1.  Write 1-2 paragraphs (2/3 page-dbl spaced) describing the work presented. Include in your overview, some background on the topic and the goals and rationale of the study. 

2.  Write 1-2 paragraphs (2/3 page-dbl spaced) describing the experimental design used to address the goal/problem and list the major results from each approach. Be sure to include the two key areas (proteomics results and central metabolism results) and how they connected the results from these two areas to make a complete story. 

3.  Answer the following questions (~1 1/2 page). 

1. Given what you know about regulation of central pathways, why did the authors not identify pathways that were required during infection in their proteomics experiments? 

2. Could an enzyme/pathway be important during infection but not show an effect when it is knocked-out? Why? 

3. What are the main findings in the paper? [hint: what does E. coli eat in the urinary tract?] 

4. What is an auxotroph? How did the author demonstrate auxotrophic phenotypes in vitro? What did the in vivo results from the 2 auxotrophs tell us about the urinary tract environment and how did these data support the main findings of the paper? 

Fitness of Escherichia coli during Urinary Tract Infection
Requires Gluconeogenesis and the TCA Cycle
Christopher J. Alteri, Sara N. Smith, Harry L. T. Mobley*

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America

Abstract

Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium’s ability to
elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and
cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to
persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the
expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment
and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using
two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that
UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P,0.005).
Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for
the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for
the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine
were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants
defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection
model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary
tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the
Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary
carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish
metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could
be considered critical components of virulence for pathogenic microbes.

Citation: Alteri CJ, Smith SN, Mobley HLT (2009) Fitness of Escherichia coli during Urinary Tract Infection Requires Gluconeogenesis and the TCA Cycle. PLoS
Pathog 5(5): e1000448. doi:10.1371/journal.ppat.1000448

Editor: Jorge E. Galán, Yale University School of Medicine, United States of America

Received October 6, 2008; Accepted April 27, 2009; Published May 29, 2009

Copyright: � 2009 Alteri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Funding was provided by Public Health Service grants AI43363 and AI059722 from the National Institutes of Health. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: hmobley@umich.edu

Introduction

Traditional studies of bacterial pathogenesis have focused on

pathogen-specific virulence properties including toxins, adhesins,

secretion, and iron acquisition systems, and mechanisms to avoid

the innate and adaptive immune response. Examining bacterial

metabolism during the course of an infection is also critical to

further our understanding of pathogenesis and identifying

potential targets for new antimicrobial agents. Infectious diseases

represent a serious threat to global health because many bacteria

that cause disease in humans such as Staphylococcus aureus,

Mycobacterium tuberculosis, and E. coli are steadily developing

resistance to many of the available treatments [1–3]. Since the

introduction of antibiotics in the last century, the emergence of

bacteria that resist these compounds has rapidly outpaced the

discovery and development of new antimicrobial agents [4]. The

need to understand bacterial physiology during infection of the

host is critical for the development of new antimicrobials or

antibiotics that will reduce their burden upon human health.

Among common infections, urinary tract infections (UTI) are

the most frequently diagnosed urologic disease. The majority of

UTIs are caused by E. coli and these uropathogenic E. coli (UPEC)

infections place a significant financial burden on the healthcare

system by generating annual costs in excess of two billion dollars

[5,6]. Because UTIs are a significant healthcare burden and E. coli

is one of the best studied model organisms for studying

metabolism, these traits can be exploited to understand and

identify metabolic pathways that are required for the growth of the

bacterium during infection of the host.

Despite being arguably the most studied organism, E. coli

metabolism during colonization of the intestine has only recently

been explored [7,8]. Commensal E. coli acquires nutrients from

intestinal mucus, a complex mixture of glycoconjugates, and

subsequently expresses genes involved in the catabolism of N-

acetylglucosamine, sialic acid, glucosamine, gluconate, arabinose

and fucose [8,9]. E. coli mutants in the Entner-Doudoroff

and glycolytic central metabolic pathways have diminished

colonization levels reflecting the importance of sugar acid

catabolism [8]. These findings suggest that commensal E. coli uses

multiple limiting sugars for growth in the intestine [8].

Together, this developing body of evidence supports the

assertion that E. coli grows in the intestine using simple sugars

released by the breakdown of complex polysaccharides by

anaerobes [9,10].

PLoS Pathogens | www.plospathogens.org 1 May 2009 | Volume 5 | Issue 5 | e1000448

Much less is known about the metabolism of enteric pathogens

during colonization of the gastrointestinal tract. Enterohemor-

rhagic E. coli (EHEC) O157:H7 requires similar carbon metabolic

pathways as do commensal strains, however, mutations in

pathways that utilize galactose, hexuronates, mannose, and ribose

resulted in colonization defects only for EHEC [9]. It was also

found that multiple mutations in a single EHEC strain had an

additive effect on colonization levels suggesting that this pathogen

depends on the simultaneous metabolism of up to six sugars to

support the colonization of the intestine [9]. When faced with

limiting sugars due to consumption by other colonizing bacteria,

EHEC may switch from glycolytic to gluconeogenic substrates to

sustain growth in the intestine [11]. Synthesis and degradation of

glycogen, an endogenous glucose polymer, plays an important role

for EHEC and pathogenic Salmonella during colonization of the

mouse intestine presumably by functioning as an internal carbon

source during nutrient limitation [12–14]. Although it is not

known which external carbon sources are used by S. enterica serovar

Typhimurium during colonization it has been demonstrated that

full virulence requires the conversion of succinate to fumarate in

the tricarboxylic acid (TCA) cycle [15,16]. These studies have

contributed much to the understanding of the in vivo metabolic

requirements of EHEC colonization; however, these studies were

done in an animal model that is not suitable for studying

pathogenesis because these animals do not exhibit signs of EHEC

infection [9,11,13].

In contrast to the nutritionally diverse intestine, the urinary tract

is a high-osmolarity, moderately oxygenated, iron-limited envi-

ronment that contains mostly amino acids and small peptides

[17,18]. The available studies on UPEC metabolism during UTI

has revealed that the ability to catabolize the amino acid D-serine

in urine, which not only supports UPEC growth, appears

important as a signaling mechanism to trigger virulence gene

expression [19,20]. Metabolism of nucleobases has been demon-

strated to play a role for UPEC colonization of the urinary tract;

signature-tagged mutagenesis screening identified a mutant in the

dihydroorotate dehydrogenase gene pyrD that was outcompeted by

wild-type UPEC in vivo [21] and in a separate transposon screen a

gene involved in guanine biosynthesis, guaA, was identified and

found to be attenuated during experimental UTI [22].

To better understand bacterial metabolism during infection, we

used a combination of comparative proteomics and systematic

disruption of central metabolism to identify pathways that are

required for UPEC fitness in vivo. By examining the expression of

UPEC cytoplasmic proteins during growth in human urine, we

confirmed that E. coli is scavenging amino acids and peptides and

found that disruption of peptide import in UPEC significantly

compromised fitness during infection. Consistent with the notion

that peptides are a key in vivo carbon source for UPEC, only

mutations ablating gluconeogenesis and the TCA cycle demon-

strated reduced fitness in vivo during experimental UTI. These

findings represent the first study of pathogenic E. coli central

metabolism in an infection model and further our understanding

of the role of metabolism in bacterial pathogenesis.

Results

Proteomic profile for uropathogenic E. coli growing in
urine

Culturing UPEC in human urine partially mimics the urinary

tract environment and has proven to be a useful tool to identify

bacterial genes and proteins involved in UTI [18,22–24]. Because

it is well established that urine is iron-limited and our previous

studies clearly demonstrated that the majority of differentially

expressed genes and proteins are involved in iron acquisition

[18,23], we determined the protein expression profile of E. coli

CFT073 during growth in human urine and compared that with

bacterial cells cultured in iron-limited LB medium to unmask

proteins involved in processes other than iron metabolism. Using

this strategy and 2D-DIGE it was possible to visualize 700

cytoplasmic protein spots, 84 of which were differentially

expressed (P,0.05) between urine and iron-limited LB medium

(Fig. 1). Of these, 56 were more highly expressed in human urine

(green) than in iron-limited LB medium, while 28 demonstrated

greater expression in iron-limited LB medium (red) than in urine

(Fig. 1).

Proteins induced in human urine with .2-fold differences from

expression levels in iron-limited LB medium were identified by

tandem mass spectroscopy (Table 1). The results indicate that E.

coli growing in urine are expressing proteins involved in the

catabolism of pentose sugars; XylA (xylose isomerase), AraF (high-

affinity arabinose-binding protein), and the non-oxidative pentose

Figure 1. Fluorescence difference in gel electrophoresis (2D-
DIGE) of UPEC cytoplasmic proteins during growth in urine.
Soluble proteins (50 mg) from E. coli CFT073 cultured in urine were
labeled with Cy3 (green), from CFT073 grown in LB with Cy5 (red), and
the pooled internal standard representing an equal amount of urine
and LB soluble proteins with Cy2 (blue). The labeled proteins (150 mg)
were pooled and applied to a pH 4–7 IPG strip and second dimension
10% SDS-PAGE. Green spots indicate protein features induced in urine;
red spots represent proteins induced in LB medium.
doi:10.1371/journal.ppat.1000448.g001

Author Summary

Bacteria that cause infections often have genes known as
virulence factors that are required for bacteria to cause
disease. Studying virulence factors such as toxins, adhe-
sins, and secretion and iron-acquisition systems is a
fundamental part of understanding infectious disease
mechanisms. In contrast, little is known about the
contribution of bacterial metabolism to infectious disease.
This study shows that E. coli, which cause most urinary
tract infections, utilize peptides as a preferred carbon
source in vivo and requires some, but not all, of the central
metabolic pathways to infect the urinary tract. Specifically,
pathways that can be used to replenish metabolites,
known as anaplerotic reactions, are important for uro-
pathogenic E. coli infections. These findings help explain
how metabolism can contribute to the ability of bacteria
to cause a common infection.

UPEC Metabolism during UTI

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phosphate pathway enzyme TalA (transaldolase) were induced

5.25-, 2.02-, and 5.66-fold, respectively (P,0.001) (Table 1). Other

proteins that were induced are the involved in metabolism of the

sugar acids gluconate (UxuA, mannonate dehydratase), glucono-

lactone (YbhE, 6-phosphogluconolactonase), sialic acid (NanA, N-

acetylneuraminate lyase), and fructose (FruB, fructose-specific

Table 1. UPEC cytoplasmic proteins differentially expressed in human urine.

Name ORF Function Fold-Change P-Value

OmpF c1071 outer membrane protein F precursor 7.84 2.50E-11

OmpF c1071 outer membrane protein F precursor 5.97 2.33E-05

TalA c2989 transaldolase 5.66 0.00021

XylA c4385 xylose isomerase 5.25 6.90E-07

TpiA c4871 triosephosphate isomerase 4.58 1.30E-07

SerA c3494 D-3-phosphoglycerate dehydrogenase 4.44 3.40E-09

SpeB c3522 agmantinase 4.06 3.90E-07

UxuA c5402 mannonate dehydratase 3.76 7.20E-03

NanA c3979 N-acetylneuraminate lyase subunit 3.64 4.50E-06

ArgG c3929 argininosuccinate dehydrogenase 3.41 5.80E-03

FklB c5306 peptidyl-prolyl cis trans isomerase 3.38 6.00E-04

NanA c3979 N-acetylneuraminate lyase subunit 3.37 4.50E-06

AtpA c4660 ATP synthase subunit A 3.34 6.30E-05

XylA c4385 xylose isomerase 3.32 5.60E-05

NmpC c1560 outer membrane protein NmpC precursor 3.3 6.10E-05

FruB c2704 PTS system, fructose-specific IIA/FPr component 2.93 3.40E-06

RpoA c4056 DNA-directed RNA polymerase 2.84 4.40E-04

GlyA c3073 serine hydroxymethyl transferase 2.72 1.50E-10

LivK c4248 leucine-specific binding protein 2.72 2.90E-08

FruB c2704 PTS system, fructose-specific IIA/FPr component 2.71 3.20E-04

DppA c4361 dipeptide substrate-binding protein 2.63 5.20E-04

SurA c0066 peptidyl-prolyl cis trans isomerase 2.61 3.10E-07

YliJ c0923 hypothetical GST protein 2.61 4.00E-04

HisJ c2851 histidine-binding protein precursor 2.55 1.90E-04

ArgG c3929 argininosuccinate dehydrogenase 2.41 2.60E-02

OppA c1707 oligopeptide substrate-binding protein 2.39 7.80E-03

OppA c1707 oligopeptide substrate-binding protein 2.34 2.10E-04

SerA c3494 D-3-phosphoglycerate dehydrogenase 2.28 1.90E-05

YghU c3726 hypothetical GST-like protein 2.27 1.10E-05

YbhE c0844 6-phosphogluconolactonase 2.2 9.90E-03

SucC c0805 succinyl-CoA synthetase beta chain 2.14 1.50E-04

GlpA c2782 anaerobic glycerol-3-phosphate dehydrogenase 2.13 3.50E-07

XylA c4385 xylose isomerase 2.11 1.30E-02

MalK c5005 maltose/maltodextran ATP-binding 2.1 6.90E-03

DppA c4361 dipeptide substrate-binding protein 2.09 8.40E-03

NmpC c1560 outer membrane protein NmpC precursor 2.03 1.80E-03

AraF c2314 L-arabinose-binding protein 2.02 2.80E-06

UxuA c5402 mannonate dehydratase 1.94 8.30E-04

AsnS c1072 asparaginyl-tRNA synthetase 1.9 1.20E-02

GlnH c0896 glutamine-binding protein 1.68 1.40E-03

GroEL c5227 chaperonin 22.07 8.90E-08

GroEL c5227 chaperonin 22.07 7.10E-05

NusA c3926 transcription elongation factor 22.1 3.30E-02

BasR c5118 transcription factor 22.91 5.50E-03

HdeB c4320 acid resistance protein precursor 23.71 2.50E-04

doi:10.1371/journal.ppat.1000448.t001

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IIA/FPr PTS system component). Multiple isoforms of the

periplasmic dipeptide and oligopeptide substrate-binding proteins

DppA and OppA were also induced (.2-fold, P,0.009) in urine

confirming the notion that amino acids and small peptides are

being acquired from this milieu (Table 1). Proteins involved in

amino acid metabolism were also identified and include SerA (D-

3-phosphoglycerate dehydrogenase) that is involved in serine

biosynthesis and two enzymes in the arginine biosynthesis

pathway, ArgG (argininosuccinate dehydrogenase) and SpeB

(agmatinase) (Table 1). As expected, none of the proteins identified

were involved in iron uptake or metabolism, although DppA has

been reported to bind heme albeit with less affinity than dipeptide

substrates [25].

Notably, there was an increase in abundance for two central

metabolism enzymes, TalA, as mentioned above, and TpiA that

was increased 4.58-fold (P,0.0001) in urine (Table 1). TalA, a

non-oxidative pentose phosphate pathway enzyme, converts

sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate to

erythrose-4-phosphate and fructose-6-phosphate. Due to the

transfer of the glycolytic intermediate glyceraldehyde-3-phosphate

by TalA, this enzyme is an important link between the pentose

phosphate pathway and glycolysis [26]. TpiA is a glycolytic

enzyme that catalyzes the reversible isomerization of glyceralde-

hyde-3-phosphate and dihydroxyacetone phosphate [27]. The

induction of TalA and TpiA suggested that the coupling of the

pentose phosphate pathway and glycolysis or gluconeogenesis via

the transfer and isomerization of glyceraldehyde-3-phosphate may

be an important route of carbon flux through these central

pathways during the bacterium’s growth in human urine.

Contribution of genes induced in urine to UPEC fitness in
vivo

To determine whether some proteins identified by 2D-DIGE

are required for UPEC fitness during UTI, CFT073 mutants were

constructed in the genes: talA, xylA, tpiA, serA, speB, uxuA, nanA,

argG, araF, dppA, and oppA. For these studies, an experimental

competition between each mutant strain and wild-type parental

CFT073 was performed. Wild-type UPEC and the mutant strain

were prepared in a 1:1 ratio and transurethrally inoculated into

the bladders of mice. The number of mutant (kanamycin-resistant)

and wild-type (kanamycin-sensitive) bacteria recovered from the

bladder and kidneys was determined by plating the tissue

homogenates for CFU on both LB agar and LB agar containing

kanamycin. Mutants containing defects in genes that affect fitness

in vivo are out-competed by the wild-type strain when inoculated

into the same animal. This was determined by comparing the ratio

of colony forming units (CFU) of bacteria recovered from the

infection to the ratio of bacteria contained within the inoculum to

obtain a competitive index (CI). A CI.1 indicates the wild-type

out-competes the mutant strain and a CI,1 indicates the wild-

type is out-competed by the mutant. In these series of

experimental infections, only mutants defective in peptide

transport (DdppA and DoppA) were dramatically out-competed by
wild-type UPEC in vivo, CI.50, P,0.005 for the bladder (Table 2).

One additional mutant, DtpiA, that functions in both glycolysis and
gluconeogenesis, was out-competed by wild-type in the kidneys at

48 hpi, CI = 2.54, P = 0.0206 (Table 2).

Despite the lack of attenuation in vivo for the many of the mutants,

these results reveal a number of important findings. The agmatinase

mutant DspeB out-competed wild-type in the bladder at 48 hpi,
CI = 0.14, P = 0.0122 (Table 2). Agmatinase is part of arginine

metabolism and catalyzes the formation of the polyamine putrescine

and urea from agmatine and H2O. This suggests that accumulation

of agmatine or reduced production of urea and putrescine by the

mutant may provide a modest advantage over wild-type UPEC

during infection of the bladder. CFT073 DargG was unable to grow
in MOPS defined medium unless supplemented with 10 mM

arginine (Fig. 2A), validating the expected auxotrophic phenotype.

Similarly, the DserA serine auxotroph required supplementation
with either 10 mM serine or glycine in MOPS, D-serine was unable

to rescue the in vitro growth defect (Fig. 2B). Lack of arginine or

serine biosynthesis had little effect upon the ability of UPEC to grow

logarithmically in human urine, although the DargG mutant
consistently entered stationary phase at a lower cell density, with

an O.D.600 of 0.4560.04 compared to 0.5960.03 for wild-type

(P = 0.051) (Fig. 2C). When tested for in vivo fitness, neither the

DargG nor DserA strain were significantly out-competed by wild-type
UPEC at 48 hpi (Fig. 2D, 2E, and Table 2). Additionally, there was

no preference for serine over arginine or vice versa for UPEC

colonization at 48 hpi. When the auxotrophic strains were co-

inoculated into the same mice both mutants were recovered at

similar levels (Fig. 2F). These data clearly demonstrate that there are

sufficient concentrations of arginine, serine and/or glycine in the

urinary tract to support growth of these auxotrophic strains.

As mentioned, deletion of the genes encoding periplasmic

peptide substrate-binding proteins, dppA and oppA, had the greatest

impact on UPEC fitness in vivo of the CFT073 mutants in genes

whose products were induced during growth in human urine

(Table 2). The dipeptide transport mutant, DdppA, failed to
maintain colonization in the bladder at 48 hpi, 11/11 bladders

had undetectable levels (,200 CFU/g) for this mutant, while wild-

type levels from the same bladders reached a median of 10
4

CFU/

g (P = 0.0020) (Fig. 3A). Because these mice had low levels of

recoverable UPEC from the kidneys it was not possible to

determine the contribution of dipeptide transport for kidney

colonization. Import of oligopeptides via the OppA substrate-

binding protein is also required for UPEC fitness in vivo. CFT073

DoppA was out-competed nearly 500:1 wild-type:mutant in the
bladder (Table 2) with a 3-log reduction in the median CFU/g

from bladder tissue at 48 hpi (P = 0.0047) (Fig. 3B). In these co-

challenge infections, wild-type UPEC colonized 10/16 (62%) of

kidneys, while DoppA was detectable in 4/16 (25%) of kidneys at

Table 2. In vivo fitness for select 2D-DIGE mutants.

Bladder Kidneys

CI
a P-Valueb CIa P-Valueb

talA 0.150 0.1282 0.660 0.3829

xylA 1.66E202 0.0625 0.233 0.0649

tpiA 0.841 0.4050 2.540 0.0206

serA 5.310 0.4206 1.58 0.5476

speB 0.140 0.0122 2.248 0.3652

uxuA 0.397 0.0667 0.608 0.1750

nanA 0.659 0.1875 1.240 0.4075

argG 0.160 0.0625 1.970 0.3750

araF 0.854 0.4401 0.297 0.4507

dppA 56.33 0.0020 1.408 0.5625

oppA 4.77E+02 0.0047 1.56E+02 0.0420

aCompetitive Index, determined by dividing the ratio of wild-type to mutant at
48 hpi by the ratio present in the inoculum. Significant CI.1 indicates mutant
has a fitness defect.

b
P-values determined by Wilcoxon matched pairs test. Significant P-values are
bolded.

doi:10.1371/journal.ppat.1000448.t002

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48 hpi. The ratio of wild-type:mutant recovered from the kidneys

at this time point was 156:1 (Table 2) where wild-type UPEC had

3-logs greater CFU/g than DoppA (P = 0.0420) (Fig. 3B). Together,
the in vivo fitness defect for CFT073 harboring a deletion of either

dppA or oppA suggests that peptides may be an important carbon

source for UPEC during urinary tract infection.

Previously, we have shown that the low copy pGEN plasmid is

maintained in CFT073 in the absence of antibiotic pressure for up

Figure 2. In vivo contribution of UPEC arginine and serine biosynthesis. Demonstration of auxotrophic phenotypes for (A) DargG and (B)
DserA in MOPS defined medium containing 0.2% glucose and 10 mM of the indicated amino acid. (C) Growth in human urine. Growth curves
represent the average measurement at each time point from triplicate experiments. Individual female mice were transurethrally inoculated with
26108 CFU of a 1:1 mixture of wild-type and mutant bacteria. In vivo fitness at 48 h post infection (hpi) for UPEC mutants defective in (D) arginine and
(E) serine biosynthesis. (F) In vivo competition between arginine and serine auxotrophy. At 48 hpi, bladders and kidneys were aseptically removed,
homogenized, and plated on LB or LB containing kanamycin to determine viable counts of wild-type and mutant strains, respectively. Each dot
represents the log CFU/g from an individual animal. Bars represent the median CFU/g, and the limit of detection is 200 CFU. Significant differences in
colonization levels (P,0.05)

were determined using a two-tailed Wilcoxon matched pairs test.

doi:10.1371/journal.ppat.1000448.g002

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to 48 h [28]. Using this ampicillin resistant plasmid system, we

cloned the entire dppA gene including 200 bp upstream from the

predicted start site of translation and introduced the resulting

construct, pGEN-dppA, into the CFT073 DdppA strain. To
determine if it was possible to complement the DdppA defect in
vivo, co-challenge infections were performed as described and

modified to enumerate bacteria in tissue homogenates by plating

on agar containing ampicillin (wild-type CFT073 harboring

pGEN) or ampicillin and kanamycin (CFT073 DdppA containing
pGEN or pGEN-dppA). The DdppA mutant containing empty
vector (pGEN-) demonstrated the expected fitness defect in

bladder colonization when co-inoculated with wild-type CFT073

(pGEN-) (P = 0.0002) while DdppA containing a wild-type copy of
dppA (pGEN-dppA) restored colonization to wild-type levels in the

bladder at 48 hpi (Fig. 3C). Although both mutant (pGEN-) and

wild-type (pGEN-) demonstrated poor colonization in the kidneys

of these animals, complementation of DdppA (pGEN-dppA) resulted
in a 2-log increase in median kidney CFU/g at 48 hpi (Fig. 3D).

Fitness of UPEC central carbon metabolism mutants
during UTI

The requirement for peptide transport for UPEC fitness during

infection implicates peptides as an important carbon source in vivo.

This predicts that certain central metabolism pathways that

operate during catabolism of amino acids or peptides may be more

important for in vivo growth of UPEC than pathways that function

primarily to catabolize sugars. To test the role of central metabolic

pathways during an actual infection mutants were constructed in

UPEC strain CFT073 to produce defects in glycolysis (pgi,

phosphoglucose isomerase and tpiA, triosephosphate isomerase)

[29], the Entner-Doudoroff pathway (edd, 6-phosphogluconate

dehydratase) [10], the oxidative branch (gnd, 6-phosphogluconate

Figure 3. In vivo contribution of UPEC peptide substrate-binding proteins. Individual female mice were transurethrally inoculated with
26108 CFU of a 1:1 mixture of wild-type and mutant bacteria. In vivo fitness at 48 hpi for UPEC mutants defective in import of dipeptides (A) DdppA
or oligopeptides (B) DoppA. At 48 hpi, bladders and kidneys were aseptically removed, homogenized, and plated on LB or LB containing kanamycin
to determine viable counts of wild-type and mutant strains, respectively. In vivo complementation of DdppA was performed by inoculating mice with
a mixture of wild-type CFT073 containing pGEN empty vector and DdppA containing pGEN empty vector or pGEN-dppA. At 48 hpi, (C) bladders and
(D) kidneys were aseptically removed, homogenized, and plated on LB with ampicillin or LB containing ampicillin and kanamycin to determine viable
counts of wild-type (closed symbols) and mutant strains (open symbols), respectively. Each dot represents the log CFU/g from an individual animal.
Bars represent the median CFU/g, and the limit of detection is 200 CFU. Significant differences in colonization levels (P,0.05) are indicated and were
determined using a two-tailed Wilcoxon matched pairs test.
doi:10.1371/journal.ppat.1000448.g003

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dehydrogenase) and the non-oxidative branch (talA, transaldolase)

of the pentose phosphate pathway [26], gluconeogenesis (pckA,

phosphoenolpyruvate carboxykinase) [30], and the TCA cycle

(sdhB, succinate dehydrogenase) [31]. The in vitro growth of these

central metabolism mutants were examined and compared to

wild-type UPEC during culture in human urine, LB medium, and

MOPS defined medium containing 0.02% glucose. All of the

central metabolism mutants produced similar logarithmic growth

as wild-type when cultured in human urine (Fig. 4A) and LB

medium (data not shown) under defined inoculation conditions. As

expected, only mutants with defects in glycolysis demonstrated

diminished growth in MOPS medium containing glucose as the

sole carbon source (Fig. 4B). The Dpgi strain produced an extended
lag phase of 5.561.1 h compared with wild-type (P = 0.001) and

DtpiA failed to reach exponential phase after 18 h (Fig. 4B). These
data and the indistinguishable growth of the glycolysis mutants

from wild-type in urine supported the proteomics data and

indicated that UPEC growing in urine utilizes carbon sources

other than glucose.

To determine the role for central metabolism during E. coli

infection of the urinary tract, the ascending model of murine UTI

was used as described above to measure the impact that a lesion in

central metabolism has upon the relative fitness of the strain in vivo.

Mutants with defects in glycolysis had levels of colonization in the

bladder at 48 hpi similar to wild-type (P.0.400) (Fig. 5A and 5B).

In the kidneys, Dpgi CFU/g were comparable to wild-type
(Fig. 5A), while DtpiA demonstrated a 10-fold reduction in the
median CFU/g (P = 0.0206) (Fig. 5B). The pentose phosphate

pathway mutants, Dgnd (Fig. 5C) and DtalA (Table 2), were not
significantly out-competed by wild-type in vivo. The mutant with a

defect in the Entner-Doudoroff pathway (Dedd) also was not
impaired in the ability to infect both the bladder and kidneys as

indicated by its similar colonization to wild-type at 48 hpi

(Fig. 5D). UPEC in vivo fitness was significantly reduced in the

TCA cycle mutant DsdhB, this mutation resulted in a 50-fold
reduction in median CFU/g in the bladder (P = 0.0134) and a 1.5-

log decrease in kidney CFU at 48 hpi (P = 0.0400) (Fig. 5E). This

defect in the TCA cycle impacted fitness to a greater extent in the

bladder, where 11/15 (73%) of mice had undetectable levels of

mutant bacteria, than in the kidneys where 6/15 (40%) mice had

undetectable counts (Fig. 5E). The gluconeogenesis mutant, DpckA
had a 2-log reduction in median CFU/g in both the bladder

(P = 0.0005) and kidneys (P = 0.0322) and half of the mice (7/14)

displayed undetectable levels of DpckA at 48 hpi (Fig. 5F).
To verify that this mutation is non-polar as expected and the

defect in colonization is not due to a secondary mutation, in vivo

complementation experiments were conducted. The DpckA mutant
with the pGEN empty vector demonstrated a 2-log reduction in

CFU/g at 48 hpi (P = 0.0039) in the bladder when co-inoculated

into mice with wild-type UPEC containing pGEN (Fig. 6). When

CFT073 DpckA (pGEN-pckA) were co-inoculated with CFT073
(pGEN-) there was no significant difference in bladder CFU/g at

48 hpi between the strains (Fig. 6). Thus, by re-introducing the

pckA gene into the mutant it was possible to complement the DpckA
defect in bladder colonization at 48 hpi.

The in vitro growth and in vivo fitness for the UPEC central

metabolism mutants is summarized in Table 3. As expected, only

mutations in glycolysis had a negative effect on growth in defined

medium with glucose. Only gluconeogenesis or TCA cycle

mutants demonstrated reduced persistence at 48 hpi in both the

bladder and kidneys (Table 3). Non-oxidative and oxidative

pentose phosphate pathway and Entner-Doudoroff pathway

mutants did not demonstrate any colonization defect and of the

glycolytic mutants only the triosephosphate isomerase deletion had

a measurable defect in the kidneys but not in the bladder (Table 3).

Together, the fitness defect for the peptide transport mutants and

these data indicate UPEC could be using amino acids as the

primary carbon source during infection. Surprisingly, there was no

correlation between the ability of the central metabolism mutants

to grow in human urine ex vivo and grow in the urinary tract in vivo.

Discussion

Bacterial pathogenesis traditionally involves studying virulence

traits involved in the production of toxins and effectors, iron

acquisition, adherence, invasion, and immune system avoidance.

Although many paradigms exist that describe mechanisms of

pathogenesis, the contribution of microbial metabolism to

bacterial virulence during an infection is less understood. Much

work has been done studying E. coli as model organism for

characterizing individual central metabolism pathways and

enzymes [10,27,32–38]. We have shown here that central

metabolism studies in E. coli can be extended to investigate the

contribution of central pathways to bacterial pathogenesis using a

virulent uropathogenic E. coli strain and a well-established animal

model of UTI. It is known that commensal E. coli require the

Entner-Doudoroff pathway and glycolysis for colonization in vivo;

while the TCA cycle, pentose phosphate pathway, and gluconeo-

Figure 4. In vitro growth of UPEC central metabolism mutants.
Optical density of wild-type UPEC and central metabolism mutants
during growth in (A) pooled and sterilized human urine from 8–10
donors and in (B) MOPS defined medium containing 0.2% glucose as
the sole carbon source. Growth curves represent the average
measurement at each time point from triplicate experiments.
doi:10.1371/journal.ppat.1000448.g004

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Figure 5. In vivo fitness of UPEC central metabolism mutants. Individual female mice were transurethrally inoculated with 26108 CFU of a 1:1
mixture of wild-type and mutant bacteria. In vivo fitness at 48 hpi for UPEC mutants defective in: (A,B) glycolysis, (C) pentose phosphate pathway, (D)
Entner-Doudoroff pathway, (E) TCA cycle, and (F) gluconeogenesis. At 48 hpi, bladders and kidneys were aseptically removed, homogenized, and
plated on LB or LB containing kanamycin to determine viable counts of wild-type and mutant strains, respectively. Each dot represents the log CFU/g
from an individual animal. Bars represent the median CFU/g, and the limit of detection is 200 CFU. Significant differences in colonization levels
(P,0.05) are indicated and were determined using a two-tailed Wilcoxon matched pairs test.
doi:10.1371/journal.ppat.1000448.g005

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genesis are dispensable in the intestine [8]. In contrast, we have

shown that during E. coli infection of the urinary tract, the

pathways required for commensal colonization are dispensable

while the TCA cycle and gluconeogenesis are necessary for UPEC

fitness in vivo. Adaptation to distinct host environments has been

previously shown to involve shared traits between commensal and

pathogenic strains [39,40]. Because commensal E. coli are an

important natural component of the intestine one concern faced

when developing antimicrobials that target pathogenic strains is

how to avoid eradicating commensal bacteria. Thus, these findings

highlight important differences between commensal and patho-

genic E. coli that could be exploited for the development of

antimicrobials that target these pathways in this pathogen during

infections that may not affect commensal strains. Interestingly, in

addition to UPEC, gluconeogenesis is required for virulence in

microbes that represent an array of pathogenic lifestyles, from

intracellular bacteria and parasites [41,42], plant-pathogenic [43],

and intestinal pathogens [16]; suggesting that anaplerosis may be a

common mechanism of microbial pathogenesis.

This study comprehensively examines the role of pathogenic E.

coli central metabolism in a disease model and provides insight not

only into UPEC metabolism in vivo but also information regarding

the nutrients available to support the growth of E. coli within the

urinary tract. The proteomics experiments did reveal that UPEC

growing in human urine induces expression of multiple isoforms of

both dipeptide- and oligopeptide-binding proteins, both of which

were found to be required for UPEC to effectively colonize the

urinary tract. This indicates that these bacteria actively import

short peptides in urine and this function may indicate that peptides

are an important carbon source in vivo. Consistent with this, only

bacteria with defects in peptide transport, gluconeogenesis, or the

TCA cycle demonstrated a significant reduction in fitness in vivo in

both the bladder and kidneys. These findings suggest a model that

describes the biochemistry of E. coli during UTI. For optimal

growth during infection, short peptides are taken up by UPEC and

degraded into amino acids that are catabolized and used in a series

of anaplerotic reactions that replenish TCA cycle intermediates

and generate gluconeogenesis substrates (Fig. 7).

Certain glycolytic steps are irreversible and the reverse

gluconeogenic reaction is performed by an enzyme specific for

gluconeogenesis. Carbon flux through glycolysis and gluconeo-

genesis must be carefully controlled by the cell to avoid a futile

cycle of carbon metabolism [44]. Allosteric regulation of enzymes

that catalyze irreversible reactions in these pathways and

catabolite repression are mechanisms used to avoid the futile

cycle [45,46]. A gluconeogenic-specific enzyme subject to

allosteric regulation is phophoenolpyruvate carboxykinase that

converts oxaloacetate to phosphoenolpyruvate [47]. Deletion of

the gene pckA that encodes this enzyme resulted in a significant

reduction in UPEC fitness in vivo. Because bacteria prevent

glycolysis and gluconeogenesis from occurring simultaneously and

deletion of pckA reduced fitness in vivo, we reason that carbon flux

through gluconeogenesis during UPEC infection may be an

important indication of amino acid catabolism in vivo.

It is not surprising that, in addition to gluconeogenesis, the TCA

cycle is also required for UPEC fitness in vivo. These two pathways

are connected and collectively described as ‘‘filling in’’ or

anaplerotic reactions. The TCA cycle is necessary to provide

substrates for gluconeogenesis when cells use amino acids as a

carbon source. Gluconeogenic amino acids can be degraded to

oxaloacetate or to pyruvate that can be converted to acetyl-CoA

and enter the TCA cycle [47]. Oxaloacetate, a TCA cycle

intermediate, is converted to phophoenolpyruvate during gluco-

neogenesis by PckA as described above. A mutation in the TCA

cycle enzyme succinate dehydrogenase, sdhB, results in a UPEC

strain that has reduced fitness in vivo. This finding suggests that

UPEC are growing aerobically in the urinary tract because

succinate dehydrogenase is replaced by fumarate reductase during

anaerobic growth and therefore, future work could confirm if the

reductive TCA cycle is not operating during UPEC infection. The

requirement for peptide import and the TCA cycle for UPEC

fitness during infection is consistent with the hypothesis that acetyl-

CoA production from the degradation of amino acids could be

occurring in vivo as has been shown by another group [48].

Interestingly, with the exception of peptide-transport proteins,

up-regulation of protein expression in urine ex vivo did not correlate

with functional importance in vivo. This could be due to the fact

that many central metabolism genes are constitutively expressed

and that human urine only partially mimics the complex lifestyle

of UPEC during UTI [49]. The absence of host cells and the

Figure 6. In vivo complementation of UPEC DpckA. Individual
female mice were transurethrally inoculated with 26108 CFU of a 1:1
mixture of wild-type CFT073 containing pGEN empty vector and DpckA
containing pGEN empty vector or pGEN-pckA. At 48 hpi, bladders were
aseptically removed, homogenized, and plated on LB with ampicillin or
LB containing ampicillin and kanamycin to determine viable counts of
wild-type (closed symbols) and mutant strains (open symbols),
respectively. Bars represent the median CFU/g, and the limit of
detection is 200 CFU. Significant differences in colonization levels
(P,0.05) are indicated and were determined using a two-tailed
Wilcoxon matched pairs test.
doi:10.1371/journal.ppat.1000448.g006

Table 3. Growth of central metabolism mutants in vitro and
in vivo.

Mutant Pathway In Vitro Growth In Vivo

LB Urine Glucose
Colonization
Defect

edd Entner-Doudoroff + + + None

gnd Pentose phosphate + + + None

pckA Gluconeogenesis + + + Bladder, Kidneys

pgi Glycolysis + + 2 None

sdhB TCA cycle + + + Bladder, Kidneys

talA Pentose phosphate + + + None

tpiA Glycolysis + + 2 Kidneys

doi:10.1371/journal.ppat.1000448.t003

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immune response during growth in urine ex vivo could in part

account for this discrepancy. It also remains possible that mutants

that lack growth defects in urine but demonstrate reduced fitness in

vivo could represent genes or metabolic pathways that are required

for intracellular phases of growth during cystitis [50].

Despite these disadvantages, up-regulation of both DppA and

OppA expression was seen in urine and loss of either dppA or oppA

was found to negatively impact UPEC colonization in vivo.

Induction of dppA has been reported in a hypervirulent UPEC

strain that has a lacks a functional D-serine deaminase gene (dsdA)

[51]. Deletion of dppA in this mutant strain resulted in a loss of the

hypervirulent phenotype in vivo and significantly reduced its ability

to colonize the urinary tract in competition with wild-type [51].

Surprisingly, in contrast to our findings, this group found that

mutation of dppA alone had no effect on UPEC fitness in vivo [51].

Due to lack of complementation, it is unclear from that work why

loss of dppA dramatically attenuated a hypervirulent strain but had

no effect on wild-type. Despite this inconsistency in that work, the

importance of peptide transport for UPEC fitness in vivo is

supported by the findings that loss of either dppA or oppA

significantly reduced colonization of the urinary tract and that

the reduced bacterial colonization in the DdppA strain can be
restored to wild-type levels by complementing the mutant with a

wild-type dppA gene.

In summary, defects in the both branches of the pentose

phosphate pathway, the Entner-Doudoroff pathway, and glycolysis

had limited or no impact on UPEC fitness in vivo. On the other

hand, the TCA cycle- and gluconeogenesis-defective strains

demonstrate significant fitness reductions during UTI. The

utilization of short peptides and amino acids as a carbon source

during bacterial infection of the urinary tract is supported by the

observation that UPEC mutants defective in peptide import have

reduced fitness in vivo while auxotrophic strains do not. Together,

these findings provide compelling evidence to support the notion

that catabolism of amino acids to form TCA cycle intermediates

and gluconeogenic substrates is important for the ability of UPEC

to infect the urinary tract efficiently. This shows that anaplerotic

and central metabolism pathways are required for UPEC fitness in

vivo and suggest microbial metabolism should be considered

important for bacterial pathogenesis.

Materials and Methods

Bacteria and growth conditions
Strains were derived from E. coli strain CFT073, a prototypic

UPEC strain isolated from the blood and urine of a patient with

acute pyelonephritis [52]; its genome has been sequenced and fully

annotated [53]. Isolated colonies were used to inoculate overnight

Luria-Bertani (LB) cultures. Bacteria from overnight cultures were

collected by centrifugation, washed with sterile PBS, and 10
6

CFU

were used to inoculate pre-warmed LB or human urine. To mimic

iron-limitation in urine, LB containing 10 mM deferoxamine

mesylate (Sigma) was used as a growth medium for comparative

proteomics. For human urine cultures, mid-stream urine was

collected into sterile sample containers from 8–10 male and female

donors, pooled, and sterilized by vacuum filtration through a

0.22 mm pore filter. MOPS defined medium containing 0.2%
glucose [54] with and without 10 mM L-arginine, L-serine,

Figure 7. UPEC acquires amino acids and requires gluconeogenesis and the TCA cycle for fitness in vivo. Peptide substrate-binding
protein genes dppA and oppA are required to import di- and oligopeptides into the cytoplasm from the periplasm. Short peptides are degraded into
amino acids in the cytoplasm and converted into pyruvate and oxaloacetate. Pyruvate is converted into acetyl-CoA and enters the TCA cycle to
replenish intermediates and generate oxaloacetate. Oxaloacetate is converted to phosphoenolpyruvate by the pckA gene product during
gluconeogenesis. Mutations in the indicated genes dppA, oppA, pckA, sdhB, and tpiA demonstrated fitness defects in vivo.
doi:10.1371/journal.ppat.1000448.g007

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glycine, aspartatic acid, or D-serine (Sigma) was also used to test

growth of mutant strains. Growth curves were established in

triplicate using a Bioscreen bioanalyzer in 0.4 ml volumes; OD600
was recorded every 15 min. All cultures were incubated at 37uC;
LB overnight and MOPS cultures were incubated with aeration;

urine cultures were incubated statically. For preparation of

proteins, UPEC isolate CFT073 was grown statically to exponen-

tial phase (OD600 = 0.25) in pre-warmed LB or human urine at

37uC in 56100 ml cultures for each growth medium.

Preparation of cytoplasmic proteins
Bacteria were harvested from 500 ml of culture by centrifuga-

tion (10,0006 g, 30 min, 4uC) and lysed in a French pressure cell
at 20,000 psi. Harvested cells were washed and resuspended in

10 ml of 10 mM HEPES, pH 7.0 containing 100 U of Benzonase

(Sigma). Following two passes through the chilled pressure cell,

lysates were centrifuged (75006 g, 10 min, 4uC) to remove
unbroken cells and supernatants were ultracentrifuged (120,0006
g, 1 h, 4uC) to remove membranes and insoluble material. Soluble
proteins were quantified using the 2D Quant Kit (GE Healthcare)

following the manufacturer’s protocol and either used immediately

in DIGE-labeling procedures or stored at 280uC.

2D-DIGE and MS/MS
For fluorescence difference in gel electrophoresis (2D-DIGE)

[55], bacterial proteins were minimally labeled with cyanine-

derived fluors (CyDyes) containing an NHS ester-reactive group as

recommended by the manufacturer (GE Healthcare). To deter-

mine quantitative differences within the UPEC soluble proteome

during growth in human urine, cytoplasmic proteins prepared

from human urine cultures were labeled with Cy3, from LB broth

with Cy5, and a pooled internal standard representing equal

amounts of both urine and LB preparations with Cy2 as described

previously [23]. Briefly, 50 mg of protein was incubated with
400 pmol CyDye for 30 min and the reaction was stopped by

added 10 mM lysine. Following labeling, samples labeled with

each CyDye were pooled (150 mg total protein), mixed with an
equal volume of 26DIGE sample buffer; 7 M urea, 2 M thiourea,
10 mM tributylphosphine (TBP) (Sigma), 26 biolytes 3–10 (Bio-
Rad), 2% ASB-14 and incubated on ice for 10 min. For

rehydration, samples were brought to 0.35 ml with 16 DIGE
rehydration buffer (7 M urea, 2 M thiourea, 5 mM TBP, 16
biolytes 3–10, 1% ASB-14) and used to passively rehydrate pH 4–

7 IPG strips (Bio-Rad) overnight at room temperature. Rehydrat-

ed IPG strips were equilibrated and subjected to isoelectric

focusing for 50,000 V?h and second dimension SDS-PAGE on

10% gels within low fluorescence glass plates (Jule Biotechnologies,

Inc.) and were run at a constant current of 55 mA at 4uC for 4 hr.
Following SDS-PAGE, image acquisition and pixel intensity was

obtained using a Typhoon scanner (GE Healthcare) and

differential in-gel analysis and biological analysis of variance were

performed using the DeCyder 6.5 software suite (GE Healthcare).

Using this software, the normalized spot volume ratios from Cy3

or Cy5 labeled spots were quantified relative to the Cy2-labeled

internal standard from the same gel. The Cy2-labeled standard

was then used to standardize and compare normalized volume

ratios between the Cy3 and Cy5 labeled proteins between gels

representing three independent experiments to generate statistical

confidence for abundance changes using student’s t-test and

ANOVA. To identify the proteins, 500 mg of cytoplasmic proteins
were focused as described above and spots of interest were excised

from a colloidal Coomassie-stained 2D SDS-PAGE gel and

subjected to enzymatic digestion with trypsin. Mass spectra were

acquired on an Applied Biosystems 4700 Proteomics Analyzer

(TOF/TOF). MS spectra were acquired from 800–3500 Da and

the eight most intense peaks in each MS spectrum were selected

for MS/MS analysis. Peptide identifications were obtained using

GPS Explorer (v3.0, Applied Biosystems), which utilizes the

MASCOT search engine. Each MS/MS spectrum was searched

against NCBInr. Tryptic digestion and tandem mass spectrometry

were performed at the University of Michigan Proteome

Consortium.

Construction of UPEC metabolism mutants
Deletion mutants were generated using the lambda red

recombinase system [56]. Primers homologous to sequences

within the 59 and 39 ends of the target genes were designed and

used to replace target genes with a nonpolar kanamycin resistance

cassette derived from the template plasmid pKD4 [56]. Kanamy-

cin (25 mg/ml) was used for selection of all mutant strains. Gene
deletions begin with the start codon and end with the stop codon

for each gene. To determine whether the kanamycin resistance

cassette recombined within the target gene site, primers that flank

the target gene sequence were designed and used for PCR. After

amplification, each PCR product was compared to wild-type PCR

product and in cases where size-differences are negligible; PCR

products were digested with the restriction enzyme EagI (New

England Biolabs). Both the PCR products and restriction digests

were visualized on a 0.8% agarose gel stained with ethidium

bromide. For in vivo complementation, the dppA and pckA genes

were amplified from CFT073 genomic DNA using Easy-A high-

fidelity polymerase (Stratagene) and independently cloned into

pGEN-MCS [28,57] using appropriate restriction enzymes. The

sequences of pGEN-dppA and pGEN-pckA were verified by DNA

sequence analysis prior to electroporation into CFT073 DdppA or
DpckA mutant strains.

Experimental UTI
Six-to eight-week-old female CBA/J mice (20 to 22 g; Jackson

Laboratories) were anesthetized with ketamine/xylazine and

inoculated transurethrally over a 30 sec period with a 50 ml
bacterial suspension per mouse using a sterile polyethylene

catheter (I.D. 0.28 mm6O.D. 0.61 mm) connected to an infusion
pump (Harvard Apparatus). To measure relative fitness, overnight

LB cultures for CFT073 and the mutant strain were collected by

centrifugation and resuspended in sterile PBS, mixed 1:1 and

adjusted to deliver 26108 CFU per mouse. Dilutions of each
inoculum were spiral plated onto LB with and without kanamycin

using an Autoplate 4000 (Spiral Biotech) to determine the input

CFU/mL. After 48 hpi, mice were sacrificed by overdose with

isoflurane and the bladder and kidneys were aseptically removed,

weighed, and homogenized in sterile culture tubes containing 3 ml

of PBS using an OMNI mechanical homogenizer (OMNI

International). Appropriate dilutions of the homogenized tissue

were then spiral plated onto duplicate LB plates with and without

kanamycin to determine the output CFU/g of tissue. Plate counts

obtained on kanamycin were subtracted from those on plates

lacking antibiotic to determine the number of wild-type bacteria.

Competitive indices were calculated by dividing the ratio of wild-

type to mutant at 48 hpi by the ratio of wild-type to mutant input

CFU/mL. Groups of 5 mice per co-challenge were used to

determine defects in fitness, when a defect was apparent the co-

challenge was repeated two more times with groups of 5 mice.

Statistically significant differences in colonization (P-value,0.05)

were determined using a two-tailed Wilcoxon matched pairs test.

All animal protocols were approved by the University Committee

on Use and Care of Animals at the University of Michigan

Medical School.

UPEC Metabolism during UTI

PLoS Pathogens | www.plospathogens.org 11 May 2009 | Volume 5 | Issue 5 | e1000448

Acknowledgments

The authors would like to thank Daniel Reiss for assisting with the

proteomics experiments.

Author Contributions

Conceived and designed the experiments: CJA. Performed the experi-

ments: CJA SNS. Analyzed the data: CJA HLTM. Wrote the paper: CJA

HLTM.

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