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: email@example.com
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.
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