Differential Clearance and Host-Pathogen Interactions of YopE- andYopK- YopL- Yersinia pestis in BALB/c Mice
SUSAN C. STRALEY1* AND MICHAEL L. CIBULL2
Departments of Microbiology and Immunology' and Pathology,2 Chandler Medical Center,University of Kentucky, Lexington, Kentucky 40536
Received 28 November 1988/Accepted 8 January 1989
This study characterized infections in BALB/c mice by the nonpigmented Yersinia pestis KIM and itsderivatives lacking the low-Ca2+-response virulence plasmid pCD1 or failing to express selected yersinial outermembrane proteins (YOPs). The parent Y. pestis showed net growth in the spleen by 2 h and in the liver after7 h; exponential growth in both the liver and spleen culminated in death of the mice starting on day 4, with totalbacterial numbers of less than 108 in the blood, liver, and spleen together. The histopathology progressed frommicroabscesses to extensive coagulative necrosis unaccompanied by further immigration of inflammatory cells.This, together with the relatively low bacterial numbers, suggests a toxigenic mechanism. YopE- or YopK-YopL- yersiniae were cleared from the spleen but grew in the liver after an initial lag. Their growth was curbedafter 1 to 2 days and entered a plateau that lasted 5 to 6 days; viable numbers then declined rapidly. Thissuggests that these Yop- mutations distinguish, at least kinetically, between host responses in liver and spleen.Both strains caused acute inflammation in liver that evolved into structured lesions surrounded by progres-
sively mononuclear inflammation suggestive of a granulomatous response. Accordingly, YOP E and YOPs Kand L are necessary in the early days of the infection for net growth in spleen and prolonged growth in the liver;their absence is reflected morphologically by the emergence of cell-mediated immunity in the liver. The YopE-and YopK- YopL- mutants bound only slightly increased amounts of C3, suggesting that YOPs E, K, and Lare protective through mechanisms other than interfering with the binding of complement.
There are 11 yersinial outer membrane proteins (YOPs)expressed by the 75-kilobase low-Ca2+-response (Lcr) viru-lence plasmid of Yersinia pestis (31). The other two speciesof Yersinia pathogenic for humans, Y. pseudotuberculosisand Y. enterocolitica, have Lcr plasmids highly homologousto the one in Y. pestis (26) and express a similar number ofYOPs (31), some of which cross-react immunologically withthose of Y. pestis (3). However, Y. pestis expresses twoYOPs not found in Y. pseudotuberculosis, YOPs K and L(31, 32).The YOPs are a heterogeneous group of proteins with
respect to molecular weight and isoelectric point (31). Theyare organized in multiple operons scattered over the Lcrplasmids (12, 32). In Y. pestis, the YOPs have been shown tobe regulated at the transcriptional level by temperature andCa2+, being expressed maximally at 37°C in the absence ofCa2+ (32). The phagolysosome of the resident macropfiagemay be an important environment for their expression invivo, as the operons encoding YOPs K and L, E, and H havebeen shown to be strongly expressed by Y. pestis growingwithin cultured human monocyte-derived macrophages (25;M. S. Klempner and S. C. Straley, unpublished data). YOPE, which is present in the three Yersinia species as immu-nologically cross-reactive molecules (3, 29), and YOPs Kand L, unique to Y. pestis, have been shown to be necessaryfor full virulence of Y. pestis in mice (32). YOPs K and L liein a common operon (24). An insert of Mu dlI (Ap lac) at28.4 kilobases on the pCD1 map (14) eliminates expressionof only YOP L (32), whereas an insert at 31 kb eliminatesexpression of both YOPs and greatly increases the 50%lethal dose (LD50) of the bacteria in mice inoculated intra-venously (24, 32). Accordingly, YOP K and/or L is impor-tant for virulence of Y. pestis in this mouse model. An insert
* Corresponding author.
eliminating the expression of YOP J did not affect virulencein the intravenous mouse model (32).The YOPs may have several roles in pathogenesis. In Y.
pseudotuberculosis and Y. enterocolitica, Lcr-plasmid-en-coded surface components have been shown to inhibitcomplement deposition on the bacteria, thereby providingresistance to phagocytosis (17, 18, 36). Surface proteinswere implicated in this phenomenon, but these studies didnot identify which YOPs might be responsible. The high-molecular-weight YOP A not expressed by Y. pestis hasbeen implicated in serum resistance in Y. enterocolitica (1),but not in Y. pseudotuberculosis, in which serum resistanceis not dependent upon the Lcr plasmid (23). This shows thatthe presence of a YOP can have different effects in differentmolecular contexts. Both Y. pseudotuberculosis and Y.enterocolitica release some YOPs into the medium undercertain culture conditions (12, 15), which raises the possibil-ity that these proteins may function as released proteins,e.g., as immunological flak or as invasins. In vitro assess-ment of possible roles for YOPs in the pathogenesis of Y.pestis has been hampered by the degradation of theseproteins by the plasminogen activator (the plasmid-encodedfibrinolysin activity [28-30]) located in the outer membraneof this species (33).
Considerable current interest is focused on the roles of theYOPs in pathogenesis of the yersiniae; their study promisesto enlarge our understanding of the host-parasite interactionsof this genus of facultative intracellular parasites. Further, as
major immunologically cross-reacting outer membrane pro-teins expressed in vivo (3), the YOPs may form the basis ofuseful detection and diagnostic methods for the yersiniaepathogenic for humans. They also are potentially importantcomponents of an improved plague vaccine. In this study wecharacterize the infections due to Y. pestis Yop- insertionmutants to obtain clues about the functions of YOP E and of
"All Y. pestis strains were derivatives of Y. pestis KIM5 pgm-1, which is conditionally virulent due to the loss of the chromosomally encoded pigmentationvirulence property. Strains KIM5-3022, KIM5-3031, and KIM5-3073 were derived as previously described (32).
b The three plasmids present in the parent Y. pestis strain (11) are pCD1, which encodes the low-Ca2+ response responsible for the phenomenon of calciumdependence; pPCP1, which encodes plasminogen activator and coagulase activities as well as a bacteriocin pesticin; and pMT1, the cryptic ("MT') 110-kilobaseplasmid.
Lcr+, Ability to express the complete set of low-Ca2+-response virulence properties; Pac+, ability to express the pPCP1-encoded plasminogen activator andcoagulase activities: YopE+, YopK+, YopL+, and YopJ+, ability to express these various YOPs; Tox+, ability to express the plague murine toxin.
d Determined in a previous study (32).eND, Not determined.
YOPs K and L in pathogenesis of experimental plague inmice. We found that yersiniae missing YOPs K and L werecompromised in the ability to show rapid initial growth in theliver and spleen, resulting in clearance from the spleen andearly curbing of growth. Moreover, a granulomatous reac-tion was seen in response to YopE- and YopK- YopL-strains in contrast to a progressively cell-poor response seenwith the parent Yop+ strain. These findings place the func-tions of these YOPs early in infection and form the basis oftestable hypotheses for the roles of these proteins in thevirulence of Y. pestis.
MATERIALS AND METHODS
Bacteria and bacteriophages. The Y. pestis strains used inthis study are described in Table 1. They were conditionallyvirulent due to the absence of the pigmentation virulencedeterminant (Pgm-), a property genetically and biochemi-cally unlinked to the low-Ca2" response. This renders thebacteria avirulent from a peripheral route of infection, suchas subcutaneous or intraperitoneal inoculation, but theyretain full virulence when given intravenously (37). Y. pestisKIM8 (pPCP1-) was obtained from Robert R. Brubaker,Michigan State University. The pCD1- Y. pestis KIM10 wasobtained from a colony of Y. pestis KIM8 that grew at 37°Con tryptose-blood agar base (Difco Laboratories, Detroit,Mich.) with added 25 mM MgCl2 and 25 mM sodium oxalateto chelate-free Ca2+ (16). Y. pestis KIM8-3122, KIM8-3131,and KIM8-3173 contained pCD1 plasmids having transposoninsertions known to abolish expression of YOP E, YOPs Kand L, and YOP J, respectively (24, 32), but in which Mud11734 replaced the original transposon Mu dl (Apr lac)
(hereafter called Mu dlI). This replacement stabilized theinserts against further transposition, because Mu d11734lacks the Mu transposase gene (8). The pCD1::Mu dIlplasmids, previously transformed into Escherichia coli K-12X1553 (32), were converted to pCD1::Mu d11734 by homol-ogous recombination. Mu d11734 was transduced from E.coli P011734 (obtained from Malcolm J. Casadaban, Univer-sity of Chicago) into E. coli X1553 (pCD1: :Mu dIl-22,pCD1::Mu dIl-31, or pCDI1::Mu dIl-73) via bacteriophageP1L4, with selection for kanamycin resistance on Mud11734. The resulting pCD1::Mu dI1734, small enough topackage within P1L4, was then transduced into Y. pestisKIM10 with selection for kanamycin resistance and screen-ing for loss of ampicillin resistance carried by Mu dlI. Thefinal Y. pestis strains were confirmed to have the expectedplasmid profiles and BamHI and HindlIl restriction patternsfor their isolated plasmids. In Y. pestis KIM5-3022, KIM5-3031, and KIM5-3073, the Mu dlI inserts had been stabilizedagainst high-frequency transposition by the presence of aTn9 insert in the Mu b gene (32).
Cultivation of bacteria. Bacterial stock cultures were main-tained and growth was initiated for experiments as previ-ously described (34). E. coli was grown at 30°C in Luriabroth or on Luria agar (20). Y. pestis cells to be injected intomice were grown at 26°C in xylose- and MgCI2-supple-mented heart infusion broth (Difco) and were washed andsuspended in phosphate-saline buffer as described previ-ously (34). Phosphate-saline buffer was solution "a" ofDulbecco phosphate-buffered saline (10). Appropriate doseswere obtained by diluting the bacteria in phosphate-salinebuffer, using the relationship A620 = 1 corresponds to
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approximately 5.8 x 108 CFU/ml. To determine the actualnumber of CFU per dose, every dose was serially diluted inphosphate-saline buffer and plated in triplicate on tryptose-blood agar base incubated at 30°C. Yersiniae to be tested forC3 deposition were grown under conditions described pre-viously that elicit strong expression of YOPs: followingseven generations of exponential growth at 26°C in definedmedium (TMH) containing 20 mM MgCl2 and no addedCa2+, the temperature of the culture (A620 = 0.2) was shiftedto 37°C, and growth was continued for 6 h (32). Theappropriate antibiotics were included in all media at 25 ,ug/mleach during growth of all bacteria that carried drug resis-tance markers.
Infection of mice. Female BALB/cByJ mice (JacksonLaboratories, Bar Harbor, Maine), 6 to 8 weeks old, were-anaesthetized with methoxyflurane and injected intrave-nously retro-orbitally with 0.1 ml of bacterial suspension.For determinations of the LD50, 5 (for Y. pestis KIM5-3031)or 10 (for Y. pestis KIM5-3022) mice were used per dose.They were caged in groups of 5 and observed for 14 days (forY. pestis KIM5-3031) or 16 days (for Y. pestis KIM5-3022).The LD50 was calculated by the method of Reed and Muench(27). For determinations of infection kinetics, mice receiveda dose of approximately 5 x 103 yersiniae.A preliminary experiment using Y. pestis KIM5, KIM6,
KIM5-3022, and KIM5-3031 indicated that the distribution ofbacteria within organs was nonuniform and that separateanimals would be required for histopathology and CFUdeterminations. A second preliminary experiment using Y.pestis KIM5 showed that of the lungs, liver, spleen, andlymph nodes (cervical, axial, inguinal, and brachial) frommice dying of the infection, the most significant histologicalabnormalities were seen in the liver and spleen. Accord-ingly, we restricted our CFU determinations to the blood,liver, and spleen. The organs from five mice (unless other-wise specified) were pooled for each datum point. Mice wereanaesthetized with Metofane, and 0.1 ml of blood wascollected by heart puncture and mixed with 0.5 ml of ice-coldsterile distilled water. Water was used as a diluent topromote release of bacteria from any infected macrophagespresent. Control tests had shown that the viability of Y.pestis is not significantly affected by incubation for 1 to 2 hin water. In initial experiments, the water contained 10 U ofheparin per ml, but this was discontinued after we found thatclotting did not occur if the collection tube was held on iceand samples (or dilutions in phosphate-saline buffer) wereplated in triplicate (tryptose-blood agar base incubated at300C) as soon as a total of 0.5 ml of blood had been pooledfrom five mice. The limit of detection (total of one colony onthe three plates) was approximately 3 CFU/ml of blood.Following blood collection, each mouse was killed by cervi-cal dislocation, and its liver and spleen were asepticallyremoved and placed in weighed petri dishes. The organswere then weighed, minced with scalpel blades, transferredto sterile bags containing 10 ml of distilled water, and placedon ice. The bags were sealed with tape and homogenized inthe Stomacher 80 Lab-Blender (Tekmar Co., Cincinnati,Ohio) for 90 s. The tonicity was restored by the addition of1 ml of 1Ox phosphate-saline buffer, and the suspensionswere diluted with phosphate-saline buffer and plated intriplicate (tryptose-blood agar base incubated at 300C). Thelimit of detection was approximately 6 CFU/g of liver and 60CFU/g of spleen. Histopathological observations were madeon the liver, lungs, spleen, kidney, and heart, and, in somecases, intestines. Mice were killed by cervical dislocation,incisions were made in their organs to permit efficient
penetration of fixative, the organs were placed in room-temperature 10% neutralized Formalin, and portions wereprocessed through graded alcohol and xylene and embeddedin paraffin. Sections (4 pLm) stained with hematoxylin andeosin were examined. Photographs were taken with anOlympus Vanox microscope and Kodak Panatomic-X film.C3 deposition. Female BALB/cByJ mice, 6 to 7 weeks old,
were exsanguinated by heart puncture. Serum was collectedby centrifugation and pooled, using glass tubes and pipettes.The pooled serum was held on ice and used within 6 h or wasdispensed into glass tubes and frozen overnight at -20°C.The frozen serum was transferred to -70°C and used within2 weeks. Frozen serum was thawed once. Serum that washeated a-t 56°C for 30 min on the day of the experiment wasused as a negative control. Yersiniae grown so as to elicitstrong expression of YOPs were washed once and sus-pended in phosphate-saline buffer (at room temperature),and 0.5-ml samples containing ca. 2 x 108 bacteria wereadded to 0.5-ml volumes of serum in glass tubes (13 by 125mm). These were placed at 37°C for 1 h with occasionalshaking. The bacteria were then pelleted and washed twicewith phosphate-saline buffer in a microfuge at room temper-ature. They were suspended in 0.5 ml of ice-cold goatanti-mouse C3 coupled to fluorescein isothyocyanate (Or-ganon Teknika, Malvern, Pa.) which had been reconstitutedas instructed by the vendor and then diluted 1:20 withphosphate-saline buffer. The tubes were incubated on ice for30 min, and then the bacteria were pelleted and washedtwice with phosphate-saline buffer. They were suspended in0.5 ml of cold 1% paraformaldehyde, pH 7.4, freshly pre-pared in phosphate-saline buffer, and refrigerated overnight.Samples in which the bacteria were clumped even aftervigorous (30 s at full speed) vortex mixing (Vortex-Genie;Scientific Instruments, Inc., Bohemia, N.Y.) were subjectedto 40 strokes in a Dounce homogenizer (piston A). Thesuspensions were diluted in phosphate-saline buffer to give afinal concentration of ca. 106 bacteria per ml. Forward-scattered fluorescein isothiocyanate fluorescence was mea-sured in a fluorescence-activated cell sorter (FACStar cy-tometer; Becton-Dickinson FACS Systems, Mountain View,Calif.) with a 560-nm dichroic mirror and set for 488-nmexcitation and 400-mW power.
RESULTS
Growth of Yop- Y. pestis in mice. A goal of this study wasto learn how YOPs E, K, and L contribute to pathogenesis ofY. pestis. Our approach was to compare the infectionsproduced in mice by Y. pestis strains lacking YOP E orYOPs K and L with that caused by the parent strain. Weinfected BALB/c mice intravenously with 5 x 103 CFU ofthese bacteria and measured the numbers of CFU in theliver, spleen, and blood as a function of time. For the parentstrain (Y. pestis KIM5), this challenge was approximately 50LD50 doses (Table 1). Under these conditions, the parent Y.pestis had initiated growth in liver and spleen by the firstsampling time, 1.4 days after infection (Fig. 1A). The bacte-ria continued to grow in these organs, and mice began to dieon day 4. We detected Y. pestis KIM5 in blood only late inthe infection (4.4 days; 1.9 x 103 CFU/ml of blood). Thisprobably reflects the direct rapid localization of bacteria tothe liver and spleen following retro-orbital injection and therelatively low bacterial numbers in organs when the micedied. (We had no difficulty detecting the large numbers ofCFU in blood characteristic of a different infection model[intraperitoneal challenge of mice previously injected intra-
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Yop- YERSINIA PESTIS IN MICE 1203
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FIG. 1. Infection kinetics of Y. pestis Yop- mutants and refer-ence strains in mice. BALB/cByJ mice were infected intravenously(retro-orbitally). At daily intervals thereafter, groups of mice (five,unless otherwise specified) were sacrificed and CFU were deter-mined for their pooled livers (O and 0) and spleens (El and *). Datain parentheses represent less than 30 colonies counted in the averagefrom three replicate plates. (A) Open symbols, parent strain Y.pestis KIM5 (dose, 7 x 103 CFU); closed symbols, Y. pestisKIM5-3073 (YopJ-) (dose, 5 x 103 CFU, not confirmed by plating);half-closed symbols, Y. pestis KIM6 (pCD1-) (dose, 2 x 103 CFU).For Y. pestis KIM5-3073, the numbers of mice pooled for eachdatum point were five (day 1), four (day 2), three (day 3), and two(day 4). (B) Y. pestis KIM5-3022 (YopE-). Closed symbols, Dose of5 x 103 CFU; open symbols, dose of 4 x 105 CFU (four mice usedper datum point). (C) Y. pestis KIM5-3031 (YopK- YopL-). Closedsymbols, Dose of 6 x 103 CFU (on day 12, no CFU were detectedfor either the liver or spleen); open symbols, dose of 4 x 105 CFU(four mice used per datum point).
peritoneally with an emulsion of FeSO4 and peanut oil {S. C.Straley and M. L. Cibull, manuscript in preparation}]).These infection kinetics for the parent Y. pestis are essen-tially identical to those previously reported for mice infectedintravenously via the tail vein with the same Yersinia strain(37, 38).We found similar kinetics of infection for a strain lacking
YOP J (Y. pestis KIM5-3073; Fig. 1A), previously shown tobe fully virulent in this intravenous mouse model (32). Thesedata show that the presence of a MudIlb::Tn9 insert in pCD1had no significant effect on the development of the infection.
Yersiniae lacking pCD1 (Y. pestis KIM6) were clearedfrom both liver and spleen (Fig. 1A), as shown previously(37). The YopE- and YopK- YopL- mutants showedinfection kinetics intermediate between those for the parentY. pestis KIM5 and the pCD1- Y. pestis KIM6. Theiravirulence was revealed dramatically by their low numbersof CFU in the liver, their complete clearance from the spleen(Fig. 1B), and their absence from blood. After an apparentlag, both the YopE- and the YopK- YopL- strains wereable to initiate growth in the liver, but their numbers reacheda plateau after 3 or 4 days and began to decline after about 8days (Fig. 1B and C). However, the YopK- YopL- strainwas not fully eliminated from the animals during the 2-weekexperiment; after declining to undetectable numbers 12.4days after infection, small numbers of these bacteria wereagain recovered in the liver on day 14. In contrast, theYopE- bacteria were completely cleared from the miceduring the 14-day observation period. Both mutants were
106
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104
103
102
101
1000 12 24 36 48
HOURS AFTER INFECTION
FIG. 2. Early infection kinetics of the parent Y. pestis KIMS andYopE- Y. pestis KIM5-3022 in mice. BALB/cByJ mice wereinfected and analyzed for the number of viable yersiniae as in theexperiment shown in Fig. 1. 0 and *, CFU in the liver; O and *,CFU in the spleen. Parentheses denote data based on fewer than 30colonies in the average from three replicate plates. Closed symbols,Parent Y. pestis KIM5 (dose, 4 x 103 CFU); open symbols, Y. pestisKIM5-3022 (YopE-) (dose, 2 x 103 CFU).
cleared from the spleen, with the YopE- strain showing themost rapid disappearance. This clearance apparently was adelayed response, because the bacterial numbers recoveredfrom spleens were comparable to the input numbers as lateas 34 h after infection (Fig. 1). These data show that theYop- mutations affected early events in the infection;growth in spleen was essentially prevented, and net growthin liver was initially retarded compared with that shown bythe parent and then lasted only 1 to 2 days.These initial kinetics were confirmed by measurements of
viable bacterial numbers of the parent and the YopE- strainat earlier times after infection (Fig. 2). There was no detect-able lag (i.e., less than 2 h) in growth of the parent Y. pestisin the spleen (Fig. 2), whereas the YopE- strain did notshow net growth in the spleen (not plotted). The slight lag inthe growth of the YopE- strain relative to that of the parentin the liver was seen clearly by 18 h and probably wasoccurring from the beginning (Fig. 2). This suggests that theYopE- bacteria were avirulent because they were unable tocounteract an element of the natural defense system thatfunctions early in infection.Because none of the plate counts for CFU in the spleen
contained greater than 30 colonies for the 5 x 103 dose of theYopE- strain, we repeated the determinations for the spleenusing a dose of 4 x 105 CFU (approximately the LD50; Table1), but the bacteria were still cleared (Fig. 1B). At the LD50,
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TABLE 2. Times of death and appearance of enterocolitis-associated symptoms in mice infected with Y. pestis
KIM5-3022 (YopE-)Doseb
Daya 102 103 104 105 106
Deaths EAS Deaths EAS Deaths EAS Deaths EAS Deaths EAS
a Day after infection.6 A total of 10 BALB/cByJ mice were injected retro-orbitally with the
designated number of CFU's of Y. pestis KIM5-3022. EAS, Enterocolitis-associated symptoms (see text). Values are the numbers of mice that weredead or that showed EAS on a given day.'-, None.d Only a faint stain remained around anus; mouse was recovering.
we would have expected some of the mice to have infectionscomparable to those seen with the parent strain, resulting inmarkedly retarded clearance of the bacteria or even a netincrease. When mice were infected with the YopK- YopL-strain at the LD50, this was in fact observed (Fig. 1C), andthese mice, as with the parent Y. pestis KIM5, died betweendays 4 and 6. To characterize the apparently differentoutcome of infection by YopE- Y. pestis, we observedgroups of 10 mice infected by various doses of these yersin-iae and found a curious biphasic character to the disease(Table 2). The mice died between days 4 and 6 only afterreceiving the highest dose tested (106 CFU). Additionaldeaths occurred during days 8 to 13, associated with anenterocolitis manifested outwardly by some or all of thefollowing symptoms: wasting; a red, swollen anus; andmatted, stained fur around the anus. This phenomenon wasnot seen in infections with other strains of Y. pestis used inthis study. This secondary condition was seen in fewer thanhalf of the animals and was dose dependent, with an optimaldose of around 104 CFU (Table 2). Symptoms were firstnoticeable on days 5 to 8 after infection, and the timing andcharacter of the secondary condition were reproducible (fourexperiments). We were not able to recover Y. pestis from themice showing the intestinal symptoms using the followingtechniques: (i) swabbing the anus of a symptomatic mouse 10days after infection and culturing on tryptose-blood agar plusampicillin; (ii) similarly culturing rectal/colonic lavages madewith phosphate-saline buffer from two mice showing dif-ferent degrees of symptoms on day 8 after infection; and (iii)washing (with phosphate-saline buffer) the excised ceca andcolons from four symptomatic mice on day 6 after infection,plating the resulting suspensions on MacConkey agar sup-plemented with potassium gluconate as the carbon source(no antibiotics), and probing the colonies that appeared afterincubation at 30°C by a colony blot procedure, using nick-translated plasmids from Y. pestis KIM (pCD1, pMT1, andpPCP1) as the hybridization probe. We did not furthercharacterize this YopE--associated enterocolitis. However,these preliminary studies indicate that there are two distinctphases to disease in mice infected with Y. pestis KIM5-3022,giving rise to a net LD50 that is comparable to the one for Y.pestis KIM5-3031.
Histopathology elicited by Y. pestis infection. Infectionswith the parent strain of Y. pestis, the YopE- strain, and theYopK- YopL- strains demonstrated both qualitative andquantitative differences (Fig. 3). The parent strain (KIM5)caused microabscesses containing predominantly polymor-phonuclear neutrophils (PMN), scattered randomly withinthe liver parenchyma by 12 h and in the marginal zone of thespleen by 24 h. These progressed to large areas of necrosis,with a disproportionate loss of accompanying inflammation,such that on day 3, inflammation exceeded necrosis (Fig. 3A)but by day 5 necrosis far exceeded inflammation in bothorgans (Fig. 3C). The spleen was particularly hard hit, withessentially all of the red pulp and a portion of the white pulpbeing necrotic by day 5 (Fig. 3F). No evidence of dissemi-nated intravascular coagulation was observed in any of theorgans studied. These histological observations confirm onespreviously reported for mice infected with Y. pestis KIM5via the tail vein (5, 39).The earliest lesions in the liver seen in mice infected with
YopE- or YopK- YopL- yersiniae were similar to thoseseen with the parent strain but were delayed slightly inonset. Later lesions associated with infection by the YopE-bacteria (KIM5-3022) were fewer but larger and more local-ized than those seen with KIM5 (Fig. 3D). The lesions wereassociated with an intense early polymorphonuclear re-sponse (day 3) which later evolved a more granulomatouscharacter. In contrast to the extensive necrosis of the spleenseen with the parent strain, the spleen in the mice infectedwith KIM5-3022 showed only rare, poorly formed granulo-mas in the white pulp by day 3 and only mild congestion withscattered PMNs in the red pulp thereafter through day 9. Thehistological features of the late (days 8 to 10) enterocolitiswere characterized primarily by abscesses involving thelymphoid tissue (Peyer's patches) of the ileum and colon(Fig. 4B).The hepatic lesions seen in mice infected with the YopK-
YopL- strain (KIM5-3031) were similar to but smaller thanthose seen in the infection by YopE- yersiniae and lessnumerous than those seen with the parent strain (Fig. 3E).Moreover, the progression from a PMN (Fig. 3B) to apredominantly mononuclear response (Fig. 3E) was morerapid than that seen in the infection by YopE- bacteria.However, like the latter, it culminated in granulomatouslesions (Fig. 4A). Surprisingly, the cycle was apparentlyreinitiated with microabscesses associated with PMN re-sponse reappearing on day 12 and progressing to a mixedPMN-mononuclear response by day 14. These histologicfindings were associated with a reappearance of viablebacteria by culture (Fig. 1). No splenic lesions were detect-able in the mice infected with Y. pestis KIM5-3031.C3 deposition by the parent and Yop- Y. pestis grown in
vitro. The data described above had shown that early eventsin the host-parasite interaction were altered in the infectionswith the Yop- yersiniae. Laboratories studying relatedsystems have reported that the absence of the Lcr plasmid orof all of the YOPs resulted in increased complement depo-sition and enhanced phagocytosis of the bacteria (17, 18, 36).Complement deposition is a component of the host defensesystem that might affect the fate of Y. pestis early in aninfection, perhaps by altering the "visibility" of the bacteriato PMNs via opsonization. Accordingly, we measured C3deposition by yersiniae exposed to normal serum fromBALB/cByJ mice. The necessary handling for this assayremoved the yersiniae from conditions inductive of YOPexpression long enough to permit the plasminogen activatorto degrade the YOPs that had been inserted in the outer
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FIG. 3. Histopathology in mice infected with the parent Y. pe.stis KIM5 or with mutants lacking YOP B or YOPs K and L. Mice wereinfected alongside the others of the experiment shown in Fig. 1 and were processed at intervals thereafter for histological examination. Bar,100 p.m in all panels except B, where it represents 20 p.m. (A) Liver, KIM5, day 3. The early lesions for all three strains were characterizedby scattered microabscesses with a predominantly polymorphonuclear response. (B) Liver. KIM5-3031 (YopK- YopL-), day 3, showing onesuch early lesion at high power. (C, D, and B) Liver, day 5. (C) KIM5; (D) KIM5-3022 (YopE-); and (E) KIM5-3031 (YopK- YopL-). Thelesion due to the parent KIM5 (C) shows a lack of inflammation associated with extensive necrosis in striking contrast to the activeinflammation associated with the lesions due to KIM5-3022 and KIM5-3031. Lesions due to the YopE- strain (D) were considerably largerthan those caused by the YopK- YopL- strain (E), but both were circumscribed and rimmed by an intense inflammatory response composedof PMNs and mononuclear cells. (F) Spleen, KIM5, day 5. The spleen shows almost complete necrosis of the red pulp, with remaining viableislands of white pulp lymphocytes.
membrane (28; R. J. Jacob and S. C. Straley, unpublisheddata). Accordingly, to obtain stable expression of YOPsin the outer membranes of in vitro-grown Y. pestis (28),we used the pPCPI- (Pac-) derivatives Y. pestis KIM8,KIM8-3122 (YopE-), KIM8-3131 (YopK- YopL-), and
KIM8-3173 (YopJ-), as well as the Pac+ Y. pestis KIM5,KIM5-3022, KIM5-3031, and KIM5-3073. The latter bacteriaserve as effective Yop- reference strains in these experi-ments.
Incubation in high concentrations of normal mouse serum
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:e .# ~~~~~~~~~~~*< s < t * p<
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et''b r , v 2 < i ¢* * < :0 k......................................................0. .
k&o ; } 'etFam __*W , :
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twes;rsPS*' ;2N ;y *r<X#eeeSK4w44
FIG.4.Latehistologicalfeatureof infections with YopE- and YoK- YopL- Y. pestis. (A) Granulomtous lesion in liver, day 9 afteinetinbyYp-oL-Y psi (I5-01. a, 0,u.() nenePM nilrtei ymhidtssesbjcntt itsinlmuoady9aftr in .feto byYp-Y ets(l532) a,10,m
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Yop- YERSINIA PESTIS IN MICE 1207
150
(I)I~-zwwlL0C:w
z
50
- = PARENT
= YopJ-
= YopE-
00too
- - = YopK- YopL- FLUORESCENCEFIG. 5. Effects of YOPs expression and of YopE- and YopK- YopL- mutations on C3 binding by Y. pestis. Yersiniae grown so as to
induce YOP expression were incubated in normal mouse serum and then in fluorescein isothiocyanate-conjugated goat anti-mouse C3. Theywere tested for binding of C3 by fluorescence-activated cell sorter analysis of the fluorescence from the bound anti-C3. The vertical markerin all panels is a visual aid. (A, B, and C) The Y. pestis strains lacked pPCP1 (i.e., they were the KIM8 series of strains KIM8, KIM8-3122,KIM8-3131, and KIM8-3173) and therefore expressed YOPs in their membranes (see text); 5,000 events were recorded for each bacterialsample. (D, E, and F) The yersiniae all contained pPCP1 (i.e., they were the KIM5 series of strains KIM5, KIM5-3022, KIM5-3031, andKIM5-3073) and therefore were effectively Yop- in their membranes; 2,000 events were recorded for each bacterial sample.
(20 and 40%) caused a small shift toward greater fluores-cence (indicative of greater C3 binding) for the YopE- Pac-and YopK- YopL- Pac- bacteria as compared with theYop+ Pac- Y. pestis KIM8 or YopJ- Pac- Y. pestisKIM8-3173 (Fig. 5A, B, and C). The fluorescence by theYopE- Pac- bacteria was slightly greater than that for theYopK- YopL- Pac- strain. These differences were smallbut reproducible (five experiments). There were no differ-ences in fluorescence distribution among these strains whenlower concentrations of serum were tested (we tried 2.5, 5,10, and 15% as well as 0%, heat-inactivated 2.5%, heat-inactivated 5%, and heat-inactivated 20%; data not shown).Nor were there any further increases in the fluorescencedifferences of the strains when a higher concentration ofserum was used (65%). In contrast, the pPCP1+, effectivelyYop- strains, did not differ in their C3 binding as indicatedby fluorescence (Fig. SD, E, and F), showing that theabsence of pPCP1 was necessary for generating the fluores-cence differences of panels A to C. These data suggest thatthe YOPs are responsible for the differences in C3 binding inthe pPCP1- bacteria. The small magnitude of these differ-ences is not an artifact of using pPCP1- Y. pestis, becausesmall differences similar to those of panels A to C wereobtained for C3 binding by Y. pseudotuberculosis 43 (sero-type I) lacking its native Lcr plasmid and containing Y. pestispCD1: :MudIl-22 (YopE-), pCD1: :MudIl-31 (YopK-YopL-), or pCD1::MudI1-73 (YopJ-) (data not shown).
DISCUSSIONIn this study, we characterized the kinetics of infection by
Y. pestis strains lacking either YOP E or YOPs K and L,
with the aim of obtaining clues about the functions of theseproteins in the pathogenesis of experimental plague. Asidefrom their Yop- mutations, the Y. pestis strains that westudied are normal in their low-Ca2" response; i.e., theyexpress normal amounts of V and other YOPs, they regulatethe expression of V normally, and they show Ca2+-depen-dent growth identical to that of the parent strain (32). Ourdata reveal that absence of YOP E or YOPs K and L had astriking effect on the time course of the infections as well ason the clinical and histopathological features of the resultingdisease. These findings underscore the importance of YOPsin plague.
In our intravenous mouse model, death followed relativelylow bacterial loads of less than 108 bacteria per animal, ashad been noted previously for experimental plague in mice(37, 38, 40). We found no evidence of disseminated intravas-cular coagulation, suggesting that endotoxin is not a majorimmediate cause of death in our model. This is consistentwith previous estimates that the LD50 of Y. pestis endotoxinis too high to account for the lethality of Y. pestis in mice (4).The histopathology for the parent Y. pestis KIM5 infectionrevealed extensive necrosis in both spleen and liver despiterelatively low bacterial numbers and the absence of contin-ued influx of inflammatory cells. This is suggestive of atoxogenic disease, and we hypothesize that toxicity makes amajor contribution to death in our experimental plaguemodel.The plague murine toxin is one candidate for this effect.
This cytoplasmic membrane-associated protein toxin (21) isbelieved to function as a beta adrenergic blocker (21). It ishighly toxic for mice, with an LD50 of 0.1 to 3 ,ug, but its
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1208 STRALEY AND CIBULL
actual relevance to plague is unclear; it is thought to bereleased only upon lysis of the bacteria and is not associatedwith necrotizing pathology (7). However, its high potencyfor mice has prompted the speculation that murine toxin maybe important in the lethality of Y. pestis for mice (4), and ithas been noted that the significantly larger bacterial numbersattained in mice infected with Y. pseudotuberculosis and Y.enterocolitica compared with those seen in Y. pestis infec-tions may reflect the absence of this toxin in the formerYersinia species and its presence in Y. pestis (37).There are two other toxic components of Y. pestis that
might contribute to necrosis. The so-called pH 6 antigen is aprotein expressed by Y. pestis at temperatures above 35°Cand at pH values below 6.7. This protein, extractable fromwhole bacteria with potassium thiocyanate, is reported to becytotoxic for monocytes and also to have hemagglutinatingand dermonecrotizing activity (2). This cytotoxin is a goodcandidate for a mediator of the necrosis observed in the liverand spleen. It might be expressed by the bacteria growingwithin a phagolysosome (35), and the acidosis that accom-panies coagulative necrosis may be sufficient to induceexpression of this toxin, furthering necrosis by extracellu-larly located bacteria. A second cytotoxic component of Y.pestis and Y. pseudotuberculosis has been reported to re-quire the presence of an Lcr plasmid and growth of thebacteria at 37°C (13).An important histopathological feature of the parent strain
infection confirmed in our study was the absence of contin-ued influx of inflammatory cells into the enlarging lesions (5,39). Normally, the inflammatory response continues with theelicitation of macrophage and lymphocyte immigration. Incontrast, the YopE- and YopK- YopL- mutants that westudied elicited a response beginning as acute inflammationand evolving into the accumulation of mononuclear cells intogranulomalike foci.The parent and YopE- or YopK- YopL- strains also
differed strikingly in their abilities to initiate growth in thespleen and maintain growth in the liver, suggesting a role forYOP E and YOPs K and L early in infection. The parentinitiated growth in the spleen with an undetectable lag (i.e.,less than 2 h), whereas the two Yop- mutants failed to shownet growth for longer than a day and subsequently wereeliminated. In liver, the mutants showed a lag, followed by 1to 2 days of rapid growth, followed by a plateau in growththat coincided with the peak of acute inflammation. How-ever, this host response was unable to clear the bacteriafrom liver. An alternative host defense mechanism devel-oped after about a week of infection and brought about theultimate decline in viable numbers of the YopE- and YopK-YopL- Y. pestis. This coincided with the appearance ofgranulomas and probably reflected the induction of cell-mediated immunity.The V antigen also may play a role early in infection.
Indirect evidence indicates that the V antigen is necessaryfor survival of Y. pestis in the spleen and for prolongedgrowth in the liver (38, 39). We favor the view that V andYOPs function to overcome growth-limiting phenomena thatotherwise "buy time" for the host to develop the antibacte-rial responses manifested as killing in the spleen, curbing ofgrowth in the liver, formation of granulomas, and ultimatelythe clearance of bacteria. Accordingly, elimination of any ofthese virulence factors ultimately permits the developmentof cell-mediated immunity.
Curiously, the clearance of the YopE- and YopK-YopL- Y. pestis from the spleen occurred during the timethat net growth was taking place in the liver. This is a
significant finding; these mutant yersiniae distinguish, atleast kinetically, between antibacterial responses of the liverand spleen. Perhaps there is an antibacterial cell type in thespleen that proliferates before effective numbers of thesecells can accumulate in the liver. YOPs E, K, and L mightfunction to counteract this elicited cell, and the absence ofthese YOPs would result in the inability to grow in the spleenand, later, in cessation of growth in the liver. For the parentyersiniae, extensive growth in the spleen might cause suffi-cient cytotoxicity to prevent development of this defensemechanism in the liver. A precedent for a localized antibac-terial response is seen in murine infections by Salmonellatyphimurium, in which the ability of the salmonellae to showinitial exponential growth is modulated by the Ity genefunction which is a property of resident macrophages (22).However, subsequent growth is limited by a different, local-ized macrophage response manifested only after severaldays of infection (19).We do not know if the antihost function of YOPs E, K,
and L occurs when yersiniae are within phagolysosomes ofmacrophages. It is possible that these YOPs are necessaryfor survival or growth within splenic macrophages but notwithin the macrophages resident in the liver and peritonealcavity. This hypothesis is attractive because we have previ-ously observed that the yop genes, including those for YOPE and YOPs K and L, are strongly expressed when thebacteria are within human monocyte-derived macrophagesin culture (25; M. S. Klempner and S. C. Straley, unpub-lished data). However, the YOPs (and the entire pCD1) arenot necessary for survival or growth within cultured residentmouse peritoneal macrophages or cultured human mono-cyte-derived macrophages (25, 34). The resolution to thisapparent paradox may be that there are qualitatively dif-ferent classes of macrophages, some being lethal for Yop-bacteria, while others allow proliferation of Yop- as well as
Yop+ yersiniae.Another way YOPs E, K, and L could function early in
infection is to prevent uptake of yersiniae by cells nonper-missive for growth, such as PMNs and monocytes (9).Evidence is accumulating that some surface protein(s) pre-sumably encoded by the Lcr plasmids of Y. pseudotubercu-losis and Y. enterocolitica may inhibit phagocytosis byinhibiting complement deposition on the bacteria (17, 18,36). We found reproducible differences in C3 deposition(presumably as C3b) by the YopE- and YopK- YopL-mutants compared with the parent or YopJ- yersiniae.Accordingly, YOPs E, K, and L may contribute to an
inhibition of phagocytosis that is important for survival of Y.pestis in mice; the capsule (the so-called fraction 1 antigen)may play a less pivotal role in mice than in other species (4,6, 41). Small differences in C3 deposition should also reflectdifferences in amounts of the chemoattractant C3a producedin response to the parent and mutant yersiniae, with result-ing effects on immigration of PMNs. Perhaps the slight lag ininitial growth of the YopE- Y. pestis in the liver and theintense acute inflammation surrounding lesions in that organreflected these consequences of slightly increased comple-ment activation for the mutant compared with the parent Y.pestis. It will be of interest to determine how the differencesin C3 deposition as reflected by fluorescent-antibody mea-
surements translate into differences in efficiency of phago-cytosis in direct phagocytic assays. However, we are notconvinced that the small differences in C3 deposition are
sufficient to account for all of the differences between parentand mutant infections found in our study.There were significant differences between the host re-
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Yop- YERSINIA PESTIS IN MICE 1209
sponses to the YopE- and YopK- YopL- Y. pestis strainsthat point to differences in the functions of the missingYOPs. The frequent, small lesions induced in the liver onday 3 by the YopK- YopL- strain resembled those inducedby the parent strain in distribution and character, whereasthe YopE- Y. pestis had induced few, strikingly large,circumscribed lesions which enlarged further during thesucceeding week. These differences in histopathology werereflected in significantly different times of death and symp-toms of disease caused by the YopE- and YopK- YopL- Y.pestis. In contrast to the parent or YopK- YopL- Y. pestis,the YopE- strain killed mice between days 4 and 6 onlywhen the dose was at least 106; a dose of 4 x 105 was stillcleared by the mice. A secondary enterocolitis correlatedwith deaths on days 8 through 13 for mice infected with theYopE- strain and was not observed in the disease caused bythe YopK- YopL- Y. pestis. It is premature to speculateabout the mechanisms underlying these phenomena; how-ever, they will help guide future tests of the interaction ofour Y. pestis strains with splenic and hepatic phagocytes andhelp us uncover the mechanisms of action of YOPs E, K,and L.
ACKNOWLEDGMENTSWe thank Ruth Ann Bivins and Clarissa Cowan for expert
technical help. We also thank Norman Goodman of the Departmentsof Pathology and Microbiology and Immunology for identifyingisolates from mice suffering from colitis-associated symptoms. Thefluorescence-activated cell sorter was operated by CatherineNowack of the University of Kentucky Flow Cytometry CoreFacility.
This study was supported by Public Health Service grant A121017and National Science Foundation grant DCB-8409128.
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