Salmonella Typhimurium undergoes distinct genetic adaption during chronic infections of mice
© Søndberg and Jelsbak. 2016
Received: 13 August 2015
Accepted: 25 February 2016
Published: 8 March 2016
Typhoid fever caused by Salmonella enterica serovar Typhi (S. Typhi) is a severe systemic human disease and endemic in regions of the world with poor drinking water quality and sewage treatment facilities. A significant number of patients become asymptomatic life-long carriers of S. Typhi and serve as the reservoir for the disease. The specific mechanisms and adaptive strategies enabling S. Typhi to survive inside the host for extended periods are incompletely understood. Yet, elucidation of these processes is of major importance for improvement of therapeutic strategies.
In the current study genetic adaptation during experimental chronic S. Typhimurium infections of mice, an established model of chronic typhoid fever, was probed as an approach for studying the molecular mechanisms of host-adaptation during long-term host-association.
Individually sequence-tagged wild type strains of S. Typhimurium 4/74 were used to establish chronic infections of 129X1/SvJ mice. Over the course of infections, S. Typhimurium bacteria were isolated from feces and from livers and spleens upon termination of the experiment. In all samples dominant clones were identified and select clones were subjected to whole genome sequencing. Dominant clones isolated from either systemic organs or fecal samples exhibited distinct single nucleotide polymorphisms (SNPs). One mouse appeared to have distinct adapted clones in the spleen and liver, respectively. Three mice were colonized in the intestines by the same clone containing the same non-synonymous SNP in a transcriptional regulator, kdgR, of metabolic genes. This likely indicates transmission of this clone between mice. The mutation was tracked to have occurred prior to 2 weeks post infection in one of the three mice and had subsequently been transmitted to the other two mice. Re-infection with this clone confirmed that it is superior to the wild type for intestinal colonization.
During 4 to 6 weeks of chronic infections, S. Typhimurium acquired distinct SNPs in known regulators of metabolic and virulence genes. One SNP, the kdgR-SNP was confirmed to confer selective advantage during chronic infections and constitute a true patho-adaptive mutation. Together, the results provide evidence for rapid genetic adaptation to the host of S. Typhimurium and validate experimental evolution in the context of host infection as a strategy for elucidating pathogen host interactions at the molecular level.
Salmonella enterica are Gram-negative intracellular pathogens able to cause a variety of infections in both human and animals . The species S. enterica constitutes >2500 different serovars, and where serovars with a broad host range like Salmonella enterica serovar Typhimurium (S. Typhimurium) mainly cause relatively mild gastrointestinal infections, host restricted serovars like S. Typhi (human restricted), typically cause severe systemic disease (typhoid fever) of their hosts. Furthermore, about 1–6 % of human typhoid patients become asymptomatic chronic carriers  and sporadically excrete bacteria in their stool thus serving as the reservoir for the disease. S. Typhimurium causes a typhoid-like systemic infection in mice and functions as a model for typhoid fever. Depending on the mouse strain it is possible to study both acute and chronic S. Typhimurium infections. Specifically, a mouse strain containing the wild type allele of the Slc11a1 gene serves as a robust model for studying asymptomatic chronic Salmonella infections . During systemic chronic infections of these mice, S. Typhimurium reside in macrophages of the spleen, liver and mesenteric lymph nodes for up to 1 year with sporadic excretion of S. Typhimurium bacteria in the feces . A negative genetic screen has shown that virulence factors associated with systemic acute infections, including the ones encoded by Salmonella Pathogenicity Island 2 (SPI2) , are also essential for establishing chronic infections . However, the specific contributions of individual factors and the metabolic requirements during long-term host association are not fully understood.
In a few natural chronic bacterial infections of humans, the temporal occurrence of adaptive mutations over the course of months to years of infection has recently been delineated [6, 7]. For example, during chronic lung infections of Cystic Fibrosis patients, it has been documented how Pseudomonas aeruginosa acquire adaptive mutations facilitating persistence and survival in the host over the course infection . The adapted clones retain the ability to transmit to and infect new patients and represent examples of bacterial evolution and host adaptation studied in real time. These studies have provided new insight into specific adaptive strategies favoring successful chronic infections by revealing the molecular signatures of pathogen host interactions. Similar studies exploring the level of within host adaptive evolution of S. Typhi during chronic infections are lacking, partly due to difficulties in identifying chronic carriers and maintaining an extended temporal sampling protocol, as these patients are often asymptomatic. In the present paper, using a previously established tagged strain approach , the level of genetic adaptation of S. Typhimurium during experimental chronic infections of mice was investigated. To this end, dominant clones isolated 4 to 6 weeks post infection were subjected to whole genome sequencing to identify potential adaptive mutations. In all mice, dominant S. Typhimurium clones isolated from either systemic organs or the fecal samples exhibited distinct single nucleotide polymorphisms (SNPs). Three mice were colonized in the intestines by the same clone containing the same non-synonymous SNP in a transcriptional regulator (kdgR) of metabolic genes, indicating transmission of this clone between mice. Further investigations confirmed that this clone was superior to the wild type for intestinal colonization presumably due to changes in the regulatory activity of the affected protein.
Together, these results provide evidence for rapid genetic adaptation to the host of S. Typhimurium and validate experimental evolution in the context of host infection as a strategy for elucidating pathogen host interactions at the molecular level.
Results and discussion
Dominant Salmonella clones emerge during chronic infections
S. Typhimurium CFU of samples following i.p. infection
Per gram feces, 2 weeks post infection
Per gram feces, 4 weeks post infection
Per gram feces, 6 weeks post infection
4 × 105
1.2 × 104
3.3 × 104
6.6 × 103
6.5 × 104
1.6 × 104
1.4 × 104
4.6 × 104
3.7 × 104
5 × 106
1.5 × 104
2 × 103
1 × 104
1.4 × 104
2.2 × 105
5.4 × 104
7.5 × 103
6 × 106
7.6 × 104
6.8 × 105
Dominant clones have acquired potential patho-adaptive mutations
Whole genome sequencing results
SRA accession numbera
Coordinates in ST4/74 genomec
Gene affected and amino acid effectd,e
Feces, 6 weeks
G- > T
Feces, 4 weeks
A- > G
G- > A
Synonymous, located in STM474_0020 Synonymous, located in maeB
Feces, 6 weeks
G- > T
Feces, 4 weeks
G- > T
A- > G
Promoter region of yicE Synonymous, located in STM474_0057
C- > A
Feces, 6 weeks
C- > A
In conclusion, during this relatively short-term experimental chronic infection numerous mutations are detected in isolated clones. Most of these clones are associated with the intestinal environment; however, in one mouse mutations are also detected in isolates from systemic organs. Furthermore, in this mouse distinct mutations are detected in the liver- and spleen-clones, respectively, providing evidence for niche specific adaptation within the same host with limited migration between organs. Finally, the sequencing results also reveal that three mice are colonized by the same clone in the intestines implying transmission of the dominant clone between these mice via the fecal-oral route.
The ptsN-SNP clone is present and dominant in the fecal sample of mouse 5E at four weeks post infection
To investigate the role of ptsN during chronic infections and to determine if the ptsN-SNP confers any advantages for survival inside the host as suggested by the dominance of the clone, two new groups of 5 mice each were infected IP to establish chronic infections with a 1:1:1 mixture of ST4/74-WITS1 (wt-WITS1), the ptsN-SNP-WITS3-clone and an isogenic strain deleted for ptsN (ΔptsN). The infection was allowed to proceed for 5 weeks and S. Typhimurium bacteria were isolated from fecal samples every week and from livers and spleens upon termination. Isolated bacteria were genotyped (wt-WITS1, ptsN-SNP-WITS3 or ΔptsN) using a PCR/sequencing strategy. Only two mice were colonized by S. Typhimurium in the intestines prior to 3 weeks post infection (data not shown). This is in accordance with the first experiment (Table 1). At no point during the infection, from either the fecal or the organ samples of all 10 mice, was the ΔptsN strain isolated. This signifies that similar to acute infections, ptsN is essential for chronic infections. Only wt-WITS1 clones were isolated from the fecal samples with the exception of mouse 5 of group 2 where the fecal sample was dominated by the ptsN-SNP clone throughout the experiment (data not shown). In the spleen, the ptsN-SNP clone was present in an average of 37 %+/−7.% of analyzed clones, and in the liver it was significantly less prevalent with a 20 %+/−8.4 % recovery rate (p = 0.0002) (Fig. 2b). These results could suggest that the ptsN-SNP is less fit than the wt-WITS1 in both the spleen and liver during this infection and does not corroborate the observation from the original experiment from which the ptsN-SNP clone was isolated. Further investigations are required to elucidate the specific roles of PtsN during chronic infections and the consequence of the ptsN-SNP mutation.
The kdgR-mutation originated in mouse 5D at an early time-point post infection
The kdgR-SNP mutant exhibits rapid intestinal colonization upon re-infection
The kdgR-SNP mutant has constitutive repression of its target gene kdgK
The KdgR-protein is a repressor of kdgK and kdgT, two metabolic genes of the KDG-branch involved in transport and catabolism of 2-keto-3-deoxygluconate (KDG) . KDG can be imported by KdgT or result from degradation of starch or glucoronate. KDG is then converted to KDG-6-phosphate (KDGP) by the KdgK kinase. At this point, the KDG-branch converges with the Entner-Doudoroff (ED) pathway catabolizing gluconate-6-phosphate to first KDGP and then to pyruvate and D-glyceraldehyde-3-phosphate by the KDGP aldolase encoded by eda. KdgR also represses expression from one of three promoters controlling the eda gene, encoding a central enzyme of the ED-pathway. However, this effect of KdgR on eda expression is negligible . The ED-pathway functions as an alternative to the Embden-Meyerhof-Parnas glycolytic pathway , and in E. coli the ED-pathway is essential for intestinal colonization of mice .
In the present study, the level of genetic adaptation was investigated during a relative short S. Typhimurium infection of a mouse model of chronic infections. This revealed that even within a very short time frame (2 to 4 weeks post infection) S. Typhimurium acquires distinct patho-adaptive mutations conferring selective advantage during chronic infections. Together with the recent availability of whole genome sequencing as a common tool in molecular microbiology this study serves as a proof-of-concept for further investigations into the molecular and evolutionary mechanisms of host-adaptation. For example, data presented here point to that even one SNP in the genome of S. Typhimurium confers a clear advantage for intestinal colonization and growth with indications of increased transmission as a consequence. Further investigations of the effect of this SNP will provide insight into the metabolism of S. Typhimurium during intestinal colonization. Other mutations identified were synonymous or located at intergenic regions. Although, the effect of these mutations is more difficult to infer from the sequence alone, they may still provide selective advantage by altering the regulation and expression of neighboring genes . In one mouse, distinct adapted clones were isolated from the liver and spleen, respectively, pointing to potential within-host niche specific adaptation. Overall, the results presented here provide novel insight into host-pathogen interactions and present a new approach for studying this at the molecular level. Of note, the rapid occurrence of adaptive mutations reported here should be taken into account in future studies investigating long-term bacteria-environmental interactions.
Bacterial strains and growth conditions
Strains and plasmids used in the study
S.Typhimurium strain ST4/74. Reference strain.
S.Typhimurium strain ST4/74, WITS1, kanR
S.Typhimurium strain ST4/74, WITS2, kanR
S.Typhimurium strain ST4/74, WITS3, kanR
S.Typhimurium strain ST4/74, WITS4, kanR
S.Typhimurium strain ST4/74, WITS5, kanR
S.Typhimurium strain ST4/74, WITS6, kanR
S.Typhimurium strain ST4/74, WITS7, kanR
S.Typhimurium strain ST4/74, WITS8, kanR
S.Typhimurium strain ST4/74, ΔkdgR, kanR
S.Typhimurium strain ST4/74, ΔptsN, kanR
E. coli cloning strain
TOPO-cloning vector, ampR, kanR
Cloning vector, ampR, kanR
Plasmid with λ-Red recombinase expressed from arabinose inducible promoter
Template plasmid for λ-Red mutagenesis, kanR
Construction of tagged strains
All primers used in the study can be found in Additional file 1: Table S1. To construct 8 individually tagged strains the kanamycin gene from pACYC177 were used as template in PCR reactions with 8 individual forward primers (Tag1kanfwd, Tag2kanfwd, Tag3kanfwd, Tag4kanfwd, Tag5kanfwd, Tag6kanfwd, Tag7kanfwd, Tag8kanfwd), each with a unique sequence tag on the linker and one common reverse primer (Tagkanrev2). The 8 PCR products were TOPO-cloned into the pCR®2.1-TOPO vector from Invitrogen according to the manufactures recommendations. A fragment containing an individual 40-bp tag and the kanamycin resistance cassette was amplified from the individually tagged TOPO-vectors, using the primers Tag1malXYfwd, Tag2malXYfwd, Tag3malXYfwd, Tag4malXYfwd, Tag5malXYfwd, Tag6malXYfwd¸ Tag7malXYfwd¸ Tag8malXYfwd, and Tagsmalxyrev2. Approximately 1 μg of each linear PCR product was used for integration into malXY pseudogene locus of the chromosome of ST4/74 S. Typhimurium using the Lambda Red method as described previously . Transformants were selected by plating onto selective media. Correct integration of the tag-sequence and the kanamycin gene into the malXY locus was confirmed using a PCR strategy with the primers malXYcon and Tagkanrev2. The tags were then reintroduced into a wild-type S. Typhimurium ST4/74 background via transduction with phage P22 HT105/1 int201 using previously described protocols. Gene deletions and concomitant insertions of an antibiotic resistance cassette were constructed using Lambda Red mediated recombination as described elsewhere . All constructs were verified by PCR and re-introduced into a wt ST4/74 background via P22 phage transduction as previously described .
Infections of mice
Seven weeks old female 129X1/SvJ mice, 16–18 g, were used to establish chronic Salmonella infections. Ten mice were purchased from Jackson Laboratories. Upon arrival, mice were randomly distributed into two groups of five mice each. Throughout mice had unlimited access to food and water, and cages were supplied with bedding for environmental enrichment. For individual assessment, all mice were earmarked. The infections were carried out by the section for experimental medicine (AEM) at Copenhagen University, where they have specialized facilities for animal housing and expertise for animal handling. To prepare the inoculums each WITS strain were grown for 16 h, 200 rpm at 37 °C in LB media. The 8 WITS strains were mixed 1:1:1:1:1:1:1:1 before the infection to give a challenge dose of 104 bacteria in total. 5 mice caged in the individually ventilated cage (IVC) were inoculated via the intraperitoneal route. A control group of 5 mice was administered physiological saline. The route of infection was chosen as the aim of the study was to establish an asymptomatic systemic infection. The exact bacterial number and ratio between individually tagged strains was determined by plating and by a PCR based sequencing approach as described below. Mice were manually inspected for symptoms of distress every 8 h during the first week post infection, and every 12 h for the remainder of the experiment. Over the course of the infection, fecal samples were collected from individual mice and S. Typhimurium bacteria were recovered and enumerated after plating a dilution series on to LB agar containing kanamycin. Mice were killed at 6 weeks post infection by cervical dislocation. In case a mouse displayed symptoms of illness, it was euthanized prior to this time point. The spleens and livers were removed aseptically and bacteria were recovered and enumerated after plating a dilution series on to LB agar containing kanamycin. Up to 96 individual S. Typhimurium clones from each sample (fecal and organ) were collected and frozen in glycerol stocks at −80 °C. Re-infections were carried out as described above in groups of five mice in one group. For re-infections isolated adapted clones were mixed 1:1 with the wild type and mice were infected IP with a challenge dose of 104 bacteria in total. Re-infections were terminated after 4 weeks of infections.
All mouse experiments were reviewed and approved by the Copenhagen University animal experimentation unit and conducted with permission from the Animal Experiments Inspectorate (http://www.dyreforsoegstilsynet.dk) under license number 2013-15-2934-00761 in accordance with Danish law LBK 474 af 15/05/2014 (Animal experimentation and welfare act).
PCR and sequencing of WITS clones
Distribution of WITS in the inoculums and isolated clones recovered from fecal samples and systemic organs after infections were determined by a PCR based sequencing approach. For each sample the WITS was determined in 20 to 48 individual clones by PCR amplifying the region containing the WITS followed by Sanger sequencing of individual PCR products. Primer sequences (Tagseqfwd and Tagseqrev) used for PCR amplification of the WITS region can be found in Additional file 1: Table S1.
Extraction of chromosomal DNA and whole genome sequencing of dominant clones
Bacterial cells were grown overnight in 5 ml LB at 37 °C with 200 rpm. The next day cultures were harvested and DNA was extracted using the ChargeSwitch gDNA Bacteria Kit from Invitrogen according to the manufacturer’s recommendation. Samples were stored at −20 °C. DNA was analyzed by Nanodrop 1000 from Thermo Fischer and shipped to BGI or Macrogen for Whole Genome Sequencing using the Illumina HiSeq 2000 platform with 100 bp paired end reads. Paired-end reads were mapped on the ST4/74 genome sequence (NCBI NC_016857, CP002488-CP002490) using the Geneious Alignment Tool giving a raw coverage depth of approximately 100–200 fold. Variant base calling relative to ST4/74 was performed with the Geneious work package. Sequence reads from all isolates are deposited in the Short Read Archive under accession number SRP070821 (accession numbers for individual samples are provided in Table 2). SNPs supported by 95–100 % of the reads, and all single nucleotide deletions and insertions (indels) were re-examined by Sanger capillary sequencing of PCR products amplified using specific primers (Additional file 1: Table S1) flanking the regions of interest.
Extraction of RNA and northern blotting
Cells were grown in M9 for 16 h, 200 rpm at 37 °C. The next day all cultures were diluted 100-fold into fresh M9 an incubated at 37 °C with shaking at 200 rpm. Growth was monitored by OD600 measurements every hour. At OD600 ~ 0.4 +/−0.1, 1 ml aliquots were harvested from each culture and immediately frozen and stored at −80 °C. At this point 2-keto-3-deoxygluconate (KDG) was added to all cultures at a final concentration of 5.6 mM (0.1 mg ml−1). The cultures were re-incubated for 1 h and 1 ml aliquots were again harvested from all cultures. Cells were lysed mechanically using the FastPrep system (Bio101; Q-biogene), and total RNA was isolated as described previously  using the RNeasy mini kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. Total RNA was quantified by Nanodrop 1000 from Thermo Fischer and 5 μg of RNA of each preparation was loaded onto a 1 % agarose gel and separated in 10 mM sodium phosphate buffer. RNA was transferred to a positively charged nylon membrane (Boehringer Mannheim) by capillary blotting. Hybridization was performed according to  using a gene-specific kdgK probe that had been labeled with [32P]dCTP using the Ready-to-Go DNA-labeling beads from Amersham Biosciences. Internal fragments of the kdgK gene were used as template in the labeling reaction. All steps were repeated in three independent experiments giving similar results.
Significant differences were determined using the 2 sample t-test with unequal variances. Error bars represent standard deviations.
This work was funded by the Danish Research Council for Technology and Production through Grant No. 12–126640 to LJ. The research council was not involved in the design of the study. We thank Mette Holm for excellent technical assistance and the Department of Experimental Medicine (AEM), Copenhagen University, for assistance with animal experiments. We are grateful to Professor John E. Olsen, Copenhagen University, for valuable discussions.
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