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Bacterial Infections

Identification of Attenuated Yersinia pseudotuberculosis Strains and Characterization of an Orogastric Infection in BALB/c Mice on Day 5 Postinfection by Signature-Tagged Mutagenesis

Joan Mecsas, Inna Bilis, Stanley Falkow
Joan Mecsas
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5402
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Inna Bilis
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5402
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Stanley Falkow
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5402
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DOI: 10.1128/IAI.67.5.2779-2787.2001
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  • Fig. 1.
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    Fig. 1.

    Comparison of number of YPIIIpIB71 CFU recovered from the cecum, PP, and MLN of mice infected with a 3:1 ratio of WT Y. pseudotuberculosis to the Yop-deficient strain YPIIIpIB71 (mixed) with the number of CFU from mice infected with only YPIIIpIB71 at doses of 3 × 108 (alone-low) and 3 × 109(alone-high). In the mixed infection experiment, a total of 3 × 109 bacteria were used, of which 7.5 × 108 were YPIIIpIB71. Four mice were infected with the mixed inoculum, and five mice were infected with the low and high doses of YPIIIpIB71. Mice were sacrificed 5 days postinfection.

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    Fig. 2.

    Phenotypes of ST strains with cells in culture. (A) Percentage of ST strains recovered after bacteria were grown at 26°C in 2xYT containing 5 mM CaCl2 and exposed to HeLa cells at a multiplicity of infection of 10:1. After a 30-min infection, gentamicin was added to the medium (first box in each pair) for an additional 90 min, the cells were washed and lysed with 1% Triton X-100, and the bacteria were plated. In a side-by-side control, gentamicin was not added to the medium cells were lysed with a final concentration of 1% Triton X-100 and bacteria were plated (second box in each pair). All experiments were done in triplicate, and each strain was tested at least two times in both assays. (B) Percentage of gentamicin-resistant bacteria of various ST strains after infection of HeLa cells with bacteria pregrown at 37°C for 2 h. HeLa cells were infected as described above and in Materials and Methods. (C) Percentage of RAW264.7 macrophage cell death due to infection with Y. pseudotuberculosis relative to complete lysis of the macrophages. The bacteria were pregrown at 37°C for 2 h, the cells were infected for 2 h, gentamicin was added to the medium, and culture supernatants were assayed 7 h after the addition of gentamicin. All experiments were performed in triplicate and repeated twice. The strain labeled lcrR is YPIIIpIB71, which does not secrete any Yops.

Tables

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  • Table 1.

    Numbers of CFU and ST strains in tissues 5 days after oral infection

    DoseMouseCecumMLNSpleen
    CFUSTaCFUSTCFUST
    8 × 10711.0 × 103NDb4.8 × 1045150ND
    2200ND6ND 0
    36.5 × 104ND1.6 × 10324 0
    8 × 10814.2 × 104274.0 × 10328 0
    28.9 × 104351.0 × 104266.8 × 103ND
    33.0 × 104351.2 × 10419 0
    3 × 10911.3 × 105309.4 × 103132.9 × 103ND
    22.0 × 106324.0 × 10425100ND
    35.0 × 104362.5 × 104222.5 × 1041
    8 × 10916.0 × 105421.7 × 10431500ND
    23.0 × 105366.0 × 104283.6 × 1036
    36.0 × 105392.8 × 104275.5 × 103ND
    • ↵a Number of different ST strains in each tissue, calculated by probing colony blots as described in Materials and Methods.

    • ↵b ND, not done.

  • Table 2.

    A strain unable to secrete the Yops (YPIIIpIB71) is outcompeted by WT Y. pseudotuberculosis after orogastric inoculation

    TissueMean log differenceaPbCompetitive index/mousec
    Cecum−2.86 ± 0.740.00120.001, 0.007, 0.003, 0.005
    PP<−2.55 ± 1.110.00680.0002, <0.006, 0.026, 0.0002
    MLN−1.74 ± 0.590.00960.12, 0.017, 0.013, 0.004
    Spleen<−2.89 ± 0.500.0014<0.002, <0.001, <0.01, <0.007
    • ↵a Average of log(CFU of YPIIIpIB71) − log(CFU of WT) recovered from the indicated tissue (n = 4) .

    • ↵b From a two-tailed, paired Student'st test.

    • ↵c (CFU of YPIIIpIB71/CFU of WT)output/(CFU of YPIIIpIB71/CFU of WT)input. < indicates no YPIIIpIB71 CFU recovered from that tissue. Limit of detection, 10 CFU/tissue.

  • Table 3.

    Phenotypes of putative ST mutant strains after orogastric and i.p. infection of BALB/c mice

    Strain/genotyped (phenotype)Orogastric infection with 2 × 109 bacteriaaMean log difference (log CFU of ST strain − log CFU of WT) after coinfection with equal mixture of WT and mutant Y. pseudotuberculosis (Pe)
    Orogastricb (n = 5)i.p.c (n = 5)
    No. dead/no. infectedDays until deathCecumPPMLNSpleen
    YPIIIpYV (wild type)6/64, 4, 5, 6, 7, 11
    425/yscH (type III secretion)0/3−2.20 ± 0.27 (<0.0009)
    426/yscU (type III secretion)0/3
    431/yscB (type III secretion)0/3
    542/yscL (type III secretion)0/3
    429/lcrR (regulator of type III secretion)0/3−2.01 ± 1.62 (0.05)−2.071 ± 1.67 (0.05)−1.40 ± 0.64 (0.008)−0.75 ± 0.18 (<0.0009)
    498/lcrR (regulator of type III secretion)0/3−2.68 ± 0.59 (<0.0009)
    427/ddhC (O-Ag biosynthesis)0/3−1.51 ± 0.12 (<0.0009)
    496/wzx (O-Ag biosynthesis)2/312, 16−2.71 ± 1.07 (0.004)−2.42 ± 0.03 (0.032)−2.67 ± 0.64 <0.0009−2.15 ± 0.83 (0.0044)
    543/gmd (O-Ag biosynthesis)4/67, 9, 21, 24−2.97 ± 1.5 (0.027)−2.02 ± 0.25 (0.005)−2.14 ± 0.63 (0.027)−1.94 ± 0.50 (0.0020)
    494/inv (invasin)2/37, 7−1.63 ± 0.26 (0.001)−1.41 ± 1.36 (0.080)−2.15 ± 0.7 (0.003)0.03 ± 0.34 (0.855)
    497/sufl (suppressor offtsI)0/3−1.70 ± 0.78 (0.008)
    499/cls (cardiolipin synthesis)2/312, 15−3.42 ± 1.57 (0.004)−3.31 ± 0.67 (0.032)−2.16 ± 0.72 (0.001)−1.21 ± 0.29 (0.0007)
    500/ksgA (kasugamycin resistant)1/324−0.59 ± 0.39 (0.027)−0.76 ± 0.68 (0.065)−2.14 ± 0.63 (0.027)−0.74 ± 0.36 (0.011)
    4283/36, 6, 10
    4303/35, 9, 10
    4934/68, 8, 10, 12−1.00 ± 1.24 (0.204)−0.41 ± 1.01 (0.47)−0.37 ± 1.50 (0.65)
    4953/36, 7, 80.40 ± 1.14 (0.47)−0.08 ± 0.9 (0.85)0.33 ± 1.3 (0.71)0.07 ± 0.49 (0.76)
    5443/37, 8, 9
    5453/36, 8, 8
    • ↵a Eight-week-old mice were infected with 2 × 109 bacteria and observed until death or for 40 days.

    • ↵b Mice were infected with an equal mixture of 2 × 109 WT Y. pseudotuberculosis and the indicated ST strain. After 5 days, the numbers of WT and ST bacteria in the cecum, PP, and MLN were determined.

    • ↵c Mice were infected i.p. with an equal mixture of 106 WT Y. pseudotuberculosis and the ST mutant. After 3 days, the numbers of WT Y. pseudotuberculosis and ST bacteria in the spleens was determined.

    • ↵d The insertions within the yscH, yscB, lcrV, ddhC, wzx, gmd, cls, and ksgA genes may be polar on downstream open reading frames.

    • ↵e From a two-tailed, paired Student'st test.

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Identification of Attenuated Yersinia pseudotuberculosis Strains and Characterization of an Orogastric Infection in BALB/c Mice on Day 5 Postinfection by Signature-Tagged Mutagenesis
Joan Mecsas, Inna Bilis, Stanley Falkow
Infection and Immunity May 2001, 69 (5) 2779-2787; DOI: 10.1128/IAI.67.5.2779-2787.2001

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Identification of Attenuated Yersinia pseudotuberculosis Strains and Characterization of an Orogastric Infection in BALB/c Mice on Day 5 Postinfection by Signature-Tagged Mutagenesis
Joan Mecsas, Inna Bilis, Stanley Falkow
Infection and Immunity May 2001, 69 (5) 2779-2787; DOI: 10.1128/IAI.67.5.2779-2787.2001
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KEYWORDS

Mouth
stomach
Yersinia Infections
Yersinia pseudotuberculosis

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