Cbl-b negatively regulates TLR/MyD88-mediated anti-Toxoplasma gondii immunity

ABSTRACT Toxoplasma gondii is an important intracellular protozoan, which needs to exploit host nutrition for successful parasitism. We previously reported that Casitas B-lineage lymphoma-b (Cbl-b) was screened as a host dependency factor of T. gondii by using lentiviral CRISPR-Cas9-single guide RNA (sgRNA) libraries. Furthermore, the detailed mechanism of Cbl-b being required by T. gondii infection was explored in this study. The proliferation of T. gondii was found to be significantly inhibited in Cbl-b knockdown cell lines, and the Cbl-b expression level increased with prolonged T. gondii infection, while the MyD88 level was significantly decreased in the T. gondii infection group. Toll-like receptor (TLR)/MyD88 is a conserved innate cellular immune signaling pathway against pathogens infection. Cbl-b was found to interact with MyD88 and mediate MyD88 ubiquitination in the fluorescence resonance energy transfer and co-immunoprecipitation experiments. A Cbl-b knockout (KO) C57BL/6J lineage was then constructed and, together with the wild-type (WT) mice, was infected with the same amount of T. gondii tachyzoites of ME49 strain. At 13 days post infection, all the mice in the WT group died, while 80% of the mice in the Cbl-b KO group still survived, even to the end of the experiment. The parasitic burden in the liver, lung, and brain of the Cbl-b KO mice was significantly lower than that of the WT mice (P < 0.05). In Cbl-b KO infected mice, the percentage of B cells was higher, whereas that of macrophages was lower, and the interferon-γ and interleukin-6 levels in the serum were higher than that in the WT infected mice; the difference was significant (P < 0.05). Furthermore, when host cells were infected by T. gondii, host’s Cbl-b was found to interact with MyD88 to ubiquitinate MyD88 for ribosome-dependent degradation. Therefore, the host’s innate immunity against T. gondii through the TLR/MyD88 pathway was negatively regulated by Cbl-b. IMPORTANCE This is the first report that a human E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (Cbl-b), functions as a host dependency factor for the intracellular protozoan Toxoplasma gondii and the mechanism for how T. gondii infection inhibits the TLR/MyD88 innate immunity pathway through MyD88 degradation mediated by Cbl-b. This finding is an impactful contribution for understanding the host cell immunity against T. gondii infection.

T he intracellular protozoan Toxoplasma gondii is widely distributed, and about 30% of worldwide population is serum positive with this parasite (1).Its infection results in non-specific symptoms in immune-competent individuals and serious diseases such as encephalitis and retinochoroiditis in immunocompromised patients (2).As an obligatory parasite, T. gondii has to exploit host factors for successful parasitism.In our previous study, Casitas B-lineage lymphoma proto-oncogene B (Cbl-b) was identified as one of the host dependency factors required by T. gondii, by using genome-wide CRISPR/Cas9 screening (3).It is found that in the host cells with Cbl-b knockdown, the survival and reproduction rates of the parasites are decreased, while the cells can still survive under the pressure of T. gondii infection (3).Cbl proteins are a small class of E3 ubiquitin ligases, which are important negative regulators of immune responses (4).Cbl-b is a member of Cbl family, which contains a highly conserved N-terminal tyrosine-kinase-binding domain and a RING finger domain, and the C-terminal half contains a ubiquitin-associated domain (5).The ring finger domain mediates the transfer of ubiquitin between E2 ubiquitin-conjugating enzymes and the substrate proteins.The main function of Cbl-b is to ubiquitinate the target proteins, transport the ubiqui tinated proteins to the lysosome for degradation, and negatively regulate the related signal pathways (6)(7)(8).Cbl-b plays a crucial role in immunosuppression by negatively regulating the activation of T cells.The expression of Cbl-b in tumor-infiltrating immune cells is significantly higher than that in normal tissues, and the expression of Cbl-b is significantly lower in the immune cells of the patients with autoimmune diseases such as regulatory T cells from systemic lupus erythematosus (7).
Toll-like receptor (TLR) signaling pathways in host cells play an important role in the immune process against T. gondii infection.TLR signaling pathways are mainly divided into MyD88-dependent and MyD88-independent pathways.In fact, all TLR family members are involved in MyD88-dependent signaling pathways except TLR3 (9).It is reported that TLR11/12/2/4 in mouse cells can recognize proteins such as glycosyl phosphatidylinositol (GPI) and profilin on the surface of T. gondii and then activate MyD88-dependent signal pathway (10)(11)(12).TLR11 is a pseudogene in human genome (13).Human cells rely on TLR2/4/9/5 to recognize GPI and profilin on the surface of T. gondii and then activate MyD88-dependent signal pathway (14).The activated TLR/ MyD88 signal pathway can enhance the secretion of interleukin-12 (IL-12), IL-6, IL-8, IL-10, interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α), and so on (15)(16)(17)(18).It is reported that Cbl-b overexpression in human embryonic kidney cells (HEK293) blocks the interaction of TLR4 and MyD88 to negatively regulate TLR4-MyD88-dependent cell signaling in the presence of lipopolysaccharide (LPS) (19).It is also found that Cbl-b interacts with MyD88 and induces the degradation of MyD88 in murine peritoneal macrophages stimulated with LPS (20).In this study, the role and the mechanism of Cbl-b functioning on host's anti-Toxoplasma gondii infection were investigated.

The proliferation of T. gondii was significantly inhibited by Cbl-b knockdown
Human foreskin fibroblast (HFF) and human monocytic cell line (THP-1) cells were edited by the CRISPR Cas 9 genome editing technique.It was found that, in HFF cells, Cbl-b expression was significantly down-regulated in Cbl-b-sgRNA2 (the single guide RNA targeting Cbl-b)-transfected group compared to that of the normal cell, Cbl-b-sgRNA1transfected group and the scrambled sequence-transfected control (SC) group (Fig. 1A).Cbl-b-sgRNA2 was then selected for subsequent experiments.
The normal and Cbl-b knockdown (KD) HFF cells were infected with T. gondii CEP-GFP fluorescent strain at multiplicity of infection (MOI) 3.At 20 h post infection (hpi), the percentage of the parasitophorous vacuoles (PVs) containing one parasite in the control group was much higher than that in the Cbl-b KD group (P < 0.05), while the percentage of the PVs with four parasites in the control group was much lower than that in the Cbl-b KD group (P < 0.05), and no significant difference was found for the percentage of the PVs with two parasites between these two groups (Fig. 1B).
The consistent results were seen in the THP-1 cells.The Cbl-b expression in the Cbl-b KD groups (#1 and #9 clones) was decreased significantly compared to that in the normal cells (Fig. 1C).The normal and Cbl-b KD THP-1 cells were infected with ME49 strain at MOI 1.At 24 hpi, T. gondii B1 gene was amplified by quantitative PCR (qPCR) to evaluate the parasitic proliferation.The results showed that the proliferation efficiency of T. gondii in Cbl-b KD group was significantly lower than that in the normal group (P < 0.05) (Fig. 1D).Furthermore, the transcription of IL-12, IFN-γ, IL-6, and IL-1β in the normal and the Cbl-b KD THP-1 cells upon T. gondii infection (MOI = 1) were detected by qRT-PCR.The results showed that the transcription levels of IL-12 in the Cbl-b KD THP-1 cells were lower than that in the normal cells, while the transcription levels of IFN-γ, IL-6, and IL-1β in Cbl-b KD THP-1 cells were higher than that in the normal cells (P < 0.01) (Fig. 1E).

T. gondii infection led to enhanced Cbl-b expression and MyD88 degradation
HeLa cells were infected with ME49 tachyzoites (MOI = 1).The Cbl-b transcription level in the ME49 infection group was increased gradually within the 24 hpi (P < 0.01) (Fig. 2A), and the Western blot (WB) showed similar results (Fig. 2B).At 0.5 h, 2 h, 4 h, 6 h, 8 h, and 12 h post infection, the amount of Cbl-b in the infected cells was increased gradually and was larger than that in the uninfected group (P < 0.05).The expression level of β-actin was consistent among these seven groups (Fig. 2B).Toll-like receptor (TLR)/ MyD88 signaling pathway of host cells plays a key role in the immune process against T. gondii infection.RAW264.7 cells were infected with RH, ME49, and VEG tachyzoites at MOI of 5.At 28 hpi, the total protein was harvested and subjected to WB.The results showed that the amount of MyD88 was decreased after T. gondii infection (Fig. 2C), and this phenomenon was not changed after adding the proteasome inhibitor MG132.However, the degradation of MyD88 was partially alleviated after adding the lysosomal degradation inhibitor 3-MA (Fig. 2C).These results suggested that MyD88 was degraded through the lysosomal pathway after T. gondii infection.

Cbl-b interacted with MyD88 and mediated the ubiquitination and degrada tion of MyD88
The interaction of Cbl-b and MyD88 was detected with fluorescence resonance energy transfer (FRET).The FRET efficiencies were measured by acceptor photo-bleach technique (Fig. 3A).The FRET efficiencies in the positive control group (cells transfec ted with pEYFP-CFP) and the negative control group (cells co-transfected with pEYFP and pECFP) were about 50% and 10%, respectively, while the FRET efficiency in the experimental group (cells co-transfected with pEYFPC1-MyD88 and pECFPN1-Cbl-b) was about 70%, which was significantly higher than that in the negative control group (P < 0.01) (Fig. 3B).pcDNA3.1(+)-Cbl-b-3×FLAGand pcDNA3.1(+)-MyD88-HAplasmids were transfected to COS7 cells separately or together.The co-immunoprecipitation (Co-IP) assay was further used to confirm the interaction of Cbl-b and MyD88.The WB result showed when MyD88 was used as a bait protein, Cbl-b-Flag could be detected in the immunoprecipitants using an anti-Flag antibody only in the dually transfected cells (Fig. 3C), which indicated the interaction of Cbl-b and MyD88.

Cbl-b deficiency enhanced host immune response against T. gondii
To further explore the role of Cbl-b in host's immunity against T. gondii, Cbl-b KO mice on C57BL/6J background were constructed.As shown in Fig. 5A, the Cbl-b gene has 18 exons, with the ATG start codon in exon 2. sgRNAs directed Cas9 endonuclease to cleave Cbl-b gene in intron 4-5 and intron 10-11 and created a double-strand break.Such breaks would be repaired by non-homologous end joining and resulted in the disruption of Cbl-b gene.The pups were genotyped by PCR and WB (Fig. 5B and C).Both the WT and Cbl-b KO mice were infected with 1,000 T. gondii ME49 tachyzoites.At 13 days post infection (dpi), all the WT mice died, while 80% of the Cbl-b KO mice still survived to the end of the experiment (more than 30 dpi) (P < 0.05) (Fig. 6A).Significantly decreased weight and enlarged spleen were observed in the T. gondii-infected mice, compared to that of the uninfected mice, regardless of whether Cbl-b was knocked out (P < 0.01) (Fig. 6B).However, Cbl-b KO made no significant difference on the weight of the mice and the weight of the spleen of the infected and uninfected mice (P > 0.05) (Fig. 6B).T. gondii B1 gene was amplified by qPCR to evaluate the parasitic burden in different organs.As shown in Fig. 6C, the parasitic burden in different organs was not significantly changed at 3 dpi, while the parasitic burden in the liver, lung, and brain of the Cbl-b KO mice was significantly lower than that in the organs of the WT mice at 7 dpi (P < 0.05).
Meanwhile, the immune cell subsets in the T. gondii-infected mice were evaluated.As shown in Fig. 6D, in both Cbl-b KO and WT mice, the proportion of B cells, dendritic cells, and macrophages increased in the infected group, while the percentage of CD4 + and CD8 + T cells decreased in the infected group, compared with that in the uninfected mice (P < 0.01).On the other hand, the percentage of B cells was increased, and that of macrophages was decreased in the Cbl-b KO infected mice, compared with that in the WT infected mice (P < 0.05).These results indicated that the differentiation and development of B cells and macrophages were regulated by Cbl-b during T. gondii infection.
Cytokines play important roles in host's immune responses against T. gondii.The IFN-γ, IL-6, TNF-α, IL-12, IL-10, IL-1β, IL-2, IL-5, IL-17, and IL-4 in the serum of the mice were detected.As shown in Fig. 6E, the level of serum IFN-γ, IL-6, TNF-α, IL-12, IL-10, IL-2, and IL-4 increased in the infected group compared with that in the uninfected group, in both Cbl-b KO and WT mice (P < 0.05).Moreover, the levels of IFN-γ and IL-6 were significantly increased in the Cbl-b KO infected mice, compared with that in the WT infected mice (P < 0.05) (Fig. 6E).These results indicated that Cbl-b was important for IFN-γ and IL-6 secretion upon T. gondii infection.

DISCUSSION
T. gondii is an intracellular parasite with numerous and widespread hosts.In this study, both the transcription and expression level of host Cbl-b were found increased with the time of T. gondii infection (Fig. 2A and B).It has been reported that the Cbl-b of peripheral blood mononuclear cells is highly expressed in the patients with helminth-induced chronic infection (21).
It is reported that C-type lectin receptors (CLRs, dectin-1, and dectin-2) play an important anti-infection role in human's body, but the expression level of this protein is gradually decreased with the extension of the infection time after fungal infection (22)(23)(24)(25).Cbl-b was thought to be a therapeutic target for fungal infectious diseases.The reason is that when cells are infected by fungi, Cbl-b interacts with and ubiquitinates dectin-1 and dectin-2, leading to their degradation through the lysosomal pathway, to negatively regulate the natural immune response mediated by CLRs (22)(23)(24)(25).In our previous study, Cbl-b was identified as a host dependence factor with the genome-wide CRISPR-Cas9 library screening technology.When Cbl-b was knocked down or knocked out, the proliferation of T. gondii was significantly inhibited (Fig. 1 and 6C).Cbl-b was thought to ubiquitinate and degrade dectin-1 and dectin-2 through lysosomal pathway to negatively regulate the natural immune response mediated by CLRs in T. gondii infection (26).However, because of the specificity of T. gondii surface antigen, dectin-1 KO did not change the efficiency of T. gondii invasion, indicating that CLR signaling pathway did not participate in the host's immune process against T. gondii (26).TLR signaling pathways in host cells play an important role in the immune process against T. gondii infection.The activated TLR/MyD88 signal pathway enhances the secretion of IL-12, IL-6, IFN-γ, IL-8, IL-10, TNF-α, and so on (15)(16)(17)(18).Our study found that Cbl-b interacted with MyD88 and mediated the ubiquitination and degradation of MyD88 (Fig. 2-4).It is reported that Cbl-b interacts with MyD88 and induces the degradation of MyD88 when stimulated by LPS in mouse peritoneal macrophages (20).Altogether, these results suggested that Cbl-b was an important negative regulator for the immune responses mediated through TLR/MyD88 signaling pathway.
Cbl-b KO mice were constructed and used to study the function of Cbl-b.The survival curves of the mice infected by T. gondii showed that, compared with the WT mice, Cbl-b KO mice could live longer (Fig. 6A), and the percentage of B cells increased and which of macrophages decreased in Cbl-b KO mice (Fig. 6D).The percentage of B cells and macrophages in the spleen of Cbl-b KO uninfected mice is not significantly different from that in the WT uninfected mice.These results indicated that Cbl-b KO resulted in increase of B cell's percentage and decrease of macrophage's percentage after T. gondii infection.B cells play a crucial role in humoral immunity which can differentiate into plasma cells, synthesize, and secrete specific antibodies (27).Apart from secreting antibodies, B cells also work as antigen presenting cells for soluble antigens (28).It has been reported that B cells play an important role in host anti-T.gondii infection and are required for vaccination-induced resistance to virulent tachyzoites (29).All of the B cell-deficient mice complemented with naïve B-1 cells died 18 days after T. gondii infection, whereas B cell-deficient mice complemented with primed B-1 cells survived about half a year post infection.More Th1-and Th2-type cytokines and nitric oxide production were found in T. gondii-infected B cell-deficient mice complemented with primed B-1 cells (30).We found in our study that the level of IFN-γ, IL-6 were significantly elevated in the infected mice regardless of whether Cbl-b was knocked out, but it was higher in the Cbl-b KO mice than that in the WT mice after T. gondii infection (Fig. 6E).These results indicated that Cbl-b functioned to suppress IFN-γ and IL-6 secretion upon T. gondii infection.IFN-γ is essential in controlling acute Toxoplasma gondii infection and preventing the reactivation of latent bradyzoites (31,32).IFN-γ is indispensable for host defense as it is essential for the expression of IFN-γ-inducible genes, which leads to clearance of intracellular microbes (33).B cells were reported to be able to promote CD4 and CD8 T cells' IFN-γ production during Th1 inflammatory response to T. gondii infection (34).Taken together, the effect of Cbl-b on mice immune response may be associated with B cells.
In summary, our study indicated that Cbl-b negatively regulated the anti-T.gondii innate immunity mediated by TLR/MyD88 signaling (Fig. 7).T. gondii infection induced the expression of host protein Cbl-b, and then Cbl-b ubiquitinated MyD88 for degrada tion through lysosomal pathway.As a result, the secretion of anti-infection cytokines including IFN-γ and IL-6 was significantly inhibited; T. gondii, therefore, can survive and multiply successfully in the host cells.

Parasites and cell lines
The tachyzoites of wild-type T. gondii ME49, RH, VEG, and CEP-GFP fluorescent strain were maintained in our laboratory.The HFF, THP-1, COS7, HEK293T, RAW264.7, and HeLa cell lines were purchased from the American Type Culture Collection (USA).The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM medium) (Gibco, New York, NY) containing 10% fetal bovine serum (FBS) (Gibco, New York, NY) in 5% CO 2 at 37°C.T. gondii tachyzoites were cultured in HFF cells in DMEM containing 1% FBS.When most of host cells were ready to be ruptured, the cells were scraped and passed through a syringe several times.The tachyzoites were then purified with a 3-µm filter (Whatman, Kent, UK) (35).

CRISPR/Cas9-mediated Cbl-b knockdown in human cells
The CRISPR-Cas9 system was used to knock down Cbl-b expression in HFF and THP-1 cells.Human Cbl-b cDNA sequence was obtained from GeneBank, and two sgRNA sequences of Cbl-b -sgRNA1 and Cbl-b -sgRNA2 (shown in Table 1) were designed online (http://www.e-crisp.org/E-CRISP/designcrispr.html).A 20-bp scrambled sequence named "SC" (shown in Table 1) was used as a negative control.
These sequences were ligated into lentiCRISPRv2 by site-directed mutation to create lentiCRISPRv2-Cbl-b-1, lentiCRISPRv2-Cbl-b-2, and lentiCRISPRv2-SC plasmids, respec tively.Then, they were transfected into HEK293T cells with lentiviral packaging vectors pCMV-dR8.2dvpr and pCMV-VSV-G with lipofectamine 2000, respectively.Viruses were collected from the supernatant of the cell culture at 72 h post transfection by passing through a 0.45 filter.HFF and THP-1 cells were infected with the packaged viruses for 72 h in the presence of 10 µg/mL polybrene (Santa Cruz).The cells with stable Cbl-b -sgRNA1, Cbl-b -sgRNA2, or SC expression were selected and maintained with 4 µg/mL puromycin in the further experiments.The expression of Cbl-b in stable cell line was analyzed by WB.

Name of sgRNA Sequence
Cbl and lysed.The cell lysate was incubated with anti-HA antibody for 2 h at 4°C.Then, the mix was incubated with protein A-agarose (Santa Cruz Biotechnology, USA) overnight at 4°C with gentle rotation.The agarose beads were collected by centrifugation at 500 × g for 5 min.The precipitation samples were analyzed by WB.

Detection of ubiquitinated MyD88
HEK293T cells grown on the T75 flask were transfected with 2-µg pcDNA3.1-Cbl-balone or co-transfected with 2-µg pcDNA3.1-MyD88,and 2 µg pcDNA3.1(+)-Ub-HAwas transfected into all the groups.Then, the cells were treated with 10 µM MG132 for 12 h.After that, the cells were washed with phosphate buffered saline (PBS) for three times and lysed.The cell lysate was collected and incubated with anti-MyD88 antibody for immunoprecipitation.The samples were subjected to WB with FK2 antibody.

Generation of Cbl-b KO mice
The Cbl-b KO C57BL/6 mice were constructed by CRISPR/Cas9-mediated genome engineering.The Cblb-201 transcript (ENSMUST00000114471.2) was taken for an example to describe the strategy.Cbl-b gene has 18 exons, with the ATG start codon in exon 2 and TAG stop codon in exon 18.The sgRNAs mediated Cas9 endonuclease cleavage in intron 4-5 and intron 10-11 of Cbl-b gene and created a double-strand break.Such a break will be repaired by non-homologous end joining, and result in the disruption of Cbl-b.The pups will be genotyped by PCR, followed by WB.

Preparation of splenocytes
Mice were intraperitoneally injected with 1,000 T. gondii ME49 tachyzoites and sacrificed at 7 dpi.Spleens were mechanically removed, homogenized, and processed through a 200-µm cell strainer (BD Falcon).Erythrocytes were lysed with RBC lysis buffer (Python bio); the cells were washed twice and resuspended in the RPMI-1640 medium containing 10% FBS.

Detection of cytokines in mouse serum with FCM
For detection of cytokines in mouse serum, the samples were assayed using the BD Cytometric Bead Assay (BD Biosciences) Mouse Flex kit according to the manufactur er's instructions.Serum samples were mixed with the beads to capture the cytokines including IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, IL-1β, IFN-γ, and TNF-α, and then, the corresponding antibodies conjugated with phycoerythrin were added.The mix was incubated for 2 h under room temperature in the dark.The beads were collected with centrifugation and washed with PBS for two times and detected by FCM.The mean fluorescence intensity for all cytokines was analyzed.

Statistics
With equal variance and normal distributions, the differences between groups were analyzed with Prism using a two-tailed Student's t-test or one-way analysis of variance with SPSS software package.Mann-Whitney U test was used with unequal variance or abnormal distributions.The statistical significance was defined as P < 0.05.

FIG 1
FIG 1 The proliferation of T. gondii was significantly inhibited in Cbl-b knockdown cells.(A) Western blot detection of Cbl-b in the cell lysates of the HFF cells with different treatments as indicated (normal cells and the cells transfected with SC, Cbl-b-sgRNA1, or Cbl-b-sgRNA2).(B) The percentages of the parasitophorous vacuoles containing 1, 2, and 4 tachyzoites in the WT and Cbl-b knockdown (KD) HFF cells infected with CEP-GFP T. gondii strain were counted at 20 h post infection.(C and D) Normal THP-1 cells and two clones of THP-1 with Cbl-b KD were harvested; Cbl-b in the cell lysates were detected with WB (C); Quantitative PCR was used to detect B1 gene copies in these THP-1 cells infected with T. gondii ME49 strain (D).(E) The transcription levels of IL-12, IFN-γ, IL-6, and IL-1β were detected with qRT-PCR in the normal and Cbl-b KD THP-1 cells infected with ME49 for 24 h, then normalized to that of GAPDH, and compared.The experiments were performed at least three times.The differences between groups were analyzed with Prism using a two-tailed Student's t-test or Mann-Whitney U test.Error bars, SEM.*P < 0.05.IB, immunoblotting.

FIG 3
FIG 3 Identification of Cbl-b-MyD88 interaction.(A and B) The interaction of Cbl-b and MyD88 was identified with FRET.COS7 cells were transfected with pEYFP-CFP for positive control, co-transfected with pEYFPC1-MyD88 and pECFPN1-Cbl-b for the experimental group, and co-transfected with pEYFP and pECFP for the negative control.The FRET efficiency was analyzed by acceptor photo-bleach technique.Before photo-bleach, the donor CFP fluorescence (F408) and acceptor YFP fluorescence (F514) were tracked upon donor excitation.After photo-bleach, acceptor YFP in situ (the red line circled areas) was eliminated without affecting CFP.The extent of dequenching of CFP fluorescence (F408) upon elimination of the energy transfer to YFP is proportional to the FRET efficiency.Ten fields were evaluated.The experiment was performed in triplicate, and the experiments were repeated three times.(A) One representative field for each group.(B) The FRET efficiencies were compared among the positive control group, the experimental group, and the negative control group.The differences between groups were analyzed with Mann-Whitney U test.Error bars, SEM.**P < 0.01.pcDNA3.1(+)-Cbl-b-3×FLAGand pcDNA3.1(+)-MyD88-HAplasmids were transfected separately or together to COS7 cells on the T75 culture flasks as indicated.The cell lysates were subjected to Co-IP assay, and the anti-HA antibody was used as the bait to capture the proteins interacting with MyD88.β-Actin was detected as the loading control.The experiment was performed at least three times.WCL, whole cell lysate.

FIG 5 8 FIG 6
FIG 5 The construction of Cbl-b knockout mice.(A) The schematic chart for constructing Cbl-b KO mice.Cbl-b gene has 18 exons, with the ATG start codon in exon 2 and TAG stop codon in exon 18.Two sgRNAs direct Cas9 endonuclease cleavages in intron 4-5 and intron 10-11 of Cbl-b gene and create a double-strand break.Such a break is repaired by non-homologous end joining and results in the disruption of Cbl-b.(B) PCR was used to identify the KO of Cbl-b with genome DNA as a template.(C) WB was used to identify the KO efficiency of Cbl-b.

FIG 6 (
FIG 6 (Continued)IL-2, IL-5, IL-17, and IL-4 in the serum of the mice were detected with flow cytometry.Six mice were prepared for each group, and the experiments were repeated at least three times.The differences between groups were analyzed with Prism using a two-tailed Student's t-test or one-way analysis of variance with SPSS software package.Mann-Whitney U test was used with unequal variance or abnormal distributions.Error bars, SEM.*P < 0.05.**P < 0.01.PECs, peritoneal cells.

FIG 7
FIG 7 Model for how Cbl-b negatively regulated the anti-T.gondii innate immunity through TLR/ MyD88 signaling.1, T. gondii infection induced the expression of host protein Cbl-b.2, Furthermore, Cbl-b ubiquitinated MyD88 for degradation, which resulted in the inhibition of anti-infection cytokines (including IFN-γ and IL-6) secretion.By this way, T. gondii can survive and multiply successfully in the host cells.3 and 4, However, when host cell Cbl-b was KO by CRISPR-Cas9 technology, T. gondii infection activated TLR/MYD88 signaling pathway to promote the secretion of IL-12, IL-6, IL-8, IL-10, IFN-γ, TNF-α, and so on.As a result, host cell anti-T.gondii responses are promoted, and tachyzoites can be eliminated.

TABLE 1
Sequences for the sgRNA