Deciphering the landscape of host barriers to Listeria monocytogenes infection

Abstract

Listeria monocytogenes is a common food-borne pathogen that can disseminate from the intestine and infect multiple organs. Here, we used sequence tag-based analysis of microbial populations (STAMP) to investigate Lmonocytogenes population dynamics during infection. We created a genetically barcoded library of murinized Lmonocytogenes and then used deep sequencing to track the pathogen's dissemination routes and quantify its founding population (N_b) sizes in different organs. We found that the pathogen disseminates from the gastrointestinal tract to distal sites through multiple independent routes and that N_b sizes vary greatly among tissues, indicative of diverse host barriers to infection. Unexpectedly, comparative analyses of sequence tags revealed that fecally excreted organisms are largely derived from the very small number of L. monocytogenes cells that colonize the gallbladder. Immune depletion studies suggest that distinct innate immune cells restrict the pathogen's capacity to establish replicative niches in the spleen and liver. Finally, studies in germ-free mice suggest that the microbiota plays a critical role in the development of the splenic, but not the hepatic, barriers that prevent L. monocytogenes from seeding these organs. Collectively, these observations illustrate the potency of the STAMP approach to decipher the impact of host factors on population dynamics of pathogens during infection.

Keywords: Listeria monocytogenes; STAMP; pathogen dissemination; pathogen transmission; population dynamics.

Fig. S1.

Validation of the STAMP approach for determination of N_b sizes. (A) In vitro growth of wt and barcoded L. monocytogenes. (B) Correspondence of N_b sizes was determined based on CFUs (x axis) and analyses of barcode frequencies using STAMP (y axis). The three colors indicate three independent experiments.

Fig. 1.

Recovered CFU and N_b′ sizes in different tissues following OG or i.v. inoculation of L. monocytogenes. BALB/c mice were orogastrically inoculated with 3 × 10⁹ CFUs of L. monocytogenes (10403S InlA^m) (A) or injected i.v. with 6 × 10⁴ CFUs (B). Bacterial loads (CFU, black squares) and N_b sizes (N_b′, red circles) in different tissues were determined 3 d after OG infection or 2 d after i.v. infection. CFU values are expressed per organ for the SI, colon, MLN, spleen, liver, GB, and brain; they are expressed per milliliter for blood and per pellet for feces. Each black square and red circle represents data from one mouse. Geometric means (horizontal lines) and SD (whiskers) are shown. SI (Gent−), gentamicin-negative (untreated SI tissues).

Fig. 2.

Genetic relatedness of L. monocytogenes populations isolated from different tissues. Genetic relatedness of L. monocytogenes populations in the indicated samples 48 h and/or 72 h after OG inoculation (A) and 48 h after i.v. inoculation (B and D). (C) Relative frequencies of barcodes in the GB, feces, and colon of two representative mice. (E) Correlation between the L. monocytogenes burden in the blood (x axis) and the genetic relatedness of L. monocytogenes populations in the spleen and liver (y axis) 48 and 72 h after OG inoculation of mice. The Pearson correlation coefficient value was used to quantify correlation. Each open circle in A, B, D, and E represents data from one mouse.

Fig. S2.

Genetic relatedness of L. monocytogenes populations isolated from different tissues after OG and i.v. inoculation. Pairwise comparisons of the genetic relatedness of L. monocytogenes populations in the indicated tissues 72 h after OG inoculation (A) or 48 h after i.v. inoculation (B) are shown. Each black circle represents data from one mouse, and the red bars represent the arithmetic means. (C) Relative frequencies of barcodes in the GB, feces, SI, colon, MLN, spleen, and liver of a representative mouse.

Fig. S3.

Recovered CFUs (black squares) and N_b (N_b′, red circles) in the indicated tissues 2 d following OG inoculation of BALB/c mice with L. monocytogenes. Each black square and red circle represents data from one mouse. Geometric means (horizontal lines) are shown. SI (Gent−), gentamicin-negative (untreated SI tissues).

Fig. 3.

Chemotaxis and motility are not required for L. monocytogenes to breach intestinal barriers and reach distal infection sites. (A) Motility of wt (WT) and ΔcheA L. monocytogenes in 0.3% agar plates. (B) CFU and N_b sizes for the indicated tissues at 3 dpi of mice inoculated orogastrically with 3 × 10⁹ CFUs of ΔcheA L. monocytogenes. Each black square and red circle represents data from one mouse. Geometric means (horizontal lines) and SD (whiskers) are shown. (C) Competitive indexes for the indicated tissues from mice inoculated orogastrically with a 1:1 mixture of barcoded ΔcheA and wt L. monocytogenes. Each black circle represents data from one mouse. (D) Relative frequencies of L. monocytogenes barcodes in the GB of infected mice; data are from two litters of mice (n = 9) that were infected on different days.

Fig. 4.

Modulation of the capacity of L. monocytogenes to establish and proliferate in the spleen, liver, and GB following treatment with monoclonal anti–Gr-1 or anti–TNF-α antibodies. Control (Ctrl) and anti–Gr-1 mAb-treated mice (A) or anti–TNF-α–treated mice (B) were infected i.v. with 6 × 10⁴ CFUs of L. monocytogenes. CFU (black squares) and N_b′ (red circles) values for the indicated tissues were determined 1 dpi in the anti–Gr-1–treated mice and 2 dpi in the anti–TNF-α–treated mice. The data are from two litters of mice (n = 10) that were infected on different days. Each black square and red circle represents data from one mouse. Geometric means (horizontal lines) and SEM (whiskers) are shown. The Mann–Whitney test was used to assess significance: *P < 0.05, **P < 0.01, ***P < 0.001. n.s., no significance.

Fig. 5.

Modulation of host barriers to L. monocytogenes infection by the microbiota. (A) GF and SPF Swiss Webster mice were inoculated i.v. with 6 × 10⁴ L. monocytogenes CFUs. CFU (black squares) and N_b′ (red circles) values in the indicated tissues were determined 2 d later. Data are from two litters of mice (n = 10) that were infected on different days. Each black square and red circle represents data from one mouse. (B) Antibiotic-treated (Abx) Swiss Webster mice and nontreated control mice were inoculated i.v. with 6 × 10⁴ L. monocytogenes CFUs. CFU (black squares) and N_b′ (red circles) values in the indicated tissues were determined 2 d later. Each black square and red circle represents data from one mouse. Geometric means (horizontal lines) and SEM or SD (whiskers, A and B, respectively) are shown. The Mann–Whitney test was used to assess significance: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. S4.

Schematic of L. monocytogenes population dynamics following OG inoculation of barcoded organisms. L. monocytogenes populations with distinct composition and complexity arise within the spleen, liver, GB, and other tissues due to organ-specific restrictions imposed by innate immune factors, as well as by the existence of multiple dissemination pathways (mean N_b values are shown in parentheses). Very few organisms establish infection in the GB, but the founder(s) ultimately replicate to very high numbers. Organisms released from the GB through the bile become the principal source of L. monocytogenes excreted in the feces.