The authors have declared that no competing interests exist.
Conceived and designed the experiments: RRC EM SEP SJP DJT JP IGC DJM MPS. Performed the experiments: EM SEP SJP DLH HMD JW PMvD AMB AJB GDP DJT GCL AKT. Analyzed the data: RRC IGC DJM MPS. Contributed reagents/materials/analysis tools: RRC SEP AKT. Wrote the paper: RRC EM SEP JP IGC DJM MPS.
Current address: Centre for Genomic Research, University of Liverpool, Liverpool, United Kingdom
Current address: Canada's Michael Smith Genome Sciences Centre, Vancouver, British Columbia, Canada
Current address: The Jenner Institute, University of Oxford, Oxford, United Kingdom
Current address: Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom
Current address: Oxford Nanopore Technologies, Oxford, United Kingdom
Current address: Discuva, Cambridge, United Kingdom
Current address: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom
¶ These authors also contributed equally to this work.
Chickens, pigs, and cattle are key reservoirs of
Although STM has provided valuable insights into
An input pool of random transposon insertion mutants is generated, and used to inoculate experimental animals. Output pools of bacteria that are capable of survival and growth in each host are isolated from an appropriate tissue. Massively parallel sequencing of the regions flanking each transposon allow the disrupted genes to be identified, and comparison of the sequence counts derived from the input and output pools allows the relative fitness of each mutant to be assessed.
To validate the quantitative nature of TraDIS, we applied it to investigate pools of Mu and mini-Tn
Though useful, previous attempts to assign comprehensively the role of
The inner two rings indicate the positions of annotated genes, coloured according to their GC content (blue = low, yellow = intermediate, red = high). The outer ring indicates the number of transposon-flanking sequence reads obtained at each position, with peaks corresponding to the presence of a transposon insertion.
TraDIS assignments of the insertion sites and fitness scores of mutants are listed in
Mutants which showed a significant change in abundance in the output relative to the input are highlighted in red. Mutants which had no reads in the output pool are assigned an arbitrary fitness score of −15.
To summarize the dataset further, genes were scored as potentially important in colonization if they were disrupted in at least one significantly attenuated mutant in any of the four host species. This enables comparisons between datasets derived from different transposon libraries (such as the mouse and chicken/pig/cattle datasets), and visualization of the data in comparison with other genome-wide datasets. For some genes, insertions at different subgenic locations can have divergent effects on the encoded protein resulting in contrasting fitness scores, so it is important to consider the genetic context of each individual transposon when interpreting the TraDIS data in detail. This is true for all transposon-based mutant screens, although earlier technologies such as STM and TMDH lacked sufficient resolution to permit such considerations. We recommend the use of the TraDIS browser (
Fitness scores were obtained for 3194 distinct genes disrupted by transposon insertions in the mouse screen, and 2715 genes in the chicken, pig and calf screens, of which fitness score existed for 2435 genes in all three food-producing animals. Fitness scores were available from all four hosts for 1935 genes, of which 1069 had a significantly attenuated mutant in at least one host. Venn diagrams showing the numbers of significantly attenuated mutants, and the numbers of genes potentially important for colonization in chickens, pigs and cattle are shown in
A) the numbers of transposon mutants which were significantly attenuated in each host B) the numbers of genes which were disrupted in the TraDIS mutant library, and which are potentially important for colonization (
Twelve genes were selected for further investigation based on the TraDIS data:
Mutation | Mean CI Day 4 | Mean CI Day 6 | Mean CI Day 10 | Chicken TraDIS |
0.187 (0.0004) | 0.171 (0.0016) | 0.002 (<0.0001) | −5.43 to −15 (5/5) | |
0.047 (<0.0001) | 0.001 (<0.0001) | −2.36 to −8.11 (4/4) | ||
0.294 (0.0026) | 0.310 (0.0309) | 0.104 (0.0001) | −2.05 to −3.38 (1/2) | |
0.957 (0.9014) | 0.469 (0.0159) | 0.820 (0.2802) | −15 (1/1) | |
0.761 (0.2741) | 0.613 (0.2588) | 0.804 (0.1625) | −3.08 to −15 (4/4) | |
SL1344_0084 | 0.727 (0.0959) | 1.062 (0.7933) | 0.430 (0.0008) | −0.82 to −9.11 (6/7) |
SL1344_4248 | 0.487 (0.0015) | 1.152 (0.7458) | 1.020 (0.8951) | −3.51 to −5.07 (4/4) |
SL1344_3128 | 1.267 (0.3367) | 1.001 (0.9963) | 1.204 (0.7051) | −1.02 to −9.20 (7/9) |
0.003 (<0.0001) | 0.013 (<0.0001) | −3.58 to −8.21 (4/4) | ||
0.347 (0.0024) | 0.638 (0.1336) | 0.416 (0.0038) | −1.48 to −7.05 (3/4) | |
0.184 (<0.0001) | 0.175 (<0.0001) | 0.033 (<0.0001) | 0.44 to −4.38 (2/6) | |
0.592 (0.0015) | 0.581 (0.7458) | 0.522 (0.8951) | −15 (1/1) |
With a single exception (SL1344_3128) all mutants were negatively-selected relative to the parent strain at day 4 post-inoculation, which corresponds to the time at which mutants were recovered for the TraDIS analysis. The difference in the mutant∶wild-type ratio was significantly different from the ratio in the inocula for 8 of the mutants at day 4. For a further 2 mutants, significant differences were detected at later time points. Taken together with comparisons to existing datasets for signature-tagged mutants of the same strain in the same animal models
The TraDIS dataset is a powerful resource for understanding intestinal colonization of a range of highly relevant hosts by
There are also attenuated mutants that harbour insertions within the other recognized
Fimbriae play a well-established role in
Many
The recent RNAseq-based analysis of the
The chicken, pig and cattle TraDIS data presented in
Although most colonization factors were necessary for infection of chickens, pigs and cattle, there were some patterns amongst the colonization factors that appeared to function in a host-specific manner that may reflect underlying differences in host biology. There are many genes associated with flagellar motility that are essential for infection of pigs but not required for chicken or calf infection, including
The serovar Typhimurium strain ST4/74 investigated here is a natural bovine isolate that elicits pathology typical of clinical salmonellosis in all the host species used in this study. However, some atypical
Our study represents the first comprehensive genome-wide survey of the role of thousands of
Many novel colonization-associated genes were identified within the
Animal experiments were conducted according to the requirements of the Animals (Scientific Procedures) Act 1986 (project license number 30/2485) with the approval of the local Ethical Review Committee.
For full details of experimental animals, bacterial strains, materials, molecular biological techniques and statistical methods see
Genomic DNA was prepared from the inocula and output samples, and fragmented to ∼300 bp. An Illumina adapter was ligated to the fragments, and PCRs were performed using an adapter-specific primer in conjunction with primers homologous to each end of the transposon. The sequences of all oligonucleotide primers used in this study are detailed in
The number of reads corresponding to each transposon in the input pool, and the number of reads mapping to the equivalent position in the output pool data, were compared using DESeq
Defined null mutants were obtained for twelve genes identified as attenuated in the TraDIS screen, and assessed in competition with wild-type ST4/74 during oral infection of chickens. The ratios of mutant∶wild-type bacteria from caecal isolates at day 4, 6 and 10 were compared with those in the inoculum, and the significance of any differences was tested using Student's t-test.
Comparison of fitness scores obtained using TraDIS with the equivalent attenuation scores obtained using TMDH. Values were obtained by investigation of pools of
(PDF)
Venn diagram showing the numbers of genes in which at least one significantly attenuated mutant was identified for each of the four host species.
(PDF)
Venn diagrams illustrating the overlap between attenuated and non-attenuated mutants from the earlier STM studies and the attenuation of mutants with transposon insertions at equivalent loci in the TraDIS datasets a) chickens, b) pigs and c) cattle
(PDF)
Illustration of the chorismate biosynthesis pathway, adapted from KEGG
(PDF)
Box plot of GC content of genes for which attenuated mutants were observed in the chicken TraDIS dataset, and genes for which no attenuated mutants were obtained.
(PDF)
Complete dataset derived from TraDIS investigation of pools of
(XLSX)
Complete dataset derived from TraDIS investigation of pools of
(XLSX)
List of all genes disrupted in the TraDIS mutant libraries from the food-producing animal and mouse experiments. For each gene, “yes” indicates that at least one significantly attenuated mutant was identified in that experiment, “no” indicates that the gene was disrupted but no evidence of attenuation was obtained.
(XLSX)
Fitness scores of mutants within sRNA genes annotated in the
(XLSX)
Oligonucleotides used in this study.
(XLSX)
Supporting data and methods.
(PDF)