IAI Accepted Manuscript Posted Online 19 January 2016 Infect. Immun. doi:10.1128/IAI.01423-15 Copyright © 2016 Grant et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
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Genes Required for the fitness of Salmonella enterica serovar Typhimurium
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During Infection of Immunodeficient gp91-/-phox Mice
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Running title: Salmonella TraDIS in immunodeficient mice
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Andrew J. Grant1#, Olusegun Oshota1, Roy R. Chaudhuri1a, Matthew Mayho2, Sarah
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E. Peters1, Simon Clare2, Duncan J. Maskell1, Pietro Mastroeni1
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1 Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, UK.
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2 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
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Cambridge, UK.
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a Current address: Department of Molecular Biology and Biotechnology, University
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of Sheffield, Firth Court, Western Bank, Sheffield, UK.
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#
To whom correspondence should be addressed:
[email protected]
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1
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ABSTRACT
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Salmonella enterica causes systemic diseases (typhoid and paratyphoid
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fever), non-typhoidal septicaemia (NTS) and gastroenteritis in humans and
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other animals world-wide.
24
infectious disease problem in Sub-Saharan Africa is NTS in children and
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immunocompromised adults. A current goal is to identify Salmonella mutants
26
that are not pathogenic in the absence of key components of the immune
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system such as might be found in immunocompromised hosts.
28
attenuated strains have the potential to be used as live vaccines. We have
29
used Transposon Directed Insertion-site Sequencing (TraDIS) to screen
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mutants of Salmonella enterica serovar Typhimurium for their ability to infect
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and grow in the tissues of wild type and immunodeficient mice. This was to
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identify bacterial genes that might be deleted for the development of live
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attenuated vaccines that would be safer to use in situations and/or
34
geographical areas where immunodeficiencies are prevalent. The relative
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fitness of each of 9,356 transposon mutants, representing mutations in 3,139
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different genes, was determined in gp91-/-phox mice. Mutations in certain
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genes led to reduced fitness in both wild type and mutant mice. To validate
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tehse results these genes were mutated by allelic replacement and resultant
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mutants were re-tested for fitness in the mice. A defined deletion mutant of
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cysE was attenuated in C57BL/6 wild type mice and immunodeficient gp91-/-
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phox mice, and was effective as a live vaccine in wild type mice.
An important but under-recognized emerging
Such
2
42
INTRODUCTION
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Salmonella enterica causes systemic diseases (typhoid and paratyphoid
44
fever), non-typhoidal septicaemia and gastroenteritis in humans and other animals
45
world-wide. Salmonella enterica serovar Typhi (S. Typhi) and S. enterica serovar
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Paratyphi (S. Paratyphi) are restricted to humans and cause typhoid and paratyphoid
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fever, respectively. These are important febrile illnesses often seen in crowded and
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impoverished populations with inadequate sanitation that are exposed to unsafe
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water and food, and in travellers visiting countries where these diseases are
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endemic (1). In 2010, typhoid and paratyphoid fevers were together estimated to
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account for 190,200 deaths (2). Mortality rates in untreated typhoid fever can be 10-
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15%. In some cases patients recover but remain carriers of the bacteria for many
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years. Invasive non-typhoidal salmonellae (iNTS) are estimated to cause over 2.1
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million illnesses and 416,000 deaths in Africa annually despite antimicrobial
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treatment. Vaccine development for iNTS disease is a high priority (3). iNTS disease
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is linked with conditions which impair the immune system such as lack of antibodies
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in young children and multiple co-morbidities such as advanced HIV infection,
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malaria, haemolysis, sickle cell disease (SCD), malnutrition and cytokine pathway
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deficiencies (4, 5).
60
Current measures for controlling Salmonella infections are far from ideal. The
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emergence of new multi-drug-resistant strains has reduced the usefulness of most
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antimicrobials (6). Prevention by implementation of hygiene measures alone is
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proving insufficient. Currently licensed vaccines are far from optimal and no effective
64
vaccine or delivery system effective against NTS is available.
3
65
Live attenuated S. enterica vaccines are more effective in many different
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infection systems than non-living ones due to their ability to generate protective T-
67
helper 1 type immunity in addition to antibody responses (7). New live attenuated
68
vaccines against Salmonella are currently being developed and tested (8). However,
69
concerns remain about the use of live vaccines in general when these are targeted
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to endemic areas with a high incidence of conditions that impair the immune system,
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and there is reluctance to use live vaccines in animals bred for food production due
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to fears of side-effects subsequent to the unlikely, but possible, spread to humans.
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Reversion to virulence would pose a serious threat to individuals with an impaired
74
immune system who may inadvertently receive the live vaccine. Immuno-
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suppression can be transient, latent or undiagnosed, possibilities that are likely to be
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more frequent in children or in adults from countries where sophisticated diagnostic
77
facilities are not always available.
78
We and others have found that some live attenuated Salmonella vaccines,
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such as aromatic-dependent (aro) mutants, htrA mutants and aroA htrA double
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mutants can cause lethal infections in immunodeficient mice such as those lacking in
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the production of reactive oxgen species (ROS), IL-12 or IFNγ and in mice lacking T-
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cells (9, 10). Bacterial mutants of these genes could therefore be dangerous if used
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in patients with similar immune defects. We have also shown that other vaccines,
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specifically Salmonella Pathogenicity Island-2 (SPI-2) mutants of S. enterica, similar
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to the ones currently being tested in humans, regain virulence in ROS-deficient
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animals (11) and could therefore prove dangerous in individuals with reduced
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efficiency of the oxygen dependent antimicrobial mechanisms of phagocytes (e.g.
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chronic granulomatous disease (CGD) or malaria patients) (12, 13). These
4
89
immunodeficiencies may very well be latent in the individual at the time of
90
vaccination.
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A current goal is to identify mutations that would allow the generation of live
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attenuated salmonellae, that would not significantly regain virulence in the absence
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of key components of the immune system. The feasibility of this approach is
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supported by the evidence that some Salmonella mutants can retain this attenuation
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in situations of serious immunodeficiency in animal models of systemic infection. For
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example, SPI-2 mutants are attenuated in gene-targeted mice lacking IFNγ, despite
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these strains being virulent in ROS-deficient animals (14).
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Recent advances in high-throughput techniques to assess simultaneously the
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genotypes and relative fitness of individuals in complex pools of bacterial transposon
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(Tn) mutants allow us to utilise a genome-wide screen for genes necessary for
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bacterial fitness in the face of particular immunological challenges.
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Directed Insertion-site Sequencing (TraDIS) is one such technique. TraDIS exploits
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very high throughput Illumina sequencing to obtain sequence reads directly from the
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region of DNA flanking each Tn insert in pools of mutants (15). This allows the
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location of the Tns to be precisely determined in a massively parallel manner. The
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number of reads obtained for each Tn allows the relative abundance of each mutant
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to be assessed, and a quantitative measure of fitness can be obtained by comparing
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data obtained before and after a selection step.
Transposon
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In the current study we have used a stringent mouse model of
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immunodeficiency to perform a genome-wide screen with the potential to identify
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genes that can disable reversion to virulence in immunodeficient hosts and that
5
112
could therefore be included in new generations of live vaccines against invasive
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salmonelloses.
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MATERIALS AND METHODS Bacterial strains, media and growth conditions.
All wild type strains,
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defined mutants and plasmids are summarised in Table 1. Preparation of
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electrocompetent Escherichia coli and S. Typhimurium cells and transformations
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were performed as previously described (21). Bacteria were grown on Luria-Bertani
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(LB) medium. Media were supplemented with the appropriate antibiotic for selection
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(ampicillin 100μg/ml, kanamycin 50μg/ml, chloramphenicol 10μg/ml, tetracycline
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12.5μg/ml).
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Recombinant DNA techniques: Standard methods were used for molecular
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cloning (22).
Chromosomal and plasmid DNA purification and routine DNA
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modifications including restriction endonuclease digestion of DNA, modifications of
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DNA and ligations were carried out using commercial kits and supplies according to
127
the manufacturers’ instructions (QIAGEN; Thermo Fisher Scientific; New England
128
Biolabs). DNA concentration and purity were measured using a Nanodrop ND-1000
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spectrophotometer.
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(http://frodo.wi.mit.edu/) and purchased from Sigma (Sigma-Genosys, UK). PCRs
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were performed in 25 μl reaction volumes in 0.2 ml Eppendorf tubes in a Perkin
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Elmer Gene Amp 2400 thermal cycler. Reactions contained 200 M dNTPs, 2 mM
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Mg2+, 0.01 volumes of Proof Start DNA polymerase (QIAGEN; 2.5 U μl-1), 0.1
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volumes polymerase buffer (10x), 1 μM forward and reverse primers and template
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DNA (~50 ng plasmid DNA or ~100 ng chromosomal DNA). Thermal cycler
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conditions were 94ºC for 10 min, then 35 cycles of 94ºC for 1 min, 55ºC for 1 min
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and 72ºC for 1 min, followed by a final extension at 72ºC for 10 min.
PCR primers (Table S1) were designed using Primer3
7
138 139
Generation of defined mutants of S. Typhimurium. Mutants were
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generated using a modification of the ET-cloning procedure (23, 24) as previously
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described (20). PCR was used to amplify the chloramphenicol resistance cassette
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from pACYC184 (18), the kanamycin resistance cassette from pACYC177 (18) or
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the tetracycline resistance cassette from pBR322 (19) with 5’ and 3’ 60 bp homology
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arms complementary to the flanking regions of the gene to be deleted.
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Approximately 1 μg of linear PCR product was used for integration into the
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chromosome using a modification of the Lambda Red method (25), as previously
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detailed (26).
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chloramphenicol, kanamycin or tetracycline as appropriate. Screening for loss of the
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pBADλred helper plasmid was as previously described (27), using MAST ID
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Intralactam circles (MAST Diagnostics, Bootle Merseyside, UK) to screen for the
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absence of beta-lactamase in bacterial colonies. (Further details of construction of
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mutants are provided in Supplemental Material).
Transformants were selected by plating onto media containing
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Mouse infections. All animals were handled in strict accordance with good
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animal practice as defined by the relevant international (Directive of the European
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Parliament and of the Council on the protection of animals used for scientific
157
purposes, Brussels 543/5) and local (Department of Veterinary Medicine, University
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of Cambridge) animal welfare guidelines.
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ethical review committee of the University of Cambridge and was licensed by the UK
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Government Home Office under the Animals (Scientific Procedures) Act 1986. Sex-
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and aged-matched 9-12 week old C57BL/6 wild type mice (Harlan Olac Ltd), and
All animal work was approved by the
8
162
gp91-/-phox mice (bred at the Wellcome Trust Sanger Institute) were infected by
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intravenous (i.v.) injection of bacterial suspensions in a volume of 0.2 ml. Static
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grown cultures of wild type S. Typhimurium SL1344 and defined mutants were grown
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overnight at 37ºC from single colonies in 10 ml LB broth, and then diluted in
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phosphate buffered saline (PBS) to the appropriate concentration for inoculation.
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Bacterial cultures for the pools of Tn5 and Mu Tn mutants (TraDIS pools) were
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grown as described previously (14). Inocula were enumerated by plating dilutions
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onto LB agar plates. Mice were killed by cervical dislocation and the livers and
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spleens were aseptically removed and homogenized in sterile water using a
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Colworth Stomacher 80. The resulting homogenate was diluted in a 10-fold series in
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PBS and LB agar pour plates were used to enumerate viable bacteria.
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TraDIS analysis of S. Typhimurium mutant pools. The Illumina sequence
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reads from the input (bacteria inoculated into mice) and output (bacteria recovered
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from infected livers and spleens) pools (European Nucleotide Archive ERA201074)
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were generated using single-end short-read sequencing with Tn specific primers and
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by massively parallel sequencing of Tn-flanking regions in the input and output
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mutant pools. Genomic DNA was prepared and 2 µg fragmented by Covaris to an
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average size of ~300 bp. Following end-repair and ‘A’ tailing a modified Illumina
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adapter, synthesized and annealed by Integrated DNA Technologies (IDT) using
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oligonucleotides SplA5_top and SplA5_bottom (Table S1), was ligated to the
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fragments for 40 min at 20oC. Ligated fragments were cleaned using Ampure XP
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beads (Beckman Coulter) with a bead to sample ratio of 0.8 to 1. PCR enrichment of
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fragments containing the Tn was performed using a primer homologous to each end
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of the Tn (Mu_AG_5’PCR, Mu_AG_3’PCR, Tn5_AG_5’PCR or Tn5_AG_3’PCR; 9
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Table S1) in conjunction with an adapter-specific primer containing an index tag
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(SplAP5.x; Table S1).
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(Beckman Coulter), quantified by qPCR and then pooled. The resultant products
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were sequenced on a HiSeq2500 (Illumina) using a specially modified recipe to
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overcome difficulties generated by the monotemplate Tn sequence. Briefly, a Tn-
192
specific sequencing primer (Mu_AG_5’seq, Mu_AG_3’seq, Tn5_AG_5’seq or
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Tn5_AG_3’seq; Table S1) anneals to the Tn 10 bases away from the genomic DNA
194
junction. Sequencing takes place with no imaging for the first 10 cycles followed by
195
imaging for the next 40 cycles. This gives a 40bp genomic DNA read. The template
196
is denatured and the same sequencing primer is re-annealed and 10 cycles of
197
sequencing takes place to give a 10 bp Tn read.
PCR products were cleaned using Ampure XP beads
198 199
Sequence Analysis. Illumina reads generated from the genomic DNA of the
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input and output pools containing Tn5 and Mu Tn insertions were stripped of the 5’
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Tn tags using Cutadapt version 1.8.1 (28), and the remainder of each sequence read
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was mapped to the S. Typhimurium SL1344 chromosome and plasmid sequences
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(Genbank accession numbers FQ312003, HE654724, HE654725 and HE654726)
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using BWA mem version 0.7.12-r1039 (29). The raw input and output read counts
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were loaded into R version 3.0.2. A step was performed to eliminate nonspecific
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background reads by filtering out the insertions with a total read count across all
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samples of 94% of the Tn mutants
259
screened were mapped). The reason for the lower number of mapped mutants
260
compared with the number of mutants in the original input pools could be due to
261
mutants not surviving the initial grow-up in the input pool, and/or due to the presence
262
of duplicate mutants in the library. A total of 3,139 different genes were disrupted by
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the 9,356 Tn insertions.
264
The sites of insertion of all Tns in the S. Typhimurium TraDIS mutant libraries,
265
the identities of the genes that are disrupted, and the fitness scores of the mutants
266
are listed in Table S2. The fitness scores of the mutants obtained during infection of
267
C57BL/6 mice showed a high proportion of seemingly attenuated mutants (Fig. S1a).
268
Additionally, the output read counts from the two replicate C57BL/6 mice did not
269
show a good correspondence (Fig. S1b), and the fitness scores did not correlate well
270
with those obtained in BALB/c mice (Fig. S1c) from our previous study (32). From
271
these observations we concluded that the C57BL/6 mice had exhibited stochastic
272
loss of mutants for reasons unrelated to their genotype (i.e. random dropout). By
273
contrast, only a small proportion of mutants were attenuated in the gp91-/-phox mice,
274
with good correspondence between the replicates and with the data obtained from
275
BALB/c mice (Fig. S2). The random dropout was not identified in the BALB/c mice,
276
probably because of the increased bacterial load used as the inoculum in this model 13
277
(~1x106 CFU in the BALB/c mice vs ~5x104 CFU in the C57BL/6 mice), or in the
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gp91-/-phox mice, due to the absence of bacterial killing in this model. Thus, the
279
attenuated mutants in the C57BL/6 dataset are likely to include false negatives due
280
to the random dropout of mutants in this model (i.e. mutants that have been
281
assigned as attenuated, but that could be colonisation proficient). This prevented an
282
in-depth comparison of the different genes that contributed to fitness in the wild type
283
C57BL/6 and immunocompromised mice. However, the data from the screen in the
284
gp91-/-phox mice were robust and provided us with solid foundations to select
285
representative genes from the ‘attenuated mutant’ lists and testing defined allelic
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replacement mutants of these genes in wild type and immunocompromised mice.
287 288
Analysis of defined allelic replacement S. Typhimurium mutants in
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C57BL/6 WT and gp91-/-phox mice. An ideal live attenuated vaccine would need to
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colonise but be attenuated in an immune-competent host and to remain attenuated
291
in conditions where the immune system may be impaired. Candidate genes for
292
generation of such vaccines will be amongst those that lead to attenuated mutant
293
phenotypes in spleens and livers of both C57BL/6 WT and gp91-/-phox mice. We
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selected 14 genes that fit this pattern: cydC, cydD, cysE, dksA, ftsK, miaA, nuoK,
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ptsI, recD, secG, seqA, sucA, thdF and yqiC and deleted them by allelic replacement
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in S. Typhimurium. We also generated a double mutant knocking out both cydC and
297
cydD, a mutant containing a deletion of the entire suc operon, a mutant containing a
298
deletion of the entire nuo operon, a mutant containing a deletion of the tol operon
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(since many different tol mutants were attenuated, and tol genes have previously
300
been shown to have a role in virulence/fitness in mice (33)), and a mutant containing
301
a deletion of the tol/pal operon. All of these were tested individually for attenuation in 14
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C57BL/6 WT mice compared to the SL1344 wild type, administered by i.v. injection,
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and at input doses as detailed in Table 2. We also included in the experiment
304
controls mutants lacking aroC, aroC/aroD/htrA, and aroC/ssaV, which have
305
established roles in virulence and have been or are being used as vaccine
306
candidates (34-36). Each gene, or genes, was inactivated separately by λRed
307
recombinase-mediated integration of linear PCR amplicons by homologous
308
recombination. Mice were killed at 7 days p.i. or earlier if clinical signs became
309
apparent (Table 2). Defined mutants in cydC, cydD, cydC/cydD, recD, seqA and the
310
suc operon were not attenuated in the C57BL/6 mice, and mice had to be killed at 3
311
days p.i., along with the mice infected with the wild type SL1344. Mice infected with
312
defined mutants in ftsK, nuoK and the nuo operon mutants were killed on day 4 p.i.
313
as the mice displayed clinical signs. Mice infected with a dksA mutant were killed on
314
day 5 p.i. Mice infected with sucA and thdF mutants were displaying clinical signs at
315
6 days p.i. and were killed, although interestingly viable bacterial counts in the livers
316
and spleens were not high enough to be consistent with the observed signs of
317
infection, indicating that the clinical signs might be due to focal infection at other
318
body sites. Mice infected with cysE, miaA, ptsI, secG, tol operon, tol/pal operon and
319
yqiC mutants as well as the controls infected with aroC, aroC/aroD/htrA and
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aroC/ssaV, did not display any visible clinical signs on day 7 p.i.
321
Given their attenuation in WT mice, mutants in cysE, miaA, ptsI, secG and
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yqiC, along with the tol operon and tol/pal operon were used to infect gp91-/-phox
323
mice. We also included aroC, aroC/aroD, and ssaV mutants as controls. Mice were
324
killed at 7 days p.i., or earlier if clinical signs became apparent (Table 3). Mice
325
infected with defined ptsI, ssaV, tol operon and tol/pal operon mutants were killed on
326
day 3 p.i. when they displayed clinical signs. Mice infected with aroC, miaA and 15
327
secG mutants were killed on day 4 p.i. Mice infected with an yqiC mutant were killed
328
on day 5 p.i. when they displayed clinical signs. Mice infected with aroC/aroD or
329
cysE mutants were killed on day 7 p.i. which was the pre-defined end point of the
330
experiment. The cysE and the aroC/aroD mutants had higher bacterial counts in the
331
organs of the gp91-/-phox mice than in the C57BL/6 WT mice. In order to determine
332
how long the gp91-/-phox mice could survive infection with a cysE mutant, we
333
performed a small pilot study, infecting gp91-/-phox mice with Log10 5.72 CFU
334
SL1344 cysE and then allowing the infection to proceed until the mice displayed
335
clinical symptoms. The mice had to be killed on day 8 p.i. when the bacterial loads in
336
the livers were Log10 8.33 + 0.11 CFU (error given as standard deviation) and the
337
bacterial loads in the spleens were Log10 7.81 + 0.05 CFU (error given as standard
338
deviation). We next proceeded to compare cysE against an aroC mutant in gp91-/-
339
phox mice (Fig. 2). At three different input doses, the time till the appearance of
340
clinical signs was consistently delayed for the cysE mutant compared to the aroC
341
mutant.
342
As a prerequisite to a vaccination study, we determined the time it took for
343
SL1344 cysE to be cleared from the systemic organs (liver and spleen) of WT
344
C57BL/6 mice (Fig. 3). We found that by day 84 p.i. mice infected i.v. had cleared all
345
SL1344 cysE from the systemic organs, while in the orally infected group it took until
346
day 91 p.i. for the organs to be clear of the SL1344 cysE.
347
We next vaccinated WT C57BL/6 mice, either i.v. or orally, with SL1344 cysE
348
and then challenged the mice, 91 days post immunisation, orally with virulent
349
SL1344 and monitored the bacterial counts in the livers and spleens at various times
350
post infection (Fig. 4). The majority of the vaccinated mice were protected from
351
challenge with the virulent SL1344. However, during the challenge experiment it was 16
352
necessary to kill six mice (five from the orally immunised group, and one from the i.v.
353
immunised group) because they were displaying signs of infection. Thus, 97% of the
354
SL1344 cysE i.v. vaccinated C57BL/6 mice, and 86% of the SL1344 cysE orally
355
vaccinated C57BL/6 mice, were protected against challenge with virulent SL1344.
17
356
CONCLUSION
357
Here we have presented a report of fitness of random Tn mutants of S.
358
Typhimurium in vivo and described attenuated mutants based on the TraDIS
359
analysis of pools of Tn mutants screened in C57BL/6 WT and gp91-/-phox mice. Our
360
study provides proof-of-principle data on the feasibility of using TraDIS analysis in
361
combination with infections of gene-targeted animals to allow identification of
362
bacterial mutants as live vaccine candidates that would be safe in situations where
363
the immune system is impaired.
364
We show that through TraDIS analysis it is possible to identify novel
365
mutations in S. Typhimurium genes that still allow colonisation in WT mice but
366
importantly also reduce bacterial growth rate in vivo in severly immunodeficient gp91-
367
/-
phox mice. It is worth noting that the variance of defined mutants from their TraDIS
368
fitness scores (Tables 2 and 3) is likely to be due to differences in competition
369
dynamics for a given mutant relative to co-screened mutant bacteria, as we
370
previously demonstrated (32), and the differences in time-points at which the
371
bacteria were enumerated p.i. between the TraDIS screen and the defined mutant
372
experiments. Thus, some of the attenuated mutants identified in the screen are
373
virulent when tested individually, while some others retain the attenuation, and
374
therefore the screen is useful, but requires further evaluation of mutations
375
individually.
376
Through the TraDIS screens and validation of defined mutants, we identified
377
that SL1344 cysE was attenuated for growth, and cleared, in WT C57BL/6 mice and
378
had reduced growth in gp91-/-phox mice, although these mice succumbed to the
379
infection 8 days p.i. SL1344 cysE offered protection when given i.v. or orally as a
380
live vaccine to C57BL/6 WT mice, however, a few of the vaccinated mice succumbed 18
381
to the challenge with the virulent SL1344, indicating that cysE does not offer full
382
protection to all vaccinated animals.
383
Our work did not identify a single-gene S. Typhimurium mutant that would
384
colonise WT animals at sufficient levels to be predicted to be immunogenic and that
385
would also be completely unable to cause disease in the severly immunodeficient
386
animals used in this study. The attractive possibility that different combinations of
387
mutations could yield this ‘ideal’ vaccine candidate remains to be explored. However,
388
even mutations that reduce growth in immunocompromised hosts would be
389
advantageous and could be used to construct live strains in a way that would
390
crucially enable more time for medical intervention should an immunocompromised
391
vaccinee develop signs of infection. Furthermore, the animal model that we used
392
was of extreme immunodeficiency, while it is likely that in the field one would
393
encounter situations where macrophage function is only partially impaired.
394
Therefore it remains to be established whether some of the mutants identified from
395
this study would be safer in models of partial immunodeficiency.
396
Extending the approach reported here to other gene-targeted mouse strains
397
has the potential in the future to generate a more comprehensive picture of the
398
functional interactions between the bacterial and host genomes and to identify
399
bacterial mutants as live vaccine candidates that would be safe across a wide range
400
of immunodeficiencies.
19
401
FUNDING INFORMATION
402
This work was supported by a Medical Research Council (MRC) grant
403
G1100102. Tn libraries were previously constructed with funding from the
404
Biotechnology and Biological Sciences Research Council grant APG19115. O.O.
405
was supported by a Newton Trust grant awarded to A.J.G. M.M. was supported by
406
the Wellcome Trust grant number WT098051. The authors have no conflicting
407
financial interests. The funders had no role in study design, data collection and
408
interpretation, or the decision to submit the work for publication.
20
409 410 411
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29
610
FIGURES AND TABLE LEGENDS
611 612
Figure 1: Relative fitness scores (log2-fold change) plotted against normalised mean
613
read counts. Fitness scores represent the log2-fold change in the sequence read
614
counts of output pools compared with the input pools. Output pools were obtained
615
from the livers of gp91-/-phox, C57BL/6 and BALBc mice (32). Significantly
616
attenuated mutants, represented as red closed circles, are a population of mutants
617
showing negative fitness scores and an adjusted P-value (Benjamini-Hochberg) of
618
P≤0.05. Mutants with fitness scores which were not significantly different from zero
619
are shown as black circles. The wide range of fitness scores exhibited in the
620
C57B/L6 mice is indicative of large scale stochastic loss ("random dropout") of
621
mutants during those infections.
622 623
Figure 2: Time to presentation of clinical signs in gp91-/-phox mice infected with
624
various doses of SL1344 cysE or SL1344 aroC. gp91-/-phox mice were infected i.v.
625
with Log10 5.85 CFU, Log10 4.85 CFU or Log10 3.85 CFU of either SL1344 aroC (grey
626
symbols) or SL1344 cysE (black symbols) and the time to clinical signs was
627
monitored.
628 629
Figure 3: WT C57BL/6 mice were infected i.v. (circles) or orally (triangles) with Log10
630
5.80 CFU or Log10 8.68 CFU, respectively, of SL1344 cysE, and bacterial counts in
631
the systemic organs (livers and spleens) were monitored at various times post
632
infection until the bacteria had cleared from the organs. 30
633 634
Figure 4: Vaccination study, immunising C57BL/6 WT mice with SL1344 cysE and
635
subsequent challenge with WT SL1344. A) WT C57BL/6 mice were vaccinated with
636
SL1344 cysE administered at either Log10 5.7 CFU i.v. or Log10 8.5 orally. At 91 days
637
post-immunisation the vaccinated mice and an unvaccinated group of mice (naive
638
control) were challenged orally with Log10 8.8 CFU of virulent SL1344. Bacterial
639
counts in the systemic organs (livers and spleens) were monitored at various times
640
post-challenge (N.B. one mouse from the orally vaccinated group had to be killed on
641
day 9 post challenge, since it was displaying signs of infection). B) WT C57BL/6
642
mice were vaccinated with SL1344 cysE administered at either Log10 5.8 CFU i.v. or
643
Log10 8.7 orally. At 91 days post-immunisation the vaccinated mice were challenged
644
orally with Log10 8.8 CFU or virulent SL1344. Bacterial counts in the systemic organs
645
(livers and spleens) were monitored at various times post challenge (N.B. one
646
mouse from both the orally vaccinated and the i.v. vaccinated group had to be killed
647
on day 9 post challenge, one mouse from the orally vaccinated group had to be killed
648
on day 18, day 19 and day 34 post challenge, since they were displaying signs of
649
infection).
650 651
Table 1: Bacterial strains and plasmids used in this study.
652 653
Table 2: CFU in the organs of C57BL/6 WT mice infected with defined mutants of S.
654
Typhimurium, the upper and lower TraDIS fitness scores (in C57BL/6 mice) and the
655
number of mutants in each gene identified by TraDIS (in C57BL/6 mice).
31
656 657
Table 3: CFU in the organs of gp91-/-phox mice infected with defined mutants of S.
658
Typhimurium, the upper and lower TraDIS fitness scores (in gp91-/-phox mice) and
659
the number of mutants in each gene identified by TraDIS (in gp91-/-phox mice).
32
660
-/-
gp91 phox
Mu
Tn5
C57BL/6
Mu
Tn5
BALBc
Mu
Tn5
661 662
Figure 1: Relative fitness scores (log2-fold change) plotted against normalised mean
663
read counts. Fitness scores represent the log2-fold change in the sequence read
664
counts of output pools compared with the input pools. Output pools were obtained
665
from the livers of gp91-/-phox, C57BL/6 and BALBc mice (32). Significantly 33
666
attenuated mutants, represented as red closed circles, are a population of mutants
667
showing negative fitness scores and an adjusted P-value (Benjamini-Hochberg) of
668
P≤0.05. Mutants with fitness scores which were not significantly different from zero
669
are shown as black circles. The wide range of fitness scores exhibited in the
670
C57B/L6 mice is indicative of large scale stochastic loss ("random dropout") of
671
mutants during those infections.
34
672
673 674
Figure 2: Time to presentation of clinical signs in gp91-/-phox mice infected with
675
various doses of SL1344 cysE or SL1344 aroC. gp91-/-phox mice were infected i.v.
676
with Log10 5.85 CFU, Log10 4.85 CFU or Log10 3.85 CFU of either SL1344 aroC (grey
677
symbols) or SL1344 cysE (black symbols) and the time to clinical signs was
678
monitored.
35
679
680 681
Figure 3: WT C57BL/6 mice were infected i.v. (circles) or orally (triangles) with Log10
682
5.80 CFU or Log10 8.68 CFU, respectively, of SL1344 cysE, and bacterial counts in
683
the systemic organs (livers and spleens) were monitored at various times post
684
infection until the bacteria had cleared from the organs.
36
685
A
686 687
B
688 689
Figure 4: Vaccination study, immunising C57BL/6 WT mice with SL1344 cysE and
690
subsequent challenge with WT SL1344. A) WT C57BL/6 mice were vaccinated with
691
SL1344 cysE administered at either Log10 5.7 CFU i.v. (circles) or Log10 8.5 orally 37
692
(triangles). At 91 days post-immunisation the vaccinated mice and an unvaccinated
693
group of mice (naive control; squares) were challenged orally with Log10 8.8 CFU of
694
virulent SL1344. Bacterial counts in the systemic organs (livers and spleens) were
695
monitored at various times post-challenge (N.B. one mouse from the orally
696
vaccinated group had to be killed on day 9 post challenge, since it was displaying
697
signs of infection). B) WT C57BL/6 mice were vaccinated with SL1344 cysE
698
administered at either Log10 5.8 CFU i.v. (circles) or Log10 8.7 orally (triangles). At 91
699
days post-immunisation the vaccinated mice were challenged orally with Log10 8.8
700
CFU or virulent SL1344. Bacterial counts in the systemic organs (livers and spleens)
701
were monitored at various times post challenge (N.B. one mouse from both the orally
702
vaccinated and the i.v. vaccinated group had to be killed on day 9 post challenge,
703
one mouse from the orally vaccinated group had to be killed on day 18, day 19 and
704
day 34 post challenge, since they were displaying signs of infection).
38
705 Strain or Plasmid S. Typhimurium SL1344
Relevant genotype or description
Source/Reference
Virulent in mice with an i.v. LD50 of
