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Published in final edited form as: Cell Host Microbe. 2019 Aug 22;26(3):426–434.e6. doi: 10.1016/j.chom.2019.08.001

Genome-Wide Analysis of Salmonella enterica serovar Typhi in Humanized Mice Reveals Key Virulence Features

Joyce E Karlinsey a,#, Taylor A Stepien b,#, Matthew Mayho e, Larissa A Singletary a,$, Lacey K Bingham-Ramos c, Michael A Brehm f, Dale L Greiner f, Leonard D Shultz g, Larry A Gallagher d, Matt Bawn e,h, Robert A Kingsley e,i, Stephen J Libby c, Ferric C Fang a,b,c,*
PMCID: PMC6742556  NIHMSID: NIHMS1537086  PMID: 31447308

SUMMARY

Salmonella enterica serovar Typhi causes typhoid fever only in humans. Murine infection with S. Typhimurium is used as a typhoid model, but its relevance to human typhoid is limited. Non-obese diabetic-scid IL2rγnull mice engrafted with human hematopoietic stem cells (hu-SRC-SCID) are susceptible to lethal S. Typhi infection. In this study, we use a high-density S. Typhi transposon library in hu-SRC-SCID mice to identify virulence loci using transposon-directed insertion site sequencing (TraDIS). Vi capsule, LPS and aromatic amino acid biosynthesis were essential for virulence, along with the siderophore salmochelin. However, in contrast to the murine S. Typhimurium model, neither the PhoPQ two-component system nor the SPI-2 pathogenicity island were required for lethal S. Typhi infection, nor was the CdtB typhoid toxin. These observations highlight major differences in the pathogenesis of typhoid and nontyphoidal Salmonella infections and demonstrate the utility of humanized mice for understanding the pathogenesis of a human-specific pathogen.

Graphical Abstract

Virulence determinants of the human-restricted pathogen Salmonella Typhi are incompletely understood. Karlinsey et al. use a high-density transposon library to identify genes required for S. Typhi virulence in humanized mice engrafted with human immune cells. The screen reveals key differences between S. Typhimurium and S. Typhi pathogenesis.

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INTRODUCTION

Salmonella enterica serovars Typhi and Paratyphi cause enteric fever and infect only humans. S. Typhi infrequently causes diarrhea, instead producing a severe systemic illness with bloodstream invasion and fever, headache and prostration (Raffatellu et al., 2008). A hallmark of typhoid is the development of a carrier state in which infected individuals exhibit prolonged fecal S. Typhi shedding (Gunn et al., 2014). The World Health Organization estimates that 11–20 million cases of typhoid fever result in 128,000–161,000 related deaths per year, mainly in Asia and Africa (WHO, 2018). Recent emergence of drug-resistant S. Typhi strains poses a major public health challenge (Levine and Simon, 2018; Klemm et al., 2018). Understanding the molecular and immunological mechanisms of S. Typhi pathogenesis can lead to more effective typhoid vaccines and therapies.

Insight into how S. Typhi causes human disease has been hampered by the lack of a small animal model. Non-human primates are resistant to typhoid and exhibit only mild self-limited illness after S. Typhi challenge (Edsall et al., 1960; Gaines et al., 1968). Non-typhoidal S. Typhimurium causes an acute systemic infection in most laboratory mice and is commonly described as a model for typhoid. This model has uncovered many important Salmonella virulence determinants. However, the relevance of these observations to human typhoid is uncertain, as S. Typhimurium causes enteritis rather than enteric fever in humans, and S. Typhi and S. Typhimurium have numerous genetic differences (Sabbagh et al., 2010).

We previously reported that hu-SRC-SCID mice develop lethal S. Typhi infection, along with characteristic pathology and cytokine responses of typhoid fever (Libby et al., 2010). A small number of candidate virulence loci were identified in a transposon site hybridization (TraSH) screen in this model (Libby et al., 2010). However, each pool was limited to approximately 103 insertions and yielded a number of false-positive results due to genetic bottlenecks. We have subsequently employed TraDIS (transposon-directed insertion site sequencing, Langridge et. al., 2009), a high-throughput method that allows the parallel analysis of high-complexity transposon libraries in individual mice. Here we report a genome-wide analysis of loci required for S. Typhi infection following intraperitoneal (i.p.) inoculation of hu-SRC-SCID mice. This systematic analysis of S. Typhi virulence in an infection model demonstrates the unique ability of a humanized mouse model to allow the comprehensive identification of virulence determinants of a human-specific pathogen.

RESULTS

Identification of S. Typhi Genes Required for Virulence in hu-SRC-SCID Mice

A high-density transposon (Tn) library of S. Typhi with 50x genome coverage by a Tn5-based transposon (Table S1) was used to infect hu-SRC-SCID mice. After i.p. inoculation of 4×105 colony-forming units (cfu), mice were monitored for signs of illness and euthanized when moribund. As seen previously (Libby et al., 2010), most animals rapidly developed illness and were euthanized 24 h post infection (p.i.), although some were less severely ill and were euthanized 64 to 79 h p.i. Variation in severity is expected due to differences in donor genetic backgrounds and levels of engraftment in individual mice. Organism burdens in liver and spleen of infected hu-SRC-SCID mice were higher than in infected non-engrafted NOD-scid IL2rγnull mice (Figure S1A). Two “input” libraries, representing the inoculum (T2, JZ01) and one “output” library per mouse, generated from spleen cfu outgrowth, were constructed and sequenced on an Illumina 2500 platform (STAR Methods; Langridge et al., 2009; Barquist et al., 2016). Sequences were analyzed with the Bio-TraDIS analysis pipeline (Barquist et al., 2016), and results summarized in Table S2. The input S. Typhi library contained over 185,370 unique insertions, whereas the output libraries from spleens had 53,154 to 125,183 unique insertions. Underrepresentation of mutants within specific genes in the output pool (“counter-selection”) identifies genes essential for virulence in the hu-SRC-SCID model (Table S2).

Initial analysis of differences in reads between input and output libraries showed variable and indiscriminate counter-selection for mice euthanized 64 to 79 h p.i. Therefore, only mice euthanized 24 h p.i. were used for comparison of input and output pools (T3,T4,T5,T6,T13,T14,T15,T16,T17). Analysis of the nine output pools and two input pools revealed 72 counter-selected genes that met pre-determined criteria of a false discovery rate of <0.05 and log fold change <-2; these genes are categorized into functional groups in Table 1. Counter-selected loci include genes for purine and amino acid biosynthesis (pur, aro), LPS (lipopolysaccharide) and O-antigen synthesis (waa, rfb), DNA repair (rec), protein folding (dsb, fkp), iron acquisition (ent, iro) and Vi capsular synthesis (vex, tvi) (Tables 1, S2, and S3).

Table 1.

S. Typhi Ty2 Genes Counter-Selected in hu-SRC-SCID Mice 24 h p.i.

Gene name Locus tag Function logFC
LPS biosynthesis; O-antigen
rfbD/rmlD t0776 dTDP-4-dehydrorhamnose reductase −5.03
rfbC/rmlC t0778 dTDP-4-dehydrorhamnose 3,5-epimerase −4.9
rfbI/ddhD t0779 putative reductase Rfbl −3.52
rfbG/ddhB t0781 CDP-glucose 4,6-dehydratase −3.5
rfbH/ddhC t0782 putative dehydratase RfbH −3.12
rfbE/tyv t0784 CDP-tyvelose-2-epimerase −2.89
rfbX/wzx t0785 putative O-antigen transporter −2.9
rfbV/wbaV t0786 putative glycosyl transferase −2.79
rfbN/wbaN t0788 putative rhamnosyltransferase −4.55
rfbM/manC t0789 mannose-1-phosphate guanylyltransferase −3.27
rfbK/manK t0790 phosphomannomutase −3.82
rfbP/wbaP t0791 undecaprenyl-phosphate galactosephosphotransferase −3.77
wzxE t3371 putative lipopolysaccharide biosynthesis protein −4.47
waaL t3806 O-antigen ligase −4.67
LPS biosynthesis; Outer core
waaB t3800 lipopolysaccharide 1,6-galactosyltransferase −4.51
waaI t3801 lipopolysaccharide 1,3-galactosyltransferase −3.92
waaJ t3802 lipopolysaccharide 1,2-glucosyltransferase −4.11
waaK t3805 lipopolysaccharide 1,2-N-acetylglucosaminetransferase −4.61
Metabolism and enzymes
purL t0291 phosphoribosylformylglycineamide synthetase −5.38
purN t0357 phosphoribosylglycinamidine myltransferase −5.53
purM t0358 phosphoribosylformylglycinamidine cyclo-ligase −5.57
cvpA t0501 colicin V production protein −6.39
purF t0502 amidophosphoribosyltransferase −5.54
pabB t1053 para-aminobenzoate synthase component l −3.47
gpmA t2115 phosphoglycerate mutase 1 −3.78
purE t2327 phosphoribosylaminoimidazole carboxylase catalytic subunit −5.45
purH t3455 phosphoribosylaminoimidazolecarboxamide formyltransferase −3.45
purD t3456 phosphoribosylglycineamide synthetase −4.47
gntK t3980 putative gluconokinase −4.24
purA t4417 adenylosuccinate synthetase −3.93
Vi antigen biosynthesis
vexE t4344 Vi polysaccharide export protein −4.61
vexD t4345 Vi polysaccharide export inner-membrane protein −5.5
vexC t4346 Vi polysaccharide export ATP-binding protein −4.99
vexB t4347 Vi polysaccharide export inner-membrane protein −5.16
vexA t4348 Vi polysaccharide export protein −5.69
tviE t4349 Vi polysaccharide biosynthesis protein TviE −5.39
tviD t4350 Vi polysaccharide biosynthesis protein −5.21
tviC t4351 Vi polysaccharide biosynthesis protein, epimerase −5.77
tviB t4352 Vi polysaccharide biosynthesis protein, UDP-glucose/GDP-mannose dehydrogenase −4.83
tviA t4353 Vi polysaccharide biosynthesis protein −4.75
Iron acquisition/utilization/cluster repair
entA t2270 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase −4.55
ybdZ t2281 conserved hypothetical protein −5.52
iroC t2669 putative ABC transporter protein −5.14
iroD t2670 putative ferric enterochelin esterase −5.66
iroE t2671 putative exported protein −2.32
iroN t2672 TonB-dependent outer membrane siderophore receptor protein −4.9
exbD t3078 biopolymer transport ExbD protein −4.58
exbB t3079 biopolymer transport ExbB protein −4.4
yggX t3024 conserved hypothetical protein probable Fe(2+)-trafficking protein −3.74
Unclassified
t1103 t1103 putative regulator sirB1 −4.15
t2640 t2640 conserved hypothetical protein −4.05
t3486 t3486 hypothetical protein −5.02
yhgE t4007 putative membrane protein DUF4153 domain-containing protein −5.33
t4182 t4182 entericidin B precursor −6.21
Cell membrane
yfgC/bepA t0363 putative Zn-dependent protease, contains TPR repeats −4.14
yejM/pbgA t0626 putative sulphatase cardiolipin transport −3.74
ybiS t2050 putative exported protein L,D-transpeptidase −5.87
yrfF/igaA t4011 negative regulator Rcs regulatory system −5.09
Amino acid synthesis
aroC t0480 chorismate synthase −6.96
aroA t1956 3-phosphoshikimate 1-carboxyvinyltransferase −5.02
serC t1957 phosphoserine aminotransferase −3.65
Transcription
nagC t2193 N-acetylglucosamine repressor −2.61
greA t3216 transcription elongation factor −5.26
fabR t3498 HTH-type transcriptional repressor FabR −4.68
Chaperone
fkpA t4052 FKBP-type peptidyl-prolyl isomerase −3.51
ytfN/tamB t4464 autotransporter assembly complex protein TamB −5.1
Respiration and ATP synthesis
ndh t1709 NADH dehydrogenase −5.04
corE/ypjD t2633 putative membrane protein −3.23
Transport
tolB t2128 tolB protein precursor −4.68
tolQ t2131 tolQ protein −4.51
Cell redox homeostasis
dsbA t3623 thiol:disulfide interchange protein −4.17
DNA replication, modification
recA t2730 recA protein −5.95
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Comparison of Sequence Reads in Input and Output Libraries for S. Typhi Virulence Genes

Regions of counter-selected Tn insertions were visualized with Artemis software (Carver et al., 2011). Figure 1 shows read maps from representative input and output samples; counter-selected loci include Vi capsule (Figure 1A), salmochelin (Figure 1B) and LPS biosynthetic operons (Figure 1C). Counter-selected genes had high read numbers of input but not output samples, whereas flanking genomic regions showed similar read numbers in both input and output samples, indicating that chromosomal location did not affect counter-selection. Unexpectedly, some genes important during murine S. Typhimurium infection were not significantly counter-selected in S. Typhi-infected hu-SRC-SCID mice. The importance of Salmonella Pathogenicity Island-1 (SPI-1) and SPI-2 genes for S. Typhimurium invasion, macrophage survival and systemic infections in mice is well-documented (Zhang et al., 2018; Hensel et al.,1995; Hensel et al., 1998). However, S. Typhi carrying transposon insertions in SPI-1 or SPI-2 genes were not counter-selected in hu-SRC-SCID mice (Figure 1D and 1E). The PhoPQ two-component system is a regulator of SPI-2 (Bijlsma and Groisman, 2005) and other genes required for S. Typhimurium virulence (Groisman, 2001; Dalebroux and Miller, 2014), but was only weakly counter-selected in S. Typhi-infected humanized mice (logFC phoP=2.1 phoQ=-0.27 Table S3). In addition, the cdtB locus encoding the typhoid toxin was not counter-selected (Figure 1F).

Figure 1. Selection of S. Typhi Loci Identified by TraDIS in hu-SRC-SCID Mice.

Figure 1.

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The frequency and distribution of mapped sequence reads were generated using Artemis. Counter-selected loci: (A) Vi antigen (B) salmochelin biosynthesis and utilization and (C) LPS biosynthesis. Non-counter-selected loci: (D) Salmonella Pathogenicity Island 1 region (E) Salmonella Pathogenicity Island 2 region and (F) typhoid toxin. The y-axis shows frequency of mapped reads within window size 3. Maximum read maps are shown in the upper right except where * indicates map reads greater than shown. Input S. Typhi transposon library (blue) and a representative output S. Typhi transposon library from hu-SRC-SCID mouse 24 h p.i. (red) are shown.

Confirmation of Selected Mutants in hu-SRC-SCID Mice

Validation of the TraDIS analysis was performed by competitive infections of wild-type and selected mutant strains in hu-SRC-SCID mice. Wild-type and isogenic mutant S. Typhi Ty2 strains in a 1:1 ratio (105 cfu of each strain) were administered by i.p. inoculation. Mice infected with wild-type and iroCDEN, ssrB, phoP or cdtB mutants were euthanized 24 h p.i.; wild-type and vexA- or entA-infected mice were euthanized after 72 h. Competitive indexes were determined from liver and spleen cfu. Mutants lacking Vi antigen (vexA) or enterobactin synthesis (entA) were significantly outcompeted by wild-type S. Typhi in both liver and spleen (Figure 2A), confirming that Vi- or enterobactin-deficient mutant S. Typhi have reduced virulence. An iroCDEN mutant was outcompeted by wild-type in the three mice from which sufficient numbers of bacteria were recovered (Figure S1B). Therefore, the conversion of enterobactin to salmochelin appears to be essential for virulence after i.p. infection. A phoP mutant was outcompeted only in the spleen. Mutants lacking ssrB (SPI-2) or cdtB (typhoid toxin) were recovered as well as wild-type (Figure 2A), confirming that these genes are not essential for virulence in hu-SRC-SCID mice. Hu-SRC-SCID mice challenged with up to 2.5×106 cfu of an aroA mutant of S. Typhi did not succumb to infection, unlike mice infected with wild-type (Figure S2).

Figure 2. Confirmation of S. Typhi Mutants in hu-SRC-SCID Mouse and Human Macrophage Infections.

Figure 2.

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(A) Competitive indexes (CI) of S. Typhi vexA, entA, iroCDEN, ssrB, phoP or cdtB mutants compared to wild-type were measured in hu-SRC-SCID mice. S. Typhi mutants counter-selected or not counter-selected in the TraDIS analysis are indicated with a (+) or (-) respectively. Six hu-SRC-SCID mice for each group were infected with an equal mixture of wild-type and mutant strains. Solid lines represent median CI in the livers (blue circles) and spleens (red squares). Dotted line represents the expected CI if neither strain has a competitive disadvantage. Significance of the CI was determined by Wilcoxson signed rank test to a hypothetical median of 1; * indicates p=0.03; # indicates animals with too few colonies isolated for CI determination (see Figure S1B). (B) Uptake and (C) survival of WT (black bars), vexA (magenta bars), aroA (yellow bars), entA (cyan bars), iroCDEN (orange bars), invA (purple bars), ssrB (blue bars), phoP (red bars), and cdtB (green bars) S. Typhi strains in THP-1 macrophages. Bar graphs show values of mutant strains relative to wild-type (WT) and error bars from at least 3 biological replicates. Statistical significance p was determined by paired two-tailed Student’s t test.

Effect of Virulence Genes on S. Typhi Persistence in Macrophages

Selected S. Typhi mutants were also assayed for survival in THP-1-derived human macrophages. Uptake was measured by comparing intracellular cfu at t = 0 with the infective dose (Figure 2B), and survival was measured by comparing intracellular cfu at t = 0 and t = 24 h p.i. (Figure 2C). Only a vexA mutant showed a difference in uptake, an expected finding given the ability of Vi to interfere with phagocytosis (Robbins and Robbins, 1984). Mutants lacking aroA, entA, iroCDEN or phoP demonstrated reduced survival in macrophages (Figure 2C). However, invA (SPI-1) or ssrB (SPI-2) mutants were not impaired in macrophage survival (Figure 2C), corroborating the TraDIS results. Mutants lacking the PhoP-regulated typhoid toxin CdtB (Charles et al., 2009) were also able to survive in macrophages (Figure 2C). Expression of virulence-associated genes was measured in S. Typhi and S. Typhimurium during macrophage infection using regulated GFP reporter plasmids (Figure S3). Modest differences in expression fold-change from 0 to 24 h were seen between S. Typhi and S. Typhimurium, including salmochelin (iroB), SPI-2 (ssaG), and the PhoP-regulon (mig14).

S. Typhi Is More Sensitive to Iron Limitation Than S. Typhimurium

Mutants deficient in the synthesis or transport of the siderophores enterobactin and salmochelin (ent and iro) were counter-selected in hu-SRC-SCID mice (Table 1, Figure 1B), consistent with host iron restriction during S. Typhi infection. S. Typhi and S. Typhimurium in vitro growth was measured in LB medium with or without the divalent metal chelator 2,2-dipryridyl (DP). Although both serovars exhibited reduced growth rates with DP, S. Typhi growth was more severely restricted, indicating greater sensitivity to iron limitation (Figure 3). Growth of both S. Typhi and S. Typhimurium in DP-chelated medium was rescued by FeCl3, confirming that iron limitation was responsible for impaired growth in DP-chelated medium (Figure 3).

Figure 3. Growth of Salmonella enterica Under Iron Limitation.

Figure 3.

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Wild-type S. Typhimurium (STm, black circles) and S. Typhi (STy, red squares) were grown in LB or with 300 μM 2,2-dipyridyl (DP) (STm, black open circles; STy, red open squares) or with 300 μM DP and 800 μM FeCl3 (STm, green circles; STy, green squares), and OD600 was measured for 18 h. Means and error bars representing standard deviation of three biological samples of OD600 are shown for each sample.

DISCUSSION

Human host-specificity has posed a formidable challenge in identifying unique determinants of S. Typhi virulence. Murine infection with S. Typhimurium is used as a model of human typhoid, but major differences between typhoidal and non-typhoidal Salmonella serovars limit what can be inferred about typhoid pathogenesis from this model. The hu-SRC-SCID mouse model is a unique research tool to study the pathology and immunology of human typhoid (Libby et al., 2010). S. Typhi virulence in mice engrafted with human hematopoietic cells suggests human mononuclear cells are essential for typhoid pathogenesis. Here we report the genome-wide analysis of S. Typhi virulence determinants in humanized mice, confirming some expectations but also revealing unexpected findings.

Biosynthesis of aromatic amino acids, Vi capsular polysaccharide and the siderophore salmochelin appears to be particularly important for S. Typhi virulence in hu-SRC-SCID mice. Aromatic amino acid biosynthesis has long been known to be required for Salmonella virulence, as these amino acids are not freely available in the mammalian host (Hoiseth and Stocker, 1981). S. Typhimurium and S. Typhi strains deficient in aromatic amino acid biosynthesis are attenuated for virulence in mice and humans, respectively, and have been used as live attenuated vaccines (Dougan et al.,1988; Hone et al., 1992). Our results confirm that aroA mutant S. Typhi is strongly counter-selected in hu-SRC-SCID mice.

The Vi (virulence) antigen was initially described in association with highly virulent strains of S. Typhi Ty2 (Felix and Pitt, 1934) and later shown to be a capsular polysaccharide (Robbins and Robbins, 1984). Although Vi expression may be lost during lab passage, bloodstream isolates from patients with typhoid are nearly always Vi-positive (Wain et al., 2005), suggesting that Vi is important for S. Typhi virulence. A variety of functions for Vi have been described from in vitro studies, including prevention of complement-mediated clearance (Wilson et al., 2011), antibody-binding (Hart et al., 2016) and Tlr4 signaling (Wilson et al., 2008). Here we show that Vi is essential for systemic infection of humanized mice. Genes required for biosynthesis, transport and assembly of Vi capsular polysaccharide were highly counter-selected in hu-SRC-SCID mice.

In addition to protection conferred by Vi, S. Typhi requires specific LPS modifications for resistance to antibody and complement (Kintz et al., 2017). LPS biosynthetic genes, especially those involved in O-antigen synthesis and modifications of the outer core, were highly counter-selected in hu-SRC-SCID mice. O-antigen is important for S. Typhi serum resistance and macrophage survival (Hoare et al., 2006; Da Silva et al., 2018), while the outer core is required for host cell invasion (Lyczak et al., 2001, Hoare et al., 2006). The outer core terminal glucose plays a critical role in S. Typhi internalization by host cells (Hoare et al., 2006) and is added by the WaaB glucosyltransferase, which was identified by TraDIS. Interestingly, this terminal residue is not required for internalization of S. Typhimurium, highlighting a key difference between these serovars.

Mutants deficient in iron acquisition and utilization or iron-sulfur cluster repair were counter-selected in hu-SRC-SCID mice, including the siderophore salmochelin. The importance of salmochelin was confirmed by competitive infection and shown to be required for S. Typhi survival in macrophages. Iron acquisition is known to be required for Salmonella survival in macrophages (Nairz et al., 2015). Enterobactin is glucosylated by iro genes to produce salmochelin, which counteracts the neutralizing effects of host-derived lipocalin-2 (Crouch et al., 2008; Fischbach et al., 2006; Flo et al., 2004). Reduced survival of an S. Typhi iroCDEN mutant in macrophages demonstrates the importance of evading lipocalin-2 neutralization for S. Typhi survival. Although salmochelin loss attenuates S. Typhimurium virulence in mice (Crouch et al., 2008; Raffatellu et al., 2009; Nagy et al., 2013), salmochelin is not required for S. Typhimurium growth in murine macrophages unless other iron uptake systems are also impaired (Nagy et al., 2013), and salmochelin loss has a more profound impact on S. Typhi virulence, suggesting that non-glucosylated enterobactin may be less effective as a siderophore in S. Typhi than in S. Typhimurium.

Studies in murine S. Typhimurium infection have highlighted the importance of SPI-2 for macrophage survival and systemic infection (Hensel et al.,1995; Hensel et al., 1998). It was therefore surprising that our TraDIS analysis did not show significant counter-selection of SPI-2. Dispensability of SPI-2 was confirmed by competitive infection. Although previous studies have shown that S. Typhi SPI-2 genes are upregulated in human THP-1 macrophages (Faucher et al., 2006), SPI-2 was not required for macrophage survival (Forest et al., 2010 and Figure 2C). Collectively, these observations suggest a marked difference in the dependence of S. Typhi and S. Typhimurium on SPI-2.

An additional factor long recognized as essential for S. Typhimurium virulence is the transcriptional regulator PhoP, which was only weakly counter-selected in hu-SRC-SCID mice. PhoP is required for S. Typhimurium survival in murine macrophages (Fields et al., 1986; Miller et al., 1989), and we observed reduced survival of phoP mutant S. Typhi in THP-1 cells. However, PhoP-regulated genes were not highly counter-selected in our study, and a phoP mutant had only a modest competitive disadvantage in humanized mice. This suggests that PhoP may be less important during acute typhoid fever than in non-typhoidal Salmonella infections, at least during the early infection stages examined in this model. An analysis of PhoP-regulated proteins in S. Typhi and S. Typhimurium found some S. Typhi-specific proteins including the typhoid toxin (CdtB) (Charles et al., 2009). We failed to observe counter-selection of mutants lacking cdtB or the associated genes pltA and pltB, and competitive infection showed that a cdtB mutant has no defect in hu-SRC-SCID mice. A previous study similarly showed increased replication of cdtB mutant S. Typhi in humanized Rag2−/−γc−/− mice engrafted with human fetal liver and hematopoietic progenitor cells (Song et al., 2010). In an experimental challenge study, a cdtB mutant was not attenuated for virulence in human subjects (Gibani et al., 2019). Collectively, these observations indicate that the typhoid toxin is not required for S. Typhi virulence during early acute typhoid infection in hu-SRC-SCID mice and in humans.

A limitation of our study is the acute nature of S. Typhi infection in hu-SRC-SCID mice, which differs from the prolonged systemic infection that characterizes typhoid fever. While the presence of human mononuclear cells allows S. Typhi to proliferate, the residual presence of murine immune cells creates a chimeric immune environment in which artificial interactions may be detrimental to the host. In addition, the absence of non-hematopoietic human cells prevents S. Typhi interactions with other human cell types. For example, although human hematopoietic stem cells used to engraft hu-SRC-SCID mice express the typhoid toxin receptor CD45, toxin interactions with human epithelial cells cannot be studied using this model (Galan, 2016). Oral inoculation of hu-SRC-SCID mice is infeasible due to the absence of IL-2 receptor gamma chain molecule, which prevents the development of mucosal lymphoid tissue (Pearson et al., 2008). Finally, the labor-intensive generation of hu-SRC-SCID mice limits sample size in this and similar studies, and subject-to-subject variation is expected due to differences in engraftment levels and donor heterogeneity. Despite these constraints, we observed consistent counter-selection of many genes across all mice analyzed, validating the importance of these genetic loci for S. Typhi virulence. The hu-SRC-SCID model is unique in its ability to support lethal S. Typhi infection and recapitulates important aspects of typhoid pathogenesis such as systemic spread, histopathological findings and cytokine profile (Libby et al., 2010), suggesting that our observations are relevant to human typhoid.

Live attenuated S. Typhi strains are presently under investigation, both as typhoid vaccines and as carriers to elicit immunity to heterologous antigens (Levine et al., 1990). Our studies in hu-SRC-SCID mice confirm that mutations in pathways required for aromatic amino acid biosynthesis reduce the virulence of vaccine strains. However, mutations in SPI-2 genes or phoP are also used in vaccine strains but may not have attenuating effects in S. Typhi. Hu-SRC-SCID mice provide an improved model to assess candidate virulence-attenuating mutations and a new starting point from which to investigate typhoid pathogenesis.

STAR METHODS

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for resources and reagents, including strains and plasmids generated in this study, should be directed to and will be fulfilled by the Lead Contact, Ferric C. Fang (fcfang@uw.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Bacterial Strains and Growth Conditions

Bacterial strains used in this study are listed in the Key Resources Table. S. enterica cultures were grown in Miller’s Luria Broth (LB) medium at 37°C with shaking at 250 rpm. Medium was supplemented with “aromix” (40 μg ml−1 L-phenylalanine, 40 μg ml−1 L-tryptophan, 10 μg ml−1 2,3-dihydroxybenzoic acid, 10 μg ml−1 p-amino benzoic acid), ampicillin (100 μg ml−1), carbenicillin (100 μg ml−1), kanamycin (50 μg ml−1), X-gal (40 μg ml−1) or 2,6-diaminopimelic acid (0.4 mM), as indicated.

Cell Culture

Human THP-1 monocytes were obtained from ATCC and cultured in RPMI 1640 medium (Corning Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Millipore-Sigma), sodium pyruvate (Corning Inc.), non-essential amino acids (Gibco), 50 U ml−1 penicillin and 50 μg ml−1 streptomycin (Corning Inc.) at 37°C in 5% CO2.

Experimental Animals

Mouse experiments in this study were approved by University of Washington Institutional Animal Care and Use Committee (IACUC) and performed as described in protocol 3373–01. Investigators were not blinded, and animal studies were non-randomized. No statistical method was used to predetermine sample size. NOD-PrkdcscidIL2rgtm1Wjl (NSG) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and engrafted with human CD34+ hematopoietic stem cells derived from umbilical cord blood (Hasgur et al., 2016; Pearson et al., 2008). Umbilical cord blood was obtained from donors that were consented under an approved IRB protocol at the UMass Memorial Medical Center, Department of General Obstetrics and Gynecology (Worcester, MA), and all samples used for engraftment were de-identified. Mice were not used in any other procedures and were accompanied by a certificate of good health. Mice were maintained under ABSL-2 containment at the University of Washington Animal Care and Research Facility on a 14-hour light cycle and housed up to 5 animals per cage in Allentown cages with micro-isolator tops. Mice were checked daily during infection studies, and veterinary care was provided 7 days a week.

METHOD DETAILS

Bacterial Strain and Plasmid Construction

Plasmids and primers used in this study are listed in the Key Resources Table and Table S4, respectively. Primers were purchased from Integrated DNA Technologies (IDT, Skokie, IL). Mutant alleles of S. enterica serovars were constructed using λ-Red recombination as described (Datsenko and Wanner, 2000; Karlinsey 2007) with primers sets listed in Table S4. All plasmids were constructed using NEBuilder® HiFi Assembly Master mix (NEB, Ipswich, MA) with the following vectors and PCR products using primer sets listed in Table S4 as follows: pJK741, pJK745, pJK747, pJK749 and pJK750 with pMPMA3Δnull-gfp digested with EcoRI and PCR products generated with gDNA from S. Typhi Ty2; pJK744, pJK746, pJK748 and pJK754 with pMPMA3Δnull-gfp and PCR products generated with gDNA from S. Typhimurium 14028s; pJK753 with pWSK130 digested with BamHI and PCR products generated with pDNA from pFPVmCherry. All mutant strains and plasmid constructs were confirmed by DNA sequencing (Genewiz, South Plainfield, NJ).

S. Typhi Transposon Library Construction

Transposon mutagenesis in S. Typhi Ty2 was performed by conjugal mating of pLG100 containing transposable element T22 (ISlacZ-Tn2/FRT with a selectable kanamycin marker) (Gallagher et al., 2007) as follows: The donor strain FLS232 (Rho3/pLG100) was grown in LB broth with carbenicillin and 2,6-diaminopimelic acid to OD600~1.0. This was mixed with recipient strain S. Typhi Ty2 grown in LB broth with “aromix” to OD600~1.0 at a ratio of (0.1:1), then spotted onto a sterile 0.45μM nitrocellulose membrane filter seeded on a LB plate. Eleven independent matings were performed, and after 1 h incubation at 37°C, the filters were added to 1 ml LB broth with “aromix,” vortexed, then pooled together. The pooled matings were subsequently plated onto ten QTrays (240×240×20mm) plates (Molecular Devices, San Jose, CA) containing LB agar with “aromix” and kanamycin (lacking DAP) and incubated at 37°C for ~18 h. Each plate was harvested with 15 ml of LB broth, and all plate harvests were pooled together for a total of ~300,000 kanamycin-resistant mutant colonies. DMSO was added to 10% and 1 ml aliquots were frozen at −80°C. One aliquot was thawed, and the viable titer of the transposon library was found to be at 8.7×109 cfu ml−1. The construction of an S. Typhi transposon library for Illumina-based sequencing was performed as described below, and the subsequent sequences were processed using the Bio-TraDIS analysis pipeline (Barquist et al., 2016). Eighty-one percent of the total mapped reads of 7,067,870 matched to the transposon tag, to which 64% of these sequences were mapped to S. Typhi genome sequence AE014613.1. A total of 253,439 unique insertion sites were found with an average of one insertion site for every 19 bp of sequence. Read and insertion counts for each gene, as well as a list of essential genes as determined by the Bio-TraDIS analysis pipeline, are listed in Table S1.

Libraries for Illumina-based TraDIS

Construction of S. Typhi transposon libraries for Illumina-based TraDIS is based on the TdT method (Gallagher et al., 2015) modified for transposon T22 (Gallagher et al., 2007), outlined in Figure S4A and the primer design in Figure S4B. Genomic DNA (gDNA) from transposon library outgrowths were isolated from ~1×109 cells using a DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD), per the manufacturer’s protocol. The gDNA was additionally purified by ethanol precipitation and resuspended in 100 μl TE (10mM Tris-HCL,1 mM EDTA pH 8.0). The gDNA was quantified with the Invitrogen Qubit ds DNA BR assay kit on a Qubit fluorometer (Invitrogen, ThermoFisher Scientific, Waltham, MA).

One to 1.5 μg of gDNA in a final volume 130 μl TE was transferred to a Covaris microtube AFA Fiber Crimp-cap and sheared to fragment size ~300bp using an LE220 focused-ultrasonicator with Rack PN500282 and duty factor 30%, (W) 450 and cycles 200, for 60 s (Covaris, Woburn, MA). The sheared gDNA was end-repaired using NEBNext End Repair in a final volume of 155 μl per the manufacturer’s protocol (New England Biolabs, Ipswich, MA). The end-repaired DNA was purified using a MinElute PCR Clean up kit (Qiagen, Germantown, MD), and the DNA was eluted twice with 10 μl EB. The purified end-repaired DNA was C-tailed with Terminal Transferase (TdT) in the following reaction: 18.6 μl end-repaired DNA, 2.8 μl freshly prepared solution of 9.5 mM dCTP (and 0.5 mM ddCTP (Millipore-Sigma, St. Louis, MO), 5.6 μl of 5X TdT reaction buffer, 1.0 μl Terminal Transferase 30U/μl (Promega, Madison, WI). The C-tailing reaction was incubated at 37°C for 60 m then 75°C for 20 m. C-tailed DNA was purified using a Performa DTR gel filtration column per the manufacturer’s protocol (EdgeBio, SanJose, CA) and quantified by fluorometry.

The first PCR reaction (PCR1) was performed using a transposon-specific primer T22–87_Left and primer olj376 specific to the C-tailed end (Figure S4 and Table S4) in the following reaction: 7.4 μl of EDGE-purified C-tailed DNA (~150 ng), 25 μl of 2X KAPA HiFi Hot Start Ready Mix (Kapa Biosystems, Wilmington, MA), 3 μl of 10 μM primer olj376, 1 μl of 10 μM primer T22–87_Left, 0.25 μl of 100x SYBR Green 1 (Invitrogen, ThermoFisher Scientific, Waltham, MA), 13.35 of μl PCR-grade water. Thermocycling was performed in a BioRAD CFX96 with the following conditions: 95°C-2:00; 24X{98°C-0:30, 64°C-0:30, 72°C-1:30, read} 72°C-2:00; 10°C hold. Under these conditions using 150 ng of C-tailed DNA, the inflection point was observed at approximately 24 cycles. As a control, no amplification was seen in a PCR1 reaction where a C-tailed DNA sample was performed without TdT enzyme. Subsequent PCR purification was not required after the PCR1 reaction.

The second PCR reaction (PCR2) added the P5-Tn and P7-Index primers (Figure S4 and Table S4) in the following reaction: 1.2 μl of PCR1 product, 25 μl of 2X KAPA HiFi Hot Start Ready Mix, 3 μl of 10 μM primer T22_PAIR_AmpF_Left, 3 μl of 10μM primer TdT_Index_XX, 0.25 μl of 100x SYBR Green 1, 17.55 μl PCR grade water. Thermocycling was performed in a BioRAD CFX96 with the following conditions: 95°C-2:00; ~14X{98°C-0:30, 64°C-0:30, 72°C-1:30, read} 72°C-2:00; 10°C hold. To determine the number of cycles to run, a PCR2 reaction was piloted for 25–30 cycles to calculate the inflection point for each sample. Two reactions of PCR2 were performed per PCR1 sample with the number of cycles (inflection point) determined in the pilot PCR2 reaction. Twenty-four indexed TdT_Index_XX primers (Table S4) were used for multiplexing up to 24 libraries for subsequent Illumina sequencing.

The PCR2 reactions were purified by SPRI beads using a ratio left-right 0.8–0.61 size selection method for a range of 230–660 bps, per the manufacturer’s protocol (Beckman Coulter Life Sciences, Indianapolis, IN). This method first selects the right side of the bp range, then the left. Size selection was performed in a 96-well round bottom microtiter plate (Costar 3795). Ninety-five μl of PCR2*(0.61X) SPRI beads = (58 μl) were mixed 10X, then incubated for 1 m at RT. The microtiter plate was placed on an Agencourt SPRIPlate 96R ring super magnet plate (Beckman Coulter) for 2 m. One-hundred forty-five μl of supernatant were removed into a new well. One hundred forty-five μl of (0.8X−0.61X)=0.19X SPRI beads = (27.6 μl) were added, mixed 10X, and incubated for 1 m at RT. The plate was placed on the magnet for 2 m, and supernatants were discarded. While the plate was on the magnet, 180 μl of freshly made 85% ethanol were added before incubation for 30 s at RT. Ethanol was discarded before air-drying for 2 m at RT. The plate was removed from the magnet and 35 μl EB (Qiagen) added before mixing and incubation for 5 m at RT. The plate was then placed on the magnet for 2 m and supernatant transferred to a new 0.65 ml Eppendorf tube for storage at −20°C.

Quantification of the purified libraries was performed by three different methods as follows: One μl of SPRI sized select library was quantified using a High Sensitivity DNA Kit on an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). Quantification of the purified libraries was also performed by qPCR with the KAPA Library Quantification kit Illumina platform (KAPA Biosystems, Wilmington, MA), per the manufacturer’s protocol. Finally, libraries were quantified using an Invitrogen Qubit ds DNA HS assay kit on a Qubit fluorometer.

For multiplexing, the libraries were pooled to a final concentration of 2 nM of each library. The libraries were sequenced at the Fred Hutch Cancer Research Center Genomics Facility on platform HiSeq 2500 Rapid Run (65°C) (2-lanes if multiplexing), 50SR with a custom primer T22_custom_1stRead_SEQ_Left (Table S4) with a 15 pM final library concentration, and a 6% spike-in of 12.5 pM PhiX library.

Library Infection in Humanized Mice

Non-engrafted NSG mice and human hematopoietic stem cell-engrafted hu-SRC-SCID mice (Hasgur, 2016; Pearson et al., 2008) were infected with a total of ~4 − 105 cfu of an S. Typhi transposon library by i.p. injection with 0.5 ml of ~8 − 105 cfu ml−1 in PBS. In addition, 0.5 ml of the inoculum was added to a 125 ml Erlenmeyer flask containing 25 ml LB broth with “aromix” and kanamycin before incubation for ~18 h at 37°C with shaking at 200 rpm. DMSO was added to the outgrowth “input” sample to 10% and aliquots stored at −80°C for subsequent transposon-specific library construction and Illumina sequencing.

The infected mice were closely monitored for signs of illness, and moribund animals were euthanized. The majority of the animals were euthanized at approximately 24 h p.i.; however, a small number of animals showed less severe signs of illness and were euthanized at 64 to 79 h p.i. Livers and spleens were harvested and homogenized in PBS. A portion of each organ was plated for cfu counts and the remainder of the homogenates were transferred to a 125 ml Erlenmeyer flask containing 25 ml LB broth with “aromix” and kanamycin before incubation for ~18 h at 37°C with shaking at 200 rpm. The outgrowth “output” samples were passed through a 70 μM cell strainer, DMSO was added to 10%, and aliquots stored at −80°C for subsequent transposon-specific library construction for Illumina sequencing. Sequence reads with a transposon tag were identified, mapped to S. Typhi strain Ty2 whole genome sequence (Accession number AE014613.1) with SMALT (smalt-0.7.6) and quantified using the Bio-TraDIS analysis pipeline (Barquist et al., 2016). Insertion counts for each gene in the libraries are listed in Table S2.

Competitive Infections in Humanized Mice

Human HSC-engrafted NSG (hu-SRC-SCID) (Hasgur et al., 2016; Pearson et al., 2008) mice were infected with an equal ratio of wild-type and mutant strains of S. Typhi Ty2. For each competitive infection, six hu-SRC-SCID mice were inoculated i.p. with a total of 2 − 105 cfu in PBS each. Mice were closely monitored for illness and euthanized when moribund (e.g., exhibiting reduced spontaneous movement or oral intake, hypothermia). Livers and spleens were harvested and homogenized in PBS. Dilutions of the homogenates were plated for cfu on LB plates with “aromix” and incubated overnight at 37°C. Colonies were enumerated and 100 colonies patched onto LB plates with “aromix” and the appropriate antibiotic to determine the ratio of wild-type to mutant bacteria. The competitive index (CI) was calculated as a ratio of (mutant/wild-type)output / (mutant/wild-type)input. Statitsical significance was determined using a Wilcoxson signed rank test using Prism v. 8.1.2 (GraphPad).

Macrophage Infections

Human THP-1 monocytes were differentiated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 48 h; the medium was changed to PMA- and antibiotic-free RPMI 24 h prior to infection. Salmonella strains were grown in LB broth with “aromix,” amplicillin and kanamycin for 18 h with shaking at 37°C, then a djusted to OD600 1.0 and washed twice with sterile PBS. Salmonella were mixed with equal parts human pooled serum (MP Biomedicals LLC) and incubated at 37°C for 20 m to opsonize the bacteria. Opsonized bacteria were used to infect THP-1 human macrophage-like cells at a MOI of 10:1. Infected monolayers were centrifuged for 5 m at 1000 rpm to synchronize infection, then incubated at 37°C for 1 h to promot e internalization. Following internalization, monolayers were washed with RPMI supplemented with 20 g ml−1 gentamicin to kill extracellular bacteria. All macrophage infections were performed in biological triplicate. For survival studies, infected macrophages were lysed in 1% Triton X-100 at 0 h and 24 h post-infection. Lysates were serially diluted and plated on LB plates with “aromix” to determine the number of intracellular cfu. For gene expression analysis, infected macrophages were lysed after 24 h with 1% Triton X-100. Lysates were gently centrifuged at 2500 rcf for 5 m, supernatants were removed, and bacteria pelleted by centrifugation at 15000 rpm for 5 m. Bacterial pellets were resuspended in 2.5% paraformaldehyde and fixed for 15 m at 37°C. F ixed bacteria were washed once in PBS then analyzed for GFP expression using a BD LSRII flow cytometer (Becton, Dickinson) at the Pathology Flow Cytometry Core Facility (Department of Pathology, University of Washington, Seattle, WA) with the following gating scheme: All Salmonella strains assayed by flow cytometry had a constitutive expressing mCherry plasmid (pJK743) as well as various promoter fusions to GFP to assay for expression. Bacteria were first gated on forward and side scatter to logarithmic amplification, then mCherry fluorescence was detected after 561nM excitation and emission collected through a 595LP, 610/20 nm filter. A total of 10,000 mCherry counts were collected and GFP fluorescence was detected after 488 nM excitation and emission collected through a 505LP, 530/30 nm filter. Data were processed using FlowJo v. 10.3 software (Treestar, Inc.). Total GFP fluorescence was calculated from the GFP-positive cells and statistical significance calculated using a Student’s unpaired two-tailed t-test with software Excel v. 16.19 (Microsoft) or Prism v. 8.1.2 (GraphPad).

Salmonella Growth Under Iron Limitation

Bacterial strains were grown overnight in 5 mL LB, diluted to OD600 = 1.0, diluted 1:10 in fresh LB, then diluted 1:10 into LB or LB supplemented with 2,2-dipyridyl (DP) (Millipore Sigma) to a final concentration of 300 μM in a 300 μL volume in a microtiter plate. In iron add-back experiments, FeCl3 was added to a final concentration of 800μM. Cells were grown in a Labsystems Bioscreen C (Growth Curves USA) that measured OD600 every 30 m, and experiments were performed in triplicate.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical method and sample size for experiments are indicated in the corresponding figure legends. The TraDIS input and output libraries were analyzed using the tradis_comparison.R script in the BioTraDIS pipeline, which applies an edgeR package (Robinson el al., 2010) to quantitatively analyze significant differences of read counts in genes between two conditions. Genes that were counter-selected in hu-SRC-SCID mice with a false discovery rate (Q value) of <0.05 and log fold change (logFC) of <-2 were deemed significant. Statistical analysis of the competitive infections in mice and macrophage infections was performed using Prism v. 8.1.2 software (GraphPad). An unpaired two-tailed Student’s t-test was performed on the means of parametric data, and a Wilcoxson signed rank test was performed on the means of non-parametric data. Statistical significance was defined as p<0.05. Error bars on figures show standard deviation.

DATA AND CODE AVAILABILITY

The datasets generated during this study are available at Sequence Read Archive https://www.ncbi.nlm.nih.gov/sra/PRJNA546274.

Supplementary Material

1
2
3

Supplemental Table S1 Related to STAR Methods. Read and insertion counts of S. Typhi Ty2 transposon library sample T1 using Bio::TraDIS pipeline analysis. The high-density S. Typhi Ty2 transposon library of was constructed as described in STAR Methods.

4

Supplemental Table S2 Related to STAR Methods. Read and insertion counts of input and output libraries from Bio::TraDIS pipeline analysis. Input S. Typhi transposon libraries (T2 and JZ01). Output transposon libraries from hu-SRC-SCID mice 24 h p.i. (T3, T4, T5, T6, T13, T14, T15, T16, and T17). Read counts highlighted in blue, insertion counts highlighted in red.

5

Supplemental Table S3 Related to STAR Methods. S. Typhi genes counter-selected in hu-SRC-SCID mice 24 h p.i. Data was analyzed using Bio::TraDIS pipeline script tradis_comparison.R (Barquist et al., 2016) with input S. Typhi transposon libraries (T2 and JZ01) and output transposon libraries from hu-SRC-SCID mice 24 h p.i. (T3, T4, T5, T6, T13, T14, T15, T16, and T17). Genes found counter-selected in hu-SRC-SCID mice with a false discovery rate (Q value) of <0.05 and log fold change (logFC) of <-2 were deemed significant.

HIGHLIGHTS.

  • Genes required for S. Typhi virulence were identified by TraDIS in humanized mice

  • Iron acquisition by the siderophore salmochelin is required for S. Typhi virulence

  • Some differences between the S. Typhimurium and S. Typhi mouse models were observed

  • S. Typhi-infected humanized mice are a useful model for the study of typhoid pathogenesis

ACKNOWLEDGEMENTS

The authors thank Dr. Gemma Langridge of Quadram Institute Bioscience for advice on TraDIS methodology; Davin Hoover and James Januik for dedicated laboratory assistance; Dr. Thea Brabb of UW Comparative Medicine for assistance with animal care; Dr. Jeffery Delrow, Andy Marty and Qing Zhang at the Fred Hutchinson Cancer Research Center Genomics Facility for help with Illumina sequencing and analysis; Chris Frazer and Dr. Debra Nickerson of UW Genome Sciences for use of the Covaris LE220; Dr. Hillary Hayden and Dr. Samuel Miller of UW Microbiology for use of the Qubit; Dr. Rafael Hernandez and Dr. Lakshmi Rajagopal at Seattle Children’s Research Institute for use of the Agilent 2100 bioanalyzer; and Dr. Olivia Steele-Mortimer at NIAID for the pMPMA3 ΔPlac PssaG GFP and pMPMA3 ΔPlac null GFP expression plasmids. This work was supported by NIH grants AI112640 (F.C.F.), AI132963 (M.A.B., L.D.S.), OD018259 (L.D.S.) and CA034196 (L.D.S.).

Footnotes

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DECLARATION OF INTERESTS

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1537086-supplement-1.pdf (1.4MB, pdf)
2
NIHMS1537086-supplement-2.docx (53.5KB, docx)
3

Supplemental Table S1 Related to STAR Methods. Read and insertion counts of S. Typhi Ty2 transposon library sample T1 using Bio::TraDIS pipeline analysis. The high-density S. Typhi Ty2 transposon library of was constructed as described in STAR Methods.

NIHMS1537086-supplement-3.xlsx (418.2KB, xlsx)
4

Supplemental Table S2 Related to STAR Methods. Read and insertion counts of input and output libraries from Bio::TraDIS pipeline analysis. Input S. Typhi transposon libraries (T2 and JZ01). Output transposon libraries from hu-SRC-SCID mice 24 h p.i. (T3, T4, T5, T6, T13, T14, T15, T16, and T17). Read counts highlighted in blue, insertion counts highlighted in red.

NIHMS1537086-supplement-4.xlsx (4.8MB, xlsx)
5

Supplemental Table S3 Related to STAR Methods. S. Typhi genes counter-selected in hu-SRC-SCID mice 24 h p.i. Data was analyzed using Bio::TraDIS pipeline script tradis_comparison.R (Barquist et al., 2016) with input S. Typhi transposon libraries (T2 and JZ01) and output transposon libraries from hu-SRC-SCID mice 24 h p.i. (T3, T4, T5, T6, T13, T14, T15, T16, and T17). Genes found counter-selected in hu-SRC-SCID mice with a false discovery rate (Q value) of <0.05 and log fold change (logFC) of <-2 were deemed significant.

NIHMS1537086-supplement-5.xlsx (379.7KB, xlsx)

Data Availability Statement

The datasets generated during this study are available at Sequence Read Archive https://www.ncbi.nlm.nih.gov/sra/PRJNA546274.

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