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Infect Immun, March 1998, p. 907-911, Vol. 66, No. 3
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
D-Lactate Production and
[14C]Succinic Acid Uptake by Adherent and Nonadherent
Escherichia coli
K.
McCabe,
M. D.
Mann,* and
M. D.
Bowie
Institute of Child Health, University of Cape
Town, Red Cross War Memorial Children's Hospital, Rondebosch 7700, South Africa
Received 15 May 1997/Returned for modification 15 July
1997/Accepted 11 December 1997
 |
ABSTRACT |
Escherichia coli isolates of different adherence
phenotypes produced different amounts of D-lactate.
Alterations of culture conditions did not influence the amount of
D-lactate produced. The observed pH decreases in tissue
culture medium corresponded with increases in D-lactate
concentration. Very little [14C]succinic acid was
incorporated into cells during the in vitro incubation of adherent and
nonadherent E. coli with HeLa cells, but the amounts of
tracer removed from the culture medium by adherent and nonadherent
strains differed. The results are further evidence of a difference in
the metabolic behavior of adherent and nonadherent E. coli.
| |
INTRODUCTION |
One of the virulence associated
properties of enteropathogenic Escherichia coli (5, 13,
14) is the ability to adhere to small intestinal mucosa (3,
11, 12, 21, 24, 26, 27). Although this adherence is an important
event in the induction of diarrhea, the mechanism by which adherent
E. coli mediates pathogenicity remains uncertain
(1, 2, 7, 18, 26, 27).
Several studies have shown that the in vitro adherence of E. coli to HEp-2 or HeLa cells in tissue culture can be used as a
marker of enteroadherence (4, 6, 8, 9, 15, 16, 19, 22, 23, 28,
29). We used the HeLa assay (20) to detect this
virulence characteristic in E. coli isolates from infants with acute diarrhea and, during the 3-h assay, observed E. coli-induced changes in the pH of the tissue culture
medium (17). The pH changes induced by organisms with
different adherence phenotypes differed. Since the characteristic end
products of E. coli fermentation include lactic acid,
succinic acid, and acetic acid, the pH changes could be explained by
differences in the production of organic acids. Other plausible
explanations are differences in the removal of organic acids from the
medium and interactions between bacteria and HeLa cells during
adherence.
This paper describes two sets of experiments, one based on the
production of lactic acid and the other on the removal of succinic acid
from the medium. The objectives were to determine (i) whether there is
a metabolic difference between localized, diffuse, and nonadherent
isolates in the amount of lactate produced or succinate removed from
the incubation medium, (ii) whether E. coli changes from aerobic to anaerobic metabolism during incubation periods of up to
5 h under different culture conditions, (iii) whether an increase
in lactate production or succinate removal coincides with the drop in
pH previously observed, and (iv) whether the pH changes can be
attributed to differences in bacterial growth rates between isolates
with different in vitro adherence patterns and nonadherent strains.
| |
MATERIALS AND METHODS |
Bacterial strains.
For the experiments on lactate
production, three E. coli isolates from children with
acute diarrhea were chosen as index strains after their in vitro
adherence to HeLa cells had been tested by the method of Nataro et al.
(20). One isolate displayed diffuse adherence, and one was
nonadherent. The third strain adhered in a localized manner and on
serotyping was found to belong to O127K63 (Wellcome Diagnostics). None
of the three strains hybridized with enzyme-labelled oligonucleotide
probes (Du Pont) that detect heat-labile and heat-stable enterotoxins,
and none demonstrated verocytotoxicity in cell culture assays with Vero
cells. Three well-characterized enteropathogenic E. coli strains were used as controls: E2348/69, CVD206, and JPN15.
E2348/69 and CVD206 are an isogenic pair consisting of a wild-type,
adherent (localized) strain and its attaching and effacing
lesion-deficient (eaeA) mutant, respectively. JPN15, a
derivative of E2348/69, has a depletion of the E. coli
adherence factor plasmid usually associated with initial adherence. To
assess the consistency of D-lactate production by adherent
strains, 18 additional isolates from children with acute diarrhea were
also studied. Five E. coli isolates displayed diffuse
adherence, eight showed localized adherence, and five showed
aggregative adherence.
Removal of succinic acid from the medium was tested in the three index
strains.
Measurement of pH, lactate, and bacterial growth.
The
E. coli strains were cultured overnight at 37°C in
tryptone water and diluted with phosphate-buffered saline (PBS) to yield a concentration of 1 × 108 to 2 × 108 bacteria/ml. Eagle's minimum essential medium (MEM)
containing 1% D-mannose was purged with the appropriate
gas mixture and dispensed into 48-well Costar tissue culture clusters.
A 510-µl volume of MEM-mannose was inoculated with 20 µl of dilute
E. coli suspension. Bacteria were cultured for 0, 1, 2, 3, 4, or 5 h at 37°C in 5% CO2-95% air, 5%
CO2-95% O2, or 5% CO2-95%
N2. pH was measured at the end of each incubation period
with a pH meter (Radiometer) using a pH microelectrode with a
sensitivity range of pH 0 to 14 and 0 to 80°C. Following incubation,
the culture medium was collected and centrifuged twice in a Beckman
Microfuge. The E. coli-free supernatant fluid (SNF) was
used for enzymatic analysis of lactic acid. Each individual assay was
carried out three times.
Assays for
D- and
L-lactic acid were performed
with a commercial kit (catalog no. 149993; Boehringer Mannheim) with
the following
modifications to the manufacturer's recommended method.
For the
determination of
D-lactate levels,
D-lactate dehydrogenase was
added (catalog no. 10694;
Boehringer Mannheim) at a concentration
of 75 U/ml. The sample volume
was increased fourfold. Final absorbances
were read at 340 nm after 25 min to ensure that the reaction had
run to completion. For the
measurement of
L-lactate, the sample
volume was increased
40-fold. The sensitivity and linearity of
the assay were established
with a commercial
L-lactate standard
(catalog no. 125440;
Boehringer Mannheim) over a concentration
range of 5 to 100 µmol/liter. Bacterial concentrations in cultures
before and after
incubation were estimated by measuring the optical
density (OD) at 600 nm, where 1 OD unit is 8 × 10
8 cells/ml. The
viability of bacterial suspensions was measured
by surface colony
counts on agar plates.
To check the effect of depletion of the medium, E2348/69 was incubated
in MEM-mannose in 5% CO
2-95% air as described above
for
2 h. The bacteria and medium were separated by centrifugation.
The
pellet was resuspended in either fresh medium or the medium
in which it
had been cultured for the previous 2 h. After a further
2 h
of incubation the
D-lactate concentration was measured. The
experiment was done in triplicate.
Removal of succinic acid from the medium.
The HeLa cell
adherence assay was performed with monolayers of HeLa cells as
described by Nataro et al. (20). Briefly, 48-well tissue
culture clusters (Costar) were seeded with HeLa cells at 105/ml. The cells were cultured overnight at 37°C in 5%
CO2 in MEM supplemented with 2.2 g of
NaHCO3/liter, 10% fetal calf serum, 30 µg of
penicillin/ml, and 50 µg of streptomycin/ml. E. coli test strains were grown overnight in tryptone water and subsequently diluted with PBS (pH 7.4) to a concentration of 1 × 108 to 2 × 108 bacteria/ml. Semiconfluent
monolayers of HeLa cells were washed, and 500 µl of MEM containing
1% D-mannose was added to each well. Twenty microliters of
the diluted E. coli suspension and 10 µl of
[14C]succinic acid (approximately 5,000 cpm) were added
to HeLa cells. The succinic acid tracer was either
[2,3-14C]succinic acid (code CFA 142) or
[1,4-14C]succinic acid (code CFA 66; Radiochemical
Centre, Amersham, United Kingdom).
The [
14C]succinic acid tracer studies were carried out in
the absence and presence of HeLa cells and in the absence and presence
of adherent or nonadherent
E. coli. All samples were
analyzed
in duplicate. Culture medium,
E. coli, and
HeLa cells were harvested
at the beginning of the incubation period (0 h) and after 3 h
of incubation. Culture medium and
E. coli were aspirated off and
centrifuged in a Beckman Microfuge for
5 min. [
14C]succinic acid was extracted from the SNF. The
E. coli pellet
was washed once and resuspended in PBS.
Wells containing HeLa
cells were also washed once with PBS. The cells
were dissociated
with trypsin-EDTA and resuspended in PBS.
From all SNFs, methyl derivatives of [
14C]succinic acid
were prepared and extracted into chloroform (
10). Aliquots
of the
chloroform extracts as well as samples of the water phase after
extraction were assessed for
14C counts. The amount
recovered was the sum of the two counts.
Direct
14C
counting was performed on the HeLa cells and
E. coli
suspensions.
In the calculations of recovery of
14C, counts in the SNFs,
HeLa cells, and
E. coli were expressed as percentage of
the total
counts added to each well, i.e., disintegrations per minute
from
10 µl of [
14C]succinic acid in 520 µl of culture
medium. The mean percent
recoveries in SNF and cells were obtained from
three separate
experiments using [2,3-
14C]succinic acid
and from five using [1,4-
14C]succinic acid.
The difference in the percent recovery at the beginning and after
3 h of incubation was calculated for each
E. coli
SNF and
its matching cellular component. The amount lost, presumably as
CO
2, was calculated from the difference between the
percentage
of label removed from the SNF and that recovered in the
cellular
compartment.
Statistical analysis.
Analysis of variance was used to test
for differences in lactate production, pH, and bacterial growth. The
succinic acid data was analyzed by analysis of variance in a
23 factorial design (25) to determine the
significance of each factor and interactions between them. The three
factors were time, HeLa cells, and E. coli, and the two
levels were the presence and absence of each factor.
| |
RESULTS |
Lactate production and pH measurements.
No detectable
L-lactate was produced by any of our index isolates, but
all produced D-lactate.
Tables
1 and
2 summarize the mean
D-lactate levels and pH values for our localized adherent,
diffuse adherent, and nonadherent
E. coli strains and
for E2348/69, CVD206, and JPN15 in 5% CO
2 and 95% air.
The trends in pH decreases and
D-lactate increases in our
localized adherent strain and in E2348/69 were similar. Very little
D-lactate could be detected up to 2 h of incubation.
By 3 h
D-lactate
levels had started to rise and pH had
started to fall. A further
progressive increase in
D-lactate levels and drop in pH occurred
between 3 and
5 h (Tables
1 and
2). At 4 and 5 h
D-lactate
levels
were higher in E2348/69 than in our localized adherent strain.
Unlike the parent strain, E2348/69, JPN15, which lacks the
E. coli adherence factor plasmid, produced very small
amounts of
D-lactate, and CVD206, the
eae
mutant, produced only trace amounts
during the entire 5-h incubation
period. The pH drop was small
in CVD206 and JPN15, and pHs remained
above 7.0 at the end of
5 h of incubation.
Our diffuse and nonadherent isolates produced similar concentrations of
D-lactate up to 3 h of incubation, but at 4 and 5
h, the diffuse adherent isolate showed approximately twice as
much
D-lactate as the nonadherent strain. With both strains,
concentrations
of
D-lactate were higher than those found
with the localized adherent
isolate.
The amount of
D-lactate produced by our three index
isolates was not influenced by the introduction of different gases
during
culture periods of up to 3 h. In 5 h, the diffuse
adherent isolate
produced the same amount of
D-lactate
under anaerobic and aerobic
conditions (1,145 ± 108 and
1,140 ± 81 µmol/liter, respectively)
and a smaller amount in
air (Table
1). With the nonadherent strain,
the pattern was similar,
with 918 ± 81 µmol/liter being produced
in N
2,
992 ± 49 µmol/liter being produced in O
2, and less
being
produced in air (Table
1). In N
2 and O
2,
the localized adherent
isolate produced amounts of
D-lactate similar to that produced
in 5%
CO
2-95% air (Table
1). The magnitudes of the increase in
D-lactate levels and the drop in pH were consistent for
each isolate
regardless of culture conditions.
The increase in lactate concentration was not due to depletion of the
medium. The mean lactate level of 93.7 µmol/liter (standard
error
[SE], 6.1) for E2348/69 resuspended in the medium in which
it had
been cultured for the first 2 h was similar to the 97.1
µmol/liter (SE, 7.8) found after 4 h of standard incubation
(Table
1). The concentration was higher when the strain was resuspended
in fresh medium, 158.9 µmol/liter (SE, 6.2).
Table
3 shows
D-lactate
concentrations obtained from the 18 additional isolates. At the end of
the 5-h incubation period,
the highest levels were obtained in the
diffuse adherent group,
whereas the lowest concentrations were detected
in the localized
adherent category. This is consistent with
D-lactate values shown
by the two index strains with these
adherence patterns. Isolates
in the aggregative adherent group showed
lower levels than the
diffuse adherent strains but higher levels than
the localized
adherent strains.
Bacterial growth.
Bacterial concentrations measured by OD
correlated well with viability counts. All isolates displayed active
growth within 0.5 h at 37°C. Active multiplication continued
throughout the 5 h of incubation (Table
4), whereas pH changes occurred only after 2 h (Table 2).
Removal of succinic acid.
Table
5 summarizes the mean changes in recovery
and SEs when [2,3-14C]succinic acid was used as the
tracer. The decrease in recovery from the SNF was smallest with the
localized adherent strain of E. coli (3.33%) and
greatest with the nonadherent strain (12.44%). The diffuse adherent
E. coli strain had decreased recovery (8.99%). There
was no interaction between the HeLa cells and either the diffuse
adherent or the nonadherent strains, but there was a significant interaction between the localized adherent strain and the HeLa cells.
Incorporation into E. coli varied between 2.81% for
the nonadherent strain and 3.68% for the diffuse adherent strain. With
the localized adherent strain most of the tracer removed from the SNF
(3.33%) was recovered from the E. coli cells (3.05%). There were much larger differences in tracer recovery (i.e., tracer lost, presumably as CO2) with the diffuse adherent strain
(SNF, 8.99%, and E. coli, 3.05%) and nonadherent
strain (SNF, 12.44%, and E. coli, 2.81%).
The mean recoveries with [1,4-
14C]succinic acid was used
as the tracer are shown in Table
6. The
pattern was the same as that
seen with [2,3-
14C]succinic
acid. Recovery from SNF was greatest with the localized
adherent strain
and lowest with the nonadherent strain, with no
interaction between any
of the
E. coli strains and the HeLa cells.
Removal of
[1,4-
14C]succinic acid from the SNF was much greater than
removal of
[2,3-
14C]succinic acid, but the amounts of
14C incorporated into the
E. coli and HeLa
cells were slightly lower.
The difference between the amount of label removed from the SNF and the
amount recovered from the cells was greatest with
nonadherent
E. coli, less with the diffuse adherent strain, and
least with the localized adherent isolate. The pattern was the
same for
both tracers, although all losses were greater with
[1,4-
14C]succinic acid than
[2,3-
14C]succinic acid (Table
7).
| |
DISCUSSION |
It is widely accepted that the adherence of enteropathogenic
E. coli to intestinal epithelial cells in infants is
associated with the production of diarrhea. However, information which
elucidates actual metabolic events during bacterial adhesion remains
incomplete. Our previous investigation into E. coli-induced pH changes pointed towards a difference in metabolic
behavior between adherent and nonadherent E. coli
strains during their adherence to HeLa cells. This study confirmed that
the amounts of D-lactate produced by our localized
adherent, diffuse adherent, and nonadherent E. coli index strains after 3 h of incubation were not the same. The fact that D-lactate concentrations differ in isolates displaying
different adherence patterns was also shown with 18 additional, freshly collected isolates. Mean D-lactate concentrations in the
three groups were not the same. Further support for the postulate that the E. coli strains are inherently different comes from
the very low D-lactate levels detected in CVD206 and JPN15,
two genetically engineered strains, whose adherence patterns no longer
resemble that of the parental, localized adherent strain, E2348/69.
This finding suggests a difference in metabolism between localized adherent, diffuse adherent, aggregative adherent, and nonadherent isolates.
D-Lactate production by the E. coli strains
coincided with pH decreases in tissue culture medium. For example, with
E2348/69, the increase in D-lactate concentration coincided
with a decrease in pH between 2 and 5 h of incubation. In
contrast, the low D-lactate levels found with CVD206 and
JPN15 corresponded to minimal changes in pH over the same period.
The pattern of lactate production and the pH changes cannot be
explained purely by bacterial growth. Lactate levels and pH showed
little change in the first 2 h, while bacterial counts increased
by a factor of about 30. Between 3 and 5 h, lactate production of
some strains led to a 10-fold or greater increase in lactate
concentration and the colony counts increased two to four times.
Further differences in growth rates of isolates with different
adherence phenotypes do not account for the differences in lactate
production. For example, when the lactate concentration is expressed in
terms of colony count instead of per liter, E2348/69 produced about
82.7 µmol, JPN15 produced 5.7 µmol, and CVD206 produced 0.6 µmol
per 108 colonies at 5 h.
HeLa cells did not remove [14C]succinic acid from the
tissue culture medium irrespective of whether E. coli
adhered, but the E. coli removed significant amounts of
[14C]succinic acid from the SNF; the amount varied from
strain to strain. The nonadherent strain removed the largest amount of
[2,3-14C]succinic acid, and the localized adherent
isolate removed the smallest amount. Uptake of the tracer by all three
E. coli isolates was low but significant, the greatest
percentage being recovered from diffuse adherent E. coli. The amount of tracer presumed lost as CO2
differed among the isolates used. When [1,4-14C]succinic
acid was used as the tracer there was the same order in the amount of
label removed from the medium by the three E. coli
isolates. However, larger quantities of
[1,4-14C]succinic acid than
[2,3-14C]succinic acid were removed by all isolates. This
indicates that the metabolism of [14C]succinic acid by
E. coli occurs first at the terminal carbon positions.
The pH changes previously observed when adherent and nonadherent
E. coli strains were incubated in the presence of HeLa
cells cannot be attributed to the removal of organic acids by HeLa
cells or to interactions between the E. coli and HeLa
cells. Rather, the E. coli cells removed
[14C]succinic acid from the culture medium, and the
amounts removed by nonadherent E. coli differed from
those removed by adherent bacteria. This is another metabolic
difference between attaching and nonattaching isolates. The previous
studies into E. coli-induced pH changes showed a
greater drop in pH in tissue culture medium derived from adherent
E. coli than in that from nonadherent E. coli. The metabolic difference observed then, in the form of pH differences, is confirmed by the different metabolic activities in the
production and removal of organic acids from the culture medium during
incubation.
From our studies we conclude that inherent metabolic differences
between adherent and nonadherent E. coli strains exist.
These differences may play a role in the pathogenic mechanism.
| |
ACKNOWLEDGMENTS |
We express our sincere appreciation to J. P. Nataro of the
Center for Vaccine Development, University of Maryland, Baltimore, for
providing strains E2348/69, JPN15, and CVD206 and for helpful discussions in the course of this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Paediatrics and Child Health, Red Cross Children's Hospital,
Klipfontein Rd., Rondebosch 7700, South Africa. Phone: 27-21-658-5302. Fax: 27-21-689-1287. E-mail: mmann{at}ich.uct.ac.za.
Editor: P. E. Orndorff
| |
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Infect Immun, March 1998, p. 907-911, Vol. 66, No. 3
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
