Glucose and Glycolysis Are Required for the Successful ...· Salmonella is a widespread zoonotic enteropathogen

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INFECTION AND IMMUNITY, July 2009, p. 31173126 Vol. 77, No. 70019-9567/09/$08.000 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:arthur.thompson@bbsrc.ac.uk.

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 ml1, chloram-phenicol (Cm) (Sigma Aldrich), 12.5 g ml1; and kanamycin (Km) (SigmaAldrich), 50 g ml1. 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 at37C. 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 ......................................................AACC