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Article
Novel Template Plasmids pCyaA’-Kan and pCyaA’-Cam for
Generation of Unmarked Chromosomal cyaA’ Translational
Fusion to T3SS Effectors in Salmonella
Paulina A. Fernández 1, Marcela Zabner 1, Jaime Ortega 1, Constanza Morgado 1, Fernando Amaya 1,
Gabriel Vera 1, Carolina Rubilar 1, Beatriz Salas 1, Víctor Cuevas 2, Camila Valenzuela 1,3 ,
Fernando Baisón-Olmo 1, Sergio A. Álvarez 1and Carlos A. Santiviago 1, *
Citation: Fernández, P.A.; Zabner,
M.; Ortega, J.; Morgado, C.; Amaya,
F.; Vera, G.; Rubilar, C.; Salas, B.;
Cuevas, V.; Valenzuela, C.; et al.
Novel Template Plasmids
pCyaA’-Kan and pCyaA’-Cam for
Generation of Unmarked
Chromosomal cyaA’ Translational
Fusion to T3SS Effectors in Salmonella.
Microorganisms 2021,9, 475. https://
doi.org/10.3390/microorganisms903
0475
Academic Editor: Charles Dozois
Received: 6 February 2021
Accepted: 15 February 2021
Published: 25 February 2021
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1Laboratorio de Microbiología, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias
Químicas y Farmacéuticas, Universidad de Chile, 92101 Santiago, Chile;
fernandez.oyarzun.paulina@gmail.com (P.A.F.); maarce.z96@gmail.com (M.Z.); j.ortb2@gmail.com (J.O.);
constanza.morgado.ruiz@gmail.com (C.M.); fernando.amaya@ug.uchile.cl (F.A.);
gabriel.vera@ug.uchile.cl (G.V.); carolina.rubilar@ug.uchile.cl (C.R.); beatriz.salas.v@gmail.com (B.S.);
kamo.valenzuela@gmail.com (C.V.); fernando.baisonolmo@gmail.com (F.B.-O.); salvarez@uchile.cl (S.A.Á.)
2
Facultad de Medicina y Ciencia, Universidad San Sebastián, 92101 Santiago, Chile; victorcuevase@gmail.com
3Dynamics of Host-Pathogen Interactions Unit, Institut Pasteur, 75015 Paris, France
*Correspondence: csantiviago@ciq.uchile.cl; Tel.: +56-2-2978-1681
Abstract:
The type III secretion systems (T3SS) encoded in pathogenicity islands SPI-1 and SPI-2 are
key virulence factors of Salmonella. These systems translocate proteins known as effectors into eukary-
otic cells during infection. To characterize the functionality of T3SS effectors, gene fusions to the CyaA’
reporter of Bordetella pertussis are often used. CyaA’ is a calmodulin-dependent adenylate cyclase that
is only active within eukaryotic cells. Thus, the translocation of an effector fused to CyaA’ can be
evaluated by measuring cAMP levels in infected cells. Here, we report the construction of plasmids
pCyaA’-Kan and pCyaA’-Cam, which contain the ORF encoding CyaA’ adjacent to a cassette that
confers resistance to kanamycin or chloramphenicol, respectively, flanked by Flp recombinase target
(FRT) sites. A PCR product from pCyaA’-Kan or pCyaA’-Cam containing these genetic elements can
be introduced into the bacterial chromosome to generate gene fusions by homologous recombination
using the Red recombination system from bacteriophage
λ
. Subsequently, the resistance cassette can
be removed by recombination between the FRT sites using the Flp recombinase. As a proof of concept,
the plasmids pCyaA’-Kan and pCyaA’-Cam were used to generate unmarked chromosomal fusions
of 10 T3SS effectors to CyaA’ in S. Typhimurium. Each fusion protein was detected by Western blot
using an anti-CyaA’ monoclonal antibody when the corresponding mutant strain was grown under
conditions that induce the expression of the native gene. In addition, T3SS-1-dependent secretion of
fusion protein SipA-CyaA’ during
in vitro
growth was verified by Western blot analysis of culture su-
pernatants. Finally, efficient translocation of SipA-CyaA’ into HeLa cells was evidenced by increased
intracellular cAMP levels at different times of infection. Therefore, the plasmids pCyaA’-Kan and
pCyaA’-Cam can be used to generate unmarked chromosomal cyaA’ translational fusion to study
regulated expression, secretion and translocation of Salmonella T3SS effectors into eukaryotic cells.
Keywords:
Salmonella; T3SS; SPI-1; SPI-2; effector; translational fusion; cyaA’; adenylate cyclase;
translocation; secretion
1. Introduction
Salmonella enterica comprises over 2500 serotypes that are able to infect a wide range
of animal hosts causing a variety of diseases ranging from gastroenteritis to systemic
infections [
1
,
2
]. During the course of infection, S. enterica injects effector proteins into the
cytoplasm of host cells using type III secretion systems (T3SS). This process is relevant for
Salmonella virulence, as most T3SS effectors subvert host cellular functions through their
Microorganisms 2021,9, 475. https://doi.org/10.3390/microorganisms9030475 https://www.mdpi.com/journal/microorganisms
Microorganisms 2021,9, 475 2 of 11
enzymatic activities and physical interactions, promoting bacterial survival and coloniza-
tion (reviewed in [
2
]). S. enterica harbors two independent T3SS encoded in pathogenicity
islands SPI-1 and SPI-2 (T3SS-1 and T3SS-2, respectively). At least 7 effectors are known to
be secreted through T3SSI-1, 22 through T3SS-2, and 9 through both systems (reviewed
in [
3
]). Effectors include a signal sequence at the N-terminal region (first 20–30 residues)
required for secretion through T3SS. These sequences lack a discernible consensus, which
hinders the identification of possible effector proteins [4].
The analysis of translational fusions has proven to be very useful to evaluate effector
translocation into eukaryotic host cells. Sory and coworkers described a technique using the
catalytic adenylate cyclase domain of the bifunctional CyaA toxin from Bordetella pertussis
(CyaA’) [
5
]. CyaA’ is a calmodulin-dependent adenylate cyclase that catalyzes conversion of
ATP into cyclic AMP (cAMP). Since calmodulin is ubiquitous in eukaryotic cells but absent
in bacteria, the translocation of an effector fused to CyaA’ can be evaluated by measuring
the cAMP levels in infected cells.
Recently, Ramos-Morales and coworkers developed a protocol to generate site-specific
cyaA’ translational fusions in the chromosome of S. enterica [
6
] based on the Red recombina-
tion system from bacteriophage
λ
[
7
]. Although useful, this method presents an important
limitation: because of the structure of the mutant allele encoding each fusion, undesirable
polar effects may arise from antibiotic resistance gene expression. This is particularly
relevant when genes encoding effector proteins are located in operons or nearby genes or
operons encoding structural components of the associated T3SS. In this work, we describe a
method that allows the generation of unmarked cyaA’ translational fusions in the bacterial
chromosome using the
λ
Red recombination system. To this end, we constructed tem-
plate plasmids pCyaA’-Kan and pCyaA’-Cam that are used to amplify the PCR products
required for recombination. As a proof of concept, we generated unmarked cyaA’ trans-
lational fusion to genes encoding several T3SS effectors in the chromosome of S. enterica
serovar Typhimurium (S. Typhimurium). The production of each fusion was evaluated by
Western blot using an anti-CyaA’ monoclonal antibody, and occurred only in response to
growth conditions that induce the expression of the corresponding native gene. In addition,
T3SS-1-dependent secretion of a SipA-CyaA’ fusion during
in vitro
growth was evidenced
by Western blot analysis of culture supernatants. Finally, translocation of the SipA-CyaA’
fusion into HeLa cells was confirmed by measuring cAMP levels in infected cells.
2. Materials and Methods
2.1. Bacterial Strains and Growth Conditions
The bacterial strains used in this study are listed in Table S1. All S. Typhimurium
strains are derivatives of the wild-type, virulent strain 14028s [
8
,
9
]. Bacteria were routinely
grown in Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl)
at 37
◦
C with agitation (180 rpm). If bacteria harbored a temperature-sensitive plasmid
(i.e., pKD46 or pCP20), incubations were performed at 30
◦
C. When required, media
were supplemented with ampicillin (Amp, 100 mg/L), kanamycin (Kan, 75 mg/L), or
chloramphenicol (Cam, 20 mg/L). Media were solidified by the addition of agar (15 g/L).
For SPI-1-inducing conditions, bacteria were grown at 37
◦
C without agitation in LB
medium containing 300 mM NaCl. For SPI-2-inducing conditions, bacteria were grown
at 37
◦
C with agitation (180 rpm) in N-minimal medium (5 mM KCl, 0.5 mM (NH
4
)
2
SO
4
,
0.5 mM K
2
SO
4
, 1 mM KH
2
PO
4
, 10
µ
M MgCl
2
) [
10
] buffered in 100 mM MES (pH 5.8) and
supplemented with 0.1% casamino acids and 0.4% glucose as carbon source.
2.2. Standard DNA Techniques
Plasmid DNA was obtained using the QIAprep Spin Miniprep kit (Qiagen, German-
town, MD, USA). PCR products were purified using the QIAquick PCR Purification kit
(Qiagen, Germantown, MD, USA). DNA digestions using restriction endonucleases BamHI,
XhoI and SpeI (New England BioLabs, Ipswich, MA, USA) and ligations using T4 DNA
ligase (New England BioLabs, Ipswich, MA, USA) were conducted as recommended by
Microorganisms 2021,9, 475 3 of 11
the manufacturer. When required, DNA fragments from digestions were purified from
1% agarose gels prepared in Tris-acetate-EDTA (TAE) buffer using the QIAquick Gel Ex-
traction kit (Qiagen, Germantown, MD, USA). DNA samples were routinely analyzed by
electrophoresis in 1% agarose gels prepared in TAE buffer and visualized under UV light
after GelRed (Biotium Inc., Fremont, CA, USA) staining. Primers used in this study are
listed in Table S2.
2.3. Construction of Plasmids pCyaA’-Kan and pCyaA’-Cam
To generate pCyaA’-Kan, the cyaA’ region was amplified from pUTmini-Tn5cyaA’ [
6
]
using primers cyaA(F)-BamHI and cyaA(R)-XhoI (Table S2), and the PCR product was
cloned into pGEM-T Easy (Promega, Madison, WI, USA) as recommended by the manufac-
turer to generate pGEM-T::cyaA’. The Kan resistance cassette flanked by Flp recombinase
target (FRT) sites was amplified from pCLF4 (GenBank EU629214) [
11
] using primers
pCLF4(F)-XhoI and pCLF4(R)-BamHI-XhoI (Table S2), and the PCR product was cloned
into pGEM-T Easy as recommended by the manufacturer to generate pGEM-T::Kan. A
DNA fragment containing the Kan resistance cassette flanked by FRT sites was obtained by
digestion of pGEM-T::Kan with XhoI, and cloned into the unique XhoI site downstream of
cyaA’ in pGEM-T::cyaA’ to generate pGEM-T::cyaA’-Kan. The orientation of the insert in
the resulting plasmid was checked by PCR using primers cyaA(F)-BamHI and pCLF4(R)-
BamHI-XhoI (Table S2). A DNA fragment containing oriR6K and bla gene was amplified
from pKD4 (GenBank A Y048743) [
7
] using primers pCLF4(F)-BamHI and pCLF4(R)-
BamHI (Table S2). The PCR product was purified and digested with BamHI. Finally, this
fragment was ligated to a fragment containing cyaA’ and the Kan resistance cassette flanked
by FRT sites obtained by digestion of pGEM-T::cyaA’-Kan with BamHI.
To generate pCyaA’-Cam, the backbone of pCLF2 (GenBank HM047089) was amplified
using primers pCLF4(F)-XhoI and pCLF4(R)-SpeI (Table S2) to incorporate unique XhoI
and SpeI sites. The PCR product was purified, digested with XhoI and SpeI, and ligated
to a DNA fragment containing cyaA’ obtained by digestion of pGEM-T::cyaA’ with XhoI
and SpeI.
Plasmids pCyaA’-Kan and pCyaA’-Cam carry the R6K
γ
replication origin, which
requires the trans-acting
π
protein (encoded by pir) for replication. So, they were propagated
in Escherichia coli DH5
α λ
pir. Derivatives of plasmid pGEM-T Easy were propagated in
E. coli DH5α.
2.4. Generation of cyaA’ Translational Fusions
Derivatives of S. Typhimurium 14028s containing chromosomal fusions of cyaA’ to
genes encoding effectors secreted by T3SS-1 (sipA,sptP, and sopB), T3SS-2 (sifA,sseJ,sopD2,
steC, and sseG), or both T3SS-1 and T3SS-2 (spvB and gtgE) were constructed by the Red-
swap recombination method [
7
], with modifications. Briefly, a DNA fragment including
the cyaA’ ORF and a Kan resistance cassette was amplified from plasmid pCyaA’-Kan using
specific primers (“xxx_H1 + C1” and “xxx_H2 + C2”) designed for each fusion (Table S2).
Alternatively, the same primers were used to amplify a DNA fragment including the cyaA’
ORF and a Cam resistance cassette from plasmid pCyaA’-Cam. S. Typhimurium 14028s
carrying the temperature-sensitive plasmid pKD46, which expresses the
λ
Red recombinase
system, was grown to an OD
600
of 0.5 at 30
◦
C in LB medium supplemented with Amp
and L-arabinose (10 mM). Bacteria were made electrocompetent by sequential washes
with ice-cold sterile 15% glycerol, and transformed with ~500 ng of each PCR product.
Transformants were selected on LB agar supplemented with Kan or Cam at 37
◦
C. The
presence of each chromosomal fusion was confirmed by PCR amplification using specific
“forward” primers (“xxx_Out5”) designed for each effector together with “reverse” primer
CyaRev that hybridizes within cyaA’ (Table S2).
To obtain non-polar unmarked cyaA’ translational fusions, the corresponding antibi-
otic resistance cassette was removed by transforming each mutant with the temperature-
sensitive plasmid pCP20, which encodes the Flp recombinase [
7
,
12
]. Transformants were
Microorganisms 2021,9, 475 4 of 11
selected on LB agar supplemented with Amp at 30
◦
C. Next, individual colonies were
replica-plated on LB agar, LB agar supplemented with Amp, and LB agar supplemented
with Kan or Cam, and incubated at 37
◦
C. Transformants that had lost pCP20 and the
corresponding antibiotic resistance cassette were identified as those unable to grow in the
presence of Amp and Kan or Cam. The absence of the antibiotic resistance cassette was
confirmed by PCR amplification using primers cyaA(F)-BamHI and pCLF4(R)-BamHI-
XhoI (Table S2). Finally, phage P22 HT105-1 int-201 was used to transduce mutant alleles
∆
invA::Kan and
∆
ssaD::Kan into a derivative of S. Typhimurium 14028s harboring an
unmarked sipA-cyaA’ chromosomal fusion to inactive T3SS-1 or T3SS-2, respectively. The
presence of each mutant allele was confirmed by PCR amplification using primers flanking
the sites of substitution (Table S2).
2.5. Western Blot Analyses
Different bacterial strains were grown at 37
◦
C for 5 h under
in vitro
conditions that
induce the expression of SPI-1 or SPI-2 genes (10 mL cultures). For preparation of whole
cell lysates, bacteria recovered from 1 mL of each culture were suspended in phosphate-
buffered saline (PBS) and adjusted to an OD
600
of 2. Next, 75
µ
L of each bacterial suspension
was mixed with 25
µ
L of 4
×
Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and
the mix was boiled for 10 min and stored at
−
20
◦
C until further use. For preparation
of secreted proteins, the supernatant from each remaining culture was passed through a
0.2-
µ
m filter to remove residual bacteria. Proteins from the supernatants were precipitated
with trichloroacetic acid (10% v/v) and washed 3 times with ice-cold acetone. The pellet
was air dried and subjected to metanol-chloroform precipitation [
13
] to remove salts, as
described [
14
]. The final pellet was suspended in 40
µ
L of 4
×
Laemmli sample buffer
(Bio-Rad, Hercules, CA, USA) and the mix was boiled for 10 min and stored at
−
20
◦
C
until further use.
Samples of each lysate (10
µ
L) or preparations of secreted proteins (20
µ
L) were re-
solved by SDS-PAGE in 12% polyacrylamide gels using a Mini-Protean III system (Bio-Rad,
Hercules, CA, USA). The electrophoresis was conducted at 120 V (constant) using 1
×
run-
ning buffer (1.44% glycine, 0.3% Tris, 0.1% SDS). Transfer of proteins from polyacrylamide
gels to polyvinylidene fluoride (PVDF) membranes was performed in a Mini Trans-Blot
system (Bio-Rad, Hercules, CA, USA) for 90 min at 300 mA in transfer buffer (1.44% glycine,
0.3% Tris, 20% methanol). Membranes were incubated for 2 h at room temperature in a
blocking solution containing 5% BSA (Sigma-Aldrich, St. Louis, MA, USA) in Tris-buffered
saline supplemented with 0.1% Tween-20 (TBST). After blocking, the membranes were
incubated overnight at 4
◦
C with the mouse anti-CyaA’ monoclonal antibody 3D1 (Santa
Cruz Biotechnology, Dallas, TX, USA) diluted in blocking solution (1:10,000) or the mouse
anti-DnaK monoclonal antibody [8E2/2] ab69617 (Abcam, Cambridge, MA, USA) diluted
in blocking solution (1:10,000). After three washes with TBST, the membranes were in-
cubated for 2 h at room temperature with an anti-mouse IgG antibody conjugated with
horseradish peroxidase (Cell Signaling Technology, Danvers, MA, USA) diluted in blocking
solution (1:10,000). Finally, the protein bands were revealed by using the SuperSignal
West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA) as
recommended by the manufacturer. Digital images were collected using a Dyversity 4
imaging system (Syngene, Cambridge, UK) equipped with the GeneSys 1.2.5.0 software
(Syngene, Cambridge, UK).
2.6. Mammalian Cell Culture and Infection Assays
HeLa cells were routinely grown in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) at 37
◦
C in the presence of 5% CO
2
.
Monolayers for infection were prepared by seeding ~2
×
10
5
cells per well in a 24-well
plate and incubating for 18 h at 37
◦
C in the presence of 5% CO
2
. Prior to infection, each
monolayer was washed three times with sterile PBS. Bacteria were grown overnight at
37
◦
C under
in vitro
conditions that induce SPI-1 genes, washed three times with sterile
Microorganisms 2021,9, 475 5 of 11
PBS, suspended in 400
µ
L of DMEM-FBS, and added to monolayers of HeLa cells at a
multiplicity of infection (MOI) of 100 bacteria/cell. The plate was centrifuged at 200
×
gfor
5 min (to facilitate the interaction of bacteria and cells) and then incubated at 37
◦
C in the
presence of 5% CO
2
. After 1 h of incubation, the cells were washed two times with sterile
PBS and incubated for 1 h in DMEM-FBS supplemented with gentamicin (200
µ
g/mL)
to kill extracellular bacteria. Finally, the cells were washed three times with sterile PBS
and further incubated for 1, 3, or 6 h post infection in DMEM-FBS supplemented with
gentamicin (20 µg/mL).
2.7. Intracellular cAMP Measurement
Infected cell monolayers were washed three times with sterile PBS. Next, the cells were
lysed using 130
µ
L of 1
×
Sample Diluent supplied with the DetectX cAMP Direct Immunoassay
kit (Arbor Assays, Ann Arbor, MI, USA). Each lysate was then transferred to a microcentrifuge
tube and centrifuged at 4000
×
gfor 15 min at 4
◦
C. The supernatant was transferred to a clean
microcentrifuge tube and stored at
−
20
◦
C until further use. Finally, the cAMP levels in each
sample were determined by using the DetectX cAMP Direct Immunoassay kit (Arbor Assays,
Ann Arbor, MI, USA) following the manufacturer instructions.
3. Results
3.1. Rationale and Design
Plasmids pCyaA’-Kan and pCyaA’-Cam (Figure 1) were constructed for generation
of chromosomal cyaA’ translational fusions to evaluate translocation of S. enterica T3SS
effectors during infection of eukaryotic cells. These plasmids harbor a DNA fragment
encoding the adenylate cyclase domain of the cyaA gene from B. pertussis (cyaA’) upstream
of an antibiotic resistance cassette flanked by FRT sites (Figure 1).
Microorganisms 2020, 8, x FOR PEER REVIEW 5 of 11
HeLa cells were routinely grown in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) at 37 °C in the presence of 5% CO2.
Monolayers for infection were prepared by seeding ~2 × 105 cells per well in a 24-well plate
and incubating for 18 h at 37 °C in the presence of 5% CO2. Prior to infection, each mono-
layer was washed three times with sterile PBS. Bacteria were grown overnight at 37 °C
under in vitro conditions that induce SPI-1 genes, washed three times with sterile PBS,
suspended in 400 μL of DMEM-FBS, and added to monolayers of HeLa cells at a multi-
plicity of infection (MOI) of 100 bacteria/cell. The plate was centrifuged at 200 × g for 5
min (to facilitate the interaction of bacteria and cells) and then incubated at 37 °C in the
presence of 5% CO2. After 1 h of incubation, the cells were washed two times with sterile
PBS and incubated for 1 h in DMEM-FBS supplemented with gentamicin (200 μg/mL) to
kill extracellular bacteria. Finally, the cells were washed three times with sterile PBS and
further incubated for 1, 3, or 6 h post infection in DMEM-FBS supplemented with gen-
tamicin (20 μg/mL).
2.7. Intracellular cAMP Measurement
Infected cell monolayers were washed three times with sterile PBS. Next, the cells
were lysed using 130 μL of 1× Sample Diluent supplied with the DetectX cAMP Direct
Immunoassay kit (Arbor Assays, Ann Arbor, MI, USA). Each lysate was then transferred
to a microcentrifuge tube and centrifuged at 4000× g for 15 min at 4 °C. The supernatant
was transferred to a clean microcentrifuge tube and stored at −20 °C until further use.
Finally, the cAMP levels in each sample were determined by using the DetectX cAMP
Direct Immunoassay kit (Arbor Assays, Ann Arbor, MI, USA) following the manufacturer
instructions.
3. Results
3.1. Rationale and Design
Plasmids pCyaA’-Kan and pCyaA’-Cam (Figure 1) were constructed for generation
of chromosomal cyaA’ translational fusions to evaluate translocation of S. enterica T3SS
effectors during infection of eukaryotic cells. These plasmids harbor a DNA fragment en-
coding the adenylate cyclase domain of the cyaA gene from B. pertussis (cyaA’) upstream
of an antibiotic resistance cassette flanked by FRT sites (Figure 1).
Figure 1. Scheme of template plasmids pCyaA’-Kan and pCyaA’-Cam for generation of unmarked chromosomal cyaA’
translational fusion to T3SS effectors in Salmonella. C1 and C2 are the priming sites for amplification of a DNA fragment
including the cyaA’ ORF and the adjacent antibiotic resistance gene flanked by FRT sites.
To generate the translational fusions (Figure 2), a linear DNA fragment containing
the cyaA’ ORF and a Kan or a Cam resistance cassette flanked by FRT sites is obtained by
PCR amplification using template plasmid pCyaA’-Kan or pCyaA’-Cam, respectively.
Figure 1.
Scheme of template plasmids pCyaA’-Kan and pCyaA’-Cam for generation of unmarked chromosomal cyaA’
translational fusion to T3SS effectors in Salmonella. C1 and C2 are the priming sites for amplification of a DNA fragment
including the cyaA’ ORF and the adjacent antibiotic resistance gene flanked by FRT sites.
To generate the translational fusions (Figure 2), a linear DNA fragment containing the
cyaA’ ORF and a Kan or a Cam resistance cassette flanked by FRT sites is obtained by PCR
amplification using template plasmid pCyaA’-Kan or pCyaA’-Cam, respectively. Primers
can be designed at any position in the ORF of the target chromosomal gene, although in
the case of T3SS effectors it is important to maintain the signal required for secretion at the
N-terminal region. We designed primers including 40-nt homology extensions (H1 and
H2) and 20-nt priming sequences (C1 and C2) for pCyaA’-Kan or pCyaA’-Cam (Table S2).
Extensions are homologous to regions immediately upstream (H1) and downstream (H2) of
the stop codon in the target gene in order to get an in-frame translational fusion to the cyaA’
ORF. Site-directed integration of the PCR product into the chromosome of S. Typhimurium
Microorganisms 2021,9, 475 6 of 11
is mediated by the
λ
Red recombination system encoded in plasmid pKD46 [
7
]. Finally, to
eliminate possible polar effects the antibiotic resistance cassette can be removed from the
chromosome by recombination between the flanking FRT sites using the Flp recombinase
encoded in plasmid pCP20 [7,12].
Microorganisms 2020, 8, x FOR PEER REVIEW 6 of 11
Primers can be designed at any position in the ORF of the target chromosomal gene, alt-
hough in the case of T3SS effectors it is important to maintain the signal required for se-
cretion at the N-terminal region. We designed primers including 40-nt homology exten-
sions (H1 and H2) and 20-nt priming sequences (C1 and C2) for pCyaA’-Kan or
pCyaA’-Cam (Table S2). Extensions are homologous to regions immediately upstream
(H1) and downstream (H2) of the stop codon in the target gene in order to get an in-frame
translational fusion to the cyaA’ ORF. Site-directed integration of the PCR product into the
chromosome of S. Typhimurium is mediated by the λ Red recombination system encoded
in plasmid pKD46 [7]. Finally, to eliminate possible polar effects the antibiotic resistance
cassette can be removed from the chromosome by recombination between the flanking
FRT sites using the Flp recombinase encoded in plasmid pCP20 [7,12].
Figure 2. Schematic representation of the procedure to generate unmarked cyaA’ translational fu-
sions in the chromosome of Salmonella. C1 and C2 are the priming sites for amplification of a frag-
ment of template plasmids pCyaA′-Kan and pCyaA′-Cam. H1 and H2 are specific homology regions
required for insertion of the amplified fragment into a defined site in the chromosome.
3.2. Generation and Immunodetection of CyaA’ Translational Fusions to S. Typhimurium T3SS
Effectors
As a proof of concept, we used pCyaA’-Kan to generate derivatives of S. Typhi-
murium 14028s harboring chromosomal cyaA’ translational fusions to genes encoding the
effector proteins SipA, SopB, SptP, SifA, SseJ, SopD2, SteC, SseG, GtgE, and SpvB. The
presence of the corresponding mutant alleles was evidenced by the acquisition of the an-
tibiotic resistance encoded in the mutagenic cassette recombined in the genome of each
Figure 2.
Schematic representation of the procedure to generate unmarked cyaA’ translational fusions
in the chromosome of Salmonella. C1 and C2 are the priming sites for amplification of a fragment
of template plasmids pCyaA
0
-Kan and pCyaA
0
-Cam. H1 and H2 are specific homology regions
required for insertion of the amplified fragment into a defined site in the chromosome.
3.2. Generation and Immunodetection of CyaA’ Translational Fusions to S. Typhimurium
T3SS Effectors
As a proof of concept, we used pCyaA’-Kan to generate derivatives of S. Typhimurium
14028s harboring chromosomal cyaA’ translational fusions to genes encoding the effector
proteins SipA, SopB, SptP, SifA, SseJ, SopD2, SteC, SseG, GtgE, and SpvB. The presence
of the corresponding mutant alleles was evidenced by the acquisition of the antibiotic
resistance encoded in the mutagenic cassette recombined in the genome of each recipient
strain, and further confirmed by PCR using primers flanking selected recombination join
points (see Materials and Methods section). Next, each mutant strain generated was grown
independently under culture conditions that induce the expression of genes associated
with SPI-1 (i.e., LB medium containing 300 mM NaCl) or SPI-2 (i.e., N-minimal medium
adjusted to pH 5.8). Lysates of bacteria recovered from each culture were subjected to SDS-
PAGE and each fusion protein was detected by Western blot using a commercial anti-CyaA’
Microorganisms 2021,9, 475 7 of 11
monoclonal antibody. Of note, the expression of each fusion protein was only detected
when the corresponding mutant strain was grown
in vitro
under culture conditions that
induce the expression of the native gene. Thus, fusions SipA-CyaA’, SopB-CyaA’, and
SptP-CyaA’ were detected under SPI-1-inducing conditions; fusions SifA-CyaA’, SseJ-
CyaA’, SopD2-CyaA’, SteC-CyaA’, and SseG-CyaA’ were detected under SPI-2-inducing
conditions; and fusions GtgE-CyaA’ and SpvB-CyaA’ were detected under both SPI-1- and
SPI-2-inducing conditions (Figure 3). Identical results were obtained when a mutant strain
expressing SipA-CyaA’ generated using plasmid pCyaA’-Cam as template was analyzed
(Figure S1). In all cases, these proteins were not detected in lysates prepared from cultures
of the wild-type strain (Figure 3and Figure S1). These observations indicate that genes
encoding the fusion proteins analyzed respond to environmental cues that regulate the
expression of the corresponding wild-type genes in S. Typhimurium.
Microorganisms 2020, 8, x FOR PEER REVIEW 7 of 11
recipient strain, and further confirmed by PCR using primers flanking selected recombi-
nation join points (see Materials and Methods section). Next, each mutant strain generated
was grown independently under culture conditions that induce the expression of genes
associated with SPI-1 (i.e., LB medium containing 300 mM NaCl) or SPI-2 (i.e., N-minimal
medium adjusted to pH 5.8). Lysates of bacteria recovered from each culture were sub-
jected to SDS-PAGE and each fusion protein was detected by Western blot using a com-
mercial anti-CyaA’ monoclonal antibody. Of note, the expression of each fusion protein
was only detected when the corresponding mutant strain was grown in vitro under cul-
ture conditions that induce the expression of the native gene. Thus, fusions SipA-CyaA’,
SopB-CyaA’, and SptP-CyaA’ were detected under SPI-1-inducing conditions; fusions
SifA-CyaA’, SseJ-CyaA’, SopD2-CyaA’, SteC-CyaA’, and SseG-CyaA’ were detected un-
der SPI-2-inducing conditions; and fusions GtgE-CyaA’ and SpvB-CyaA’ were detected
under both SPI-1- and SPI-2-inducing conditions (Figure 3). Identical results were ob-
tained when a mutant strain expressing SipA-CyaA’ generated using plasmid
pCyaA’-Cam as template was analyzed (Figure S1). In all cases, these proteins were not
detected in lysates prepared from cultures of the wild-type strain (Figures 3 and S1). These
observations indicate that genes encoding the fusion proteins analyzed respond to envi-
ronmental cues that regulate the expression of the corresponding wild-type genes in S.
Typhimurium.
Figure 3. Immunodetection of CyaA’ fusion proteins expressed by S. Typhimurium mutant strains
grown under SPI-1- and SPI-2-inducing conditions. Bacterial strains expressing individual CyaA’
fusion proteins were grown in vitro under conditions that induce the expression of SPI-1 genes (i.e.,
LB medium containing 300 mM NaCl) or SPI-2 genes (i.e., N-minimal medium adjusted to pH 5.8).
Bacterial lysates prepared from each culture were subjected to SDS-PAGE in 12% polyacrylamide
gels. Proteins from gels were transferred to PVDF membranes, and CyaA’ fusion proteins were de-
tected by Western blot using a commercial mouse anti-CyaA’ monoclonal antibody and anti-mouse
IgG conjugated with horseradish peroxidase as secondary antibody.
Figure 3.
Immunodetection of CyaA’ fusion proteins expressed by S. Typhimurium mutant strains
grown under SPI-1- and SPI-2-inducing conditions. Bacterial strains expressing individual CyaA’
fusion proteins were grown
in vitro
under conditions that induce the expression of SPI-1 genes (i.e.,
LB medium containing 300 mM NaCl) or SPI-2 genes (i.e., N-minimal medium adjusted to pH 5.8).
Bacterial lysates prepared from each culture were subjected to SDS-PAGE in 12% polyacrylamide gels.
Proteins from gels were transferred to PVDF membranes, and CyaA’ fusion proteins were detected
by Western blot using a commercial mouse anti-CyaA’ monoclonal antibody and anti-mouse IgG
conjugated with horseradish peroxidase as secondary antibody.
A number of the generated fusions are encoded within SPI-1 or SPI-2; therefore,
we decided to remove the antibiotic resistance cassette associated to the structure of
each mutant to avoid polar effects on neighboring genes. This is particularly relevant
in the case of genes located in operons, or adjacent to operons encoding regulators or
structural components that are essential for T3SS-1 or T3SS-2 function. After removal
of the resistance cassette, all unmarked mutant strains (including those obtained using
template plasmids pCyaA’-Kan and pCyaA’-Cam) were grown under SPI-1- and SPI-2-
Microorganisms 2021,9, 475 8 of 11
inducing conditions and lysates of bacteria recovered from each culture were subjected to
SDS-PAGE and Western blot to detect each fusion protein. As expected, all fusion proteins
were detected only when the corresponding unmarked mutant strain was grown under
conditions inducing the expression of the native gene. In addition, these proteins were not
detected in lysates prepared from cultures of the wild-type strain (Figure S2). These results
indicate that removal of the antibiotic resistance cassette does not affect the regulated
expression of each fusion.
3.3. Secretion of a Selected CyaA’ Fusion Protein by S. Typhimurium during In Vitro Growth
To evaluate if the fusion proteins generated by our method can be secreted to the
culture medium, we analyzed culture supernatants and bacterial lysates obtained from an
unmarked mutant expressing SipA-CyaA’ and derivative strains harboring mutant alleles
∆
invA::Kan and
∆
ssaD::Kan (to inactivate T3SS-1 and T3SS-2, respectively) grown under
SPI-1- and SPI-2-inducing conditions. Samples of culture supernatants and bacterial lysates
were subjected to SDS-PAGE and Western blot to detect SipA-CyaA’. As expected, the
fusion protein was detected in bacterial lysates from cultures grown under SPI-1-inducing
conditions, and not detected in lysates from bacteria grown under SPI-2-inducing condi-
tions (Figure 4). On the other hand, SipA-CyaA’ was detected in culture supernatants only
when bacteria harboring an active T3SS-1 were grown under SPI-1-inducing conditions
(Figure 4). Of note, the cytosolic protein DnaK was not detected in culture supernatant
samples, indicating that detection of SipA-CyaA’ in culture supernatants was not a conse-
quence of bacterial lysis. Finally, SipA-CyaA’ was not detected in lysates and supernatants
from cultures of the wild-type strain (Figure 4). Thus, our results indicate that CyaA’ fusion
proteins generated by our method can be secreted to the culture medium by Salmonella in a
T3SS-dependent manner during in vitro growth.
Microorganisms 2020, 8, x FOR PEER REVIEW 8 of 11
A number of the generated fusions are encoded within SPI-1 or SPI-2; therefore, we
decided to remove the antibiotic resistance cassette associated to the structure of each mu-
tant to avoid polar effects on neighboring genes. This is particularly relevant in the case
of genes located in operons, or adjacent to operons encoding regulators or structural com-
ponents that are essential for T3SS-1 or T3SS-2 function. After removal of the resistance
cassette, all unmarked mutant strains (including those obtained using template plasmids
pCyaA’-Kan and pCyaA’-Cam) were grown under SPI-1- and SPI-2-inducing conditions
and lysates of bacteria recovered from each culture were subjected to SDS-PAGE and
Western blot to detect each fusion protein. As expected, all fusion proteins were detected
only when the corresponding unmarked mutant strain was grown under conditions in-
ducing the expression of the native gene. In addition, these proteins were not detected in
lysates prepared from cultures of the wild-type strain (Figure S2). These results indicate
that removal of the antibiotic resistance cassette does not affect the regulated expression
of each fusion.
3.3. Secretion of a Selected CyaA’ Fusion Protein by S. Typhimurium During in Vitro Growth
To evaluate if the fusion proteins generated by our method can be secreted to the
culture medium, we analyzed culture supernatants and bacterial lysates obtained from an
unmarked mutant expressing SipA-CyaA’ and derivative strains harboring mutant alleles
∆invA::Kan and ∆ssaD::Kan (to inactivate T3SS-1 and T3SS-2, respectively) grown under
SPI-1- and SPI-2-inducing conditions. Samples of culture supernatants and bacterial ly-
sates were subjected to SDS-PAGE and Western blot to detect SipA-CyaA’. As expected,
the fusion protein was detected in bacterial lysates from cultures grown under SPI-1-in-
ducing conditions, and not detected in lysates from bacteria grown under SPI-2-inducing
conditions (Figure 4). On the other hand, SipA-CyaA’ was detected in culture superna-
tants only when bacteria harboring an active T3SS-1 were grown under SPI-1-inducing
conditions (Figure 4). Of note, the cytosolic protein DnaK was not detected in culture su-
pernatant samples, indicating that detection of SipA-CyaA’ in culture supernatants was
not a consequence of bacterial lysis. Finally, SipA-CyaA’ was not detected in lysates and
supernatants from cultures of the wild-type strain (Figure 4). Thus, our results indicate
that CyaA’ fusion proteins generated by our method can be secreted to the culture me-
dium by Salmonella in a T3SS-dependent manner during in vitro growth.
Figure 4. T3SS-1-dependent secretion of fusion protein SipA-CyaA’. Bacterial strains expressing SipA-CyaA’ were grown
in vitro under conditions that induce the expression of SPI-1 genes (i.e., LB medium containing 300 mM NaCl) or SPI-2
genes (i.e., N-minimal medium adjusted to pH 5.8). Proteins from culture supernatants and bacterial lysates were sub-
jected to SDS-PAGE in 12% polyacrylamide gels and transferred to PVDF membranes. SipA-CyaA’ fusion protein and
DnaK were detected by Western blot using a commercial mouse anti-CyaA’ monoclonal antibody or a commercial mouse
anti-DnaK monoclonal antibody. In both cases, an anti-mouse IgG conjugated with horseradish peroxidase was used as
secondary antibody.
Figure 4.
T3SS-1-dependent secretion of fusion protein SipA-CyaA’. Bacterial strains expressing SipA-CyaA’ were grown
in vitro
under conditions that induce the expression of SPI-1 genes (i.e., LB medium containing 300 mM NaCl) or SPI-2 genes
(i.e., N-minimal medium adjusted to pH 5.8). Proteins from culture supernatants and bacterial lysates were subjected to SDS-
PAGE in 12% polyacrylamide gels and transferred to PVDF membranes. SipA-CyaA’ fusion protein and DnaK were detected
by Western blot using a commercial mouse anti-CyaA’ monoclonal antibody or a commercial mouse anti-DnaK monoclonal
antibody. In both cases, an anti-mouse IgG conjugated with horseradish peroxidase was used as secondary antibody.
3.4. Translocation of a Selected CyaA’ Fusion Protein during S. Typhimurium Infection of
HeLa Cells
To further evaluate if the fusion proteins generated by our method can be expressed
and subsequently translocated into eukaryotic cells, we conducted
in vitro
infection assays
Microorganisms 2021,9, 475 9 of 11
using HeLa cells and our unmarked mutant expressing SipA-CyaA’. At different times of
infection, the cells were lysed and the level of cAMP in the lysates was quantified using
an ELISA kit (see Materials and Methods section). Efficient translocation of fusion protein
SipA-CyaA’ into HeLa cells was evidenced by increased intracellular levels of cAMP at
different times of infection in comparison to infections conducted with the wild-type strain
(Figure 5). Thus, our results indicate that CyaA’ fusion proteins generated by our method
can be translocated by Salmonella into eukaryotic cells during infection.
Microorganisms 2020, 8, x FOR PEER REVIEW 9 of 11
3.4. Translocation of a Selected CyaA’ Fusion Protein During S. Typhimurium Infection of HeLa
Cells
To further evaluate if the fusion proteins generated by our method can be expressed
and subsequently translocated into eukaryotic cells, we conducted in vitro infection as-
says using HeLa cells and our unmarked mutant expressing SipA-CyaA’. At different
times of infection, the cells were lysed and the level of cAMP in the lysates was quantified
using an ELISA kit (see Materials and Methods section). Efficient translocation of fusion
protein SipA-CyaA’ into HeLa cells was evidenced by increased intracellular levels of
cAMP at different times of infection in comparison to infections conducted with the wild-
type strain (Figure 5). Thus, our results indicate that CyaA’ fusion proteins generated by
our method can be translocated by Salmonella into eukaryotic cells during infection.
Figure 5. Translocation of fusion protein SipA-CyaA’ into eukaryotic cells during infection. Mono-
layers of HeLa cells were infected with an unmarked S. Typhimurium mutant strain expressing
SipA-CyaA’ using a multiplicity of infection (MOI) of 100 bacteria/cell. At different times, infected
cells were lysed and the level of cAMP in the lysates was determined using a commercial ELISA kit.
Graph shows mean values ± SEM from an independent assay performed in duplicate.
4. Discussion
In the present study, we describe the construction of novel template plasmids
pCyaA-Kan and pCyaA-Cam (Figure 1) for the generation of chromosomal cyaA’ transla-
tional fusions based on site-directed integration of a PCR product using the λ Red recom-
bination system [7]. Unlike protocols to generate chromosomal cyaA’ fusions described
previously [6,15], our method allows the removal of the antibiotic resistance cassette by
Flp-mediated recombination [7,12], resulting in an unmarked chromosomal gene fusion
(Figure 2).
Reporter gene fusions have been used as a tool to identify new effector proteins in
pathogenic bacteria and for monitoring their expression, secretion, and translocation into
infected cells. Several approaches involving chromosomally-encoded fusions have been
developed as they are advantageous over plasmid-encoded fusions since they result in a
single-copy gene fusion whose expression depends on native promoters [6,15–17]. In this
context, cyaA’ has arisen as an appealing reporter gene for studying the expression, secre-
tion, and translocation of effector proteins in the case of several pathogenic bacteria
[5,15,18–22]. The advantage of using a CyaA’ enzymatic tag is that the expression and
secretion of the corresponding fusion protein can be detected in bacterial cultures in vitro
by immunoblotting [18,21,22], and also translocation of these fusions into host cells can be
Figure 5.
Translocation of fusion protein SipA-CyaA’ into eukaryotic cells during infection. Mono-
layers of HeLa cells were infected with an unmarked S. Typhimurium mutant strain expressing
SipA-CyaA’ using a multiplicity of infection (MOI) of 100 bacteria/cell. At different times, infected
cells were lysed and the level of cAMP in the lysates was determined using a commercial ELISA kit.
Graph shows mean values ±SEM from an independent assay performed in duplicate.
4. Discussion
In the present study, we describe the construction of novel template plasmids pCyaA-Kan
and pCyaA-Cam (Figure 1) for the generation of chromosomal cyaA’ translational fusions based
on site-directed integration of a PCR product using the
λ
Red recombination system [
7
]. Unlike
protocols to generate chromosomal cyaA’ fusions described previously [
6
,
15
], our method
allows the removal of the antibiotic resistance cassette by Flp-mediated recombination [
7
,
12
],
resulting in an unmarked chromosomal gene fusion (Figure 2).
Reporter gene fusions have been used as a tool to identify new effector proteins in
pathogenic bacteria and for monitoring their expression, secretion, and translocation into
infected cells. Several approaches involving chromosomally-encoded fusions have been
developed as they are advantageous over plasmid-encoded fusions since they result in
a single-copy gene fusion whose expression depends on native promoters [
6
,
15
–
17
]. In
this context, cyaA’ has arisen as an appealing reporter gene for studying the expression,
secretion, and translocation of effector proteins in the case of several pathogenic bacte-
ria [
5
,
15
,
18
–
22
]. The advantage of using a CyaA’ enzymatic tag is that the expression and
secretion of the corresponding fusion protein can be detected in bacterial cultures
in vitro
by immunoblotting [
18
,
21
,
22
], and also translocation of these fusions into host cells can be
monitored by its calmodulin-dependent adenylate cyclase activity, measuring the cAMP
levels in infected cells [5,15,18–22].
Regarding S. enterica, a strategy based on the Red recombination system from bacte-
riophage
λ
has been successfully used for the generation of site-specific cyaA’ translational
fusions in the chromosome [
6
]. However, the main advantage of using our template
plasmids is the possibility to generate unmarked cyaA’ translational fusions by removal
Microorganisms 2021,9, 475 10 of 11
of the antibiotic resistance cassette via Flp-mediated recombination. This novel feature
is highly desirable when target genes encoding effector proteins are located in operons
or nearby genes encoding structural components of the associated T3SS, where undesir-
able polar effects may arise from strong promoter driving the expression of the antibiotic
resistance cassette.
We tested the functionality of our novel template plasmids by constructing cyaA’
fusions to 10 genes encoding T3SS effectors in the chromosome of S. Typhimurium. In all
cases, the fusion protein was detected by immunoblot in bacterial lysates from cultures
grown under the corresponding expression-inducing conditions (Figure 3and Figure S1).
This validates the use of CyaA’ as an epitope tag for studying expression and regulation of
genes encoding T3SS effector proteins. Of note, removal of the antibiotic resistance cassette
did not affect protein detection and regulated expression of the corresponding translational
fusion (Figure S2). Furthermore, using a S. Typhimurium strain carrying an unmarked
cyaA’ fusion, we confirmed the T3SS-1-dependent secretion of SipA-CyaA’ during
in vitro
growth by Western blot analysis of culture supernatants (Figure 4). We also confirmed the
translocation of SipA-CyaA’ into eukaryotic cells by measuring the levels of cAMP in HeLa
cells infected with the mentioned mutant strain (Figure 5). Although not tested in this work,
detection of translocated fusion proteins into eukaryotic cells could also be performed by
immunoblotting after subcellular fractionation, as reported [
23
], which can be an attractive
alternative when using host cells with elevated phosphodiesterase activity. In addition,
anti-CyaA antibodies can be used to detect the intracellular localization of a given T3SS
effector fused to CyaA’ by immunofluorescence microscopy, as described [24,25].
Even though this study was focused on the characterization of S. Typhimurium T3SS
effectors, our procedure for generation of unmarked chromosomal cyaA’ translational
fusions can be applied to any bacterial species that uses T3SS or other secretion systems to
translocate effector proteins into eukaryotic cells (e.g., T4SS and T6SS), and that allows the
function of λRed and Flp recombinases (e.g., E. coli and Shigella, among others).
Supplementary Materials:
The following are available online at https://www.mdpi.com/2076-2
607/9/3/475/s1, Table S1: Bacterial strains used in this study, Table S2: Primers used in this study,
Figure S1: Immunodetection of SipA-CyaA’ fusion protein expressed from a marked S. Typhimurium
mutant strain constructed using plasmid pCyaA’-Cam as template. Figure S2: Immunodetection of
CyaA’ fusion proteins expressed from unmarked S. Typhimurium mutant strains grown under SPI-1-
and SPI-2-inducing conditions.
Author Contributions:
Conceptualization, C.A.S., C.V., S.A.Á. and F.B.-O.; methodology, C.A.S., C.V.
and S.A.Á.; resources, C.A.S., S.A.Á. and F.B.-O.; investigation, P.A.F., M.Z., J.O., C.M., F.A., G.V.,
C.R., V.C., B.S. and C.V.; formal analysis, C.A.S., S.A.Á., P.A.F., M.Z., J.O., C.M., F.A., G.V., C.R., V.C.,
B.S., C.V. and F.B.-O.; validation, C.A.S.; supervision, C.A.S. and P.A.F.; project administration, C.A.S.
and P.A.F.; funding acquisition, C.A.S. and F.B.-O.; visualization, C.A.S. and P.A.F.; writing—original
draft preparation, C.A.S. and P.A.F.; writing—review and editing, C.A.S., P.A.F., S.A.Á., C.V., F.A.,
C.M., M.Z. and F.B.-O. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by FONDECYT grants 1171844 (to C.A.S.) and 3180437 (to
F.B.-O.). P.A.F., C.V., and F.A. were funded by CONICYT/ANID doctoral fellowships 21140692,
21140615, and 21191925, respectively.
Acknowledgments:
We are indebted to Francisco Ramos-Morales for the generous gift of plasmid
pUTmini-Tn5cyaA0.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Microorganisms 2021,9, 475 11 of 11
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