JBJIJournal of Bone and Joint InfectionJBJIJ. Bone Joint Infect.2206-3552Copernicus PublicationsGöttingen, Germany10.5194/jbji-6-241-2021The Staphylococcus aureus toxin–antitoxin system YefM–YoeB is associated with antibiotic tolerance
and extracellular dependent biofilm formationYefM–YoeB is associated with antibiotic tolerance
and extracellular dependent biofilm formationQiXinyuBrothersKimberly M.MaDongzhuMandellJonathan B.DoneganNiles P.CheungAmbrose L.RichardsonAnthony R.UrishKenneth L.urishk2@upmc.eduArthritis and Arthroplasty Design Group (AAD Lab), Department of
Orthopaedic Surgery, College of Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania, USADepartment of Orthopedic Surgery, the First Affiliated Hospital of
Traditional Chinese Medicine of Guangzhou University, Guangzhou, Guangdong,
ChinaDepartment of Microbiology and Immunology, Geisel School of Medicine
at Dartmouth, Hanover, NH, New Hampshire, USADepartment of Microbiology and Molecular Genetics, University of
Pittsburgh, Pittsburgh, Pennsylvania, USA
The high antibiotic tolerance of Staphylococcus aureus biofilms is associated with challenges
for treating periprosthetic joint infection. The toxin–antitoxin system,
YefM–YoeB, is thought to be a regulator for antibiotic tolerance, but its
physiological role is unknown. The objective of this study was to determine
the biofilm and antibiotic susceptibility phenotypes associated with S. aureus yoeB
homologs. We hypothesized the toxin–antitoxin yoeB homologs contribute to
biofilm formation and antibiotic susceptibility. Disruption of yoeB1 and
yoeB2 resulted in decreased biofilm formation in comparison to Newman and JE2
wild-type (WT) S. aureus strains. In comparison to yoeB mutants, both Newman and JE2 WT
strains had higher polysaccharide intercellular adhesin (PIA) production.
Treatment with sodium metaperiodate increased biofilm formation in Newman
WT, indicating biofilm formation may be increased under conditions of
oxidative stress. DNase I treatment decreased biofilm formation in Newman
WT but not in the absence of yoeB1 or yoeB2. Additionally, WT strains had a higher
extracellular DNA (eDNA) content in comparison to yoeB mutants but no
differences in biofilm protein content. Moreover, loss of yoeB1 and yoeB2 decreased
biofilm survival in both Newman and JE2 strains. Finally, in a neutropenic
mouse abscess model, deletion of yoeB1 and yoeB2 resulted in reduced bacterial
burden. In conclusion, our data suggest that yoeB1 and yoeB2 are associated with
S. aureus planktonic growth, extracellular dependent biofilm formation, antibiotic
tolerance, and virulence.
Introduction
Periprosthetic joint infection (PJI) is one of the most challenging
complications following total joint arthroplasty. The current treatment
strategy involves irrigation and debridement of the infected area as well as
administration of intravenous antibiotics. Treatment failure of PJI is high, at
around 50 %–60 % (Biau et al., 2010). The major pathogen associated with
PJI in the United States is the gram-positive bacteria Staphylococcus aureus (Del
Pozo, 2018), with up to 50 % of cases involving the extremely difficult to
treat methicillin-resistant S. aureus (MRSA) (Nodzo et al., 2017).
PJI infections are primarily caused by antibiotic-tolerant biofilms on the
surface of the implant (Tande and Patel, 2014; Mooney et al., 2018; Urish
et al., 2016; Del Pozo, 2018). These infections are difficult to eradicate
and often involve removal of the infected device, creating a huge economic
burden on healthcare and impact on the wellbeing and daily life of
patients. The scientific literature suggests antibiotic tolerance arises
from antibiotic-resistant persister cells, a decreased bacterial metabolism
when in the biofilm state, and large production of exopolysaccharide (EPS)
that coats the biofilm and prevents drug diffusion to bacteria (Neut et
al., 2007).
The mechanisms of S. aureus biofilm formation are poorly understood. Bacterial toxin–antitoxin (TA) systems are believed to play a critical role in biofilm
antibiotic tolerance and resistance (Wen et al., 2014; Kedzierska and
Hayes, 2016; Thomopoulos et al., 2015). This system consists of a toxin that
will disrupt a cellular process (translation, etc.) and an antitoxin that
prevents toxin activation. Under conditions of environmental stress such as
high temperature shock, oxidative stress, and exposure to antibiotics, the
antitoxin disassembles, and the toxin becomes activated. This activation
leads to disruption in bacterial metabolism, inducing a state of dormancy
(Biau et al., 2010; Wang et al., 2012; Fasani and Savageau, 2013). When
environmental stressors are no longer present, the antitoxin system is
reactivated, binding the toxin, and bacteria are once more susceptible to
these stressors.
Probably the most well-studied TA system in S. aureus is the type II TA system MazEF.
Our previous work identified the role of MazEF in biofilm antibiotic
tolerance (Ma et al., 2019). The second type II TA system in S. aureus is YefM–YoeB,
a ribosome-dependent RNase that cleaves close to the start codon
(Schuster and Bertram, 2016). YoeB is the toxin, and YefM is the
antitoxin. In contrast to the MazEF system that only has one toxin MazF,
there are two types of yoeB toxin genes: yoeB1 and yoeB2 (Chan et al.,
2012). The YefM antitoxin inhibits the toxin YoeB through protein–protein
interactions (Schuster and Bertram, 2016). Overproduction of YoeB
inhibits Streptococcus pneumoniae cell growth and viability (Bakar et al., 2015); however, it is
still unclear how yoeB1 and yoeB2 affect S. aureus growth and biofilm formation.
The objective of this study was to identify the phenotype and physiological
role of S. aureus yoeB in planktonic growth, biofilm formation, antibiotic
susceptibility, and virulence.
Materials and methods
For this study to measure differences between the wild type (WT) and yoeB mutants,
we conducted 12 different experiments in two different strain
backgrounds: (1) planktonic growth assays, (2) biofilm bacterial burden
assays, (3) polysaccharide intracellular adhesion (PIA) quantification, (4) biofilm quantitation after treatment with sodium metaperiodate, (5) biofilm
extracellular DNA quantification (eDNA), (6) biofilm quantitation after DNAse I treatment, (7) biofilm extracellular protein quantification, (8) biofilm
quantitation after proteinase K treatment, (9) MIC (minimum inhibitory concentration) determination, (10) planktonic antibiotic susceptibility assays, (11) biofilm antibiotic
susceptibility assays, and (12) in vivo infection in a murine abscess model. All
experiments have been divided into the major headings listed below.
Bacterial strains and growth conditions
Newman WT, yoeB1, and yoeB2 Staphylococcus aureus deficient mutants were kindly provided by Niles Donegan. The WT USA300 JE2 strain was purchased from the American Type
Culture Collection (ATCC). The USA300 JE2 strain yoeB1 and yoeB2 mutants came
from the Nebraska Transposon Mutant Library. All S. aureus cultures were grown
overnight in trypticase soy broth (TSB) medium at 37 ∘C with
shaking.
Planktonic growth assay
Bacterial growth assays were performed according to Kato et al. (2017). Briefly, after 16 h incubation, an overnight culture of the
WT reference strain and yoeB mutants (Newman and USA300 JE2 strain backgrounds)
was grown as described in Sect. 2.1 and normalized using a 0.5 McFarland
standard (Hardy Diagnostics). Normalized cultures were diluted to 1×107 colony-forming units (CFU) in TSB medium. An amount of 100 µL of each
diluted bacterial suspension was added to a 96-well plate and incubated at
37 ∘C. Absorbance at OD600 was measured every hour for 6 h with a Multiskan plate reader (Infinite 200 Pro, Tecan). Results are
expressed as a mean value for each time point ± standard deviation.
Biofilm bacterial burden assay
Sterile titanium rods (10mm×1mm) were placed into a six-well plate (Costar,
USA) containing 4 mL TSB medium. Overnight cultures were grown as described
in Sect. 2.1 and normalized to 1×105 CFU/mL using the 0.5 McFarland
standard as described in Sect. 2.2. To form mature biofilms, plates were
incubated for 24 to 72 h at 37 ∘C. Wells were replaced with
TSB medium every 24 h to remove any planktonic bacteria. At the
experimental endpoints (24, 48, or 72 h), titanium rods were washed
three times in PBS, sonicated, and plated onto TSA II blood agar plates
(Thermo Fisher Scientific, USA) to enumerate bacteria by CFU. Results are
expressed as a mean value ± standard deviation.
Biofilm polysaccharide intracellular adhesin (PIA) quantification and
changes in biofilm formation after sodium metaperiodate treatment
PIA detection was performed as described by Cerca et al. (2006). Biofilm samples were grown and
collected following the PBS washing step as described in Sect. 2.3. Samples
were resuspended in 50 µL of 0.5 M EDTA (pH 8.0) and incubated for 5 min at 100 ∘C. Next, samples were centrifuged for 10 min
at 5000×g. An amount of 40 µL of the supernatant was collected and incubated
with 10 µL proteinase K (20 mg/mL) for 30 min at 37 ∘C.
Samples were then mixed with 10 µL of 20 mM Tris pH 7.4, 150 mM NaCl,
and 0.1 % bromophenol blue. An amount of 3 µL of the preparation and
dilutions was spotted onto a nitrocellulose filter, blocked with 3 % BSA
in TBS 0.1 % Tween (TBST) for 2 h at room temperature. Then samples
were incubated with PIA antibody (L3892; Sigma) overnight at 4 ∘C.
The filter was washed five times in TBST. PIA was detected by ECL
chemiluminescent detection reagent (Thermo Scientific) according to the manufacturer's instructions. Images were developed and quantified using a
ChemiDoc Touch imaging system (Bio-Rad). In an independent biofilm assay,
bacteria were treated with 10 µM sodium metaperiodate or PBS 2 h
prior to experimental endpoints. Crystal violet was used to stain the
biofilm, then it was dissolved in 30 % acetic acid. The absorbance of the biofilm
was measured at 600 nm. All results are expressed as a mean value ± standard deviation.
Biofilm extracellular DNA quantification (eDNA) and changes in biofilm
formation after DNase treatment
WT and yoeB mutant mature biofilms were grown on titanium rods and harvested as
described in Sect. 2.3. For eDNA quantification, biofilm samples were
treated with 5 µg/mL proteinase K and 20 µg/mL N-glycanase for
1 h at 37 ∘C. Samples were filter-sterilized with a 0.45 µM filter (Fisher Scientific). The filtered resuspension was diluted 1:2
with 2 µM SYTOX green (Thermo Fisher Scientific). Fluorescence was
measured using a Multiskan plate reader (Infinite 200 Pro, Tecan), with
excitation and emission wavelengths of 465 and 510 nm, respectively. The
amounts of eDNA relative to wild-type Newman or USA300-JE2 were calculated.
To analyze any resulting changes in biofilm formation after DNase treatment,
biofilms were grown and washed as described in Sect. 2.3. A period of 2 h prior
to quantification, biofilms were treated with 100 U/mL DNase I or PBS. Crystal
violet staining was performed and quantified as described in Sect. 2. All
results are expressed as a mean value ± standard deviation.
Biofilm protein quantification and changes in biofilm formation after
proteinase K treatment
Biofilms were grown and harvested as described in Sect. 2.3. Biofilm
protein concentration was determined with a bicinchoninic acid (BCA) assay
reagent kit (Thermo Scientific) according to the manufacturer's specifications.
Prior to protein quantification, biofilms were treated with 5 µg/mL
proteinase K or PBS 2 h before the BCA assay. In a separate biofilm
assay, crystal violet staining and quantification as described in Sect. 2.4 was used to quantify any changes in biofilm after proteinase K
treatment. All results are expressed as a mean value ± standard
deviation.
Minimum inhibitory concentration (MIC) assay
MIC assays were performed according to the manufacturer's instructions.
Overnight cultures were diluted in TSB medium to achieve a specified
inoculum turbidity by normalizing OD600 absorbance to a 0.5 McFarland
standard (∼1.5×108 colony-forming unit
(CFU)/mL bacteria). The diluted bacteria were plated onto a blood agar plate
using a plate spreader to evenly distribute the inoculum. Plates were
air-dried for 20 min at room temperature. Next, a cefazolin or
vancomycin Etest strip (Liofilchem, Italy) was applied to the agar plate.
Plates were incubated overnight at 37 ∘C. After a 24 h
incubation, MIC values were read and recorded.
Planktonic susceptibility assay
Cefazolin and vancomycin were purchased from Sigma Aldrich (USA) for
antibiotic susceptibility assays. S. aureus strains and mutants were cultured in TSB
medium overnight, normalized to the same 0.5 McFarland standard as described
above, diluted to 1×107 CFU, and grown for 16 h at
37 ∘C. Pre-treatment bacterial concentrations were
determined by CFU assay on TSA II plates. Next, 10X MIC of cefazolin and
vancomycin were added to the inoculum for all strains, incubated at
37 ∘C with shaking for 24 h, and plated to identify surviving
bacteria by CFU assay. Percent planktonic cell survival was calculated and
compared to pre-treatment CFU values. Results are expressed as percent
survival ± standard deviation.
Biofilm susceptibility assay
For biofilm susceptibility assays, sterile titanium rods for biofilm
formation were prepared as described above. In order to grow mature biofilm,
bacteria were incubated for 72 h to quantify the bacterial burden prior
to antibiotic treatment. The remaining titanium rods were treated with 10X
MIC of vancomycin or cefazolin calculated from planktonic cultures and
incubated for an additional 48 h. Plates were replaced with media and
antibiotics every 24 h. After 48 h, titanium rods were removed,
washed in PBS, sonicated, and plated as described above. Biofilm percent
survival was calculated and compared to the pre-treatment CFUs. Results are
expressed as a percent survival ± standard deviation.
In vivo infection in a murine abscess model
Based on our previous work (Ma et al., 2019), S. aureus USA300-JE2 strain
background was selected for in vivo experiments. A number of 8- to 12-week-old
C57BL/6 mice were purchased from the Jackson laboratory (Bar Harbor, ME).
All animal protocols used for these experiments were approved by the
University of Pittsburgh's Institutional Animal Care and Use Committee
(IACUC). All experiments were performed in accordance with relevant
guidelines and regulations. To create a model for neutropenia, mice were
injected with 100 µL cyclophosphamide (150 mg/kg 3 d prior to
infection and 100 mg/kg 1 d prior to infection). After anesthetizing the
mice with 2 % isoflurane, leg hair was removed and washed with 3 %
betadine. An amount of 100 µL of 1×106 CFU of JE2-WT, JE2-yoeB1, or JE2-yoeB2
was injected into the thigh to create an abscess (n=8 per group). All mice
were monitored for any signs of lack of grooming, loss of appetite,
dehydration, weight loss, swelling, or signs of sepsis until experimental
endpoints. At 72 h (3 d) post-inoculation, animals were euthanized. A
∼5×5 mm piece of thigh muscle from the infection area was
obtained and placed in 1 % PBS tween (PBST) on ice. Abscess bacterial
burden was used as a measure of virulence
(Kobayashi et al., 2015). In order to quantify
bacterial burden in CFU/mL, infected samples were sonicated for 10 min,
serially diluted 1:10 in PBS, plated onto blood agar, and incubated
overnight at 37 ∘C. Results are expressed as a mean value ± standard deviation.
Statistical analysis
Statistical analysis was based on the number of populations and comparisons.
A paired Student t test was used for two populations. One-way analysis of
variance (ANOVA) and Tukey's post hoc analysis with alpha set to 0.05 were
used for multiple comparisons. Repeated measure analysis was used for
analysis of differences over time. For all statistical tests used, p values
<0.05 were considered statistically significant.
YoeB suppresses S. aureus planktonic growth. (a) Newman and (b) JE2 (ns: not significant, * p<0.05).
ResultsPlanktonic growth assay
In both strain backgrounds, increased planktonic growth in yoeB mutants in
comparison to WT was observed in the early log phase (at 5 and 6 h) (Fig. 1). In the Newman strain, differences between WT and yoeB mutants were not
observed until the 4 h time point, with a mean OD600 of 0.20
and 0.22 for yoeB mutants in comparison to WT at a mean of 0.18 (Fig. 1a, p<0.05).
These differences became more evident by the 5 h time point, with a mean of 0.27 for WT, 0.33 for yoeB1, and 0.34 for yoeB2 (Fig. 1a, p<0.05). At 6 h, the differences were even greater, with a mean of 0.38 for WT, 0.43 for yoeB1, and 0.41 for yoeB2 (Fig. 1a, p<0.05). In the JE2 strain, differences in bacterial
growth were first observed between WT and yoeB1 by the fourth hour of growth,
with a mean of 0.16 for the WT and a mean of 0.18 for yoeB1 (Fig. 1b, p<0.05).
Differences between WT and both yoeB mutants occurred starting at the 5 h
time point, with a mean of 0.25 for the WT, 0.31 for yoeB1, and 0.31 for yoeB2 (Fig. 1b, p<0.05). At the 6 h time point, the WT had a mean of 0.30, yoeB1 0.41, and yoeB2 0.40
(Fig. 1b, p<0.05).
Loss of yoeB decreases biofilm. (a) Newman and (b) USA300-JE2 bacterial burden (CFU/mL) (* p<0.05).
Biofilm bacterial burden assay
When bacterial burden of biofilm growth after 72 h (day 3) in the Newman
strain background was measured, yoeB1 and yoeB2 had a decrease in biofilm formation,
with a mean CFU/mL of 5.92×106 and 5.80×106 respectively in
comparison to WT at 1.2×107 CFU/mL (Fig. 2a, p<0.001). Similar
results were observed in the JE2 strain background, with yoeB mutants
demonstrating a lower biofilm bacterial burden with a mean of 5.58×105 for yoeB1 and a mean of 5.25×105 for yoeB2 in comparison to 3.0×106 CFU/mL for the WT (Fig. 2b, p<0.001).
YoeB is associated with extracellular polymeric substance (EPS)
biofilm formation. In the (a) Newman strain, sodium metaperiodate (SM)
treatment of yoeB mutants has no effect on biofilm formation but increases
biofilm in WT. No differences were observed in (b) USA300-JE2. After DNase I
treatment (c), Newman WT demonstrates a reduction in biofilm. However, no
changes were observed in yoeB mutants. No differences in biofilm were observed in
(d) USA300-JE2. Reductions in biofilm formation were observed after proteinase
K treatment for (e) Newman and (f) USA300-JE2 (ns: not significant,
* p<0.05).
Biofilm PIA quantitation and changes in biofilm formation after sodium
metaperiodate treatment
PIA content in yoeB mutants in the Newman strain background was much lower in
comparison to WT (mean 22 325, 100 %), with a mean of 14 001 for yoeB1 (62.7 % of
WT) and 4567 for yoeB2 (20.5 % of WT) (Fig. S1a, p<0.001). Similar
findings for PIA content were observed in the JE2 strain background in
comparison to WT (mean 23 481, 100 %), with a mean of 4255 for yoeB1 (18.1 % of
WT) and 839 for yoeB2 (3.6 % of WT) (Fig. S1b, p<0.001). When biofilms
were treated with sodium metaperiodate, no reduction in biofilm was observed
in yoeB mutants for both Newman (OD600 WT 0.35 yoeB1 0.17, yoeB2 0.17) and JE2
strain backgrounds (OD600 WT 0.28, yoeB1 0.31, yoeB2 0.29) (Fig. 3a and b). However,
an increase in biofilm in the Newman WT strain was observed after treatment
with sodium metaperiodate (OD600 WT 0.35 before treatment, 0.49 after
treatment) (Fig. 3a, p=0.002).
Biofilm eDNA quantitation and changes in biofilm formation after DNase
treatment
More eDNA release was observed in the Newman WT strain (100 %) in
comparison to yoeB mutants (yoeB1 27 % of WT, yoeB2 25 % of WT) (Fig. S2a, p=0.002). In
the JE2 strain background, no differences in eDNA were observed between WT
and yoeB mutants (WT 100 %, yoeB1 88 %, yoeB2 103 %) (Fig. S2b). After Newman biofilm
were treated with DNase, a reduction was observed in the WT (mean OD600
before treatment 0.71, after treatment 0.45) (Fig. 3c, p<0.001), but no changes were observed in yoeB mutants (mean OD600 before
treatment yoeB1 0.31, after treatment 0.34, yoeB2 before treatment 0.30, after treatment
0.31) (Fig. 3c). In JE2, no reduction in biofilm was observed after DNase
treatment in the WT (before treatment 0.88, after treatment 0.82) or yoeB mutants
(yoeB1 before treatment 0.38, after treatment 0.39, yoeB2 before treatment 0.35, after
treatment 0.35) (Fig. 3d).
Biofilm protein quantitation and changes in biofilm formation after
proteinase K treatment
In both strain backgrounds, no differences in extracellular protein were
observed between WT and yoeB mutants (Newman WT 21.8, yoeB1 24.5, yoeB2 19.3, JE2 WT 21.0,
yoeB1 19.1, yoeB2 19.2 µg/mL) (Fig. S3). However, notable decreases in biofilm for
both WT and yoeB mutants were observed after treatment with proteinase K in both
Newman (before treatment OD600 WT 0.31, yoeB1 0.21, yoeB2 0.22, after treatment
WT 0.08, yoeB1 0.08, yoeB2 0.06) and JE2 strain backgrounds (before treatment WT 0.36,
yoeB1 0.19, yoeB2 0.20, after treatment WT 0.12, yoeB1 0.10, yoeB2 0.09) (Fig. 3e, p<0.001 and
3f, p<0.001).
Minimum inhibitory concentration (MIC) assay
No differences in MIC were found between WT and yoeB mutants in both strain
backgrounds. The MICs for cefazolin and vancomycin in the Newman strain
background for both WT and yoeB mutants were 0.38 and 3 µg/mL,
respectively. MICs for cefazolin and vancomycin in the JE2 strain background
for both WT and yoeB mutants were 1 and 1 µg/mL, respectively.
Loss of yoeB1 and yoeB2 increases planktonic antibiotic susceptibility. With
the exception of the yoeB2 mutant, reduction of planktonic bacterial survival in
yoeB mutants was observed in the Newman strain background for both (a) 10X MIC
cefazolin and (b) 10X MIC vancomycin. Similar results were observed for
USA300-JE2, with more notable differences observed in yoeB mutants in comparison
to WT for both (c) cefazolin and (d) vancomycin (ns: not significant,
* p<0.05).
Planktonic susceptibility assay
In the Newman strain background, a reduction of planktonic bacterial
survival after antibiotic treatment was observed in both yoeB mutants after
treatment with 10X MIC cefazolin (OD600 WT 0.05, yoeB1 0.03, yoeB2 0.02) (Fig. 4a,
p=0.03, p=0.02). After 10X MIC vancomycin treatment, a decrease in Newman
yoeB1 planktonic survival was observed (OD600 WT 0.14, yoeB1 0.03) (Fig. 4b,
p<0.001). No differences were observed between WT and yoeB2 (OD600 0.08) (Fig. 4b). In the JE2 strain background, decreases in planktonic
bacterial survival were observed in both yoeB mutants after treatment with
cefazolin (OD600 WT 0.46, yoeB1 0.02, yoeB2 0.15) (Fig. 4c, p<0.001,
p=0.003) and vancomycin (OD600 WT 0.17, yoeB1 0.02, yoeB2 0.05) (Fig. 4d,
p<0.001).
Loss of yoeB1 and yoeB2 increases biofilm antibiotic susceptibility. Deletion
of yoeB results in a decrease in biofilm survival in the Newman strain
background after treatment with both (a) 10X MIC cefazolin and (b) 10X MIC
vancomycin. No reduction in biofilm survival was observed when JE2 WT and
yoeB mutants were treated with (c) cefazolin, but differences in biofilm survival
were observed between WT and yoeB mutants when biofilms were treated with (d) vancomycin (ns: not significant, * p<0.05).
Biofilm susceptibility assay
When biofilms in both strain backgrounds were treated with cefazolin and
vancomycin, similar results were observed as in the planktonic assay.
Noticeable reductions in yoeB mutants after cefazolin treatment were observed in
the Newman strain background (OD600 WT 4.7, yoeB1 2.2, yoeB2 1.2) (Fig. 5a,
p<0.001), while no differences were observed in the JE2 strain
background (OD600 WT 3.8, yoeB1 0.77, yoeB2 1.4) (Fig. 5c). After treatment with
vancomycin, reductions in biofilm for yoeB1 and yoeB2 mutants were observed for both
Newman (OD600 WT 3.7, yoeB1 0.41, yoeB2 0.70) (Fig. 5b, p<0.001) and JE2
strain backgrounds (OD600 WT 11.4, yoeB1 7.4, yoeB2 4.4) (Fig. 5d, p=0.001,
p<0.001).
In vivo infection in a murine abscess model
In our neutropenic mouse abscess model, by the third day of infection, WT
JE2-infected mice had a mean bacterial burden of 9.26×108 CFU/mL.
Mice infected with yoeB1 and yoeB2 had a decreased abscess burden, with a mean CFU/mL of
2.27×108 and 1.94×108 respectively (Fig. 6, p=0.006,
p=0.004).
Deletion of yoeB results in reduced bacterial burden in a neutropenic
murine abscess model. Bacterial abscess burden in a neutropenic mouse model
(n=8 per group) for S. aureus USA300-JE2. In comparison to WT, yoeB mutants have a
decreased bacterial burden (* p<0.05).
Discussion
The S. aureus type II TA systems have been well studied by our group and others
(Schuster and Bertram, 2016; Ma et al., 2019; Chan et al., 2012).
However, very little is known about the S. aureus YefM–YoeB type II TA system. The objective of
this study was to determine the role of yoeB in S. aureus planktonic growth, biofilm
formation, components of biofilm EPS, antibiotic susceptibility, and
virulence.
It is well understood that the toxin–antitoxin complex is separated under
environmental stressors (Chan et al., 2012). Under
normal growth conditions, the toxin is able to cause growth arrest (Hayes
and Van Melderen, 2011; Chan et al., 2012). Bakar et al. (2015) reported
that overproduction of yoeB inhibits cell growth and viability (Bakar et al.,
2015). We hypothesized that yoeB contributes to bacterial growth. Our results in both
strain backgrounds indicate that deletion of both yoeB1 and yoeB2 increases growth of
planktonic cells, indicating that the toxin YoeB plays an important role in
suppressing planktonic growth.
To investigate the role of yoeB in biofilm formation using clinically relevant
materials, we performed a biofilm assay using titanium rods commonly used in
orthopaedic surgical procedures. Reductions in biofilm bacterial burden were
observed in both yoeB mutants by 72 h of growth. This is in agreement with work
by Chan et al. (2018) that demonstrated deletion of yoeB resulted in a reduction in
Streptococcus pneumoniae biofilm. However, Kato et al. (2017) demonstrated that deletion of
yoeB1 and yoeB2 had no effect on biofilm formation. However, this group only looked at
biofilms up to 16 h of growth (Kato et al., 2017). Our work focused
on a more mature biofilm after 3 d of growth and indicates that YoeB plays an
important role in mature biofilm formation.
In order to elucidate the mechanism for decreased biofilm formation in
yoeB mutants, we investigated the components of biofilm extracellular polymeric
substances (EPS): polysaccharide (PIA), extracellular DNA (eDNA), and
protein (Flemming and Wingender, 2010).
Because PIA has been widely studied in relation to biofilm formation
(Rohde et al., 2010; Dotto et al., 2017; You et al., 2014), we quantified
the biofilm-associated PIA. However, it is unclear whether PIA production is
associated with yoeB in S. aureus. Similar to previous studies (Rohde et al., 2010;
Dotto et al., 2017; You et al., 2014), S. aureus WT had a greater PIA concentration in
comparison to yoeB mutants in both strain backgrounds. When we compared the
corresponding biofilm after treatment with the oxidant sodium metaperiodate,
no differences in biofilm were observed in yoeB mutants. Thus, it can only be
concluded that in the absence of yoeB, there is a decrease in
polysaccharide production, with no correlation in regards to biofilm
formation. Interestingly, we did observe an increase in biofilm formation
only in the Newman WT strain after treatment with sodium metaperiodate.
These results suggest the increase in biofilm may be a response to oxidative
stress specific to MSSA strain backgrounds.
Extracellular DNA is another important component of biofilm EPS. Work by
other groups has demonstrated that the eDNA component of EPS is essential for
biofilm formation (Flemming and Wingender, 2010; Okshevsky and Meyer,
2015) and plays an important role in biofilm architecture in the early
stages of infection (Pakkulnan et al., 2019).
Previous studies have demonstrated that cellular lysis and the S. aureus murein hydrolase
regulator contribute to eDNA release (Gao et al., 2013; Rice et al.,
2007). eDNA has been reported to serve as a net to hold bacterial cells
together, leading to large aggregate formation (DeFrancesco et al., 2017).
Other groups have identified that eDNA plays a critical role in biofilm formation
but only in the very early stages (Pakkulnan et al., 2019).
Interestingly, in the Newman strain, we observed that yoeB mutant biofilms had a much
lower eDNA content in comparison to WT, suggesting YoeB may play role in eDNA
production, even at the later stages of biofilm formation. Additional work is
needed to confirm this phenotype. No differences were observed in the MRSA
USA300 JE2 background, indicating possible differences in TA phenotypes
between MSSA and MRSA strains that warrant further study. Our results imply
S. aureus yoeB may be involved in the release of eDNA, which contributes to eDNA-associated
biofilm formation. Because of this, we analyzed biofilm formation before and
after treatment with DNase. When Newman WT biofilms were treated with DNase,
a reduction in biofilm occurred, but no changes in yoeB mutants were observed.
Our results demonstrate that YoeB plays a role in biofilm eDNA, but its role in
biofilm formation requires further study.
The extracellular-protein-associated biofilm between WT and yoeB mutants was
quantified as a final measure of biofilm EPS. No significant differences in
extracellular protein concentration were found between WT and yoeB mutants for
both Newman and JE2 strain backgrounds. Previous studies have demonstrated
that proteinase K treatment leads to a dramatic reduction in Staphylococcus aureus biofilms (You et
al., 2014; Goormaghtigh et al., 2018). We observed similar findings in our
studies for both WT and yoeB mutants, with a reduction in biofilms after treatment
with proteinase K in both strain backgrounds. Given our protein and
quantitation and biofilm results after proteinase K treatment, it can be
concluded that yoeB has no effect on the production of biofilm extracellular protein
but does play a role in biofilm formation.
Previous groups have found a correlation between TA genes and changes in MIC
(Hemati et al., 2014; Wen et al., 2014). In order to determine if yoeB1 and yoeB2
are involved in antibiotic tolerance, we determined the cefazolin and
vancomycin MIC of WT and yoeB mutants in both strain backgrounds. Similar to our
previous work with S. aureus type II TA systems (Ma et al., 2019), no differences
in MIC were observed between WT and yoeB mutants during stationary phase growth.
Since TA systems have been demonstrated to play a role in response to
planktonic antibiotic stress (Ma et al., 2019; Goormaghtigh et al., 2018;
Salzberg and Helmann, 2007; Costa et al., 2009), the role of yoeB in antibiotic
susceptibility was explored. Loss of yoeB1 and yoeB2 resulted in increased
susceptibility to cefazolin in both strain backgrounds. When comparing
strain backgrounds for vancomycin treatment, susceptibility was only
observed for yoeB1 in Newman, while vancomycin susceptibility was observed in
both yoeB mutants for JE2, indicating possible differences between MSSA and MRSA
strain backgrounds.
It is still unclear whether TA systems affect biofilm formation under
conditions of antibiotic stress. Previous work indicates that deletion of type II
TA systems leads to decreased antibiotic resistance in Escherichia coli; however, this work
did not determine a link between the induction of TA systems and antibiotic
resistance (Goormaghtigh et al., 2018). Salzberg and Helmann (2007) demonstrated that
yoeB is a cell-wall-associated gene that is able to protect Bacillus subtilis from cell-wall-targeted antibiotic stress. Our previous
work in another S. aureus type II TA system demonstrated that the toxin component of the
system is susceptible to antibiotics (Ma et al., 2019). This is in
agreement with our current results, where yoeB toxin mutants are more susceptible
to cell-wall-targeting antibiotics under biofilm growth conditions.
Finally, we investigated the role of yoeB in the context of a live host. S. aureus infections
are not chronic, unless the infection has developed over an extended period
of time such as in surgical cases. In these types of infection, biofilms
play a critical role (Urish et al., 2018; Ma et al., 2018). Bacterial
growth, antibiotic susceptibility, and biofilm formation are all crucial for
the establishment of infection and virulence (Costerton, 1999; Donlan, 2001).
We used a neutropenic mouse abscess model and quantified bacterial burden as
a measure of virulence, as has been demonstrated in other studies
(Kobayashi et al., 2015; Tram et al., 2018). Loss of both yoeB1 and yoeB2 resulted in
decreased bacterial burden in our mouse abscess model. In addition, bacteria
in the infected tissue samples were predominantly in the biofilm phenotype.
These findings were similar to our in vitro biofilm data but are in contrast to our
previous findings in another S. aureus type II TA system (Ma et al., 2019),
suggesting a different role of the YefM–YoeB TA system in virulence. In
addition, our animal data conflict with our planktonic growth results, where
we observed increased growth in yoeB mutants over time. Additional work is needed
to identify the mechanism of the phenotypic differences between planktonic
and biofilm forms of growth.
There were some limitations to these studies. With the exception of the
neutropenic mouse model, all other experiments were completed in vitro. Thus, it is
difficult to compare these results to a clinical scenario. The interaction of
microbial growth, nutrient utilization, nutrient diffusion, biofilm
formation, antibiotic killing, and development of resistance is complex. In
addition, rifampicin is widely used in combination with other antibiotics
for staphylococci biofilm infections (Zimmerli and Sendi, 2019) and is
the focus of future studies used in combination with vancomycin and
cefazolin. The MIC values used to test biofilm susceptibility to antibiotics
were determined in bacteria grown in the planktonic state only. A more
effective measure of biofilm susceptibility would be to test antibiotic
concentrations at the minimum biofilm inhibitory concentration. However, our
data did demonstrate differences between WT and yoeB mutants under the currently
tested conditions after 48 h of growth. Our current results show
contrasting differences between our animal results, biofilm formation, and
the planktonic cell growth; therefore more work is warranted to identify the TA
mechanism of these phenotypic differences.
Our current data demonstrate the importance of the YefM–YoeB toxin–antitoxin
system in planktonic growth, biofilm formation, components of biofilm EPS,
planktonic antibiotic tolerance, and biofilm antibiotic tolerance. Our data
also highlight the importance of toxin–antitoxin systems in infection, but
the role of YefM–YoeB in virulence and infection progression requires
further study.
Ethical statement
All strains for this study came from the ATCC or from our collaborators. No
patient data or strains were used for this publication.
Software code used for statistical analysis is available upon reasonable request from the corresponding author.
Data availability
For access to raw data, contact the corresponding author (Kenneth L. Urish:
urishk2@upmc.edu).
The supplement related to this article is available online at: https://doi.org/10.5194/jbji-6-241-2021-supplement.
Author contributions
XQ, DM, JBM, NPD, ALC, ARR, and KLU designed the study. XQ, DM, and JBM
performed the experiments and gathered the data. XQ and KMB wrote the paper.
KMB, JBM, and KLU critically reviewed data interpretation and the article.
Competing interests
The authors declare that they have no conflict of interest.
Disclaimer
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We thank the China Scholarship Council (CSC) for financial support for Xinyu Qi's sabbatical in Kenneth L. Urish's lab. Kenneth L. Urish is supported in part
by the National Institute of Arthritis and Musculoskeletal and Skin Diseases
(NIAMS K08AR071494), the National Center for Advancing Translational Science
(NCATS KL2TR0001856), and the Orthopaedic Research and Education Foundation
(OREF).
Review statement
This paper was edited by Alex McLaren and reviewed by four anonymous referees.
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