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\begin{document}

\title{Myh7 regulates SDRC protein and biofilm formation to alleviate
osteomyelitis induced by Staphylococcus aureus}



\author[1]{Baochuang  Qi}%
\author[1]{Lili Yang}%
\author[1]{Minzheng Guo}%
\author[1]{Chen Meng}%
\author[1]{Hongxin Shi}%
\author[1]{Tao Chen}%
\author[1]{Huan Luo}%
\author[1]{Longjun Su}%
\author[1]{Chuan Li}%
\author[2]{Yongqing Xu}%
\affil[1]{Affiliation not available}%
\affil[2]{920th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
SDRC is implicated in the pathogenesis of Staphylococcus aureus
osteomyelitis. However, the precise mechanism by which SDRC contributes
to the progression of this infection remains unclear. To explore SDRC's
role in osteomyelitis, experiments were conducted on mouse models
infected with Staphylococcus aureus strains containing either the wild
type SDRC sequence or a knockout (KO-SDRC) sequence. Levels of
inflammatory factors like IL-6 and TNF-\selectlanguage{greek}α \selectlanguage{english}were measured using Elisa.
Furthermore, changes in osteogenic indicators such as ALP, OST, and
Runx2 were detected using qPCR and Western blot analysis. Additionally,
transcriptome sequencing was employed to gain deeper insights into the
potential molecular mechanisms underlying SDRC's promotion of
Staphylococcus aureus-induced osteomyelitis. The study revealed that the
ability of Staphylococcus aureus to form biofilms was significantly
weakened in strains where the SDRC protein was knocked out. Moreover,
mice infected with the KO-SDRC strain exhibited enhanced ossification
processes. Transcriptome sequencing demonstrated significant
overexpression of the Myh7 gene in the SDRC knockout osteomyelitis
mouse. Subsequent knockout of the Myh7 gene led to notable reductions in
mRNA and protein expressions of osteogenic indicators including Runx2,
ALP, OSX, and Osteocalcin. Overall, these findings suggest that SDRC
plays a crucial role in promoting the malignant progression of
osteomyelitis caused by Staphylococcus aureus. Its main mechanism
involves enhancing the formation of Staphylococcus aureus biofilms.
Conversely, Myh7 may exert an inhibitory effect on the SDRC protein,
reducing biofilm formation and mitigating the severity of osteomyelitis.
These findings offer novel insights into potential therapeutic targets
for the treatment of osteomyelitis.%
\end{abstract}%



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\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/760918/articles/736813-myh7-regulates-sdrc-protein-and-biofilm-formation-to-alleviate-osteomyelitis-induced-by-staphylococcus-aureus}

\textbf{Introduction}

Osteomyelitis is an inflammatory disease characterized by bacterial
infection causing bone destruction and secondary bone proliferation. It
can be caused by aerobic or anaerobic bacteria, branched bacteria, and
fungi. Epidemiological studies have shown that Staphylococcus aureus is
the most common and predominant pathogen causing
osteomyelitis\textsuperscript{{[}1, 2{]}}. Bone tissue is prone to
chronic transformation and difficult to completely cure after infection
with Staphylococcus aureus, leading to recurrent
infections\textsuperscript{{[}3{]}}. Studies have shown that
Staphylococcus aureus forms a biofilm mainly on the pits of cortical
bone or dead bone tubules after infecting bone tissue, and secretes a
large amount of immune-regulatory proteins, toxins, and other virulence
factors, causing immune cell death, complement activation disorders, and
immune evasion of Staphylococcus aureus\textsuperscript{{[}4, 5{]}}.
Therefore, osteomyelitis caused by Staphylococcus aureus has a high
recurrence rate, and remains a challenging medical problem in China and
worldwide.

Staphylococcus aureus is a gram-positive pathogen widely present in the
population, mainly found on the human skin and nasal
cavity\textsuperscript{{[}6, 7{]}}. The cell wall of Staphylococcus
aureus is composed of 90\% peptidoglycan and 10\% wall teichoic acid,
which gives it a strong ability to adapt to the environment and resist
adverse factors\textsuperscript{{[}8-10{]}}. It spreads widely in the
population and causes a series of diseases in the human body, such as
vascular, lower respiratory tract, soft tissue, and skin infections,
endocarditis, and osteomyelitis\textsuperscript{{[}11{]}}. It is well
known that Staphylococcus aureus mainly infects the host through its
surface recognition proteins, anchors itself to the host cell surface by
mediating some adhesion mechanisms, releases toxins to induce host
immune reactions, and ultimately infects the host\textsuperscript{{[}12,
13{]}}. Studies have found that Staphylococcus aureus can express up to
24 cell wall-anchored proteins, which can be divided into the MSCRAMM
family, G5-E repeat domain protein family, Three-Helical Bundles family,
NEAT motif family, legume lectin domain protein family, nucleotidase
motif, etc., based on their secondary structure
characteristics\textsuperscript{{[}14, 15{]}}. Among them, the MSCRAMM
family mainly consists of proteins located on the surface of
Staphylococcus aureus that can recognize and adhere to various host
components\textsuperscript{{[}16{]}}. It is crucial for the adhesion,
invasion, and immune evasion mechanisms of the pathogen and as a member
of the MSCRAMM family, SDRC protein's biological effects are
immeasurable\textsuperscript{{[}17, 18{]}}.

SDRC protein mainly plays a biological role by mediating the formation
of Staphylococcus aureus biofilm\textsuperscript{{[}19{]}}. BARBU E M et
al. used a phage library to screen the interaction between amino acids
247-251 and 288-292 at the C-terminus of the N2 domain of SDRC protein
and found that SDR protein can promote biofilm
formation\textsuperscript{{[}19-21{]}}. Meanwhile, inhibiting the
interaction of SDRC protein itself inhibits biofilm formation, fully
demonstrating the critical role of SDRC protein in Staphylococcus aureus
infectious diseases.

In our study, we first knocked out the SDRC protein in the structure of
Staphylococcus aureus in vitro, and explored the specific role of SDRC
protein in osteomyelitis by constructing a mouse osteomyelitis model
infected with Staphylococcus aureus. At the same time, we also combined
transcriptome sequencing technology to further explore the specific
mechanism by which the SDRC protein affects the progression of
osteomyelitis, aiming to elevate the treatment of osteomyelitis to a new
level.

\textbf{Material and Method}

1.1 Knockout of the SDRC protein sequence of Staphylococcus aureus

The standard strain ATCC25923 of Staphylococcus aureus used in this
study was purchased from the bacterial library of the Chinese Academy of
Sciences. Genetic engineering technology was used to knock out the Sdrc
protein region in Staphylococcus aureus. First, design upstream and
downstream homologous arm sequence primers for SDRC protein based on the
genome sequence of Staphylococcus aureus (Primer Schedule 1). And
through overlapping PCR amplification, mutant plasmid construction,
plasmid transformation, cloning, and other operations, we ultimately
successfully constructed a mutant strain of Staphylococcus aureus with
SDRC knockout. And we further validated the knockout effect of strain
SDRC using next-generation sequencing technology.

Schedule 1 The primers of SDRC knockout primers\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Gene & Forward(5'-3') & Reverse(5'-3')\tabularnewline
\midrule
\endhead
RN4220-sdrc-up & ATTCTGGAATATTGAAAAATGCA &
GTTAAGTTAATCATAAATCAATGTTGTCCCTACTAA\tabularnewline
RN4220-sdrc-down & GGGACAACATTGATTTATGATTAACTTAACCAGGTCCA &
TTTCATCATTACTCTTGATAGCA\tabularnewline
Sdrc-pkoR1 & GCCTCGGAACCGGTACCATTCTGGAATATTGAAAAATG &
GTGAGCGCAACGCAATTTTCATCATTACTCTTGATAG\tabularnewline
Sdrc-JD & ATGTAATAGCGAATTGAAAAC & ATTGGATACCATGCCTTTCCTT\tabularnewline
Sdrc-ter & ATGTAATAGCGAATTGAAAAC & CTTGTTCAAAGCTACCTCTAAC\tabularnewline
\bottomrule
\end{longtable}

1.2 MRSA cultivation

The standard strain of Staphylococcus aureus ATCC25923 used by the
Institute was purchased from the bacterial library of the Chinese
Academy of Sciences.50 MRSA and 50 KO sdrc (purchased from Beijing Abace
Biotechnology Co. LTD ) stored at -80  each \selectlanguage{greek}μ \selectlanguage{english}Add 15ml of soybean broth
culture medium, incubate overnight at 37  at 200r/min, and detect the
knockout efficiency of sdrc using RT-PCR. Take 5 out of MRSA and KO sdrc
recovered overnight \selectlanguage{greek}μ \selectlanguage{english}Add 5ml of soybean broth medium and culture on a
constant temperature shaker. Measure the absorbance value of OD600 at
1h, 2h, 3h, 4h, 5h, 7h, 9h, 12h, 18h, and 24h according to: \textless{}i
mtid='44'\textgreater{}hello\textless{}/i\textgreater{} 07 Calculate and
plot bacterial growth curves.

1.3 ELISA experiment

Collect samples and perform enzyme-linked immunosorbent assay according
to the manufacturer's protocol, including \selectlanguage{greek}α- \selectlanguage{english}HL, PSM, CRP, TNF- \selectlanguage{greek}α\begin{CJK}{UTF8}{gbsn}、
\end{CJK}\selectlanguage{english}IL-6.

1.4 Crystal violet staining

Remove the bacteria cultured overnight from the culture medium and add
500-1000 to it \selectlanguage{greek}μ \selectlanguage{english}Place 4\% paraformaldehyde at room temperature for
30-40 minutes, remove excess liquid, tilt the plate, dry at 37  for 1
hour, add crystal violet that can cover the bottom of the hole, stain at
room temperature for 20 minutes, add PBS to wash 2-3 times, and absorb
the remaining liquid to observe the ability of biofilm formation.

1.5 Observation of MRSA biofilm formation using scanning electron
microscopy

MRSA and KO sdrc were cultured overnight at 37  in a 24 well plate and
placed under a scanning electron microscope to observe bacterial
morphology.

1.6 RT-PCR experiment

Extract total RNA from each group separately, then perform reverse
transcription reaction, and then use 2 x Universal Blue SYBR Green qPCR
Master Mix reagent kit for qPCR reaction. The reaction procedure is: pre
denaturation at 95  for 1 minute; 95  denaturation for 20 seconds, 55
 annealing for 20 seconds, 72  extension for 30 seconds; 40 cycles.
The primer sequence is shown in Table 1:

Table 1 The sequences of the Primers\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Gene & Forward (5 ' -3 ') & Reverse (5 ' -3 ')\tabularnewline
\midrule
\endhead
GAPDH & CCTTCCGTGTTCCTACCCC & GCCCAAGATGCCCTTCAGT\tabularnewline
Myh7 & TCCTGCTGTTTCCTTACTTGC & TACTACATGCCAGAAGCCCCG\tabularnewline
Myl2 & TCGCTGAAGGGCCGACTAT & TCTCCGTGGGTGATG\tabularnewline
Tnnc1 & ATCTCTTCCGCATGTTTG & GTTCTTGTCACCGTCTT\tabularnewline
RpoA & GCACCAAAAGAAGGCGTTCAG & ATATCGGCTGCAGTCACAGG\tabularnewline
Sdrc & TTGTGGATAGTTCACCCCTG & GTTTATTGCTGTTGTGGC\tabularnewline
Runx2 & CCAACTTCCTGTGCTCCGT & TGAAACTCTCGTCC\tabularnewline
OSX & ACCCCAAGATTTTCTATAAGCCC & CGCTCTAGCTCCTGACAGTTG\tabularnewline
ALP & CCTCCCGCTGTTACTGTTG & CTTGGCGTTCTAACCACCCCA\tabularnewline
Osteocalcin & CCTCTCTCTGCTCACTCTGC & CACTACCTATTGCCCTCCT\tabularnewline
\bottomrule
\end{longtable}

1.7 Isolation and Culture of BMSC Cells

Take the femur and tibia of mice, cut off both bone ends, and use a
syringe to extract DMEM-F12 culture medium without FBS. Insert a syringe
from the other end to repeatedly rinse the bone marrow cavity into a
50ml centrifuge tube until the bone marrow cavity turns white. Then,
repeatedly blow the bone marrow cells to prepare a single cell
suspension. Cultivate and pass through in a T25 culture bottle.

Take the logarithmic growth phase BMSC cells for digestion and count,
inoculate them with a 24 well plate, 1x10 5/well, and add different
concentrations of MRSA bacteria (0, 1.5625 moi, 3.125 moi, 6.25 moi,
12.5 moi, MRSA 25 moi, MRSA 50 moi, MRSA 100 moi, MRSA 200 moi, MRSA 400
moi, and MRSA 800 moi) to the cell wall. After 24 hours, CCK8 activity
testing is performed. Add CCK-8 reagent and incubate at 37  for 2
hours. Measure the OD value at 450nm and calculate IC50. Moi=number of
MRSA/BMSCs (multiple infections=number of bacteria/number of bone marrow
stem cells)

By adding osteogenic inducers (100 mmol/L dexamethasone, 0.05 mmol \selectlanguage{ngerman}· L-1
ascorbic acid, and 10 mmol · L-1) to BMSCs cells \selectlanguage{greek}β- \selectlanguage{english}Sodium
glycerophosphate for osteogenic differentiation induction.

1.8ALP staining

Take each group of cells, clean them with PBS, add ALP fixative and fix
for 3 minutes. Add the prepared ALP incubation solution dropwise, place
it in a wet box, incubate in dark for 15-20 minutes, and re stain with
nuclear fixation red staining solution or methyl green staining solution
for 3-5 minutes. Clean the cells with PBS and perform microscopic
examination.

1.9 IF experiment

Each group of cells was incubated at room temperature in a 4\% PFA
solution for 30 minutes and fixed. Then, the samples were sealed in PBS
containing 2\% Triton X-100 (PBST) and 5\% goat serum for 1 hour. They
were incubated overnight at 4 \selectlanguage{ngerman}° C with the following primary antibodies:
Runx2, Osterix (OSX), ALP, Osteocalcin. Then, incubate the cells with
the corresponding secondary antibody at 37 ° C for 1 hour. Imaging and
analyzing cells under a fluorescence microscope.

1.10 Construction of MRSA infected osteomyelitis model

Male mouse aged 6-8 weeks were randomly divided into 7 groups, with
10mouse in each group; Drill a hole in the right tibia, inject sterile
physiological saline into Group A, and inject 1 into Groups B, C, D, E,
F, and G, respectively × 109, 1 × 108, 1 × 107, 1 × 106, 1 × 105 and 1 ×
104 CFU/mL MRSA was used for 2 weeks to observe the wound healing, skin
temperature, and the presence of redness, swelling, pus discharge, and
sinus formation. Take the right tibia and secretions for subsequent
experiments.

1.11 HE staining

After decalcification, bone tissue was fixed overnight in 4\%
paraformaldehyde, dehydrated in ethanol gradient, and embedded in
paraffin \selectlanguage{greek}μ \selectlanguage{english}Slice m thick. Seal the film after staining with hematoxylin
and eosin, and randomly select areas for optical microscopy observation.

1.12 IHC staining

After decalcification, bone tissue was fixed overnight in 4\%
paraformaldehyde, dehydrated in ethanol gradient, and embedded in
paraffin \selectlanguage{greek}μ \selectlanguage{english}Immunohistochemical staining was performed on m thick
sections. The primary antibodies used for immunohistochemistry include
Runx2, OSX, ALP, and Myh7.

1.13 Transcriptome sequencing

1.3.1 Experimental process

Use TRIzol (Thermofisher, 15596018) to separate and purify the RNA of
the total sample according to the operating plan provided by the
manufacturer. Then, NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) was
used to control the total RNA content and purity of three duplicate bone
tissue samples from the WT group, MRSA group, KO sdrc group, and MRSA+KO
sdrc group of mice, and the RNA integrity was tested using Bioanalyzer
2100 (Agilent, CA, USA); Concentration\textgreater{}50ng/ \selectlanguage{greek}μ \selectlanguage{english}L. RIN
value\textgreater{}7.0, total RNA\textgreater{}1 \selectlanguage{greek}μ \selectlanguage{english}G satisfies
downstream experiments. Using oligo (dT) magnetic beads (Dynameads Oligo
(dT), cat.25-61005, Thermo Fisher, USA), the mRNA carrying PolyA
(polyadenylate) was specifically captured through two rounds of
purification. The captured mRNA was fragmented using the NEBNextR
Magnesium RNA Fragmentation Module (cat. E6150S, USA) under high
temperature conditions, at 94  for 5-7 minutes. Fragmented RNA is
synthesized into cDNA by reverse transcriptase (Invitrogen SuperScriptTM
II Reverse Transcriptase, cat. 1896649, CA, USA). Then use E Coli DNA
polymerase I (NEB, cat. m0209, USA) and RNase H (NEB, cat. m0297, USA)
undergo two-strand synthesis, converting the composite double stranded
DNA and RNA into DNA double stranded. At the same time, dUTP Solution
(Thermo Fisher, cat. R0133, CA, USA) is added to the double stranded DNA
to make the ends of the double stranded DNA flat, and an A base is added
to each end to connect it to the junction with a T base at the end, And
use magnetic beads to screen and purify its fragment size. Using UDG
enzymes (NEB, cat. m0280, MA, US) to digest the two strands, PCR pre
denaturation was performed at 95  for 3 minutes, followed by 8 cycles
of denaturation at 98  for 15 seconds each. Annealing was performed at
60  for 15 seconds, stretching at 72  for 30 seconds, and finally
extending at 72  for 5 minutes to form a library (chain specific
library) with a fragment size of 300bp \selectlanguage{ngerman}± 50bp. Finally, we used Illumina
NovaseqTM 6000 (LC Bio Technology Co., Ltd. Hangzhou, China) to perform
double ended sequencing according to standard procedures, with
sequencing mode PE150.

1.3.1 Data quality control

The raw data generated by sequencing is preprocessed and filtered
(usually genes are screened from both the difference multiple and
significance level, with the difference multiple FC\textgreater{}=2 or
FC\textless{}0.5 (i.e. the absolute value of log2FC\textgreater{}=1) and
q value\textless{}0.05 (\textbar{} log2fc
\textbar{}\textgreater{}=1\&q\textless{}0.05) as the threshold standard
(multiple groups are compared with no difference multiple, and genes
with q\textless{}0.05 are screened as genes with statistical differences
between multiple groups) to obtain valid data (Clean Data), Compare with
the reference genome again to obtain comprehensive alignment
information. At the same time, calculate the gene location information
specified in the genome annotation file gtf separately: 1) Compare the
sequencing data with the reference genome for reads statistics; 2)
Regional distribution statistics of sequencing data compared to
reference genomes. Genome:
ftp://ftp.ensembl.org/pub/release-101/fasta/mus\_musculus/dna/ \selectlanguage{english})

1.4 Construction of Myh7 Interference Stable Transgenic Plants

The Myh7 interference plasmid was purchased from the Ono gene. A 2ul
plasmid was added to 100ul of Escherichia coli, and a single colony was
smeared onto 20ml of liquid culture medium. It was incubated overnight
on a shaker at 37 deg C and 200r. Extract plasmid RT-PCR to screen the
optimal interfering vector, transfect it into 293T cells for packaging,
and collect cell culture supernatant 48 hours after transfection.
Centrifuge 500g for 10 minutes to remove cell fragments and perform
virus titer measurement or virus concentration. Transfected mouse
osteoblasts were continuously screened with 2.5ug/ml puromycin for one
month.

1.5 WB experiment

After cracking the sample on ice for 10 minutes, centrifuge 14000 g at 4
\selectlanguage{english} for 15 minutes. Measure the protein concentration using the BCA
protein quantification kit and take 80 \selectlanguage{greek}μ \selectlanguage{english}L protein plus 20 \selectlanguage{greek}μ \selectlanguage{english}Mix the 5 x
protein loading buffer well and cook in a boiling water bath for 5
minutes before performing SDS-PAGE electrophoresis. After the membrane
transfer is completed, remove the PVDF membrane and place it in 1 \selectlanguage{ngerman}× The
strips after TBST soaking and cutting are placed in a sealing solution
(5\% skimmed milk powder) and sealed on a shaking table at room
temperature for 40 minutes. First antibody (Myh7, Myl2, Tnnc1, Runx2,
OSX \selectlanguage{greek}β- \selectlanguage{english}Actin, ALP, and osteocalcin were incubated overnight at 4 , and
the secondary antibody was incubated for 40 minutes, developed, and
photographed for preservation.

1.6 Alizarin Red Staining

Clean the cell sample with PBS, fix it with 10\% formalin for 10-30
minutes, remove the fixative, clean the sample twice with PBS, add
alizarin red staining solution dropwise, cover the sample, stain for 1-5
minutes, remove the staining solution, wash with PBS twice, observe and
take photos under the microscope.

1.7 Statistical Analysis

Statistical analysis was conducted using Prism graphpad, and the
experimental data were presented in the form of mean \selectlanguage{ngerman}± standard error
(Mean ± SEM). The comparison of inter group measurement data was
conducted using T-test, and the comparison of multi group measurement
data was conducted using one-way analysis of variance. Setting
p\textless{}0.05 has statistical differences: p\textless{}0.01 has
significant statistical differences; P\textless{}0.001 has a highly
significant statistical difference.

\textbf{2.Results}

2.1 Knocking out sdrc can inhibit the formation of MRSA biofilm

Through genetic engineering technology, we successfully knocked out the
SDRC protein sequence of Staphylococcus aureus. As shown in Figure
Attachment 1, PCR amplification and agarose gel electrophoresis results
show that the SDRC protein of Staphylococcus aureus was successfully
knocked out. To investigate the effect of sdrc knockout on the formation
of MRSA biofilm, we first validated the purchased KO sdrc. RT-PCR
results showed extremely low expression of sdrc in the KO sdrc group,
indicating that the purchased knockout bacteria were correct and could
be used for subsequent experiments (Figure 1A). Next, we plotted the
growth curves of MRSA and KO sdrc at 1h, 2h, 3h, 4h, 5h, 7h, 9h, 12h,
18h, and 24h. The results showed that knocking out sdrc had no effect on
the growth level of MRSA (Figure 1B), but there were invasive virulent
pathogenic factors on the biofilm of MRSA \selectlanguage{greek}α- \selectlanguage{english}The expression levels of HL
and PSM did significantly decrease (Figure 1C). The results of crystal
violet staining showed that the knockout of sdrc could weaken the
formation of MRSA biofilm (Figure 1D), and the same results were
obtained by scanning electron microscopy. The surface of the MRSA group
was rough, and the bacterial membrane migrated towards the blank,
showing a trend towards biofilm formation, while the KO sdrc group had a
smooth surface and reduced bacterial count (Figure 1E). It indicates
that sdrc knockout can inhibit the formation of MRSA biofilm.

2.2 Knocking out sdrc can alleviate the inhibition of MRSA on the
differentiation of BMSCs into osteoblasts

Next, we examined the effect of sdrc knockout on the inhibition of BMSCs
differentiation into osteoblasts by MRSA. Firstly, we examined the
median lethal rate of MRSA on BMSCs cells, and the results showed an
IC50 of 161.5 moi (Figure 2A). Next, we intervened with 161.5 mol of
MRSA and KO sdrc on BMSCs cells to investigate the effect of sdrc
knockout on the inhibition of BMSCs differentiation into osteoblasts by
MRSA. The osteoblast induced differentiation group was used as a
positive control, and cell culture was shown in Figure 2B. After 24
hours of intervention, CCK8 results showed that both MRSA and KO sdrc
restricted the proliferation ability of BMSCs, but MRSA was more
significant than KO sdrc (Figure 2C), and ALP staining results showed
that, Both MRSA and KO sdrc can reduce the positive rate of ALP (there
may be errors in the results here, which need to be verified multiple
times) (Figure 2D and 2E). The RT-PCR results are similar to CCK8, and
MRSA significantly inhibits the specific markers of osteogenic
differentiation Runx2, OSX, ALP, and osteocalcin compared to KO sdrc
(Figure 2F). Finally, we detected the expression of Runx2, OSX, ALP, and
osteocalcin using IF experiments and obtained the same results (Figure
3A-E). It indicates that knocking out sdrc can alleviate the inhibition
of MRSA on the differentiation of BMSCs into osteoblasts.

2.3 Knocking out sdrc can alleviate the progression of osteomyelitis

To investigate the impact of sdrc knockout on the progression of
osteomyelitis in vivo, we first constructed an osteomyelitis model
infected with MRSA. As shown in Figure 4A-B, 1 \selectlanguage{ngerman}×
10\textsuperscript{8}and 1 × The difference in osteomyelitis caused by
10\textsuperscript{9}CFU/mL MRSA is not significant, so we will choose 1
in the future × 10\textsuperscript{8} CFU/mL MRSA modeling. Research has
shown that compared to KO sdrc alone intervention, MRSA+KO sdrc mixed
intervention can more effectively alleviate the progression of
osteomyelitis. Therefore, we also set up an MRSA+KO sdrc mixed
intervention group, where serum C-reactive protein is an inflammatory
marker protein. The CRP content test results showed that compared with
KO sdrc alone intervention, the MRSA+KO sdrc mixed intervention did not
reduce the release of CRP (Figure 4D).

Next, we examined the impact of knocking out sdrc on the progression of
osteomyelitis. HE results showed that the control group had normal
morphology and intact cortical structure without pathological changes;
In the MRSA group, a large number of inflammatory cells infiltrated the
bone marrow cavity, and the bone cortex at the drilling site failed to
heal; A small amount of inflammatory cells and new bone formation were
observed in the KO sdrc group. In the MRSA+KO sdrc group, there was bone
destruction and a large amount of inflammatory cell infiltration (Figure
5A). Culture of wound exudates in each group revealed that compared to
the Control group, the MRSA group had the highest bacterial density,
followed by the KO sdrc group, and the MRSA+KO sdrc group had the lowest
density (Figure 5B). The ELISA results showed that the KO sdrc group
significantly inhibited the inflammatory factors IL-6 and IL-1 \selectlanguage{greek}β \selectlanguage{english}And
TNF- \selectlanguage{greek}α \selectlanguage{english}The MRSA+KO sdrc group did not achieve the expected results
(Figure 5C). Indicating that knocking out sdrc can alleviate the
progression of osteomyelitis.

2.4 Knocking out sdrc can alleviate the inhibition of bone formation by
MRSA

To investigate the effect of knocking out sdrc on the inhibition of bone
formation by MRSA, we detected the expression of bone formation marker
proteins Runx2, OSX, and ALP using IHC. The results showed that compared
with the MRSA group, the expression of Runx2, OSX, and ALP in the
KO-sdrc group was significantly increased; Compared with the KO sdrc
group, the expression of Runx2, OSX, and ALP in the MRSA+KO sdrc group
decreased (Figure 6A-C). It indicates that knocking out sdrc can
alleviate the inhibition of bone formation by MRSA, while the mixed
intervention of MRSA+KO sdrc cannot more effectively alleviate the
inhibition of bone formation by MRSA.

2.5 Transcriptome sequencing reveals that knocking out sdrc alleviates
osteomyelitis progression by promoting Myh7 expression

We performed transcriptome sequencing on three right tibia tissue
samples from each of WT, MRSA, KO sdrc, and MRSA+KO sdrc mice, with a
threshold setting of \textbar{} log2 (FoldChange \textbar{} [?] 1, and
pvalue\textless{}0.05). The results showed that compared with the WT
group (Figure 7A (a)), a total of 457 significantly differentially
expressed genes were screened, including 138 upregulated genes and 319
downregulated genes. Compared with the KO sdrc group and the WT group
(Figure 7A (b)), a total of 290 significantly differentially expressed
genes were screened, including 180 upregulated genes and 110
downregulated genes. Compared with the KO sdrc group and MRSA group
(Figure 7A (c)), a total of 513 significantly differentially expressed
genes were screened, including 312 upregulated genes and 201
downregulated genes. Comparing the KO sdrc+MRSA group with WT (Figure 7A
(d)), a total of 628 significantly differentially expressed genes were
screened, including 202 upregulated genes and 426 downregulated genes.
We will use the Top100 gene with the lowest q value as an example of a
heatmap. Venn analysis was performed on the significantly differentially
expressed genes in the MRSA group Vs WT group, KO sdrc group Vs WT
group, and KO sdrc group Vs MRSA group, as shown in Figure 7B. A total
of 5 common genes were screened to identify the reasons for
downregulation in the WT group Vs MRSA group, upregulation in the WT
group Vs KO sdrc, and upregulation in the KO sdrc group Vs MRSA group.
It was found that these five genes Myh7, Tnnc1, Myl2, Igkv4-59, and
CT010467 all met the conditions (Figure 7B). Through literature review,
it can be concluded that Myh7 and Tnnc1 genes are associated with
skeletal muscle. After removing the new genes Igkv4-59 and CT010467,
this study focuses on three genes: Myh7, Myl2, and Tnnc1, and their
expression multiples are shown in 7C.

Next, we validated the sequencing results. RT-PCR results showed that
all three genes, Myh7, Myl2, and Tnnc1, did not match the sequencing
results (compared to the WT group, they were all down regulated in the
MRSA group and up regulated in the KO sdrc group) (Figure 8A). The WB
results showed that only Myh7 followed the trend (Figure 8B), and we
also validated it with IHC, which was the same as the WB results.
Therefore, we speculate that the knockout of sdrc may be by increasing
the expression of Myh7, Relieve the progression of osteomyelitis.
Compared with the KO sdrc group, the MRSA+KO sdrc mixed intervention
group did not increase the expression of Myh7.

2.6 In vitro validation: Knocking out sdrc can promote bone formation by
promoting Myh7 expression

To further verify that knocking out sdrc promotes bone formation by
promoting Myh7 expression, we constructed a stable osteoclast
transfection strain with MYH7 interference (Figure Attachment 2). The
results showed that compared with the KO sdrc+sh NC group, the KO
sdrc+sh Myh7 group had a decrease in osteoclast proliferation ability
(Figure 9A), and the expressions of Myh7, Runx2, OSX, ALP, and
osteocalcin were significantly reduced (Figure 9B). The positive rates
of ALP and alizarin red staining were also significantly reduced (Figure
9C-D). It indicates that knocking out sdrc indeed promotes bone
formation and alleviates the progression of osteomyelitis by promoting
MYH7 expression.

\textbf{Discussion}

Osteomyelitis leads to bone infection and destruction, mainly bacterial
infection, the most common is Staphylococcus aureus, which is a major
problem in the treatment of orthopaedic diseases\textsuperscript{{[}22,
23{]}}. Osteomyelitis often occurs in more serious open fractures, where
the bone fracture ends are exposed, which is easy to be invaded by
bacteria, leading to bone tissue infection or local residual bacterial
lesions\textsuperscript{{[}24{]}}. Due to the destruction of skin soft
tissue and blood vessels around the bone fracture end, it is very easy
to lead to bacterial growth and bacterial biofilm adhesion, resulting in
the repeated development of clinical Osteomyelitis, which is difficult
to cure\textsuperscript{{[}25, 26{]}}. At present, the clinical
treatment for chronic Osteomyelitis is mainly debridement and drainage,
and systemic sensitive antibiotics\textsuperscript{{[}23, 27, 28{]}}.
However, due to the poor local blood supply of the focus and the
formation of dead bones to varying degrees, the antibiotic treatment has
poor effect and large side effects\textsuperscript{{[}29{]}}.

As a member of MSCRAMM family, SDRC protein is mainly used to promote
the biofilm formation of Staphylococcus aureus. In Osteomyelitis
infected by Staphylococcus aureus, SDRC protein plays a huge role in the
progression and prognosis of Osteomyelitis\textsuperscript{{[}30{]}}. In
our study, we constructed a model of Staphylococcus aureus induced
Osteomyelitis by knocking out the SDRC protein sequence of
Staphylococcus aureus and infecting rat, founding that compared with
wild type Staphylococcus aureus infected Osteomyelitismouse , its IL-6,
TNF- \selectlanguage{greek}α \selectlanguage{english}The inflammatory indexes such as ALP, OST and RunX2 were
significantly reduced, the infection of bone tissue was significantly
lighter, and the osteogenic indexes such as ALP, OST and RunX2 were also
significantly increased, suggesting that SDRC protein played a huge role
in the occurrence and progress of Osteomyelitis infected by
Staphylococcus aureus. Previous studies\textsuperscript{{[}19,
31{]}}have shown that the most important biological function of SDRC
protein is its ability to promote the formation of bacterial biofilms,
which is particularly important in the process of bacterial infection of
the host\textsuperscript{{[}32, 33{]}}. Therefore, understanding the
molecular mechanism mediated by SDRC protein is of great significance
for the treatment of Staphylococcus aureus infectious diseases. Our
research results showed that after the SDRC protein is knocked out, the
biofilm forming ability of Staphylococcus aureus is significantly
weakened, and it has an invasive virulence pathogenic factor \selectlanguage{greek}α- \selectlanguage{english}HL
(P=0.1868) and PSM (P=0.0361) were significantly reduced, which
confirmed that SDRC protein played an important role in the virulence of
Staphylococcus aureus, and provided a new direction for the treatment of
Staphylococcus aureus infected Osteomyelitis.

As a member of the CWA protein family, the SDRC protein mainly functions
to promote the formation of biofilms in Staphylococcus aureus through
its own dimerization, helping bacteria to proliferate on the host
surface. Derived from human neuronal cells \selectlanguage{greek}β \selectlanguage{english}Axonal protein( \selectlanguage{greek}β-
\selectlanguage{english}Neurexin )can interact with SDRC protein, and this interaction can
inhibit SDRC protein mediated biofilm formation, slowing down the
infection of Staphylococcus aureus to the host. In current in vivo and
in vitro experimental studies, it has been found that Staphylococcus
aureus can invade osteoblasts and cause their death. Staphylococcus
aureus, which invades osteoblasts, can also live and proliferate within
it and is believed to be related to the occurrence of chronic
osteomyelitis and recurrent osteomyelitis. In terms of bone formation,
bacterial biofilms and inflammatory cytokines inhibit the osteogenic
differentiation ability of bone marrow mesenchymal stem cells, while
Staphylococcus aureus inhibits the proliferation of osteoblasts and
induces their apoptosis.

In order to further explore the mechanism of SDRC protein promoting the
occurrence and development of Staphylococcus aureus infected
Osteomyelitis, we found that Myh7 was significantly increased in the
SDRC protein knockout group through Transcriptome sequencing, and by
constructing a stable cell line with MYH7 knockdown and functional
experiments, we found that after infection with SDRC knockout
staphylococcus, MYH7 knockdown significantly reduced its osteogenic
indicators and Ossification, compared with shNC, suggesting that MYH7
may inhibit the biological function of Staphylococcus aureus SDRC
protein. The MYH7 gene is mainly located in the long arm of chromosome
14 and contains a total of 40 exons (E1-E40)\textsuperscript{{[}34,
35{]}}. It was first reported by Geisterfer Lowrance et al. in 1990. The
MYH7 gene, as the first pathogenic gene found to be associated with
hypertrophic cardiomyopathy, has been extensively reported in studies
such as myocardial injury\textsuperscript{{[}36{]}}. In China, the MYH7
gene mutation range is relatively widespread in patients with
hypertrophic cardiomyopathy, except for E6, E7, E10, E17, E24, E25, E29
Pathogenic mutations are present in all 27 exons except for E32 and E33,
with 7 mutation sites located in E22\textsuperscript{{[}36, 37{]}}. At
the same time, it has been found in Western populations that the most
common malignant mutations in patients with myocardial hypertrophy are
associated with R403Q, R453C, R719W, and R723G mutations in the MYH7
gene\textsuperscript{{[}38, 39{]}}. In our study, we found for the first
time that MYH7 was significantly reduced in the knockout of SDRC protein
in Staphylococcus aureus osteomyelitis, indicating that MYH7 plays a
potential biological role in the occurrence and development of
Staphylococcus aureus osteomyelitis, and may have a significant
correlation with the SDRC protein of Staphylococcus aureus. This
provides a solid theoretical basis for us to further explore the deep
mechanism of SDRC protein in Staphylococcus aureus osteomyelitis in the
future.

\subsection*{Acknowledgements}

{\label{acknowledgements}}

Funding

This study was funded by~National Natural Science Foundation of

China~(Grant No.~82072392);

The Grants from~Yunnan Orthopedics and Sports Rehabilitation

Clinical Medicine Research Center (Grant No.~202102AA310068);

and Yunnan Prvincial Clinical Orthopaedic Trauma Medical Center

(Grant No. ZX20191001);

Clinical Key Subject Construction Project of PLA;

Medical key subject of Joint Logistic Support Force of PLA.

\subsection*{Author contributions}

{\label{author-contributions}}

BQ, MG ,and LY conceived and designed the experiments. BQ, CM, HS, TC,
HL,

and LS performed the experiments. CL and YX analyzed the data. CL, LY,
YX

and TC contributed reagents, materials, and analysis tools. BQ and MG
wrote

the paper.

\subsection*{Data availability}

{\label{data-availability}}

The data of this study can be obtained from the corresponding author

upon reasonable request.

\subsection*{Competing interests}

{\label{competing-interests}}

The authors declare no competing interests.

\subsection*{Ethics approval and consent to
participate}

{\label{ethics-approval-and-consent-to-participate}}

The animal experiment program was approved by the Medical Ethics
Committee of

the 920th Hospital of the PLA Joint Logistics Support Force (Approval
No: Lun

shen 2019-010 (Section) -01), and carried out in accordance with the
Guidelines

for the Care and Use of Experimental Animals.

\subsection*{Footnotes}

{\label{footnotes}}

These authors contributed equally:~Baochuang Qi, Minzheng Guo, LiLi Yang

\subsection*{Contributor Information}

{\label{contributor-information}}

Yongqing Xu: Email:xuyongqingkm@163.net

Chuan Li:Email:\emph{lichuankaka@163.com}

Tao chen: Email:henpao@126.com

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Figure legend\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 1: The effect of sdrc knockout on the formation of MRSA biofilm.
A: RT-PCR was used to detect the knockout efficiency of sdrc, with rpoA
as the internal reference. B: CCK8 detection of the impact of sdrc
knockout on the growth ability of MRSA. C: ELISA detection of MRSA
virulence pathogenic factors \selectlanguage{greek}α- \selectlanguage{english}Expression of HL and PSM. D: Crystal
violet staining was used to observe the formation of MRSA biofilm. E:
Scanning electron microscopy was used to observe the changes in surface
morphology of MRSA.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 2: Effect of sdrc knockout on MRSA inhibition of BMSCs
differentiation into osteoblasts. A: CCK8 detected the median lethal
rate of MRSA on BMSCs cells (moi=number of MRSA/BMSCs). B: BMSCs cell
culture (100X), by adding osteogenic inducers (100 mmol/L dexamethasone,
0.05 mmol \selectlanguage{ngerman}· L-1 ascorbic acid, and 10 mmol · L-1) to BMSCs cells \selectlanguage{greek}β-
\selectlanguage{english}Sodium glycerophosphate for osteogenic differentiation induction. C:
CCK8 was used to detect the proliferation activity of BMSCs cells. D:
ALP staining (100X). E: Analysis of ALP staining positive rate. F:
RT-PCR was used to detect the mRNA expression levels of osteogenic
differentiation specific markers Runx2, Osterix (OSX), ALP, and
Osteocalcin, using GAPDH as an internal reference.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 3: The IF experiment detected the expression of Runx2, OSX, ALP,
and osteocalcin. A: OSX fluorescence map (200X). B: ALP fluorescence map
(200X). C: Runx2 fluorescence image (200X). D: Osteocalcin fluorescence
map (200X). E: Statistical chart of positive rates for Runx2, OSX, ALP,
and Osteocalcin.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 4: Selection of optimal MRSA infection concentration and
construction of osteomyelitis model. A: Inject 1 separately \selectlanguage{ngerman}×
10\textsuperscript{4}, 1 × 10\textsuperscript{5}, 1 ×
10\textsuperscript{6}, 1 × 10\textsuperscript{7}, 1 ×
10\textsuperscript{8} and 1 × 10\textsuperscript{9} CFU/mL MRSA was used
to construct an osteomyelitis model. B: ELISA detection of serum
C-reactive protein content. C: Construct osteomyelitis models with
optimal concentrations of MRSA, KO sdrc, and MRSA+KO sdrc. D: ELISA
detection of serum C-reactive protein content.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Figure 5: The effect of knocking out sdrc on the progression of
osteomyelitis. A: HE staining was used to observe pathological changes
(100X). B: Changes in bacterial content in wound exudate. C: ELISA
detects the content of inflammatory factors.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Figure 6. The effect of knocking out sdrc on MRSA inhibiting bone
formation. A: IHC detected the expression of Runx2 (400X). B: IHC
detected the expression of Osterix (400X). C: IHC detected the
expression of ALP (400X).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

Figure 7 Transcriptome sequencing reveals the mechanism by which
knocking out sdrc alleviates the progression of osteomyelitis. A:
Differential gene expression clustering heatmap, a is MRSA VS WT; B is
KO sdrc VS WT; C is KO sdrc VS MRSA; D is KO sdrc+MRSA VS WT.

B: Differential gene Wayne map. C: Differential gene expression multiple
plot.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

Figure 8 Transcriptome sequencing validation. A: RT-PCR was used to
detect the expression of Myh7, Myl2, and Tnnc1, with GAPDH as the
internal reference. B: WB detection of the expression of Myh7, Myl2, and
Tnnc1, in order to \selectlanguage{greek}β- \selectlanguage{english}Action as an internal parameter. C: IHC detected
the expression of Myh7 (400X).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

Figure 9: Knocking out sdrc promotes bone formation by promoting Myh7
expression. A: CCK8 was used to detect cell proliferation activity. B:
WB detection of the expression levels of Myh7, Runx2, OSX, ALP, and
osteocalcin, in order to \selectlanguage{greek}β- \selectlanguage{english}Action as an internal parameter. C: ALP
staining (100X). D: Alizarin red staining (100X).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}

Figure Attachment 1 A: PCR validation of sdrC amplification fragment,
with A and B fragments as templates, overlapping PCR amplification was
performed to fuse AB fragments. B: Amplification of sdrC homologous arm
fragments. C: 1\% agarose gel electrophoresis amplification map was
amplified by pKOR1 vector framework. D: The electrophoresis results of
sdrC-JD-F/JD-R wild bacteria and knockout bacteria showed that 1 refers
to the amplification product of knockout strain RN4220 ^ sdrc, and 2
refers to the control of wild bacteria RN4220.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image12/image12}

\end{center}
\end{figure}

Figure Attachment 2: Construction of a stable osteoclast transfection
strain interfered by Myh7. A: WB screening for the best interfering
vector to \selectlanguage{greek}β- \selectlanguage{english}Actin is an internal reference protein. B: RT-PCR
validation of the optimal interference sequence, using GAPDH as the
internal reference. C: The lentivirus was transfected into 293T cells
for packaging (100X). D: Chronic virus infection of osteoclasts (100X).

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