (52).

BMDMs prepared from ATG5flox/flox-Lyz-Cre mice or con-trol ATG5flox/flox mice were infected with Y. pestis. Immuno-blotting for LC3-I and LC3-II confirmed that the ATG5flox/flox-Lyz-Cre BMDMs were defective for autophagy (data notshown) (52). The BMDMs were infected with KIM5 or theKIM5 phoP mutant, and a CFU assay (see Materials andMethods) was performed at different time points after infec-tion. As shown in Fig. 8, KIM5 survived equally well inBMDMs from ATG5flox/flox-Lyz-Cre mice and BMDMs fromcontrol ATG5flox/flox mice. The phoP mutant showed a similarsurvival defect in both types of macrophages (Fig. 8). There-fore, although YCVs can interact with an autophagy pathwayin macrophages, recruitment of an autophagic membrane doesnot appear to be required for survival of Y. pestis in phago-somes. Instead, a primary mechanism of Y. pestis survival inmacrophages seems to be inhibition of YCV acidification.

Inhibition of phagosome acidification by Y. pestis does notrequire de novo bacterial protein synthesis. To determine if denovo bacterial protein synthesis was required for Y. pestis toinhibit phagosome acidification, J774A.1 macrophages wereinfected in the presence of the bacteriostatic antibiotic chlor-amphenicol at a final concentration of 30 �g/ml. The results ofa Lysotracker assay performed between 1.5 and 5 h postinfec-tion showed that there was no difference in the ability ofKIM5/GFP to inhibit phagosome acidification in the presence

and in the absence of chloramphenicol (data not shown). Todetermine if chloramphenicol was effectively inhibiting bacte-rial protein synthesis under the conditions used for the infec-tion assay, macrophages were infected with KIM5/GFP thathad not been preinduced to express GFP. GFP expression wasnot upregulated in bacteria in chloramphenicol-treated mac-rophages following addition of IPTG, showing that the antibi-otic effectively inhibited de novo protein synthesis. The resultsof a coinfection experiment with live and fixed KIM5 showedthat live bacteria were unable to prevent colocalization ofLysotracker with phagosomes containing fixed bacteria whenthey were in the same macrophage (data not shown). Takentogether, these results suggest that Y. pestis is able to inhibitphagosome acidification using a factor(s) that is produced bythe pathogen prior to phagocytosis and that this factor doesnot act globally within the macrophage to inhibit phagosomeacidification.

Inhibition of phagosome acidification by Y. pestis does notrequire known virulence factors. KIM10, a Y. pestis strain lack-ing pCD1, pPCP1, and the pgm locus (Table 1), was tested forthe ability to prevent phagosome acidification in J774A.1 cells.The results of a Lysotracker assay with KIM10 showed thatgenes in pCD1, pPCP1, or the pgm locus were not required toprevent phagosome acidification (data not shown). In addition,although a Y. pestis phoP mutant is defective for intracellularsurvival, phagosomes containing the KIM6� phoP mutant didnot colocalize with Lysotracker for the limited period duringwhich the bacteria remained intact within macrophages, sug-gesting that the factor(s) required to inhibit phagosome acid-ification is not under control of the PhoP regulon.

DISCUSSION

Several species of pathogenic bacteria subvert the functionof macrophages in order to survive and replicate within these

FIG. 8. CFU assay of Y. pestis survival in ATG5flox/flox-Lyz-CreBMDMs or control ATG5flox/flox BMDMs. BMDMs prepared fromATG5flox/flox-Lyz-Cre mice (Atg5�/�) or control ATG5flox/flox mice(Atg5�/�) were infected with KIM5 or the KIM5 phoP mutant at anMOI of 10. After 20 min of infection the macrophages were washed toremove nonadherent bacteria and then incubated in medium contain-ing gentamicin to kill extracellular bacteria. At the indicated timepoints washed macrophages were lysed in detergent, and serial dilu-tions were plated to enumerate CFU. The data are values from threeindependent experiments, and the error bars indicate standard devia-tions. An asterisk indicates a significant difference (P � 0.01) com-pared to the data for the 20-min time point for the same group, asdetermined by one-way analysis of variance with Dunnett’s posttest.



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cells. Examples include Francisella, Brucella, Legionella, Liste-ria, Mycobacterium, and Salmonella species. These bacteriaescape from the phagosome and replicate in the macrophagecytosol or modify the phagosome to reach a replicative niche.Pathogenic bacteria that survive within macrophage phago-somes utilize several different strategies to avoid being killed ina vacuole that could potentially mature into a phagolysosome(9, 24). M. tuberculosis survives within macrophages by stallingmaturation of its vacuole at an early stage. The mycobacterialphagosome is characterized by the absence of a number ofmarkers that are usually associated with late endosomes (37,47). For example, Mycobacterium inhibits acidification of itsvacuole by preventing the accumulation of vATPase. Maturelysosomal proteases are also excluded from the mycobacterialphagosome.

We have shown that the YCV has an unusual traffickingpattern as it can acquire markers of late endosomes (LAMP1and cathepsin D) (13) and autophagosomes (GFP-LC3) (Fig.5), yet it does not undergo acidification (Fig. 6 and 7). It isthought that acidification is required for efficient phagosome-lysosome or autophagosome-lysosome fusion (17, 28, 49). Wehypothesize that the YCV fuses with several vesicular com-partments, including late endosomes, multivesicular bodies,and autophagosomes, but fusion with lysosomes and thus fullmaturation of the YCV are inefficient due to its lack of acid-ification. The observation that Y. pestis infection results inincreased steady-state levels of LC3-II (Fig. 4) is consistentwith this model, since LC3-II turnover would be prevented innonacidified autophagosomes. We previously showed that Y.pestis could replicate in BMDMs exposed to IFN-� (36). IFN-�treatment is known to stimulate autophagy, and some patho-gens appear to hijack the autophagy pathway for intracellularreplication (26, 28). However, in some cases IFN-�-inducedautophagy is protective, leading to efficient killing (i.e., xe-nophagy) of intracellular bacteria, such as M. tuberculosis. Ourresults suggest that Y. pestis has a strategy to avoid the protec-tive role of xenophagy in either naïve or activated phagocytesand that the increase in the steady-state levels of LC3-II in Y.pestis-infected macrophages is an effect, rather than a cause, ofYCV formation.

Although results described here indicate that autophagy isnot required for survival of Y. pestis in macrophage phago-somes (Fig. 8), it is possible that recruitment of an autophagicmembrane to the YCV has important consequences for theoutcome of the pathogen-host interaction. Autophagosomesmay provide a source of membrane, along with late endo-somes, for expansion of the YCV into a spacious compartment.Alternatively, recruitment of an autophagic membrane to theYCV may prevent use of the autophagy pathway for its normalfunctions in macrophages, such as clearing damaged mitochon-dria or survival during starvation or growth factor withdrawal.There is evidence that death of macrophages infected withSalmonella enterica serovar Typhimurium can occur by auto-phagy (16). We suggest that recruitment of an autophaghicmembrane to the YCV and prevention of LC3-II turnoverallow intracellular Y. pestis to slowly compromise the viabilityof the macrophage. This could provide a mechanism for re-lease of the bacteria from the dying host cell once an intracel-lular replication cycle has been completed. In this context, weobserved that treatment with IFN-� decreased recruitment of

GFP-LC3 to the YCV (Fig. 5), which may represent a protec-tive response on the part of the macrophage to prevent seques-tration of autophagic membrane by the pathogen.

The ability to prevent phagosome acidification appears to bean attribute common to several pathogenic bacteria (17). Asdiscussed above for M. tuberculosis, the arrest in maturation ofits phagosome precludes acquisition of a sufficient density ofvATPase, and the failure to acidify is most likely a conse-quence of the block in membrane trafficking (17). It is cur-rently unclear how Y. pestis prevents phagosome acidification.It is possible that, like M. tuberculosis, Y. pestis prevents asso-ciation of the vATPase with the YCV. Alternatively, thevATPase may associate with the YCV but is inactivated, as hasbeen suggested for Y. pseudotuberculosis (46). The use of anti-vATPase antibodies in conjunction with microscopy techniquesshould allow us to distinguish between these possibilities.

Recently, Listeria monocytogenes has been shown to be ca-pable of replicating slowly in macrophage phagosomes thatexpand and do not acidify (4). The pore-forming hemolysinlisteriolysin O is required for this process, and it was suggestedthat pore formation by listeriolysin O allows dissipation of thepH gradient (4). Y. pestis is not known to secrete a pore-forming hemolysin, and analysis of the sequenced genomedoes not reveal predicted proteins with significant homology topore-forming hemolysins (unpublished observations). The pu-tative factor that Y. pestis secretes to inhibit phagosome acid-ification appears to be produced by the bacterium prior tomacrophage interaction, and it appears to act on or within thephagosome in a local manner. There is precedence for theproduction of vATPase-inhibiting molecules by microorgan-isms (e.g., bafilomycin A1), but to our knowledge there is noknown bacterial factor that can mediate this activity directly. Itis not inconceivable that a bacterial protein secreted in aphagosome could inhibit the vATPase. The V0 domain of thevATPase is exposed on the luminal side of the vacuole, andthere is previously obtained evidence that a monoclonal anti-body that is specific for the a subunit of the V0 domain caninhibit vATPase activity when this antibody resides in the en-dosomal compartment (38).

Straley and Harmon showed that genes in pCD1, pPCP1,and the pgm locus were not required for replication of Y. pestisin murine macrophages (44). Using a Lysotracker assay, wefound that these genetic elements are also not required forinhibition of phagosome acidification by Y. pestis. The phoPgene is important for survival of Y. pestis in macrophages (29).PhoP-regulated genes important for survival of S. enterica se-rovar Typhimurium in the phagosomes of macrophages in-clude genes of the pmr operon. The products of the pmroperon function to modify lipid A with aminoarabinose; thisincreases bacterial resistance to antimicrobial peptides (10,15). Genes under control of PhoP in Y. pestis that are impor-tant for survival in macrophages include ugd, pmrK, and mgtC,which are predicted to promote resistance to antimicrobialpeptides (ugd and pmrK) or low-Mg2� conditions (mgtC) of aphagosome (13). Phagosomes containing Y. pestis phoP mu-tants do not colocalize with Lysotracker for the period of timethat the bacteria remain intact within macrophages. This resultindicates that inhibition of phagosome acidification is not suf-ficient for intracellular survival of Y. pestis; resistance to anti-microbial peptides and resistance to environmental stress en-



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countered within the YCV are also important for replication ofthe pathogen in macrophages. Further understanding of themechanism by which Y. pestis inhibits phagosome acidificationrequires identification of genes essential for this process.

ACKNOWLEDGMENTS

We thank John Brumell and Joel Swanson for advice concerningexperiments, Wei-Xing Zong, Erica Ullman, and Zhixun Dou for helpwith the LC3 immunoblotting procedures, Susan VanHorn and Guo-Wei Tian for their assistance with transmission EM and confocalmicroscopy, Sylvia Samaniego for advice concerning the protocol forretroviral transduction, and Jean Celli for providing the GFP-LC3retroviral construct. We thank Jens Grabenstein for making thep207gfp3.1::kan plasmid and KIM5� and James Murtha for technicalassistance with construction of the Y. pestis ripCBA mutant. HanaFukuto is thanked for providing helpful comments to improve themanuscript.

Herbert W. Virgin IV is gratefully acknowledged for advice andfinancial support (to Z.Z.). This work was supported by NIH grant P01AI055621 (to J.B.B.) and NIH grant U54 AI057160 Project 6 (toH.W.V.).

REFERENCES

1. Achtman, M., G. Morelli, P. Zhu, T. Wirth, I. Diehl, B. Kusecek, A. J. Vogler,D. M. Wagner, C. J. Allender, W. R. Easterday, V. Chenal-Francisque, P.Worsham, N. R. Thomson, J. Parkhill, L. E. Lindler, E. Carniel, and P.Keim. 2004. Microevolution and history of the plague bacillus, Yersinia pestis.Proc. Natl. Acad. Sci. USA 101:17837–17842.

2. Alexeyev, M. F. 1995. Three kanamycin resistance gene cassettes with dif-ferent polylinkers. BioTechniques 18:52, 54, 56.

3. Amano, A., I. Nakagawa, and T. Yoshimori. 2006. Autophagy in innateimmunity against intracellular bacteria. J. Biochem. 140:161–166.

4. Birmingham, C. L., V. Canadien, N. A. Kaniuk, B. E. Steinberg, D. E.Higgins, and J. H. Brumell. 2008. Listeriolysin O allows Listeria monocyto-genes replication in macrophage vacuoles. Nature 451:350–354.

5. Cavanaugh, D. C., and R. Randall. 1959. The role of multiplication ofPasteurella pestis in mononuclear phagocytes in the pathogenesis of fleaborneplague. J. Immunol. 85:348–363.

6. Charnetzky, W. T., and W. W. Shuford. 1985. Survival and growth of Yersiniapestis within macrophages and an effect of the loss of the 47-megadaltonplasmid on growth in macrophages. Infect. Immun. 47:234–241.

7. Checroun, C., T. D. Wehrly, E. R. Fischer, S. F. Hayes, and J. Celli. 2006.Autophagy-mediated reentry of Francisella tularensis into the endocytic com-partment after cytoplasmic replication. Proc. Natl. Acad. Sci. USA 103:14578–14583.

8. Du, Y., R. Rosqvist, and A. Forsberg. 2002. Role of fraction 1 antigen ofYersinia pestis in inhibition of phagocytosis. Infect. Immun. 70:1453–1460.

9. Duclos, S., and M. Desjardins. 2000. Subversion of a young phagosome: thesurvival strategies of intracellular pathogens. Cell. Microbiol. 2:365–377.

10. Ernst, R. K., T. Guina, and S. I. Miller. 1999. How intracellular bacteriasurvive: surface modifications that promote resistance to host innate immuneresponses. J. Infect. Dis. 179:S326–S330.

11. Finegold, M. J. 1969. Pneumonic plague in monkeys. An electron micro-scopic study. Am. J. Pathol. 54:167–185.

12. Gong, S., S. W. Bearden, V. A. Geoffroy, J. D. Fetherston, and R. D. Perry.2001. Characterization of the Yersinia pestis Yfu ABC inorganic iron trans-port system. Infect. Immun. 69:2829–2837.

13. Grabenstein, J. P., H. S. Fukuto, L. E. Palmer, and J. B. Bliska. 2006.Characterization of phagosome trafficking and identification of PhoP-regu-lated genes important for survival of Yersinia pestis in macrophages. Infect.Immun. 74:3727–3741.

14. Grabenstein, J. P., M. Marceau, C. Pujol, M. Simonet, and J. B. Bliska.2004. The response regulator PhoP of Yersinia pseudotuberculosis is impor-tant for replication in macrophages and for virulence. Infect. Immun. 72:4973–4984.

15. Groisman, E. A. 2001. The pleiotropic two-component regulatory systemPhoP-PhoQ. J. Bacteriol. 183:1835–1842.

16. Hernandez, L. D., M. Pypaert, R. A. Flavell, and J. E. Galan. 2003. ASalmonella protein causes macrophage cell death by inducing autophagy.J. Cell Biol. 163:1123–1131.

17. Huynh, K. K., and S. Grinstein. 2007. Regulation of vacuolar pH and itsmodulation by some microbial species. Microbiol. Mol. Biol. Rev. 71:452–462.

18. Janssen, W. A., and M. J. Surgalla. 1969. Plague bacillus: survival within hostphagocytes. Science 163:950–952.

19. Klionsky, D. J., H. Abeliovich, P. Agostinis, D. K. Agrawal, G. Aliev, D. S.Askew, M. Baba, E. H. Baehrecke, B. A. Bahr, A. Ballabio, B. A. Bamber,

D. C. Bassham, E. Bergamini, X. Bi, M. Biard-Piechaczyk, J. S. Blum, D. E.Bredesen, J. L. Brodsky, J. H. Brumell, U. T. Brunk, W. Bursch, N.Camougrand, E. Cebollero, F. Cecconi, Y. Chen, L. S. Chin, A. Choi, C. T.Chu, J. Chung, P. G. Clarke, R. S. Clark, S. G. Clarke, C. Clave, J. L.Cleveland, P. Codogno, M. I. Colombo, A. Coto-Montes, J. M. Cregg, A. M.Cuervo, J. Debnath, F. Demarchi, P. B. Dennis, P. A. Dennis, V. Deretic,R. J. Devenish, F. Di Sano, J. F. Dice, M. Difiglia, S. Dinesh-Kumar, C. W.Distelhorst, M. Djavaheri-Mergny, F. C. Dorsey, W. Droge, M. Dron, W. A.Dunn, Jr., M. Duszenko, N. T. Eissa, Z. Elazar, A. Esclatine, E. L. Eskelinen,L. Fesus, K. D. Finley, J. M. Fuentes, J. Fueyo, K. Fujisaki, B. Galliot, F. B.Gao, D. A. Gewirtz, S. B. Gibson, A. Gohla, A. L. Goldberg, R. Gonzalez, C.Gonzalez-Estevez, S. Gorski, R. A. Gottlieb, D. Haussinger, Y. W. He, K.Heidenreich, J. A. Hill, M. Hoyer-Hansen, X. Hu, W. P. Huang, A. Iwasaki,M. Jaattela, W. T. Jackson, X. Jiang, S. Jin, T. Johansen, J. U. Jung, M.Kadowaki, C. Kang, A. Kelekar, D. H. Kessel, J. A. Kiel, H. P. Kim, A.Kimchi, T. J. Kinsella, K. Kiselyov, K. Kitamoto, E. Knecht, M. Komatsu, E.Kominami, S. Kondo, A. L. Kovacs, G. Kroemer, C. Y. Kuan, R. Kumar, M.Kundu, J. Landry, M. Laporte, W. Le, H. Y. Lei, M. J. Lenardo, B. Levine,A. Lieberman, K. L. Lim, F. C. Lin, W. Liou, L. F. Liu, G. Lopez-Berestein,C. Lopez-Otin, B. Lu, K. F. Macleod, W. Malorni, W. Martinet, K. Mat-suoka, J. Mautner, A. J. Meijer, A. Melendez, P. Michels, G. Miotto, W. P.Mistiaen, N. Mizushima, B. Mograbi, I. Monastyrska, M. N. Moore, P. I.Moreira, Y. Moriyasu, T. Motyl, C. Munz, L. O. Murphy, N. I. Naqvi, T. P.Neufeld, I. Nishino, R. A. Nixon, T. Noda, B. Nurnberg, M. Ogawa, N. L.Oleinick, L. J. Olsen, B. Ozpolat, S. Paglin, G. E. Palmer, I. Papassideri, M.Parkes, D. H. Perlmutter, G. Perry, M. Piacentini, R. Pinkas-Kramarski, M.Prescott, T. Proikas-Cezanne, N. Raben, A. Rami, F. Reggiori, B. Rohrer,D. C. Rubinsztein, K. M. Ryan, J. Sadoshima, H. Sakagami, Y. Sakai, M.Sandri, C. Sasakawa, M. Sass, C. Schneider, P. O. Seglen, O. Seleverstov, J.Settleman, J. J. Shacka, I. M. Shapiro, A. Sibirny, E. C. Silva-Zacarin, H. U.Simon, C. Simone, A. Simonsen, M. A. Smith, K. Spanel-Borowski, V. Srini-vas, M. Steeves, H. Stenmark, P. E. Stromhaug, C. S. Subauste, S. Sugimoto,D. Sulzer, T. Suzuki, M. S. Swanson, I. Tabas, F. Takeshita, N. J. Talbot, Z.Talloczy, K. Tanaka, I. Tanida, G. S. Taylor, J. P. Taylor, A. Terman, G.Tettamanti, C. B. Thompson, M. Thumm, A. M. Tolkovsky, S. A. Tooze, R.Truant, L. V. Tumanovska, Y. Uchiyama, T. Ueno, N. L. Uzcategui, I. van derKlei, E. C. Vaquero, T. Vellai, M. W. Vogel, H. G. Wang, P. Webster, J. W.Wiley, Z. Xi, G. Xiao, J. Yahalom, J. M. Yang, G. Yap, X. M. Yin, T.Yoshimori, L. Yu, Z. Yue, M. Yuzaki, O. Zabirnyk, X. Zheng, X. Zhu, andR. L. Deter. 2008. Guidelines for the use and interpretation of assays formonitoring autophagy in higher eukaryotes. Autophagy 4:151–175.

20. Lathem, W. W., S. D. Crosby, V. L. Miller, and W. E. Goldman. 2005.Progression of primary pneumonic plague: a mouse model of infection,pathology, and bacterial transcriptional activity. Proc. Natl. Acad. Sci. USA102:17786–17791.

21. Levine, B., and V. Deretic. 2007. Unveiling the roles of autophagy in innateand adaptive immunity. Nat. Rev. Immunol. 7:767–777.

22. Lilo, S., Y. Zheng, and J. B. Bliska. 2008. Caspase-1 activation in macro-phages infected with Yersinia pestis KIM requires the type III secretionsystem effector YopJ. Infect. Immun. 76:3911–3923.

23. Lukaszewski, R. A., D. J. Kenny, R. Taylor, D. G. Rees, M. G. Hartley, andP. C. Oyston. 2005. Pathogenesis of Yersinia pestis infection in BALB/c mice:effects on host macrophages and neutrophils. Infect. Immun. 73:7142–7150.

24. Meresse, S., O. Steele-Mortimer, E. Moreno, M. Desjardins, B. Finlay, andJ. P. Gorvel. 1999. Controlling the maturation of pathogen-containing vacu-oles: a matter of life and death. Nat. Cell Biol. 1:E183–E188.

25. Meyer, K. F. 1950. Immunity in plague; a critical consideration of somerecent studies. J. Immunol. 64:139–163.

26. Mizushima, N., B. Levine, A. M. Cuervo, and D. J. Klionsky. 2008. Auto-phagy fights disease through cellular self-digestion. Nature 451:1069–1075.

27. Mizushima, N., and T. Yoshimori. 2007. How to interpret LC3 immunoblot-ting. Autophagy 3:542–545.

27a.Morales, V. M., A. Backman, and M. Bugdasarian. 1991. A series of wide-host-range low-copy-number vectors that allow direct screening for recom-binants. Gene 97:39–47.

28. Ogawa, M., and C. Sasakawa. 2006. Bacterial evasion of the autophagicdefense system. Curr. Opin. Microbiol. 9:62–68.

29. Oyston, P. C. F., N. Dorrell, K. Williams, S.-R. Li, M. Green, R. W. Titball,and B. Wren. 2000. The response regulator PhoP is important for survivalunder conditions of macrophage-induced stress and virulence in Yersiniapestis. Infect. Immun. 68:3419–3425.

30. Palmer, L. E., S. Hobbie, J. E. Galan, and J. B. Bliska. 1998. YopJ of Yersiniapseudotuberculosis is required for the inhibition of macrophage TNF� pro-duction and the downregulation of the MAP kinases p38 and JNK. Mol.Microbiol. 27:953–965.

31. Pear, W. S., A. M. Scott, and G. P. Nolan (ed.). 1997. Generation of hightiter, helper-free retroviruses by transient transfection. Humana Press, To-towa, NJ.

32. Perry, R. D., and J. D. Fetherston. 1997. Yersinia pestis—etiologic agent ofplague. Clin. Microbiol. Rev. 10:35–66.

33. Perry, R. D., M. L. Pendrak, and P. Schuetze. 1990. Identification and



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

cloning of a hemin storage locus involved in the pigmentation phenotype ofYersinia pestis. J. Bacteriol. 172:5929–5937.

34. Prentice, M. B., and L. Rahalison. 2007 Plague. Lancet 369:1196–1207.35. Pujol, C., and J. B. Bliska. 2003. The ability to replicate in macrophages is

conserved between Yersinia pestis and Yersinia pseudotuberculosis. Infect.Immun. 71:5892–5899.

36. Pujol, C., J. P. Grabenstein, R. D. Perry, and J. B. Bliska. 2005. Replicationof Yersinia pestis in interferon gamma-activated macrophages requires ripA,a gene encoded in the pigmentation locus. Proc. Natl. Acad. Sci. USA102:12909–12914.

37. Rohde, K., R. M. Yates, G. E. Purdy, and D. G. Russell. 2007. Mycobacteriumtuberculosis and the environment within the phagosome. Immunol. Rev.219:37–54.

38. Sato, S. B., and S. Toyama. 1994. Interference with the endosomal acidifi-cation by a monoclonal antibody directed toward the 116 (100)-kD subunitof the vacuolar type proton pump. J. Cell Biol. 127:39–53.

39. Sebbane, F., D. Gardner, D. Long, B. B. Gowen, and B. J. Hinnebusch. 2005.Kinetics of disease progression and host response in a rat model of bubonicplague. Am. J. Pathol. 166:1427–1439.

40. Shaner, N. C., R. E. Campbell, P. A. Steinbach, B. N. Giepmans, A. E.Palmer, and R. Y. Tsien. 2004. Improved monomeric red, orange and yellowfluorescent proteins derived from Discosoma sp. red fluorescent protein.Nat. Biotechnol. 22:1567–1572.

41. Shenoy, A. R., B. H. Kim, H. P. Choi, T. Matsuzawa, S. Tiwari, and J. D.MacMicking. 2007. Emerging themes in IFN-gamma-induced macrophageimmunity by the p47 and p65 GTPase families. Immunobiology 212:771–784.

42. Simonet, M., and S. Falkow. 1992. Invasin expression in Yersinia pseudotu-berculosis. Infect. Immun. 60:4414–4417.

43. Steinberg, T. H., and J. A. Swanson. 1994. Measurement of phagosome-lysosome fusion and phagosomal pH. Methods Enzymol. 236:147–160.

44. Straley, S. C., and P. A. Harmon. 1984. Growth in mouse peritoneal mac-rophages of Yersinia pestis lacking established virulence determinants. Infect.Immun. 45:649–654.

45. Straley, S. C., and P. A. Harmon. 1984. Yersinia pestis grows within phagoly-sosomes in mouse peritoneal macrophages. Infect. Immun. 45:655–659.

46. Tsukano, H., F. Kura, S. Inoue, S. Sato, H. Izumiya, T. Yasuda, and H.Watanabe. 1999. Yersinia pseudotuberculosis blocks the phagosomal acidifi-cation of B10.A mouse macrophages through the inhibition of vacuolarH�-ATPase activity. Microb. Pathog. 27:253–263.

47. Vergne, I., J. Chua, S. B. Singh, and V. Deretic. 2004. Cell biology ofMycobacterium tuberculosis phagosome. Annu. Rev. Cell Dev. Biol. 20:367–394.

48. Viboud, G. I., and J. B. Bliska. 2005. Yersinia outer proteins: role in modu-lation of host cell signaling responses and pathogenesis. Annu. Rev. Micro-biol. 59:69–89.

49. Vieira, O. V., R. J. Botelho, and S. Grinstein. 2002. Phagosome maturation:aging gracefully. Biochem. J. 366:689–704.

50. Welkos, S., M. L. M. Pitt, M. Martinez, A. Friedlander, P. Vogel, and R.Tammariello. 2002. Determination of the virulence of the pigmentation-deficient and pigmentation-/plasminogen activator-deficient strains of Yer-sinia pestis in non-human primate and mouse models of pneumonic plague.Vaccine 20:2206–2214.

51. Welkos, S. L., A. M. Friedlander, and K. J. Davis. 1997. Studies on the roleof plasminogen activator in systemic infection by virulent Yersinia pestisstrain C092. Microb. Pathog. 23:211–223.

52. Zhao, Z., L. B. Thackray, B. C. Miller, T. M. Lynn, M. M. Becker, E. Ward,N. N. Mizushima, M. R. Denison, and H. W. T. Virgin. 2007. Coronavirusreplication does not require the autophagy gene ATG5. Autophagy 3:581–585.

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