Biochemical and Biophysical Research Communications 525 (2020) 378e383

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Treatment of Yersinia similis with the cationic lipid DOTAP enhances

adhesion to and invasion into intestinal epithelial cells e A proof-of-
principle study
€ hringer a, *, Jayaseelan Murugaiyan bc, Murat Eravci d, 1, Christoph Weise d,
Michael Bo
Uwe Roesler b, Heinrich Neubauer a, Lisa D. Sprague a
a
Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute for Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07743 Jena,
Germany
b €t Berlin, Centre for Infectious Medicine, Institute of Animal Hygiene and Environmental Health, Robert-von-Ostertag Str. 7 e 13, 14163
Freie Universita
Berlin, Germany
c
SRM University-AP, Department of Biotechnology, Mangalagiri Mandal, Guntur District, Andhra Pradesh 522 502, India
d €t Berlin, Institute of Chemistry and Biochemistry, BioSupraMol Bio-MS Unit, Thielallee 63, 14195 Berlin, Germany
Freie Universita

a r t i c l e i n f o a b s t r a c t

Article history: The monocationic quaternary surfactant DOTAP has been used for the delivery of nucleic acids and
Received 11 February 2020 peptides into mammalian cells. This study tested the applicability of DOTAP for the enhancement of
Accepted 11 February 2020 adhesion and invasion frequencies of Yersinia (Y.) similis to enable the analysis of the effects of low-
Available online 22 February 2020
pathogenic bacteria on intestinal epithelial cells.
Incubation of Y. similis with DOTAP ahead of infection of C2BBe1 intestinal epithelial cells increased
Keywords:
invasion and adhesion frequency four- and five-fold, respectively, in plating assays.
Yersinia similis
Proteomic approaches confirmed the increased bacterial load on infected cells: analysis of protein
DOTAP
Proteomics
extracts by two-dimensional difference gel electrophoresis (2D-DIGE) revealed higher amounts of bac-
C2BBe1 intestinal epithelial cells terial proteins present in the cells infected with DOTAP-treated bacteria. MALDI-TOF mass spectrometry
Invasion of selected spots from gel-separated protein extracts confirmed the presence of both bacterial and hu-
Adhesion man cell proteins in the samples.
Label-free quantitative proteomics analysis identified 1170 human cell proteins and 699 bacterial
proteins. Three times more bacterial proteins (279 vs. 93) were detected in C2BBe1 cells infected with
DOTAP-treated bacteria compared to infections with untreated bacteria. Infections with DOTAP-treated
Y. similis led to a significant upregulation of the stress-inducible ubiquitin-conjugating enzyme UBE2M
in C2BBe1 cells. This points towards a stronger impact of the stress and infection responsive transcription
factor AP-1 by enhanced bacterial load.
DOTAP-treatment of uninfected C2BBe1 cells led to a significant downregulation of the trans-
membrane trafficking protein TMED10.
The application of DOTAP could be helpful for investigating the impact of otherwise low adherent or
invasive bacteria on cultivated mammalian cells without utilisation of genetic modifications.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction

The genus Yersinia (Y.) currently comprises 19 species [1]. One

* Corresponding author.
E-mail addresses: michael.boehringer@fli.de (M. Bo €hringer), jayaseelan. cluster in the Yersinia phylogenetic tree is formed by the
murugaiyan@fu-berlin.de, jayaseelan.m@srmap.edu.in (J. Murugaiyan), murat. Y. pseudotuberculosis complex [2]. Three species within the cluster,
eravci@gmx.de (M. Eravci), chris.weise@biochemie.fu-berlin.de (C. Weise), uwe. Y. similis, Y. pseudotuberculosis, and Y. pestis display variable degrees
roesler@fu-berlin.de (U. Roesler), heinrich.neubauer@fli.de (H. Neubauer), lisa. of pathogenicity to humans which is reflected by differing adhesion
sprague@fli.de (L.D. Sprague).
1 and invasion frequencies to various cell types [3e8]. Adhesion is
Present address: University of Sussex, School of Life Sciences, Sussex Neuro-
science, Falmer, Brighton, BN1 9QG, United Kingdom. essential for the successful colonisation of epithelia, which is then

https://doi.org/10.1016/j.bbrc.2020.02.081
0006-291X/© 2020 Elsevier Inc. All rights reserved.
 €hringer et al. / Biochemical and Biophysical Research Communications 525 (2020) 378e383
M. Bo
followed by invasion. The latter enables the pathogen to evade the
humoral immune response of the host and to proliferate in a pro-
tected environment [9,10]. Enteropathogenic Yersiniae enter the
host’s body by way of Peyer’s patches, a part of the gut associated
lymphoid tissue, by translocating through the intestinal barrier via
b1 integrin-expressing M-cells [11e13]. Non-differentiated cells of
the intestinal epithelial cell (IEC) line Caco-2 have been used for
studying Y. pseudotuberculosis infection [14]. Peterson and Moose-
ker [15] evolved the Caco-2 cell line into the more homogeneous
C2BBe1 clone. Co-cultured with lymphocytes, the polarized C2BBe1
monolayer develops features of an M-cell interspersed follicle-
associated epithelium [16].
A recent study using b1 integrin-expressing C2BBe1 cells
confirmed the assumed low pathogenicity of Y. similis as they
showed a much lower adhesion and invasion frequency in com-
parison to pathogenic Y. pseudotuberculosis [17]. In order to increase
the adhesion and invasion frequency of Y. similis into C2BBe1 cells
without further confounders caused by genetic manipulation, an
alternative approach was sought for. Based on the idea that many
bacteria are negatively charged [18,19], it seemed plausible to as-
sume that a cationic lipid could be used to complex and subse-
quently transport bacteria into mammalian cells. The monocationic
quaternary surfactant DOTAP, which has been successfully used for
the delivery of nucleic acids [20,21] and peptides [22,23] into
mammalian cells, is an example of such a cationic lipid. The aim of
the present study was to test the applicability of DOTAP for the
improvement of adhesion and invasion frequencies of Y. similis to
enable the analysis of the effects of low-pathogenic bacteria on
intestinal epithelial cells.
2. Material and methods
2.1. Cell and bacterial culture, DOTAP treatment and infection
Cells of the intestinal epithelial line C2BBe1 (ATCC® CRL2102™,
purchased from LGC Standards at passage x þ 46) were cultivated at
37  C and 5% CO2 in DMEM (Thermo Fisher Scientific, # 32430),
containing 25 mM glucose, 4 mM L-Alanyl-L-Glutamine (Gluta-
MAX™), and 25 mM HEPES, supplemented with 0.1 Vol. foetal
bovine serum (FBS Gold, PAA Laboratories, Co €lbe, Germany).
Absence of mycoplasma contamination was regularly tested with
the Mycoplasma PCR kit (AppliChem, Darmstadt, Germany). Y.
similis Y228T [33] was cultivated on Columbia blood agar. 48 h
before the start of the experiment C2BBe1 cells at passages 60 to 75
were seeded at a density of 34,000/cm2 in 22 cm2 cell culture
dishes. After 24 h, the medium containing FBS was changed to
serum-free medium.
Bacteria from a single colony were grown overnight at 28  C in
LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, sup-
plemented with 1% (w/v) Glucose). The overnight culture was
centrifuged (3,500g, 5 min, RT) and resuspended in PBS to contain 4
∙ 108 bacteria/mL. After addition of 0.2 vol. DOTAP (100 mL to 500 mL
bacterial suspension; N-[1-(2, 3-Dioleoyloxy) propyl]-N,N,N-
trimethylammonium methyl-sulfate, Roche Diagnostics, Man-
nheim, Germany) the bacteria were incubated for 30 min at RT. As
control the same volume of PBS was added to the bacterial
suspension.
For infection with DOTAP-treated (INþ) or untreated bacteria (IN-
), 600 mL (containing ~2 ∙ 108 bacteria) of the suspension was added
to the C2BBe1 cells to a MOI of z100. The same volume of DOTAP or
PBS was added to C2BBe1 cells to obtain DOTAP-treated (Cþ) and
untreated control cells (C-). Additionally, bacterial suspensions were
added to cell culture media, to obtain a reference for bacteria
(DOTAP-treated: Yþ, untreated: Y-) (Fig. 1). Infected C2BBe1 cells and
controls were then incubated for 6 h at 37  C and 5% CO2.
379
2.2. Assessment of adhesion and invasion frequency
To determine the invasion frequency of bacteria into C2BBe1
cells the gentamicin protection assay (GPA) was used. After the
incubation period gentamicin (Biochrom, Berlin, Germany) was
added to the medium at a concentration of 100 mg/mL. The samples
were further incubated for 1 h, washed with PBS, and the cells lysed
in 0.2% (v/v) Triton X-100 for 10 min at RT. Serial dilutions of the
lysed samples were plated onto Columbia blood agar and counted
after overnight incubation at 28  C. For determination of the
adhesion frequency, the gentamicin treatment was omitted.
2.3. Protein extraction
For protein extraction from Y. similis infected (IN-, INþ) or non-
infected control C2BBe1 cells (C-, Cþ), cells were washed once with
2 mL ice-cold PBS. After addition of 750 mL ice-cold PBS to the
respective dishes the cells were scraped off and transferred each to
a separate reaction tube. The samples were then centrifuged at
3,000g (5 min, 4  C), and the resulting pellets resuspended in
500 mL cold 70% (v/v) ethanol. Samples containing only bacteria
(conditions Y- and Yþ) were centrifuged at 3,500g (5 min, 4  C),
washed once with cold PBS, centrifuged (6,000g, 5 min, 4  C), and
the pellet resuspended in 500 mL cold 70% (v/v) ethanol. All sus-
pensions were incubated for 10 min on ice and subsequently
centrifuged at 6,000g (5 min, 4  C). The supernatant was removed,
and the pellet resuspended in 20 mL lysis buffer/mg wet pellet [lysis
buffer 9.8 mmol/L (0.5% (w/v) n-Dodecyl b-D-maltoside, 50 mmol/L
Tris, 150 mmol/L NaCl and 20 mmol/L MgCl2, pH 7.4, and 0.01 Vol.
freshly prepared 100 mM Phenylmethanesulfonyl fluoride (PMSF,
Carl Roth, Karlsruhe, Germany)]. Samples were sonicated in an ul-
trasonic bath (Elma Schmidbauer, Singen, Germany) for 1 min, and
then incubated under continuous shaking for 30 min at 4  C. The
samples were then centrifuged for 20 min at 18,500g and the
resulting clear supernatant used for further analyses.
2.4. Label-free quantitative proteomics analyses
2.4.1. In-solution digestion and liquid chromatography-mass
spectrometry (LC-MS)
Samples containing 5 mg of protein were acetone precipitated
and reconstituted in 10 mL denaturation buffer (6 M urea and 2 M
thiourea in 10 mM HEPES, pH 8.0). Subsequently, proteins were
reduced, alkylated and digested with Lys-C and trypsin as described
elsewhere [24]. The peptides were desalted by solid phase extrac-
tion, using C18 EmporeTM disc cartridges (Supelco/Sigma-Aldrich,
Taufkirchen, Germany). The desalted peptide mixtures were then
loaded onto the Dionex Ultimate 3000 nanoLC system (Dionex/
Thermo Fisher Scientific, Idstein, Germany) using a C18 PepMap
trap column (75 mm  2 cm, Dionex) and separated by a 25 cm
fritless C18 micro-column of 100 mm internal diameter fused silica
capillary (Polymicro Technologies, Phoenix, AZ, USA) packed with
ReproSil-Pur C18-AQ 3 mm resin (Dr. Maisch GmbH, Ammerbuch-
Entringen, Germany). The eluted peptides were directly ionised
using electrospray ionisation and an LTQ Orbitrap Velos mass
spectrometer (Thermo Fisher Scientific, Bremen, Germany) oper-
ating in data-dependent acquisition mode with the Xcalibur soft-
ware (version 21.0.1140, Thermo Fisher Scientific) was used to
measure MS spectra [24].
2.4.2. Protein identification
The raw MS files were processed using the MaxQuant
Andromeda software suite (version 1.6.0.16) [25]. MS and MS/MS
data were searched against forward and backward protein se-
quences of a human reference proteome and sequences of Y. similis
380 €hringer et al. / Biochemical and Biophysical Research Communications 525 (2020) 378e383
M. Bo
Fig. 1. Schematic of the experimental setup. Y. similis bacteria cultivated overnight were incubated in PBS or PBS with 215 mmol/L DOTAP for 30 min at RT, and then transferred to
cell culture media or C2BBe1 cells grown in culture dishes. Non-infected cells were also treated with DOTAP (17.9 mg/mL, 23.1 mmol/L). The designations of the experimental
conditions, including abbreviations used in the text, are shown in the lower part of the figure.
(www.uniprot.org, accessed in May 2018). The parameter settings
were: MS mass tolerance ±7 ppm, MS/MS ions tolerance ±0.5 Da,
digestion mode was specific with trypsin/P as enzyme and a
maximum of two missed cleavages; variable modifications:
oxidation of methionine and protein N-terminal acetylation; fixed
modification: carbamidomethylation; 1% target-decoy-based false
discovery rate (FDR) for peptide and protein identification. The
minimum peptide length was set to 7 amino acids. Following the
protein identification, the data were imported into Perseus (version
1.6.1.1) [26], and the label free quantification (LFQ) intensity values
were log2 transformed. Data rows were first filtered to reduce the
matrix based on the categorical columns only identified by site,
reverse and potential contaminants and subsequently filtered
based on valid values for LFQ intensities that were present in at
least 3 out of 4 samples in any of the experimental groups. The
Student’s t-test was applied for identification of differentially
expressed proteins among the comparison groups and FDR was
corrected with the Benjamini-Hochberg procedure.
3. Results
3.1. Adhesion/invasion assay
Preliminary co-incubation experiments of bacteria and C2BBe1
cells were conducted with increasing incubation periods from 2 h
to 6 h. The difference of invasion frequencies of DOTAP-treated
bacterial compared to untreated bacterial cells increased with in-
cubation time and showed the highest value with an eight-fold
increase after 6 h incubation. Similarly, adhesion frequencies
increased two-fold between 2 h and 6 h incubation with untreated
bacteria but were ten times higher with DOTAP-treated compared
to untreated Y. similis after 6 h (data not shown). Adhesion fre-
quencies of Y. similis increased from 17.4 CFU/cell for untreated
bacteria to 88.2 CFU/cell with DOTAP-treated bacteria. Accordingly,
invasion frequencies of Y. similis increased from 0.14 CFU/cell for
untreated bacteria to 0.54 CFU/cell with DOTAP-treated bacteria
(Fig. 2). The toxicity of the 6 h DOTAP treatment was tested for
C2BBe1 cells by propidium iodide exclusion assay using flow
cytometry. The proportion of propidium iodide-stained cells
increased from 19% in the control to 30% in the DOTAP-treated
sample (Suppl. Fig. 1).
3.2. Mass spectrometry analysis of whole protein extracts
For additional verification that DOTAP treatment of Y. similis
enhanced adhesion, 24 protein samples (six experimental condi-
tions with four replicates each) were analysed by LC-MS. A total of
1869 proteins was detected by matching the found peptides to
species-specific databases, i.e. 1170 human and 699 bacterial hits.
925 of the detected human proteins were found in all four exper-
imental conditions relating to C2BBe1 cells (C-, Cþ, IN-, INþ). In
contrast, of the 699 detected bacterial proteins, 419 were found
only in the bacterial samples (Y- and Yþ). All the bacterial proteins
detected in conditions IN- and INþ were also found in conditions Y-
and Yþ. A marked difference was seen between samples of C2BBe1
cells incubated with untreated or DOTAP-treated bacteria: 93
bacterial proteins were found in cells infected with untreated Yer-
sinia (IN-), whereas 279 bacterial proteins were detected in samples
from C2BBe1 cells infected with DOTAP-treated bacteria (INþ). The
bacterial proteins detected in cells infected with DOTAP-treated
bacteria (INþ) overlapped with those from infections with un-
treated bacteria (IN-) with the exception of the product of the
Y. similis gene ptsI (Phosphoenolpyruvate-protein phospho-
transferase), which met the detection criteria (see: Label-free
quantitative proteomics analyses) only in IN- samples (Fig. 3).
Analysis of differences in protein abundance between the two
conditions, infection of C2BBe1 cells with untreated vs. DOTAP-
treated Y. similis (IN- vs. INþ), confirms the uneven distribution of
bacterial protein (Table 1 and Suppl. File). In total, 87 bacterial
proteins showed significantly higher intensity values in DOTAP-
treated samples (INþ), whereas none were significantly down-
regulated. No bacterial protein was significantly different between
 €hringer et al. / Biochemical and Biophysical Research Communications 525 (2020) 378e383
M. Bo 381

Fig. 2. Adhesion/invasion frequencies to C2BBe1 epithelial cells infected with untreated or DOTAP-treated Y. similis. Box-Whisker plots show the results of eight independent
experiments with minimal and maximal values. A: Invasion frequencies as determined by the gentamicin protection assay (Mann-Whitney U test: p ¼ 0.0104). B: Adhesion fre-
quencies assessed by plating of serial dilutions (Mann-Whitney U test: p ¼ 0.0002).

Fig. 3. Venn diagrams showing the distribution and overlap of the two sets of proteins detected by LC-MS analysis. A: Human proteins, B: Y. similis proteins. Labels denote the
experimental condition and number of detected proteins therein. Diagrams were generated with Venny (Oliveros, J.C. (2007e2015) Venny. An interactive tool for comparing lists with
Venn’s diagrams. http://bioinfogp.cnb.csic.es/tools/venny/index.html).

Table 1
Changing protein amount of C2BBe1 cells infected with untreated or DOTAP-treated
[28] to mammalian cells [21,23]. In this study, the applicability of
Y. similis (IN- vs. INþ) as assessed by LC-MS analysis. For identification of signifi- the transfection reagent DOTAP was tested to increase adhesion
cantly differentially abundant proteins among the comparison groups, the and invasion of a putative apathogenic bacterial species to human
Benjamini-Hochberg procedure was applied with a 5% False Discovery Rate intestinal epithelial cells.
correction of the Student’s t-test p-values.
This approach was used to avoid genetic manipulation to in-
Differentially expressed proteins during crease adhesion and invasion, as the required methodology and
infection of C2BBe1 cells with DOTAP- tools are (i) not readily available for all microorganisms, (ii) not
treated vs. untreated Y. similis
applicable in various lab settings and (iii) require a certain under-
Species IN þ up IN þ down standing on molecular mechanisms. To our knowledge, the delivery
Y. similis 87 0 of whole bacterial cells into human epithelial cells using DOTAP has
H. sapiens 1 3 not been described before.
Treatment of Y. similis with DOTAP prior to addition of bacteria
to human intestinal epithelial cells led to adhesion and invasion
conditions Y- and Yþ. When comparing untreated and DOTAP- frequencies comparable to the values obtained for
treated samples of C2BBe1 cells (C- vs Cþ), one human protein Y. pseudotuberculosis [17]. Increased adhesion and invasion fre-
(TMED10) was significantly downregulated in DOTAP-treated cells. quencies were confirmed not only by invasion and adhesion assays
One human protein (UBE2M, ubiquitin conjugating enzyme E2 M) but also by a proteomic approach. Mass spectrometry of whole cell
exhibited significant upregulation, three proteins (RPL8, RPL22 and extracts confirmed the increase of bacterial protein content in
AIFM1) significant downregulation in cells infected with DOTAP- C2BBe1 cells infected with DOTAP-treated bacteria. A three-fold
treated Y. similis (INþ) compared to infections with untreated increase in protein numbers was detected in samples of C2BBe1
bacteria (IN-, Table 1 and Suppl. File). cells infected with DOTAP-treated bacteria compared to cells
infected with untreated bacteria. These DOTAP-treated samples
represent almost 40% of the entirety of Yersinia proteins detected
4. Discussion over all experimental conditions (279 of 699 compared to 93 of 699
in untreated infections).
The cationic amphiphilic surfactant DOTAP [27] has been suc- The analysis of selected spots in a two-dimensional difference
cessfully used for the delivery of nucleic acids, peptides or proteins gel electrophoresis (2D-DIGE) approach (Suppl. Fig. 2) led to the
382 €hringer et al. / Biochemical and Biophysical Research Communications 525 (2020) 378e383
M. Bo
detection of both human and bacterial proteins in gel-separated
infection samples (Suppl. Table). The protein EIF1AX was detected
in all three master gels containing preparations with C2BBe1 cells
(Cþ, IN-, INþ). In contrast, the bacterial protein Tpx was recovered
from Yersinia only (Yþ) and the DOTAP-enhanced infection sam-
ples (INþ), but not from the gel containing infection samples with
untreated bacteria (IN-).
All of the proteins picked from the 2D gels and identified by
MALDI-TOF MS were also detected by LC-MS analysis of the whole
cell extracts (Suppl. Table and Suppl. File).
The mechanism of the enhanced intake of bacteria into C2BBe1
cells might be explained by the mechanism seen in DOTAP-
mediated DNA transfection [29]. The cationic lipid forms a bilayer
on the negatively charged bacterial surface, resulting in a positive
charge on the outer surface of the bacteria-lipid aggregate. This
aggregate then attaches electrostatically to the mammalian cell
surface and is subsequently taken up by an endocytic process. This
could be triggered by interaction of the cellular b1 integrin receptor
with the bacterial ligand invasin [30]. Y. similis encodes an invasin
gene homologue to the Y. pseudotuberculosis invasin, and C2BBe1
cells’ b1 integrin expression was tested positive by flow cytometry
(Suppl. Fig. 3).
It is still unclear, if the intracellular bacteria remain inside a
cellular compartment or are, like DNA, released into the cytoplasm
[20]. However, it can be assumed that bacteria are released into the
cytoplasm of the mammalian cell as viable bacteria were recovered
from the cells by GPA.
The method described here, delivery of living bacteria into
mammalian cells, could be a way to test the cellular reaction of non-
phagocytic cells to internalized bacteria despite lacking appropriate
adhesion or invasion molecules, needed for efficient (endocytic)
uptake. DOTAP treatment itself had no influence on bacterial pro-
tein expression, i.e. no differentially expressed proteins were
observed between Y. similis treated (Yþ) and untreated (Y-) sam-
ples. Moreover, DOTAP treatment had no detrimental effect on
bacterial viability, when comparing colony counts to untreated
samples (data not shown), but DOTAP treatment had an effect on
C2BBe1 cell integrity (Suppl. Fig. 1). These findings are in agreement
with the findings of Lappalainen et al. [31], who assessed the
cytotoxicity of DOTAP on cells of an adherent cancer cell line,
despite the higher concentrations used in the present study.
Only one protein (TMED10) was downregulated in untreated (C-
) compared to DOTAP-treated C2BBe1 cells (Cþ), and further
cellular proteins were differentially expressed between conditions
IN- and INþ. Here, one protein (UBE2M) was upregulated, and three
(RPL8, RPL22 and AIFM1) were downregulated in the LC-MS mea-
surements. UBE2M is a stress-inducible ubiquitin-conjugating
enzyme, upregulated by hypoxia and the transcription factor AP-1
[32]. Higher expression of this protein in C2BBe1 cells infected
with DOTAP-treated bacteria might be explained by the higher
adhesion and invasion frequency. This could subsequently cause a
stronger stress reaction due to the higher bacterial load.
Further studies are needed to assess the influence of secreted
proteins, for instance cytokines in the cell culture supernatants.
This would help to analyse the effect of higher invasion frequencies
of otherwise apathogenic Y. similis on the host cells in comparison
to highly pathogenic representatives of the Yersiniae.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
The technical assistance of Julia Haller is greatly appreciated.
This work was funded by the European Commission’s FP7-HEALTH
programme, project number 278976: “Antigone - ANTIcipating the
Global Onset of Novel Epidemics”. For mass spectrometry, M. E. and
C. W. would like to acknowledge the assistance of the Core Facility
BioSupraMol, supported by the Deutsche Forschungsgemeinschaft
(DFG).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2020.02.081.
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