Determination of heme in microorganisms using HPLC-MS/MS and cobalt(III) protoporphyrin IX inhibition of heme acquisition in Escherichia coli
Determination of heme in microorganisms using HPLC-MS/MS and cobalt(III) protoporphyrin IX inhibition of heme acquisition in Escherichia coli
Jonas Fyrestam 0 1 2
Conny Östman 0 1 2
0 Abbreviations 5-ALA 5-Aminolevulinic acid hydrochloride Co-PPIX Cobalt protoporphyrin IX LB Lysogeny broth OD
1 Division of Analytical and Toxicological Chemistry, Department of Environmental Science and Analytical Chemistry, Stockholm University , Svante arrheniusväg 16C, 106 91 Stockholm , Sweden
2 Conny Östman
One of the main threats to the achievements in modern medicine is antimicrobial resistance. Molecular targeting of bacterial acquisition mechanisms of heme has been suggested to be an alternative to antibiotics. In the present study, HPLC-MS/MS combined with a simple clean-up based on liquid-liquid extraction has been developed and evaluated for simultaneous determination of heme and porphyrin heme precursors in microorganisms. Experimental design was used to optimize the extraction parameters, to obtain a method with high recovery, low matrix effects, and high precision. The effects of additives in the culture medium on the biosynthesis of heme were studied using Escherichia coli as a model microorganism. 5-Aminolaevulinic acid and hemin increased the heme concentration in E. coli by a factor of 1.5 and 4.5, respectively. Addition of 5-aminolaevulinic acid bypassed the E. coli negative feedback control of heme biosynthesis, which led to high amounts of intracellular porphyrins. The high heme concentration obtained when hemin was used as a culture additive shows that E. coli has an uptake of heme from its surroundings. In contrast, addition of cobalt protoporphyrin IX to the growth medium reduced the amount of heme in E. coli, demonstrating this compound's ability to mimic real heme and inhibit the heme acquisition mechanisms.
Heme acquisition; Heme analysis; Escherichia coli; HPLC-MS/MS; Antimicrobial resistance; Porphyrins
Increasing antimicrobial resistance among pathogens has been
pointed out by the World Health Organization to be a problem
so serious that it threatens the achievements of modern
]. In recent years, it has been proposed that targeting
the mechanisms that take part in the acquisition of iron could
be an important complement to antibiotics. In the long run,
this could help to decrease the amount of antibiotics used
which is the main reason for increased antimicrobial resistance
against antibiotics worldwide [
Iron acts as a crucial cofactor in many important biological
processes such as respiration and DNA synthesis and is
essential for most living organisms [
]. Pathogenic bacteria are
no exception, and to cause a disease, they need to acquire iron
from their hosts . In vertebrates, the majority of iron is
present bound to a porphyrin ring, i.e., heme (Fig. 1) [
Vertebrates have developed strategies to limit bacterial access
of free iron during an infection, a process often known as
]. Transferrin and lactoferrin, two proteins
with a high affinity for iron, are synthesized in excess during
the first stage of an infection to reduce the levels of free iron
available for pathogens that are required for their survival.
These mechanisms are reducing free iron to negligible
amounts, below 10−18 M [
]. As a response to these
ironwithholding processes, bacteria have developed different
strategies to acquire iron from their host. Pathogens start to
produce and release compounds into the extracellular medium
to scavenge heme or iron from a number of sources. Gram
negative bacteria produce and excrete proteins (hemophores)
that bind to heme. These proteins have high affinity for heme
and they return to specific receptors located in the outer
membrane of the bacteria [
]. Bacteria also have heme acquisition
systems with receptors that recognize heme and transport it
into the cell via ATP-binding cassette transporters [
A number of non-iron metalloporphyrins have previously
shown to be potent antimicrobial compounds [
putative cause of this toxicity to pathogens is that these
comp o u n d s c h e m i c a l l y m i m i c r e a l h e m e . I n t h i s w a y,
metalloporphyrins can be a substrate for the heme acquisition
mechanisms and taken up by the cell. Inside the cell, these
molecules are partitioned into the cell membrane, displacing
heme and inhibiting respiration [
Accurate determinations of heme are essential to widen our
understanding of how different microbes acquire heme.
Although heme is of great importance for microbial survival,
there are currently no validated methods for selective
determination of trace levels of heme in microorganisms. Often,
unspecific methods are used such as UV-Vis absorption
]. Heme has previously been determined by HPLC in
plant cells and cyanobacteria [
], as well as in phyto- and
], but the analytical methods used in these
studies lack sufficient evaluation to accurately determine
The aim of this study was to develop and evaluate an
analytical method for the extraction, clean-up, and analysis of
heme utilizing HPLC-MS/MS using Saccharomyces
cerevisiae and Escherichia coli as model microorganisms.
The method was applied to determine heme in E. coli and its
relation to microbial synthesis and/or acquisition of heme
when grown using different culturing conditions.
Formic acid (≥ 98%), tris(hydroxymethyl)aminomethane
(Tris), ferrous sulfate, magnesium sulfate, and separate
standards of protoporphyrin IX (purity ≥ 95%), protoporphyrin IX
cobalt chloride, 5-aminolaevulinic acid hydrochloride (purity
≥ 97%), and hemin from porcine (purity ≥ 97%) were obtained
from Sigma-Aldrich (Schnelldorf, Germany). HPLC-grade
methanol, acetone, and acetonitrile were purchased from
Rathburn Chemicals Ltd. (Walkerburn, Scotland).
Analyticalgrade hydrochloric acid (37%) and dimethylformamide (DMF)
were obtained from VWR International (Fontenay-sous-Bois,
France). Ethylenediaminetetraacetic acid disodium dihydrate
salt (EDTA) and sodium chloride of reagent grade were
acquired from Scherlab S.L. (Sentmenat, Spain). A Synergy 185
water purification system from Millipore (Molsheim, France)
was used to produce deionized water at 18 MΩ cm. Porphyrin
acid chromatographic marker kit (CMK-1A) containing
10 ± 1 nmol of each of six porphyrins (mesoporphyrin IX,
coproporphyrin I, 5-carboxylporphyrin I, 6-carboxylporphyrin
I, 7-carboxylporphyrin I, and uroporphyrin I) were obtained
from Frontier Scientific Inc. (Logan, UT, USA). S. cerevisiae
was used for method validation and it was obtained from
Jästbolaget (Sollentuna, Sweden). E. coli NovaBlue was
obtained from the Department of Biochemistry and Biophysics
at Stockholm University.
Culturing and harvest of E. coli
E. coli was cultivated in sterile Miller lysogeny broth (LB) at
37 °C. To determine the influence of different additives on
heme and iron acquisition mechanisms in E. coli, the LB
medium was supplemented with five additives: Fe(II)SO4,
hemin, 5-aminolevulinic acid hydrochloride (5-ALA), and
protoporphyrin IX cobalt chloride. The influence of cultivation
time was also investigated. The different culture conditions
are listed in Table 1.
E. coli was allowed to grow to stationary phase for 60 h;
thereafter, the different additives were added for individual
experiments and grown for additional 24 h (samples 2–5 in
Table 1). In sample 5 (Table 1), cobalt protoporphyrin IX’s
(Co-PPIX) ability to inhibit E. coli hemin acquisition was
investigated. Culture was allowed to grow for 60 h, and then
Co-PPIX was added and grown for an additional 1 h. Hemin
was added and the culture was grown for 24 h. Estimation of
sample sizes was made by measuring the optical density at
600 nm (OD600) using an UV-Vis spectrophotometer (Thermo
Fisher Scientific, Stockholm, Sweden).
For the further experiments, the microorganisms were
harvested by taking 2 mL aliquots of cultures and centrifuge at
13,200×g for 5 min. After removal of the supernatant, the
pellets were washed two times with 2 mL 0.4% NaCl to
remove remaining cultivation broth. The pellets were then
subjected to the sample preparation described below.
Approximately 100 mg (wet weight) of S. cerevisiae and
3 mg (dry weight) of E. coli were put in 15-mL Falcon™
tubes together with 1 mL of Tris-EDTA buffer (pH 7.2) and
stirred for 1 h at room temperature using a shaker (IKAVXR
Vibrax, Staufen, Germany) at 1600 RPM. Samples were put
on ice and treated with ultrasonication (Sonics Vibracell,
Newtown, CT, USA) for 5 min with 1 s pulse. Three
milliliters of acetonitrile was added to the sample which then was
vortexed for 5 min and subsequently subjected to
centrifugation at 2500×g for 5 min, making a pellet of precipitated
proteins. The acetonitrile containing unpolar interfering
compounds, as well as porphyrins, was removed and can be
a n a l y z e d f o r p o r p h y r i n c o n t e n t . T h e n , 4 m L o f
acetonitrile:1.7 M HCl (8:2, v/v) was added to the pellet and
put in a shaker for 20 min, extracting heme from the proteins
into the acetonitrile. To create a two phase liquid-liquid
system, 1 mL of saturated MgSO4(aq) and 0.1 g of NaCl(s) were
added. The solution was vortexed for 5 min and centrifuged
at 2500×g for 5 min. The top organic layer was put in a vial
and, if necessary, diluted with pure acetonitrile prior to
analysis. A blank and a standard used for quantification purposes
were subjected to the same extraction and clean-up steps to
rule out any contamination during clean-up and correct for
losses. Figure 2 shows a scheme of the workflow.
Fig. 2 Scheme of the extraction and clean-up method. BStandard^ points
out were in the clean-up process the standard of heme for quantification
purposes were added
The analyses of heme, hemin, and porphyrins were performed
on a Xevo TQ-S tandem mass spectrometer (MS/MS) coupled
to a high performance liquid chromatograph Acquity I-Class
system (Waters). An ACE 3 C18 column (2.1 × 50 mm,
Advanced Chromatography Technologies Ltd., Aberdeen,
Scotland), placed in a column oven (50 °C), was used for
the separation with a gradient elution using water and
acetonitrile with 0.1% formic acid (FA) as mobile phases. Signals
from all analytes were acquired in positive electrospray mode
(ESI+) and multiple reaction monitoring (MRM) with three
compound-specific transitions for each analyte, all transitions
used as quantifier and qualifier ions. The mass spectrometer
was tuned by direct infusion of a standard solution with a
concentration of 2 μM of the analytes. The dwell time for each
transition was automatically set by the software to be 0.012 s
in order to get approximate 12 data points over a peak with a
width of 4 s.
Liquid chromatography and mass spectrometry parameters
are presented in Table 2.
Linearity and limit of detection
An 11-point calibration curve of hemin was made by
injections of a standard solution in the range of 5–250 pmol to
investigate the linear relationship between analyte
concentration and instrumental response. Each level was injected in
triplicate and analyzed in MRM mode. Limit of detection
(LOD) was determined by injections of 0.75 pmol hemin
(n = 7). The signal (S) was defined as the mean area of these
replicate injections and the noise (N) defined as the standard
deviation. Limit of detection were defined as three times the
signal to noise ratio (S/N).
Optimization of heme extraction
A full factorial central composite face design with three center
points was used to optimize the extraction conditions of heme
from S. cerevisiae with acetonitrile as the organic solvent. The
evaluated factors were time of extraction (1, 20, and 40 min),
percentage of acetonitrile (33, 66, and 99%, v/v), and molar
concentration of HCl (0.1, 1, and 2 mol/dm3) using 17
experiments. A total weight of 0.8 g S. cerevisiae was put in 3 mL of
Tris/EDTA buffer for digestion during 1 h and further lysed by
ultrasonication for 5 min (1 s pulse). Aliquots of 100 μL were
transferred to 0.5 mL micro vials and 400 μL of different
acetonitrile/HCl mixtures was added and was immediately
vortexed for 1, 20, or 40 min. Immediately after vortexing,
the micro vials were centrifuged at 13,200×g and the
supernatant transferred to vials and analyzed for heme content. The
experimental data were processed using MODDE software
(ver. 10.0.0; MKS Umetrics, Umeå, Sweden).
Protein precipitation efficiency
Five different solvent mixtures were selected for evaluation of
their ability to precipitate proteins from S. cerevisiae. Acetone,
acetonitrile, and methanol solutions were mixtures of 80%
organic solvent together with 20% 1.7 M HCl (v/v). Pure
acetonitrile as well as 1.7 M HCl were also evaluated.
S. cerevisiae was lysed as described above, and 100 μL
yeast extract was added to 200 μL of precipitation solvent in
1.5 mL micro vials, vortexed for 20 s, and left to stand for
10 min. Solutions were centrifuged at 13,200×g for 5 min.
Measurement of the protein concentration was done with a
spectrophotometer (Waters) using the Bradford micro protein
assay at a wavelength of 595 nm. Absorbances before and
after protein precipitation were used to calculate the protein
precipitation efficiency by the equation;
Absorbance before precipitation−Absorbance after precipitation
Absorbance before precipitation
Bovine serum albumin standards were used to check the
linear response of the spectrophotometer. The experiments
were performed in triplicate with three absorbance
measurements of each sample.
Stability of heme in different solvents
Storage stability of hemin in room temperature was
investigated using six solvents and solvent mixtures: 100%
acetonitrile, 1 M NaOH, 6 M formic acid, deionized water,
acetonitrile:1.7 M HCl (8:2, v/v), and the organic top layer
from the liquid-liquid extraction when using acetonitrile as
described above. Hemin standards dissolved in the six
solvents were put in the autosampler tray and analyzed daily in
duplicate during 7 days. For each analytical run, a volumetric
standard was used to account for day to day variations in
Results and discussion
Optimization of heme extraction
Acetone extraction has been used to extract heme from sample
matrices such as plants [
] and algae  and is generally
adopted as the standard procedure for heme extraction. The
extractions are normally carried out in 80% acetone
containing 20% of 0.6–2.1 M HCl [
]. However, acetonitrile has
several advantages compared to acetone as a solvent. It has
lower elution strength in reversed phase LC columns, making
it more suitable to inject without extensive band broadening of
the peaks on reversed phase columns. It also has a lower UV
cutoff wavelength, making it more suitable for the detection
with UV-vis, and it has been shown to possess better protein
precipitation properties when the aim has been to remove
proteins from the sample matrix [
]. For these reasons,
acetonitrile was selected as a candidate solvent for optimization
of heme extraction.
The experiments were evaluated using the MODDE
software, and the coefficient plot showed that only two factors
were significantly affecting the extraction efficiency of heme
(p ≤ 0.05): the molar concentration of HCl and the percentage
of acetonitrile. Surprisingly, the extraction time was found not
to influence the extraction efficiency of heme within the
experimental domain (1–40 min). There was a weak trend that
more heme was extracted when longer extraction times were
used, but the differences were small and not significant. In
Fig. 3, the concentrations of both acetonitrile and HCl are
plotted in a response surface plot when samples were
extracted for 20 min. Both HCl and acetonitrile concentrations have a
positive effect on the heme extraction with an optimum in the
selected domain. Trying to extract heme into pure acetonitrile,
not containing any HCl, resulted in no detectable amounts of
heme. Using only 1.7 M HCl resulted in poor yield of heme,
< 0.6%. A possible explanation to this is that when the
extraction is performed in 80% acetonitrile, there will be no
protonation of the heme binding amino acids and heme will still be
bonded to the proteins. When HCl, or another strong acid, is
used together with the organic solvent, heme is released from
these proteins. On the other hand, when only HCl is used,
heme is too hydrophobic to be extracted from the precipitated
proteins and into the polar solvent.
The optimum in this model was determined to be 82%
acetonitrile and 18% 1.7 M HCl when using 20 min extraction
time. These results are in agreement with previous studies
where other authors have used acetone and HCl for the
20, 23, 26, 27
]. The precipitated yeast pellet was
subjected to repeated extractions with the optimized method.
In the second extraction, 3.3% of the amount found in the first
extraction was detected. In the third extraction, less than 1%
was found, i.e., after two extractions of the sample the yield
was considered to be > 99%.
When using methanol with 20% 1.7 M HCl, as well as pure
1.7 M HCl for the extraction, the efficiencies were much lower
compared to using acetonitrile/HCl. Methanol/HCl had a
relative extraction efficiency of 66% and 1.7 M HCl only had
0.6%. Acetone:1.7 M HCl, on the other hand, demonstrated
similar extraction capabilities as acetonitrile:1.7 M HCl.
Methanol is a more polar solvent compared to acetone and
acetonitrile and 1.7 M of HCl being the most polar of the
investigated solvents. This explains the lower extraction
yields when using these solvents. When using acetonitrile
without any HCl, the heme level in the extract was below
LOD. The yield using the different solvents, together with
the protein precipitation efficiencies, is shown in Fig. 4.
Analysis of samples containing high concentrations of
proteins often requires clean-up to reduce matrix effects in the
LC-MS/MS analysis. In Fig. 4, the protein precipitation
Fig. 4 Relative yield (bars) of
heme from S. cerevisiae using
different extraction solvents and
their protein precipitation
efficiency (lines). Error bars
represent the standard deviation
efficiencies of the solvent mixtures are shown. The most
efficient precipitant was 100% acetonitrile which precipitated
100.5% (± 0.6) of the proteins present in the S. cerevisiae
extracts. This is in agreement with other studies where
acetonitrile has been reported to be the most efficient precipitant to
proteins in blood plasma [
]. Methanol:1.7 M HCl (8:2,
v/v) precipitated 82.4% (± 1.0) of the total protein content,
while acetonitrile:1.7 M HCl (8:2, v/v) had a precipitation
efficiency of 75.4% (± 4.1). Acetone:1.7 M HCl (8:2, v/v)
and 1.7 M HCl had the lowest protein precipitation efficiency
with 61.5% (± 0.2) and 62.0% (± 1.8), respectively. All
determinations of protein precipitation efficiencies are average
values from three replicates.
Clean-up and stability of heme
One of the most crucial problems to overcome in heme
analysis utilizing LC-MS/MS instrumentation is the ability
of heme to form aggregates and precipitate in aqueous
]. When a standard of hemin was analyzed
after having been dissolved in MQ water and left for 24 h at
room temperature, the hemin concentration had decreased
with 28%. Increasing as well as decreasing the pH with
NaOH and formic acid made hemin aggregate quicker.
After 24 h in 1 M of NaOH and in 6 M formic acid, the
hemin content had decreased to 52 and 45%, respectively.
This makes the analysis of heme from microorganisms
problematic since the optimized extraction solvent requires
the addition of an acid in order to extract heme from the
heme proteins. When hemin was stored in 100% of
acetonitrile, it was more stable and had only decreased 6% after
1 week of storage in room temperature. However, when
hemin was put in the solvent composition used for the
optimized extraction procedure (acetonitrile:1.7 M HCl
(8:2, v/v)), it aggregates and the concentration decreased
to 85% within 24 h, and after 1 week, only 49% of the hemin
content remained un-aggregated.
The poor yield of heme into pure acetonitrile can be used in
the clean-up process of microbiological samples. Adding
100% acetonitrile to the lysate will remove hydrophobic
interfering substances after centrifugation and removal of the
supernatant (porphyrin fraction). Heme will still be bond to
the sedimented proteins and can subsequently be extracted
with the optimized extraction solvent.
To stabilize heme in the extraction solvent, 1 mL of
saturated MgSO4(aq) and 0.1 g of NaCl(s) were added. A two-phase
system with acetonitrile in the top layer and the aqueous
solution in the bottom layer was formed. Polar interfering
substances distributed into the aqueous layer and were removed
from the sample, while heme partitioned into the organic
solvent. No heme could be detected in the lower aqueous layer. A
stability test of hemin dissolved in this organic top layer
solution showed no decrease of hemin after 1 week of storage in
room temperature. The stability of hemin in all the tested
solvents is shown in Fig. 5.
Methods for determination of heme in various biological
matrices have been presented in the literature. However, the
aggregation and precipitation of heme has not been considered
and/or the analytical methods lack sufficient validation
]. This raise questions for the accuracy in reported
values of heme concentrations. The method presented here
has solved the problem with heme aggregation and
precipitation demonstrated by stability during at least 1 week
of storage in room temperature. Further evaluation is needed if
samples are stored for a longer time and/or at higher as well as
Fig. 5 Hemin stability during
7 days when dissolved in six
different solvents. Ax is the area
ratio to a volumetric standard at
the investigated day and A0 is the
area ratio to a volumetric standard
at day 0. Organic top layer is the
solvent used in the liquid-liquid
Analysis of heme with HPLC-MS/MS
In Fig. 6, a chromatogram is shown for the HPLC-MS/MS
analysis of hemin and heme precursors (porphyrins). All
compounds are baseline separated within 4 min. Total runtime
for one sample, including pre- and post-runs to clean and
condition the column, is 6 min. In other methods where heme
has been separated with HPLC, the chromatographic run
normally takes between 15 and 45 min [
22, 33, 36, 37
Linearity and detection limit
The calibration curve showed nonlinear characteristics with a
significant curvature in the concentration range 5–250 pmol.
This pattern of hemin linearity was demonstrated on two
different mass spectrometers with electrospray ionization and is
probably explained by signal saturation in the electrospray at
high concentrations [
]. To be able to model the data in in
this concentration range, it is recommended to use a
polynomial curve fitting of second degree (R2 > 0.99). A linear
calibration curve (R2 > 0.98) was obtained when limiting the
concentration range to 5–100 pmol (Fig. S1 in Electronic
Supplementary Material (ESM)).
Limit of detection was determined at 0.2 pmol of injected
hemin. The LOD is the lowest concentration of an analyte that
the analytical process can reliably differentiate from
background levels [
]. A low LOD is of importance when
applying this method to other microorganisms that are not as easy to
culture as S. cerevisiae, thus yielding smaller sample sizes. We
have not been able to find any reported LOD values from
previous studies, but we consider 0.2 pmol to be low for this
kind of matrices.
Approximately 150 mg (wet weight) of S. cerevisiae was put
through the sample preparation steps with the only difference
that the acetonitrile used in the liquid-liquid extraction did not
contain any HCl, thus not extracting any heme from the yeast
cells. In this way, an analyte-free matrix was created close to a
real sample matrix. The analyte-free matrix was spiked with
hemin to a concentration of 15 μM. The response obtained
from the HPLC-MS/MS analysis was compared to a reference
standard dissolved in pure extraction solvent. The results
showed that the matrix effect was low, with a value of
106 ± 3% which was not significantly different from 100%
(p ≤ 0.05). The low matrix effect demonstrates that the
cleanup procedure is efficient in removing interfering compounds,
and the reproducibility and accuracy could be considered as
Precision and recovery
The intraday precision was determined at three different
concentration levels, 0.015, 0.15, and 15 μM, in an analyte-free
matrix produced as described above. Each concentration level
was injected in triplicate and the average intraday variation
was low and found to be 6 ± 5%.
Three different concentration levels, 0.15, 1.50, and
15.00 μM, were put through the clean-up steps described
above to determine the recovery. The signal responses were
compared to a standard dissolved in the same solvent
composition. The average recovery for the three different
concentration levels was found to be 89 ± 9%.
Application of the method
Determination of heme in S. cerevisiae
To demonstrate the applicability of the developed method,
samples of approximately 100 mg (wet weight) of
S. cerevisiae were put through the extraction and clean-up
steps described in the BExperimental^ section. Heme was
identified and quantified in all the samples with an average
concentration of 51 ± 5 nmol/g. A chromatogram of heme
analysis in S. cerevisiae can be seen in ESM Fig. S2. When
the porphyrin fraction in the liquid-liquid extraction was
analyzed, two isomers of coproporphyrin (I and III) and
protoporphyrin IX were detected in all samples.
Effect of additives and time of cultivation on the heme concentration in E. coli
When E. coli was analyzed with different additives and
culturing length, the heme concentrations were shown to be
affected (Fig. 7). Addition of Fe2+ ions added to the LB broth
had however no significant increase in heme concentrations
(235 ± 10 nmol/g) compared to the control (231 ± 7 nmol/g).
The length of culturing time has previously been shown to
affect the concentration of heme and porphyrins in studies of
other microorganisms [
]. When culturing E. coli for
84 h, the heme concentration increased to 418 ± 37 nmol/g
and compared to the control cultured for 16 h. This
Fig. 7 Heme concentrations in E. coli with different culturing conditions
corresponds to an increase in the heme concentration with a
factor of 1.8.
The de novo synthesis of heme is tightly regulated by a
negative feedback control. High heme concentrations inside
the cell will inhibit the 5-aminolaevulinic acid synthase activity
which is the rate-limiting step in heme biosynthesis. Adding
exogenous 5-ALA to the growth medium will bypass the
negative feedback control, and if the bacteria have all the necessary
enzymes for heme biosynthesis, a higher concentration of heme
is expected. When E. coli was grown in a medium with 5.0 mM
of 5-ALA, the heme concentration was 642 ± 41 nmol/g, which
is a 53% increase compared to the 84-h control (p ≤ 0.05). This
results show that E. coli has the ability to synthesize heme from
5-ALA, and this is further emphasized when the bacterial
extracts were analyzed for porphyrins. In all the other bacterial
experiments, only protoporphyrin IX and coproporphyrin could
be detected at low concentrations. When 5-ALA was added to
the growth medium, uroporphyrin, 7-carboxylporphyrin,
5carboxylporphyrin, coproporphyrins I and III, as well as
protoporphyrin IX were detected in high concentrations (Fig. 8).
Hemin was added to the growth medium to investigate if
E. coli has the ability to acquire heme from its surroundings.
When grown in a heme-enriched medium, the heme
concentration in E. coli increased substantially compared to the
control. A 4.5-fold increase in heme concentration was observed,
reaching a concentration of 1874 ± 161 nmol/g. The bacterial
pellets were washed twice with NaCl solution prior the
cleanup process in order to remove any heme-containing broth and
the final wash solution was analyzed for heme. No heme could
be detected in the last washing solution. High concentrations
of heme in these samples show that E. coli has a high affinity
for exogenous heme.
Fig. 8 Porphyrin concentrations
(A) and HPLC-MS/MS
chromatogram (B) of a sample
after addition of 5-ALA to E. coli
growth medium. Due to the high
response of protoporphyrin IX,
the chromatogram has been
enlarged in the range 0.00–
1.50 min to make the other peaks
visible. UP, uroporphyrin; 7P,
coproporphyrin I; CPIII,
coproporphyrin III; PPIX,
Cobalt protoporphyrin IX inhibition of heme acquisition
Cobalt protoporphyrin IX has been shown to exhibit
antimicrobial activity against Porphyromonas gingivalis,
reducing both planktonic and biofilm growth [
CoPPIX molecule is identical to heme with the exception that
iron(II) in the center of the heme molecule has been replaced
with cobalt(III). Cobalt protoporphyrin IX could chemically
mimic heme in heme-acquiring microorganisms, implying
that addition of Co-PPIX could have the potential to disturb
the heme metabolism in bacteria similar to E. coli. Cobalt
protoporphyrin IX and hemin were added in equimolar
concentrations (10 μM each) to the cultivation broth. When the
bacterial extracts were analyzed, a reduction with 52%
(905 ± 20 nmol/g) was observed, which was equivalent to
the molar ratio of hemin/Co-PPIX in the cultivation broth. A
reduction with 48% in heme concentrations was also
observed when E. coli was cultured only with Co-PPIX as an
additive (217 ± 14 nmol/g). This demonstrates that the
E. coli heme-acquiring systems have the same affinity for
Co-PPIX as for heme which potentially makes Co-PPIX a
good candidate to chemically mimic heme. To investigate if
Co-PPIX is taken up by the bacteria, all bacterial extracts
were additionally analyzed in full scan mode with a mass
range between 100 and 1500 Da. When the [M+H]+ ion of
Co-PPIX (m/z 618.4) was extracted and verified by the
chromatographic retention time with a Co-PPIX standard,
it was shown that Co-PPIX was only present in the extracts
of E. coli in which the medium had been spiked with
CoPPIX (ESM Fig. S3).
However, even though these results demonstrate that the
heme-acquiring system of E. coli confuses Co-PPIX with
heme and has an uptake of Co-PPIX, no antimicrobial effect
was observed. Starting cultures of E. coli were allowed to
grow in broth spiked with Co-PPIX, and after 24 h, no
statistical differences in bacterial number compared to a control
could be seen.
There could however be an antimicrobial effect if higher
concentrations of Co-PPIX are used, and potential
antimicrobial effect of Co-PPIX to E. coli should be studied in more
detail regarding concentrations of Co-PPIX and different
cultivation media to gain more knowledge about E. coli
susceptibility against Co-PPIX.
By combining a selective, but still simple, liquid-liquid
extraction with a selective HPLC-MS/MS analysis and applying
experimental design to find optimal extraction conditions, a
method for determination of heme as well as heme precursors,
i.e., porphyrins, in microorganisms has been developed. When
this method was applied on S. cerevisiae and E. coli, it was
shown that the heme concentrations were affected by using
different additives in the cultivation media. Furthermore, it
was shown that cobalt protoporphyrin IX was able to mimic
heme and was taken up by E. coli, leading to a reduction in
intracellular concentrations of heme. This result indicates that
heme-acquiring mechanisms in microorganisms could
potentially be a good drug target to treat bacterial infections.
However, no antimicrobial effect of Co-PPIX on E. coli could
be shown, but this will be a subject to further investigations.
Acknowledgments This material is based upon the work supported by
the Swedish Research Council, contract No. K2014-70X-22533-01-3.
We thank Dr. Christoph Loderer, Department of Biochemistry and
Biophysics at Stockholm University, for kindly providing us with E. coli.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
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