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INFECTION AND IMMUNITY, July 2009, p. 3117–3126 Vol. 77, No. 70019-9567/09/$08.00�0 doi:10.1128/IAI.00093-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Glucose and Glycolysis Are Required for the Successful Infection ofMacrophages and Mice by Salmonella enterica Serovar Typhimurium�

Steven D. Bowden,1 Gary Rowley,2 Jay C. D. Hinton,1,3 and Arthur Thompson1*Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom1; School of Biological Sciences,

University of East Anglia, Norwich NR4 7TJ, United Kingdom2; and School of Genetics and Microbiology, Trinity College,Dublin 2, Ireland3

Received 23 January 2009/Returned for modification 2 March 2009/Accepted 13 April 2009

Salmonella is a widespread zoonotic enteropathogen that causes gastroenteritis and fatal typhoidal diseasein mammals. During systemic infection of mice, Salmonella enterica serovar Typhimurium resides and repli-cates in macrophages within the “Salmonella-containing vacuole” (SCV). It is surprising that the substratesand metabolic pathways necessary for growth of S. Typhimurium within the SCV of macrophages have not beenidentified yet. To determine whether S. Typhimurium utilized sugars within the SCV, we constructed a seriesof S. Typhimurium mutants that lacked genes involved in sugar transport and catabolism and tested them forreplication in mice and macrophages. These mutants included a mutant with a mutation in the pfkAB-encodedphosphofructokinase, which catalyzes a key committing step in glycolysis. We discovered that a pfkAB mutantis severely attenuated for replication and survival within RAW 264.7 macrophages. We also show that disrup-tion of the phosphoenolpyruvate:carbohydrate phosphotransferase system by deletion of the ptsHI and crrgenes reduces S. Typhimurium replication within RAW 264.7 macrophages. We discovered that mutants unableto catabolize glucose due to deletion of ptsHI, crr, and glk or deletion of ptsG, manXYZ, and glk showed reducedreplication within RAW 264.7 macrophages. This study proves that S. Typhimurium requires glycolysis forinfection of mice and macrophages and that transport of glucose is required for replication withinmacrophages.

Salmonella is a common zoonotic enteropathogen thatcauses gastroenteritis or fatal systemic disease in mammals,including humans, cattle, and pigs. Typhoidal Salmonella sero-vars, such as Salmonella enterica serovars Typhi and Paratyphi,cause an estimated 20 million cases of salmonellosis and200,000 human deaths worldwide per year (9). Salmonella in-fections occur as a result of ingestion of contaminated foodand water. S. enterica serovar Typhimurium causes a self-lim-ited gastroenteritis in humans and results in a systemic ty-phoid-like disease in mice. Infected mice are frequently used asan experimental model for human typhoid diseases (56). Dur-ing systemic infections, S. Typhimurium penetrates the smallintestine barrier and gains access to the mesenteric lymphnodes, where the bacteria are engulfed by phagocytic cells,such as macrophages. Once inside a macrophage, S. Typhi-murium is compartmentalized into an intracellular phagosomewhich is modified to become the “Salmonella-containing vac-uole.” The Salmonella-containing vacuole acts as a shield thatprevents both lysosomal fusion and exposure to host cell anti-microbial agents (1, 21). The Salmonella bacteria must acquirenutrients for replication within macrophages.

S. Typhimurium contains a variety of virulence genes, manyof which are organized into clusters referred to as Salmonellapathogenicity islands. Much work has focused on characteriza-tion of Salmonella pathogenicity genes and the mechanisms by

which they facilitate infection (54, 55). In contrast, there is alack of information regarding the nutritional and metabolicrequirements of Salmonella during infection. It has beenshown that the complete tricarboxylic acid (TCA) cycle oper-ates during infection of mice with S. Typhimurium strain SR11(53). Surprisingly, fatty acid degradation and the glyoxylateshunt are not required to replenish the TCA cycle (53). Thesame study also showed that S. Typhimurium SR11 does notrequire gluconeogenesis to exhibit full virulence in BALB/cmice and suggested that SR11 utilizes as-yet-unidentified sug-ars for growth during infection of BALB/c mice (53). We havebeen investigating which substrates and metabolic pathwaysare required by Salmonella for infection of cultured murinemacrophages and for systemic infection of mice. Our work hasfocused on the central catabolic pathway of glycolysis, which isthe sequence of catabolic reactions that converts sugars intopyruvate with concomitant synthesis of ATP and NADH (18).Glycolysis is the foundation of both aerobic and anaerobicrespiration and is found in nearly all organisms (18). Many ofthe carbohydrates catabolized by glycolysis are imported viathe phosphotransferase (PTS) system (40). The PTS systemtransfers phosphate from the glycolytic intermediate phos-phoenolpyruvate to a cascade of enzymes, ultimately resultingin rapid phosphorylation of the transported sugar (Fig. 1).Briefly, enzyme 1 (E1) transfers a phosphate group from phos-phoenolpyruvate to enzyme 2 (EII) via HPr, and enzyme 2transports and phosphorylates the incoming sugar (Fig. 1).

Here we show that the glycolytic pathway is required forintracellular replication of S. Typhimurium in mice and mac-rophages and that glucose is the major sugar utilized by S.Typhimurium during infection of macrophages.

* Corresponding author. Mailing address: Institute of Food Re-search, Norwich Research Park, Colney, Norwich NR4 7UA, UnitedKingdom. Phone: (44) 1603 255181. Fax: (44) 1603 255288. E-mail:[email protected].

� Published ahead of print on 20 April 2009.

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MATERIALS AND METHODS

Bacterial strains, growth conditions, and reagents. The S. Typhimuriumstrains and plasmids used in this study are listed in Table 1. Strains were main-tained in Luria-Bertani (LB) broth or on plates with appropriate antibiotics atthe following concentrations; ampicillin (Sigma Aldrich), 50 �g ml�1, chloram-phenicol (Cm) (Sigma Aldrich), 12.5 �g ml�1; and kanamycin (Km) (SigmaAldrich), 50 �g ml�1. M9 minimal medium with 0.4% glucose was used whereindicated below. Oligonucleotide primers were purchased from Sigma Genosysor Illumina (California).

Mutant construction. S. Typhimurium mutants were constructed using previ-ously published procedures (11). Briefly, 60-bp oligonucleotides were designedthat contained 40 bp at their 5� end that was homologous to the DNA flankingthe target gene to be deleted from the chromosome (Table 2). The oligonucle-otides were used to PCR amplify a cassette containing the Km resistance genecarried by plasmid pKD4 or the Cm resistance gene carried by plasmid pKD3(Table 1) (11). Each resulting PCR product was then transformed by electropo-ration into S. Typhimurium 4/74 (59) carrying plasmid pKD46 (Table 1). PlasmidpKD46 encoded the �Red recombinase enzyme, which facilitated homologousrecombination of the target gene with the PCR product, resulting in complete

replacement of the target gene with the Km or Cm resistance gene (11). In orderto avoid unwanted genetic recombination events that may have occurred duringthe original transformation, the Km or Cm resistance cassette from the deletedgene was routinely transferred by P22 transduction into S. Typhimurium wild-type strain 4/74 (24). The transductants were screened on green agar plates toobtain lysogen-free colonies (50). The complete absence of the structural gene(s)was confirmed by DNA sequencing of the deleted regions of the chromosome(data not shown).

During construction of strain JH3541, the Cm resistance gene was removed viaa further recombination event by transforming JH3501 with the FLP recombi-nase expression plasmid pCP20 (8). The FLP recombinase removed the Cmresistance gene, and the strain was cured of the pCP20 plasmid by growth at37°C. In the resultant strain, JH3541 (Table 2), the manXYZ genes are com-pletely deleted, and the strain does not contain the Cm resistance cassette (11).

Plasmid construction. The pfkA gene plus 582 bp of upstream sequence wasPCR amplified from S. Typhimurium 4/74 genomic DNA using primers pfkA1(5�-TTTTAAGCTTGGGTTATCCTGGTACGGTTG) and pfkA2 (5�-TTTTGGATCCGATAAGCGTAGCGCCATCAG). The PCR product was digestedwith BamHI and HindIII, ligated into the low-copy-number vector pWSK30 (57),

FIG. 1. Mechanism underlying inducer exclusion in enteric bacteria. (Modified from reference 15 with permission.) The transport of PTScarbohydrates, including glucose, results in net dephosphorylation of the PTS proteins and inducer exclusion. The dephosphorylated EIIAGlc

permease encoded by crr blocks the import of lactose, maltose, and melibiose and the phosphorylation of glycerol by binding to the correspondingtransporter or kinase. Phosphorylated EIIAGlc activates adenylate cyclase (AC), which binds to phosphorylated as well as unphosphorylatedEIIAGlc. cAMP binds to CRP, which activates the transcription of many genes encoding catabolic enzymes and transport proteins, including ptsG.PEP, phosphoenolpyruvate.

TABLE 1. Primers used to construct S. Typhimurium gene deletion mutants

Primer Sequence

pfkaredf.......................................................CAATAGATTTCATTTTGCATTCCAAAGTTCAGAGGTAGTCGTGTAGGCTGGAGCTGCTTCpfkaredr ......................................................AGGCCTGATAAGCGTAGCGCCATCAGGCGCGCAAAAACAACATATGAATATCCTCCTTAGpfkbredf.......................................................ATTAAGTGCCAGACTGAAATCAGCCTAACAGGAGGTAACGGTGTAGGCTGGAGCTGCTTCpfkbredr ......................................................AACCGATTTTCCGTTATCCCCCTCGGCGAGGGGGAAACGACATATGAATATCCTCCTTAGptshredf .......................................................TTAGTTCCACAACACTAAACCTATAAGTTGGGGAAATACAGTGTAGGCTGGAGCTGCTTCcrrredr .........................................................AAATGGCGCCCAAAGGCGCCATTCTTCACTGCGGCAAGAACATATGAATATCCTCCTTAGglkredf .........................................................TGACAAAGACTTATTTTGACTTTAGCGGAGCAGTAGAAGAGTGTAGGCTGGAGCTGCTTCglkredr.........................................................CTTTTGTAGGCCGGATAAGGCGTTTATGCCACCATCTGGCCATATGAATATCCTCCTTAGptsIRevInt1.................................................GCAGTTCCTGTTTGTAGATTTCAATCTCTTTGCGCAGCGCCATATGAATATCCTCCTTAGcrrredf .........................................................TCCACGAGATGCGGCCCAATTTACTGCTTAGGAGAAGATCGTGTAGGCTGGAGCTGCTTCptsgredf .......................................................GAACGTAGAAAAGCACAAATACTCAGGAGCACTCTCAATTGTGTAGGCTGGAGCTGCTTCptsgredr .......................................................GCCGAATGGCTGCCTTAATTCTCCCCAACATCATTACTGCCATATGAATATCCTCCTTAGmanxredf .....................................................TGTCAAGTTGATGTGTTGACAATAATAAAGGAGGTAGCAAGTGTAGGCTGGAGCTGCTTCmanzredr.....................................................AAAAAACGGGGCCGTTTGGCCCCGGTAGTGTACAACAGCCCATATGAATATCCTCCTTAG

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and transformed into Escherichia coli strain DH5� by electroporation (58). Theresulting plasmid was designated pWSK30::pfkA and was confirmed by DNAsequencing across the multiple-cloning site using primers M13F (5�-CGCCAGGGTTTTCCCAGTCACGAC) and M13R (5�-TCACACAGGAAACAGCTATGAC) (John Innes Centre Genome Laboratory). Plasmids pWSK30 andpWSK30::pfkA were then transformed into 4/74 and JH3486 by electroporation.pWSK30::pfkA was shown to be functional because it restored growth of JH3486on M9 minimal medium with 0.4% glucose (data not shown).

Macrophage infection assays. Infection assays with murine macrophages wereperformed essentially as previously described (26). Briefly, murine macrophages(RAW 264.7; obtained from American Type Culture Collection, Rockville, MD)were grown in minimal essential medium (product no. M2279; Sigma Aldrich)(19) supplemented with 10% fetal bovine serum, L-glutamine (final concentra-tion, 2 mM; Sigma Aldrich), and 1� nonessential amino acids (product no.M7145; Sigma Aldrich). For infection, 1 � 105 macrophage cells were seededinto each well of a 12-well cell culture plate (Corning) and infected with com-plement-opsonized S. Typhimurium 4/74 and mutant strains at a multiplicity ofinfection of 1:1 (ratio of bacteria to cells) (26). To minimize Salmonella patho-genicity island 1 expression, bacteria were grown overnight on LB medium platesat 37°C and suspended in phosphate-buffered saline (PBS) before opsonization.

To increase the uptake of Salmonella, plates were centrifuged at 1,000 � g for5 min (Eppendorf 5810R), and this was defined as time zero. After 1 h ofphagocytosis, extracellular bacteria were killed by addition of 100 �g ml�1

gentamicin (Sigma). After a further 1 h, the medium was replaced with supple-mented minimal essential medium containing 10 �g ml�1 gentamicin. Incubationwas continued for 2 h and 18 h after infection. To estimate the amount ofintracellular bacteria at each time point, cells were lysed using 1% Triton X-100(Sigma), and samples were removed to determine viable counts (16). Statisticalsignificance was assessed by using Student’s unpaired t test, and a P value of�0.05 was considered significant.

Mouse infection assays. Mouse infection experiments were performed as de-scribed previously (38), with some modifications. Liquid S. Typhimurium cul-tures were grown statically in 50 ml of LB medium (supplemented with anantibiotic if required to maintain the plasmid) at 37°C overnight in 50-ml Falcontubes (Corning) (3). The following day bacteria were resuspended at a finaldensity of 1 � 104 CFU ml�1 in sterile PBS. Five female BALB/c mice (CharlesRiver U.K. Ltd.) per S. Typhimurium strain were infected with 200 �l of abacterial suspension via the intraperitoneal (i.p.) route using a final dose of 2 �103 Salmonella CFU (38). The infection was permitted to proceed for 72 h, andthen the mice were sacrificed by cervical dislocation and the spleens and livers

TABLE 2. Strains and plasmids used in this study

Strain or plasmid Relevant genotype or phenotype Method of constructiona Reference

S. Typhimurium strains4/74 Wild-type strain NA 59SL1344 rpsL hisG NA 24JH3386 4/74 pfkA::Km �Red mutagenesis; pKD4 PCR product obtained

using primers pfkaredf and pfkaredrThis study

JH3460 4/74 pfkB::Cm �Red mutagenesis; pKD3 PCR product obtainedusing primers pfkbredf and pfkbredr

This study

JH3486 4/74 pfkA::Km pfkB::Cm P22 transduced pfkB::Cm from JH3460 intoJH3386

This study

JH3494 4/74 glk::Km �Red mutagenesis; pKD4 PCR product obtainedusing primers glkredf and glkredr

This study

JH3501 4/74 manXYZ::Cm �Red mutagenesis; pKD3 PCR product obtainedusing primers manxredf and manzredr

This study

JH3502 4/74 crr::Km �Red mutagenesis; pKD4 PCR product obtainedusing primers crrredf and crrredr

This study

JH3504 4/74 ptsG::Cm �Red mutagenesis; pKD3 PCR product obtainedusing primers ptsgredf and ptsgredr

This study

JH3536 4/74 ptsHI-crr::Cm �Red mutagenesis; pKD3 PCR product obtainedusing primers ptshredf and crrredr

This study

JH3537 4/74 ptsHI::Cm �Red mutagenesis; pKD3 PCR product obtainedusing primers ptshredf and ptsIRevInt1

This study

JH3540 4/74 ptsHI-crr::Cm glk::Km P22 transduced glk::Km from JH3494 intoJH3536

This study

JH3541 4/74 manXYZ Transformed JH3501 with pCP20 to excise theCmr cassette from the chromosome; plasmidwas cured from the strain

This study

AT1011 4/74 ptsG::Cm manXYZ P22 transduced ptsG::Cm from JH3504 intoJH3541

This study

AT1012 4/74 ptsG::Cm glk::Km P22 transduced glk::Km from JH3494 intoJH3504

This study

AT1013 4/74 manXYZ glk::Km P22 transduced glk::Km from JH3494 intoJH3541

This study

AT1014 4/74 ptsG::Cm manXYZ glk::Km P22 transduced glk::Km from JH3494 intoAT1013

This study

PlasmidspKD46 �Red recombinase expression

plasmidNA 11

pKD3 Cmr cassette-containing plasmid NA 11pKD4 Kmr cassette-containing plasmid NA 11pCP20 FLP recombinase expression plasmid NA 8pWSK30 Apr low-copy-number vector,

pSC101 origin of replicationNA 57

pWSK30::pfkA Apr low-copy-number vector,pSC101 origin of replication,expresses pfkA

1,579-bp pfkA1-pfkA2 PCR product cloned intopWSK30 BamHI-HindIII sites

This study

a �Red mutagenesis is described in Materials and Methods. NA, not applicable.

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were surgically removed (38). Following homogenization of the organs in astomacher (Seward Tekmar), serial dilutions of the suspensions in PBS werespread onto LB agar plates, and bacterial CFU were enumerated after overnightincubation at 37°C (3). All animal experiments were approved by the local ethicscommittee and conducted according to guidelines of the Animal Act 1986 (Sci-entific Procedures) of the United Kingdom.

RESULTS

Glycolysis is required for intracellular replication and sur-vival of S. Typhimurium in macrophages. The enzyme phos-phofructokinase (Pfk) catalyzes a key committing step in gly-colysis and irreversibly converts -D-fructose-6-phosphate into-D-fructose-1,6-bisphosphate. In contrast to most glycolyticenzymes, phosphofructokinase is not part of the gluconeogenicpathway and instead is specific for glycolysis (18). The loss offunctional phosphofructokinase activity completely blocks gly-colysis and prevents growth of S. Typhimurium on sugars assole carbon sources (18).

In most bacteria phosphofructokinase is encoded by twogenes, designated pfkA and pfkB. (44). In order to test whetherthe glycolytic pathway is required for infection by Salmonella,we first constructed S. Typhimurium strains with completedeletions of the pfkA and pfkB genes (designated JH3386 andJH3460, respectively) and a double mutant strain which lackedboth the pfkA and pfkB genes (JH3486). The deletions wereverified by DNA sequencing and by growing the strains on M9minimal medium plates with glucose as the sole carbon source.Both the pfkA and pfkB single-mutant strains, but not the

pfkAB strain, were able to grow on glucose as a sole carbonsource (data not shown).

In RAW 264.7 macrophage replication assays neither the pfkAsingle mutant nor the pfkB single mutant was attenuated forintracellular replication compared to the wild-type strain (datanot shown). In contrast, we observed a dramatic 322-fold decreasein the number of intracellular S. Typhimurium pfkAB bacteriacompared to the number of wild-type strain bacteria after 18 h ofinfection of RAW 264.7 macrophages (Fig. 2A). Furthermore,the 34-fold decrease in the level of intracellular S. TyphimuriumpfkAB bacteria between 2 h and 18 h postinfection suggests thatthe S. Typhimurium pfkAB strain was unable to survive withinRAW 264.7 macrophages (Fig. 2A).

We next confirmed that the survival and replication defects inmacrophages infected with the S. Typhimurium pfkAB strainwere due to deletion of the pfkAB genes. We inserted the pfkAgene plus 582 bp of the upstream sequence into the low-copy-number vector pWSK30 (57), transformed the construct into theS. Typhimurium 4/74 and S. Typhimurium pfkAB strains, andperformed infection assays with macrophages. As shown in Fig.2B, the cloned copy of the pfkA gene fully complemented thepfkAB deletion in JH3486 during infection of macrophages andrestored intracellular replication of the S. Typhimurium pfkABstrain containing pWSK30::pfkA to wild-type levels. We observedthat the 4/74(pWSK30::pfkA) strain was slightly attenuated forreplication compared to 4/74 containing the empty vector (Fig.2B), which may indicate that excessive Pfk activity is detrimentalto S. Typhimurium replication within macrophages.

FIG. 2. Glycolysis is required for infection of macrophages. (A) Intracellular replication assays with S. Typhimurium 4/74 wild-type and pfkAB(JH3486) strains during infection of RAW 264.7 macrophages. (B) Complementation of the S. Typhimurium pfkAB strain in RAW 264.7macrophages. The data show the numbers of viable bacteria (expressed as percentages of the initial inoculum) inside the macrophages at 2 h and18 h after infection. Each bar indicates the statistical mean for three biological replicates, and the error bars indicate the standard deviations.



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Glycolysis is required for successful infection of mice. Dur-ing murine infections, S. Typhimurium disseminates systemi-cally from the Peyer’s patches to the liver and spleen, where itcontinues to grow within macrophages (20, 43, 48). The inabil-ity of the S. Typhimurium pfkAB strain to replicate withinmacrophages suggested that phosphofructokinase might play arole in the mouse typhoid infection model. We therefore in-fected BALB/c mice with the S. Typhimurium pfkAB mutant(JH3486) and the wild-type strain via the i.p. route. Salmonellabacteria were recovered from the spleens and livers of miceand enumerated after 72 h of infection. The results demon-strated that the S. Typhimurium pfkAB mutant is severelyattenuated (approximately 100-fold) compared to the wild-type strain in the spleens and livers of infected mice (Fig. 3A).Mice infected with the S. Typhimurium pfkAB strain exhib-ited slight ruffling of the fur after 72 h, suggesting that animmune response may have been elicited, but otherwise wereasymptomatic, whereas mice infected with the S. Typhimuriumwild-type strain showed severe symptoms of systemic typhoiddisease after 72 h. These results indicate that glycolysis is anessential central metabolic pathway required for successful in-fection of mice by S. Typhimurium.

To verify that the virulence defect of the glycolysis mutantobserved in mice was due to loss of the pfkAB genes and not toa secondary mutation, we complemented the mutation with thepWSK30::pfkA plasmid, which directs synthesis of Pfk-1. Weinfected female BALB/c mice with the wild type and JH3486,with each strain carrying pWSK30 or pWSK30::pfkA (Fig. 3B).The results showed that, as expected, JH3486 containing thepWSK30 vector was attenuated for infection of the liver andspleen compared to the wild-type strain containing the same plas-mid. However, the JH3486 strain containing pWSK30::pfkA was

not attenuated for infection of the livers and spleens of in-fected BALB/c mice compared to the wild-type strain carryingthe empty vector. This result confirms that JH3486 is attenu-ated for infection of mice, most likely because the lack of afunctional Pfk enzyme blocks glycolysis. We also observed thatwild-type S. Typhimurium expressing pWSK30::pfkA wasslightly attenuated for infection of the liver and spleen com-pared to the wild type expressing the empty vector (Fig. 3B).Similar results were obtained with macrophages (Fig. 2B), andthis suggests that excessive Pfk activity has a negative impacton S. Typhimurium infection of mice.

Carbohydrate transport is required for intracellular repli-cation of S. Typhimurium in cultured macrophages. Manydifferent carbon sources (including carbohydrates) can be ca-tabolized by glycolysis. Having confirmed that glycolysis is re-quired for successful infection of mice and macrophages by S.Typhimurium, we investigated which sugars are available toSalmonella during infection by studying sugar transport mu-tants for defects in virulence. The PTS system of E. coli and S.Typhimurium simultaneously transports and phosphorylates alarge number of carbohydrates and is a likely candidate for asystem involved in importing the glycolytic substrates requiredfor growth of S. Typhimurium within macrophages (40).

In order to determine the role of the PTS system in trans-portation of carbohydrates required for intracellular replica-tion of S. Typhimurium in macrophages, we targeted genesencoding components of the PTS system for deletion. The ptsHand ptsI genes encode the HPr and EI proteins, respectively(Fig. 1). The sugar-specific EII domains can be encoded bymore than one gene; for example, the glucose-specific EII,EIIABCGlc, is encoded by the crr gene that encodes the EIIAdomain, and ptsG that encodes the EIIBC domains (40). The

FIG. 3. Glycolysis is required for infection of mice. (A) S. Typhimurium 4/74 and pfkAB mutant (JH3486) CFU recovered from spleens andlivers at 72 h after i.p. infection of BALB/c mice. (B) Complementation of the S. Typhimurium pfkAB strain in BALB/c mice. Each bar indicatesthe statistical mean for five biological replicates, and the error bars indicate the standard errors of the means.



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crr gene is cotranscribed with ptsI and ptsH but is expressedprimarily from a second promoter located within ptsI thataccounts for approximately 80% of crr transcription in the cell(14). EIIAGlc is a multifunctional protein that is an EIIA do-main for the transport of glucose, maltose, trehalose, N-acetyl-muramic acid, and arbutin/salicin (47). In its dephosphorylatedstate, EIIAGlc is able to repress other non-PTS transportersinvolved in lactose, maltose, and galactose/glucose transportvia a process known as inducer exclusion (40, 46). In contrast,the phosphorylated EIIAGlc protein (EIIAGlc-P) is known toactivate cyclic AMP (cAMP) production via adenylate cyclase(41). The resulting increased levels of cAMP lead to activationof cAMP receptor protein (CRP), which in turn causes in-creased expression of the catabolite repression regulon (in-cluding many metabolic genes involved in ribose, trehalose,galactose, and nucleoside transport) (27, 60).

To determine the importance of the PTS system for S. Ty-phimurium virulence, we constructed an S. Typhimurium mu-tant strain (JH3536) that lacks the ptsHI-crr operon. We de-leted crr in addition to ptsHI because dephosphorylatedEIIAGlc represses several non-PTS transporters via inducerexclusion (13, 33, 35–37, 40, 46, 51) (Fig. 1). The absence of EIand HPr prevents phosphorylation of EIIAGlc, which thereforeremains in its unphosphorylated state and inhibits the function

of lactose, melibiose, and galactose/glucose transporters (33,35, 37). To determine whether inducer exclusion might alsoplay a role in S. Typhimurium virulence, we constructed amutant (JH3537) in which ptsH and the first 1,200 bp of ptsIwere deleted but which was still able to transcribe crr from thepromoter located in the 3� end of ptsI. This means that en-zymes that are subject to inducer exclusion are always inhibitedin JH3537 because EIIAGlc is permanently in the dephosphor-ylated state.

We tested the S. Typhimurium wild-type strain and ptsHI-crr (JH3536), ptsHI (JH3537), and crr (JH3502) mutants forthe ability to replicate within cultured macrophages. As shownin Fig. 4A, the number of recovered CFU of S. Typhimuriumwild-type bacteria increased 4.4-fold between 2 and 18 hpostinfection. However, the intracellular replication rates ofthe S. Typhimurium crr, ptsHI, and ptsHI-crr strains weresignificantly reduced compared to that of the wild-type strain(Fig. 4A). The S. Typhimurium crr strain was partially atten-uated for intracellular replication in macrophages (2.3-foldincrease). This may indicate that PTS sugars that require theEIIAGlc protein for transport (i.e., glucose, maltose, trehalose,N-acetylmuramic acid, arbutin, and/or salicin) are utilized ascarbon sources by S. Typhimurium during macrophage infec-tion. Alternatively, the slight attenuation of the crr mutant

FIG. 4. PTS system is required for S. Typhimurium replication within macrophages. (A) Intracellular replication assay with the S. Typhimurium4/74, crr (JH3502), ptsHI (JH3537), and ptsHI-crr (JH3536) strains during infection of RAW 264.7 macrophages. (B) Intracellular replicationassay with the S. Typhimurium 4/74, ptsHI-crr (JH3536), glk (JH3494), and ptsHI-crr glk (JH3540) strains during infection of RAW 264.7macrophages. The data show the numbers of viable bacteria (expressed as percentages of the initial inoculum) inside the macrophages at 2 h and18 h after infection. Each bar indicates the statistical mean for three biological replicates, and the error bars indicate the standard deviations.Significant differences between parental strain 4/74 and the mutant strains are indicated by asterisks, as follows: no asterisk, P � 0.05; *, P � 0.05;**, P � 0.01; and ***, P � 0.001.



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could show that EIIAGlc-P is required to promote cAMP pro-duction and that this signal molecule interacts with CRP toregulate genes that promote survival and/or replication in mac-rophages.

The S. Typhimurium ptsHI-crr strain was strongly attenu-ated for replication in macrophages, and the level of this strainincreased only approximately 1.8-fold during the experiment.The absence of the HPr and EI subunits in the S. TyphimuriumptsHI-crr strain would have prevented all PTS carbohydratesfrom entering the cell, including those that require EIIAGlc

(Fig. 1). Because the S. Typhimurium ptsHI-crr strain showedless replication than the S. Typhimurium crr strain withinmacrophages (Fig. 4A), we deduced that PTS sugars trans-ported independent of EIIAGlc are utilized by S. Typhimuriumduring infection of macrophages and are important for intra-cellular replication.

JH3537, the S. Typhimurium ptsHI strain that expressesfunctional EIIAGlc and is permanently subject to the inducerexclusion effect (see above), was more attenuated than theptsHI-crr mutant for replication in macrophages (Fig. 4A). Infact, this mutant did not replicate between 2 h and 18 h postin-fection, indicating that it was unable to multiply inside macro-phages. Our data show that inducer exclusion regulates theactivity of an S. Typhimurium transporter of a substrate avail-able inside macrophages, which could include lactose, melibi-ose, and glucose/galactose (33, 35, 37). In the case of glucoseand galactose, the inducer exclusion-dependent transporter isgalactose permease (GalP) (2, 29, 30, 33). GalP also transportsglucose (39), and its expression is induced by availability ofgalactose (7). The abundance of glucose in eukaryotic cells andour previous observation that glucose transport genes are over-expressed by S. Typhimurium in macrophages and HeLa cells(17, 22) led us to investigate glucose utilization by S. Typhi-murium in more detail.

Glucose is the major carbohydrate required for intracellu-lar replication of S. Typhimurium in macrophages. The exper-iments described above suggested that a carbohydrate thatrequires the HPr, EI, and EIIAGlc components of the PTSpermease system is required for intracellular replication of S.Typhimurium in macrophages. Glucose requires the HPr, EI,and EIIAGlc components for transport in S. Typhimurium butcan also be transported into Salmonella via the GalP andMglABC transporter. Following internalization, intracellularglucose is subsequently phosphorylated by glucokinase (25,32). Glucokinase is encoded by the glk gene, is highly specificfor glucose, and does not efficiently phosphorylate related car-bohydrates, such as galactose, mannose, or fructose (32). Inorder to determine whether glucose is required for intracellu-lar growth of S. Typhimurium, we constructed a mutant strainwith complete deletions of the ptsHI-crr and glk genes(JH3540). We confirmed that while the ptsHI-crr mutant(JH3536) grew on M9 minimal medium containing glucose asa sole carbon source, the S. Typhimurium ptsHI-crr glkstrain (JH3540) was unable to grow under the same conditions(data not shown). In this mutant, glucose that is transportedinto the cell by GalP and MglABC cannot enter glycolysisbecause the absence of glucokinase prevents production ofglucose-6-phosphate. We performed an infection assay withthe S. Typhimurium wild-type, ptsHI-crr (JH3536), ptsHI-crrglk (JH3540), and glk (JH3494) strains to determine their

abilities to replicate in macrophages (Fig. 4B). The data showthat, as expected, the S. Typhimurium glk mutant, whoseability to grow on glucose is not affected, replicated as rapidlyas the wild-type strain in infected macrophages. The ptsHI-crrmutant (JH3536), which cannot transport PTS sugars but canstill grow on glucose, was attenuated for intracellular replica-tion compared to the wild-type strain, as observed in the pre-vious experiment (Fig. 4A). However, the S. TyphimuriumptsHI-crr glk strain (JH3540), which cannot grow on glucoseat all, was unable to replicate within macrophages (Fig. 4B),reflecting the phenotype observed for the ptsHI mutant (Fig.4A). As glucokinase is highly specific for glucose, this resultstrongly suggests that glucose is the most important nutrientrequired for intracellular replication of S. Typhimurium inmacrophages. However, given the pleiotropic effects of theptsHI and crr mutations on cAMP production and cataboliterepression, it was possible that some unknown factor regulatedby CRP-cAMP could be responsible for the attenuation seen inthese mutants. We therefore decided to test mutants in whichglucose utilization is specifically affected to determine the im-pact of their mutations on virulence.

In order to confirm that glucose was definitely utilized by S.Typhimurium during infection of macrophages, we disruptedglucose transport and catabolism. To our knowledge, there arefour separate transporters for glucose in S. Typhimurium, theIIABCGlc and IIABCMan PTS systems (52), GalP (23, 39), andthe methylgalactose ABC transporter (MglABC) (12, 23).

The IIABCGlc PTS system, encoded by ptsG and crr, is theprimary PTS transporter for glucose, and its Km for glucose is3 to 10 �M (34, 52). The IIABCDMan PTS system is encodedby manXYZ and is a PTS transporter that has broad specificityfor many sugars, including mannose, glucose, 2-deoxyglucose,N-acetylglucosamine, N-acetylmannosamine, and galactos-amine (6, 42, 52); it is thought to be a scavenger system foramino sugars generated during cell wall synthesis and degra-dation, and it is an important glucose uptake system in S.Typhimurium. Glucose can also be transported into the cell viaGalP (23, 39). MglABC is an ABC transporter that is capableof transporting four carbohydrates, including glucose (5, 12,45, 49). However, glucose entering the cell via MglABC orGalP must be phosphorylated by glucokinase, IIABCGlc, orIIABCDMan in order to enter glycolysis and to be utilized as asource of carbon and ATP (10).

We also constructed S. Typhimurium mutants JH3504,JH3501, and JH3494, which have deletions in the ptsG, manXYZ,and glk genes, respectively. We were able to use P22 phagetransduction and FLP recombinase methods to engineer mu-tants with every combination of double and triple mutations inthese three genes and generated the following strains: ptsGmanXYZ strain AT1011, ptsG glk strain AT1012,manXYZ glk strain AT1013, and ptsG manXYZ glkstrain AT1014. We checked the growth of these strains on M9minimal medium plates with glucose as the sole carbon sourceand observed that while the growth of the single and doublemutants was slower than that of the 4/74 wild-type strain, onlythe triple mutant was unable to grow on this medium (data notshown). We tested these mutants to determine if the inabilityto utilize glucose as a carbon source led to decreased replica-tion during infection of macrophages (Fig. 5A). The results forthe single mutants showed that disruption of manXYZ or glk



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had no effect on S. Typhimurium replication within macro-phages compared to that of the wild-type strain. However, theptsG single mutant, JH3504, was slightly attenuated for repli-cation compared to the wild type, as previously observed forthe crr mutant (Fig. 4A). This result indicates that glucose isavailable to S. Typhimurium during infection of macrophagesbecause of the specificity of the ptsG-encoded IIBCGlc PTSsystem for glucose (40). The low level of attenuation of theptsG mutant probably reflects the fact that IIABCGlc is theprimary route of glucose transport but its loss can be mostlycompensated for by alternative glucose transporters, such asIIABCDMan. The fact that the manXYZ and glk single mutantswere not attenuated for replication indicates that these genesplay a smaller role than ptsG during growth of S. Typhimuriumon glucose. The lack of attenuation of the manXYZ mutantshows that mannose is not an important carbon source for S.Typhimurium within macrophages, because IIABCDMan is theonly efficient transport system for this sugar (40). The resultsfor the double mutants showed that the loss of both manXYZand glk had no effect on S. Typhimurium replication in mac-rophages. Therefore, IIABCDMan and glucokinase are redun-dant for utilization of glucose in the presence of a functionalIIABCGlc PTS system.

Deletion of ptsG and manXYZ led to a replication defectgreater than that of the ptsG single mutant (Fig. 5A). Thisrevealed that IIABCDMan is required to partially compensate

for the loss of ptsG and that loss of ptsG and manXYZ furtherattenuates the ability of S. Typhimurium to catabolize glucoseand replicate within macrophages. In contrast, the ptsG glkdouble mutant was as attenuated as the ptsG single mutant,showing that glucokinase is not involved in the utilization ofglucose in the presence of a functional IIABCDMan system.However, the ptsG manXYZ double mutant (AT1011)showed increased replication compared with the ptsGmanXYZ glk triple mutant (AT1014) in macrophages, show-ing that glucokinase is necessary for the utilization of glucosein the absence of the IIBCGlc and IIABCDMan systems.

The most strongly attenuated mutant was the triple mutant(AT1014), which was unable to catabolize glucose (Fig. 5A).This mutant replicated only 3.6-fold, compared to the wildtype, which replicated 10.5-fold, showing that the utilization ofglucose is important for S. Typhimurium replication withinmacrophages but is not absolutely essential. The inability ofthe ptsHI-crr glk mutant (Fig. 4B) to replicate within mac-rophages suggests that at least one other PTS sugar besidesglucose is available to S. Typhimurium within macrophages.

Although we had shown that glucose transport is importantfor S. Typhimurium replication within macrophages in a cellculture model, we had not determined if the attenuation re-flected S. Typhimurium infection in vivo. We therefore testedthe ptsG manXYZ glk triple mutant (AT1014) to deter-mine its replication during i.p. infection of BALB/c mice. The

FIG. 5. Glucose transport is required for S. Typhimurium replication within macrophages. (A) Intracellular replication assay with the S.Typhimurium 4/74, ptsG (JH3504), glk (JH3494), manXYZ (JH3501), ptsG manXYZ (AT1011), ptsG glk (AT1012), manXYZ glk(AT1013), and ptsG manXYZ glk (AT1014) strains during infection of RAW 264.7 macrophages. The data show the numbers of viable bacteria(expressed as percentages of the initial inoculum) inside the macrophages at 2 h and 18 h after infection. Each bar indicates the statistical meanfor three biological replicates, and the error bars indicate the standard deviations. Significant differences between parental strain 4/74 and themutant strains are indicated by asterisks, as follows: no asterisk, P � 0.05; *, P � 0.05; **, P � 0.01; and ***, P � 0.001. (B) S. Typhimurium 4/74and ptsG manXYZ glk mutant AT1014 recovered from spleens and livers at 72 h after i.p. infection of BALB/c mice. Each bar indicates thestatistical mean for five biological replicates, and the error bars indicate the standard errors of the means.



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results showed that AT1014, a glucose transport mutant, isattenuated at least 10-fold compared to the wild-type strain inthe spleens and livers of infected mice (Fig. 5B). This resultconfirms that glucose is also an important carbon source for S.Typhimurium during systemic infection.

In summary, we determined the abilities of 15 metabolicmutants of S. Typhimurium to thrive inside macrophages. Theresults of these experiments revealed that glycolysis and thePTS system are required for replication within macrophagesand mice. Glucose is the most important sugar for replicationin the intracellular niche.

DISCUSSION

In this study we conducted a systematic mutational analysiswhich revealed that the central metabolic pathway of glycolysisis required for infection of macrophages (Fig. 2). We estab-lished that the key committing step for sugar catabolism, theirreversible glycolytic reaction catalyzed by phosphofructoki-nase, is necessary for intracellular replication of S. Typhi-murium in macrophages.

Having established that glycolysis is required for intracellu-lar replication in macrophages, we showed that S. Typhi-murium uses glycolysis for successful infection of mice. The S.Typhimurium pfkAB strain was significantly attenuated, andthis phenotype was fully complemented by expression of pfkAin trans from a plasmid (Fig. 3), confirming that glycolysis isessential for systemic infection of mice by S. Typhimurium.Our findings are consistent with the findings of a previousstudy which suggested that sugars enter the glycolytic and glu-coneogenic pathways at or above fructose-6-phosphate duringinfection of mice (53).

The glycolytic pathway converts hexose sugars into ATP andpyruvate, and we considered the PTS system a likely route ofentry of hexoses into the bacterial cell. We constructed mu-tants in which ptsHI and/or crr was deleted to assess the im-portance of both the PTS system and inducer exclusion for S.Typhimurium replication within macrophages (Fig. 4A). Weobserved that loss of ptsHI, with or without crr, resulted inreduced intracellular replication rates, confirming that Salmo-nella requires sugar transport for growth within macrophages.As the ptsHI mutant was more attenuated than the ptsHI-crrmutant, we concluded that sugar transporters that benefit S.Typhimurium growth within macrophages are regulated byinducer exclusion. These inducer exclusion-dependent trans-porters that promote S. Typhimurium intracellular growthcould include GalP, which is known to transport glucose (23,39). In this context, we observed that a ptsHI-crr glk mutantdid not replicate within macrophages as well as the ptsHI-crrstrain (Fig. 4B), which suggested that glucose is a major nutri-ent required for growth of S. Typhimurium in macrophages.

It was still possible that the EI and HPr components of thePTS system transport other sugars that could be utilized, so weconstructed S. Typhimurium strains with deletions of the ptsG,manXYZ, and glk genes. The ptsG gene encodes the IIB andIIC domains of the glucose-specific PTS permease. ThemanXYZ genes encode the mannose PTS permease, which alsotransports several other carbohydrates, including glucose. Wecarried out macrophage infection experiments with thesestrains and found that intracellular replication of S. Typhi-

murium was severely decreased when all three genes were absentcompared to the replication of the wild type (Fig. 5A). Thisconfirmed that glucose is utilized as a carbon source by S. Typhi-murium during infection of macrophages. We then demonstratedthat the ptsG manXYZ glk mutant was also attenuated duringinfection of mice (Fig. 5B). Our data show that the attenuation ofmetabolic mutants in macrophages that was observed was notsimply an artifact of the growth conditions used to maintain thecells and did reflect the conditions encountered within the animalmodel. Glucose is therefore an important carbon source for S.Typhimurium during systemic infection of mice.

The reason why glucose utilization and glycolysis are socrucial for S. Typhimurium infection of mice and macrophagesremains an intriguing question that will benefit from furtherresearch. It is clear from previous work that S. Typhimuriumhas access to several alternative carbon sources, apart fromsugars, during systemic infection of mice (4, 28, 31, 53). Forexample, glutamine is available as a carbon source during Sal-monella infection of mice and macrophages (28). It has beensuggested that gluconeogenic substrates, such as amino acidsor TCA cycle intermediates, also need to be available to S.Typhimurium during infection of mice (31, 53).

Our discovery that S. Typhimurium requires glycolysis andglucose for successful infection of macrophages and miceshows the key role that central metabolism plays in the infec-tion of a host by a pathogen.

ACKNOWLEDGMENTS

We thank Vittoria Danino and Isabelle Hautefort for critically re-viewing the manuscript and other members of the Salmonella lab forhelpful comments.

We are grateful to the BBSRC for funding this work (grant BB/D004810/1 to A.T. and a core strategic grant to J.C.D.H.).

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