Jpn. J. Infect. Dis., 57, 2004
Neutrophil Microbicidal Activity: Screening Bacterial Mutants for
Survival after Phagocytosis Using Quantitative PCR
Henry Rosen*, Patrick J. Lewis and Cory M. L. Nitzel
Department of Medicine, University of Washington, Seattle, Washington, USA
SUMMARY: When a constant gene replacement sequence is introduced into bacteria to produce mutants and the flanking
chromosomal sequences are known, it is possible to use a quantitative polymerase chain reaction method (QPCR) to compare
the concurrent survival of the different bacterial mutants under identical conditions. We describe Escherichia coli survival
following neutrophil phagocytosis among three mutants deleted respectively for araB, dps or oxyR. Comparisons were made
both by traditional and QPCR methods with similar results and indicate that the survival defect of an oxyR and oxyS mutant
described previously can be attributed to the loss of oxyR alone. Deletion of dps, a prominent member of the regulon controlled
by the oxyR gene product does not engender a survival defect. We suggest that QPCR analysis can readily compare the
relative survival of 10 or more mutants concurrently. QPCR analysis would seem to be especially valuable when experimental
conditions are subject to a high degree of sample to sample variability or when the stress producing system involves use of
expensive or scarce resources like rare patient cells, cells from children, or the use of genetically modified animal hosts.
*Corresponding author: E-mail: firstname.lastname@example.org
Expression profiling of Escherichia coli phagocytosed by human
neutrophils identified a number of genes with increased expression
in response to stresses generated in the phagosome (1). We made
replacement mutations for a number of these genes (2) and sought
to develop an efficient method for determining the phenotype of
these mutations with respect to survival after neutrophil phagocytosis
using the quantitative polymerase chain reaction (QPCR) to estimate
concurrent survival of different mutants confronting the same micro-
bicidal conditions. This report describes aspects of these efforts.
E. coli strain EC1 is ATCC 11775 (American Type Culture Collec-
tion). Plasmid pKD46 encodes λ-phage recombinase enzymes (2).
EC1 was transformed with pKD46 to give EC218.
Previously described procedures: The gene replacement proce-
dure was as described by Datsenko and Wanner (2) with modifica-
tions (available from the authors) to address the fact that EC1 and
EC218 are heavily encapsulated and metabolize arabinose actively.
Human neutrophils and serum were prepared as previously described
(1). The traditional bacterial survival assay (1) was modified with
respect to bacterial and neutrophil cell numbers as described in the
text and Figure legends.
Competitive survival after neutrophil phagocytosis: Bacterial
colonies from Mueller-Hinton agar plates prepared during viability
assays of mixed strains (300-1,000 colonies per plate) were collected
by repeated swabbing (sterile cotton-tipped swab) and rinsing in 1
mL of TE. Genomic DNA was prepared from 0.3 mL of TE suspen-
sion by a method adapted from (3) as further modified in http://
research.umbc.edu/~jwolf/m1.htm and used as a template for quan-
titative PCR as described below.
Quantitative PCR: 10 μL of 2x master mix containing Sybr
Green (SYBR Green PCR Master Mix, Applied Biosystems) were
combined with 10 ng mixed E. coli template DNA and 25 pmol of
each appropriate primer in a final volume of 20 μL. Cycling and
detection was on a Rotor-Gene 2000 (Corbett Research): 10’ at 95°C
to activate the polymerase followed by 50 cycles alternating between
a 95°C denaturation step (10 s) and a 60°C annealing-extension step
(30 s). Fluorescence data was acquired on the FAM channel during
the 60°C step, with a gain setting of 4.
Mutants: Mutant strains of the uropathogen, EC1, were con-
structed as described in Methods. Targeted genes were those for
oxyR, dps, a prominent neutrophil-stress regulated gene of the OxyR-
regulon, and araB. The goal for constructing the araB strain was to
use it as a control strain containing a PCR detectable chromosome
modification that would not affect the response to neutrophil-
Traditional bacterial survival assay: When survival of these
strains was evaluated in a typical neutrophil microbicidal assay (Fig.
1), both the araB and dps strains were killed at rates comparable to
the wild type parent, whereas killing of the oxyR strain was consid-
erably more extensive. Thus the araB mutation did not alter bacte-
rial survival in response to neutrophil microbicidal systems and could
be used as a wild-type surrogate in further experiments. Interestingly,
although the dps mutation is known to confer a measure of hyper-
susceptibility to reagent hydrogen peroxide in E. coli (4) (con-
firmed by us in our mutant) and is a virulence factor in salmonella
pathogenesis models (5), it bears no detectable phenotype in the
neutrophil killing assay. Also, it appears that the oxyR mutation has
the same phenotype as the oxyRS mutation evaluated previously (1)
and thus oxyS appears to be unnecessary for the manifestation of the
neutrophil hypersusceptibility phenotype.
Bacterial survival assays for mixed strains: Overnight cultures
Fig. 1. Bacterial survival after neutrophil phagocytosis - traditional
Bacteria (20 ×106) were pre-opsonized with 5.5% fresh human
serum for 30 min and incubated with 5×106 neutrophils. At indicated
intervals samples were taken to assess residual bacterial viability.
Bacterial strains were wild type EC1 (n = 8, heavy solid line, diamond
symbols), and gene replacement mutants araB (n = 5, dashed line,
circles), dps (n = 4, dashed line, squares), and oxyR (n = 9, thin solid
line, diamonds). Error bars reflect standard error estimates from the
log transformed data.
min from PMN addition
Jpn. J. Infect. Dis., 57, 2004
of different strains were combined (10% araB, 10% dps, 10% oxyR
and 60% wild-type), washed, and samples were plated to obtain
individual colonies prior to the addition of serum and neutrophils
(INPUT SAMPLE). After the addition of serum and neutrophils,
further samples were plated to enumerate survivors (OUTPUT
SAMPLE). Overall survival of the bacterial mixture in response to
neutrophil phagocytosis was assessed by colony counting. The initial
inoculum of 20 ± 2 million bacteria per mL declined to 0.2 ± 0.1
(mean ± SD, triplicate samples, 1 experiment) during the 90 min
incubation: 30 min with serum and an additional 60 min with
neutrophils. Total DNA was prepared from colonies swabbed from
INPUT and OUTPUT plates as described in the methods section.
The relative abundance of each mutant in the INPUT and OUTPUT
samples was assessed by QPCR.
QPCR: Fig. 2 describes the features of INPUT and OUTPUT
DNA samples (triplicate killing assays performed on the same day,
resulting in 3 INPUT and 3 OUTPUT samples) analyzed, using the
appropriate primers and Sybr Green fluorescence, to determine the
proportionate abundance of the araB, dps, and oxyR genotypes in
the sample. The relative abundance of the araB gene disruption
sequence was highly reproducible among the 3 INPUT samples (solid
lines) and the slight shift to the left for the OUTPUT strains (dotted
lines) suggests a slight increase of the representation of the araB gene
disruption sequence in these samples. This shift can be quantified
by establishing a threshold value and determining the number of
PCR cycles required to achieve this level of fluorescence (Ct = cycles
to threshold). For the INPUT samples, with a threshold of 0.01
fluorescence units, the araB Ct was 18.8 ± 0.3 (mean ± SEM, n =
3). For the OUTPUT samples Ct was 17.8 ± 0.2, a difference of 1.0
cycles, P < 0.02.
For the dps mutation, the increased representation in the OUT-
PUT sample was less dramatic (INPUT Ct = 18.7 ± 0.1, OUTPUT
Ct = 18.1 ± 0.2) but still significant, P < 0.05. The increased repre-
sentation of the araB and dps strains in the OUTPUT samples may
be accounted for, in part, by compensation for the decline in the
representation of the oxyR strain. The Ct for the oxyR strain in the
INPUT samples averaged 18.5 ± 0.1, while the OUTPUT samples
averaged 21.2 ± 0.1, a shift of nearly 3 cycles, P < 0.005. Assuming
that each cycle reflects a 2-fold difference in template abundance
(100% PCR efficiency), the Ct differences among the strains
suggests a 7-15 fold decrease in the survival of the oxyR strain
compared to the araB and dps strains. The result is quite consistent
with the viable results described in Figure 1 and indicates that QPCR
screening can be effective at detecting strains with altered survival
compared to their peers in a competitive survival assay.
Constructing gene deletions by the facilitated homologous recom-
bination method described by Datsenko and Wanner (2) inserts a
known replacement DNA sequence at a precise location in the
chromosome. When combined with knowledge of the flanking
chromosomal sequence, it is possible to define a region of DNA
sequence, part chromosomal and part insert, that is unique to the
newly constructed mutant strain and that distinguishes it from all
other mutants constructed by this method. By locating one PCR
primer in the insert and a corresponding primer in the chromosome,
it should be possible to amplify that unique sequence in a QPCR
The number of PCR cycles required to reach an arbitrary thresh-
old of detection, typically correlates with the number of copies of
DNA template introduced into the QPCR reaction. The mutations
evaluated in this work are those for araB, oxyR and dps. The araB
gene is the first in the araBAD operon that contributes to arabinose
metabolism. There is no known relationship between arabinose
metabolism and bacterial interaction with neutrophil microbicidal
systems. Indeed neutrophils killed the araB mutant at the same rate
as the wild type parent (Fig. 1).
The oxyR gene encodes a transcription factor that is relatively
quiescent in its reduced form but becomes active following a
conformational change associated with a more oxidizing cytosolic
environment. A gene replacement mutant that eliminated both oxyR
and oxyS, was observed to be hypersusceptible to neutrophil-
mediated killing systems (1). In the current study, we have shown
that deletion of just oxyR is sufficient to produce this phenotype
Among the bacterial mRNA’s that increase dramatically in
abundance during neutrophil phagocytosis, is that for the dps
protein. The dps protein binds to DNA, sequesters iron in the bacte-
rial cytosol in a ferritin-like manner (6,7), and confers substantial
Fig. 2. QPCR of DNA from colonies arising from mixed suspensions
of E. coli. Sybr Green method.
Three INPUT samples containing DNA from a mix of wild type
(60%), araB (10%), dps (10%) and oxyR (10%) gene replacement
mutants were evaluated for relative abundance of the replaced DNA
sequences using appropriate primer sets (Table 1) and 10 ng of total
DNA from the three separate samples (solid line amplification plots).
After 30 min opsonization with serum and 60 min incubation with
neutrophils, as described in Fig. 1, OUTPUT DNA was obtained, as
described in methods (dashed line amplification plots).
25 3015 20
Table 1. PCR primers and probes
-red gene disruption
sybr Green QPCR2
1: Sequence extended by the sequence indicated by “pKD3-homology” in the first line.
2: Primers for detection of λ-red recombinants using sybr-Green fluorescence. Sequences that recognize the insertion element are in large italic font.
Jpn. J. Infect. Dis., 57, 2004
resistance to hydrogen peroxide-mediated toxicity (4). However
deletion/replacement of the dps gene did not affect EC1 survival in
neutrophils (Fig. 1).
When the three mutant strains (10% each) were mixed with wild-
type and incubated with neutrophils for 60 min, the overall mixture
experienced a 99% decline in viability. Among the survivors, the
araB and dps genotypes were slightly over-represented while the
oxyR genotype was significantly under-represented as determined
by QPCR analysis (Fig. 2). The QPCR analysis achieved the same
result in a single experiment with three replicate samples as was
observed in 4-9 experiments employing traditional methods. Based
on the 10% abundance of each mutant in the INPUT mixture, we
estimate that QPCR analysis could easily detect differences within
a mixture of 10 different strains and perhaps many more. We would
suggest that QPCR analysis could serve as an effective screening
method to select promising candidates for further study from among
Preliminary studies have indicated that single gene mutations
are insufficient to render EC1 hypersusceptible to neutrophil
microbicidal systems but that double and triple mutations acquire
a significant phenotype (unpublished). We propose to use QPCR
analysis to further screen additional mutants of EC1, examining the
contribution of key genes of the oxyR regulon individually and in
This work was supported by a grant AI049417 from the National Insti-
tutes of Health.
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mRNA expression profiles for Escherichia coli ingested by normal and
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2. Datsenko, K. A. and Wanner, B. L. (2000): One-step inactivation of
chromosomal genes in Escherichia coli K-12 using PCR products. Proc.
Natl. Acad. Sci. USA, 97, 6640-6645.
3. Maloy, S. R. (1990): Experimental techniques in bacterial genetics. Jones
and Bartlett, Sudbury, MA.
4. Almiron, M., Link, A. J., Furlong, D. and Kolter, R. (1992): A novel
DNA-binding protein with regulatory and protective roles in starved
Escherichia coli. Gene Dev., 6, 2646-2654.
5. Halsey, T. A., Vazquez-Torres, A., Gravdahl, D. J., Fang, F. C. and Libby,
S. J. (2004): The ferritin-like Dps protein is required for Salmonella
enterica serovar Typhimurium oxidative stress resistance and virulence.
Infect. Immun., 72, 1155-1158.
6. Grant, R. A., Filman, D. J., Finkel, S. E., Kolter, R. and Hogle, J. M.
(1998): The crystal structure of Dps, a ferritin homolog that binds and
protects DNA. Nat. Struct. Biol., 5, 294-303.
7. Rychlewski, L., Zhang, B. and Godzik, A. (1999): Functional insights
from structural predictions: analysis of the Escherichia coli genome.
Protein Sci., 8, 614-624.
The Role of Myeloperoxidase in the Pathogenesis of Coronary Artery Disease
Stephen J. Nicholls1,2 and Stanley L. Hazen1,2,3,4*
1Department of Cardiovascular Medicine, 2Center for Cardiovascular Diagnostics and Prevention,
3Department of Cell Biology, Cleveland Clinic Foundation and 4Department of Molecular Medicine, Cleveland
Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, USA
SUMMARY: A growing body of evidence continues to emerge implicating the role of myeloperoxidase (MPO) and its
oxidant products in the promotion of atherogenesis. A major mechanism by which MPO impacts the arterial wall is through
its modification of net cellular cholesterol flux. MPO promotes lipid peroxidation and conversion of LDL to an atherogenic
form, where it is taken up by macrophages, a critical step in foam cell formation. Emerging evidence suggests that HDL can
also be modified by MPO derived oxidants, resulting in an impairment of cholesterol efflux. In addition, modified HDL
appears to be a strong predictor of clinical risk. These features highlight MPO and its products as potential predictive markers
and targets in atheroprotection.
*Corresponding author: E-mail: email@example.com
It has become increasingly recognized that atherosclerosis is a
chronic inflammatory process, characterized by the accumulation
of lipid, inflammatory cells and necrotic material within the arterial
wall. As a result, there is great interest to identify the key factors
promoting this process. A substantial body of evidence has emerged
to implicate the role of the leukocyte derived enzyme myeloper-
oxidase (MPO) in atherogenesis. In particular, recent studies have
highlighted the potential role that MPO plays in the regulation of
cholesterol flux into the arterial wall.
Association between MPO and atherosclerosis: MPO, a mem-
ber of the heme peroxidase superfamily, generates reactive oxygen
species and diffusible radical species. It performs a physiological
role as part of the innate immune system. However, MPO also can
apparently exert a deleterious impact on the arterial wall. Immuno-
histochemical studies demonstrate the presence of MPO, its oxidant
products, and their colocalization with macrophages, in human
atheroma. Genetic studies support a protective role of MPO deficien-
cy. MPO-deficient individuals have less coronary artery disease
(CAD). In addition, a functional polymorphism in the promoter of
the MPO gene, resulting in decreased enzyme expression, was
associated with a decreased risk of CAD. Furthermore, systemic
levels of MPO and its oxidant products are associated with the preva-
lence of atherosclerotic disease. Systemic levels of MPO predict
the presence and extent of angiographic disease (1). Moreover,
levels predict the risk of clinical events in both subjects presenting
with chest pain (2) or acute coronary syndromes (3).
MPO promotes atherogenesis via a range of mechanisms: It
appears that MPO, through the generation of nitric oxide (NO)-
derived oxidants, promotes numerous pathological events in
the atherogenic cascade. In addition to generating potentially
proatherogenic species, MPO utilises the atheroprotective NO as a
substrate. These factors have been implicated in the development of
endothelial dysfunction, accumulation of foam cells in the arterial
wall and the promotion of plaque vulnerability (4). In particular,
substantial evidence suggests that MPO derived oxidants influence
the net flux of cellular cholesterol, via both an increase in its cellular
uptake and a reduction in its removal.
MPO promotes cellular accumulation of cholesterol: Oxida-
tive modification of low density lipoprotein (LDL) is a key early
event in the promotion of atherogenesis. Modification of LDL to a
high uptake form allows for its internalisation by macrophages,
which undergo morphological changes to become foam cells, a major
cellular component of the developing plaque. Evidence suggests
that MPO-generated reactive nitrogen species convert LDL into
a high uptake form, which is readily internalised by macrophages