Quantitative PCR-based competitive index for high-throughput screening of Salmonella virulence factors

Abstract

Salmonella enterica serovar Typhimurium is an intracellular pathogen and a main cause of food-borne illness. In this study, a quantitative PCR (qPCR)-based competitive index (CI) method was developed to simultaneously compare the growth of multiple Salmonella strains. This method was applied to a mixture of 17 Salmonella mutants lacking regulator genes, and their survival ratios were compared based on expression of natural resistance-associated macrophage protein 1 (Nramp1). Nramp1, as a major host innate immune component, controls the intracellular replication of pathogens. Deletion strains containing unique DNA barcodes in place of regulator genes were mixed with the parental control, and the bacteria were inoculated into congenic mice differing only at Nramp1. Most of the deletion strains were outcompeted by wild-type bacteria in either mouse strain, and the lack of Nramp1 didn't increase the tested strain/parent control replication ratios. When the same collection of mutants was tested in congenic mouse-derived primary macrophages, a major Nramp1-expressing cell type, six strains (ΔhimD, ΔphoP/phoQ, ΔrpoE, ΔrpoS, ΔompR/envZ, and Δhfq strains) grew better in Nramp1(-/-) than in Nramp1(+/+) macrophages, suggesting that these six regulators may play roles in overcoming Nramp1-mediated bactericidal activity in primary macrophages. The discrepancy in survival of macrophages and that of mice suggests either that there are differences in macrophage populations or that other cell types expressing Nramp1 control Salmonella proliferation in the host. The method described allows competitive infection analysis to be carried out on complex mixtures of bacteria and provides high reproducibility from independent biological replicates.

FIG. 1.

Scar sequences in deletion strains. The 135-nt scar sequences, replacing coding sequences of a target gene, are shown. The scar site or FLP recognition target (FRT) site left after FLP-mediated excision of the kan cassette is highlighted in dark gray. Barcode sequences inserted via SacI and AvrII are composed of 24 random nucleotides and are outlined in the schematic diagram. N, any nucleotide with either a purine or pyrimidine base; V, any nucleotide except thymine-based nucleotides. Sequences recognized by primers scarF and scarR in nested PCR are underlined and indicated by priming sites 4 and 1, respectively. T7 promoter sequences are shown between square brackets.

FIG. 2.

Schematic of the qPCR-based CI method in a mouse model. Bacterial strains were cultivated separately overnight (1) and mixed equivalently based on OD₆₀₀ measurement (2). Mixed cells corresponding to 2 × 10⁸ CFU were used directly in nested PCR to amplify the barcode sequences from the input inoculum (3). Product DNAs from nested PCR were used as templates in the following input qPCR (4). In order to compare survival rates between strains, mice were i.p. injected with 10⁴ CFU consisting of an equal mix of each of the mutant strains and the parent strain (5). The inoculum was verified by plating and counting the number of CFU. The mice were euthanized to isolate Salmonella-infected organs at the desired time point after infection (6). Organs were homogenized and spread on LB agar to grow bacteria (7). Bacterial colonies were scraped and collected the next day. The mixture was diluted in PBS buffer, and 2 × 10⁸ CFU were used as the template for nested PCR (8). Bacterial strains from the output populations were analyzed by qPCR using barcode-specific primers (9). CI_qPCR was calculated by comparing C_T values between a wild-type strain and a mutant strain in input and output qPCRs, taking into account that the amplification efficiency of individual barcodes ranged from 1.53 to 1.94.

FIG. 3.

Comparison of CI_qPCR with the traditional CI. In the CI_qPCR assay, a reference strain (the ΔSTM0314 strain) and three deletion strains (ΔSTM2209, ΔSTM3096, and ΔSTM4333 strains) with barcodes were mixed equivalently, and the mix was used to infect a group of three Nramp1^+/+ 129SvJ mice at 10⁴ CFU/mouse. For the traditional CI infection, a mixture containing a reference strain (MA6054) and each single strain at a 1:1 ratio was used to infect a group of three Nramp1^+/+ 129SvJ mice at 10⁴ CFU/mouse. Mice were sacrificed at days 2, 5, and 7 postinfection, and the intracellular bacteria residing in the spleens were enumerated using the traditional CI formula or CI_qPCR formula as described in Materials and Methods. There was no statistically significant difference between the two methods based on Student's t test.

FIG. 4.

CI_qPCR analysis of 18 strains in Nramp1^+/+ and Nramp1^−/− mice. Groups of five mice (Nramp1^+/+ and Nramp1^−/− 129SvJ) were infected with equal mixtures of 17 strains, containing mutations in the genes indicated, as well as the wild-type control. The total number of infecting bacteria was about 10⁴. The spleens were extracted at day 5 postinfection, and persistence levels of Salmonella strains in the spleen were compared via qPCR using the formula shown. CI_qPCR values were averaged from the five mice in each group and shown in white (Nramp1^+/+) and gray (Nramp1^−/−). Strains with CI_qPCR values greater than 1 indicate that they outcompeted the wild-type strain; strains with values less than 1 indicate that they were outcompeted by the wild-type strain. A Student t test was applied for statistical analysis of the results, and strains showing significant changes between Nramp1^+/+ and Nramp1^−/− mice (P < 0.05) are labeled with an asterisk.

FIG. 5.

CI_qPCR analysis of 18 strains in Nramp1^+/+ and Nramp1^−/− BMDM. Equal mixtures of the deletion strains indicated above were used to infect Nramp1^+/+ and Nramp1^−/− bone marrow-derived macrophages that were approximately 50% confluent at an input MOI of 10. Macrophages were lysed at18 h postinfection, and the survival ratios of Salmonella mutants to the reference strain were compared as described in Materials and Methods. CI_qPCR values from three independent BMDM infections were averaged, and CI_qPCR in Nramp1^+/+ (white) and Nramp1^−/− (gray) are shown. Strains with CI_qPCR values greater than 1 indicate that they outcompeted the wild-type bacteria in survival; strains with values less than 1 indicate that they were outcompeted by the wild type. Mutant strains showing significant differences in CI_qPCR values for Nramp1^+/+ and Nramp1^−/− cells are denoted by an asterisk (P < 0.05 in the Student t test).