Comparative Proteomic Analysis of Secretory Proteins of Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides Investigates Virulence and Discovers Important Diagnostic Biomarkers

Simple Summary Mycoplasma bovis (M. bovis) and Mycoplasma mycoides subsp. mycoides (Mmm) are the most important pathogenic mycoplasma species. But, the limited knowledge of the secreted proteins of Mmm and M. bovis has obstructed our understanding of mycoplasmal pathogenicity. In order to remove the interference of serum proteins, we used a serum-free medium to culture mycoplasma. The secreted proteins were extracted and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Further comparative proteomic analysis was performed to reveal homologous and unique secreted proteins and discover differential diagnostic biomarkers between M. bovis and Mmm. The findings are significant to further investigate the virulence or immunological properties of related secreted proteins for elucidating the pathogenesis and immune response of both mycoplasmas. Abstract The most important pathogenic Mycoplasma species in bovines are Mycoplasma bovis (M. bovis) and Mycoplasma mycoides subsp. mycoides (Mmm). Mmm causes contagious bovine pleuropneumonia (CBPP), which is a severe respiratory disease widespread in sub-Saharan Africa but eradicated in several countries, including China. M. bovis is an important cause of the bovine respiratory disease complex (BRD), characterized worldwide by pneumonia, arthritis, and mastitis. Secreted proteins of bacteria are generally considered virulence factors because they can act as toxins, adhesins, and virulent enzymes in infection. Therefore, this study performed a comparative proteomic analysis of the secreted proteins of M. bovis and Mmm in order to find some virulence-related factors as well as discover differential diagnostic biomarkers for these bovine mycoplasmas. The secretome was extracted from both species, and liquid chromatography-tandem mass spectrometry was used, which revealed 55 unique secreted proteins of M. bovis, 44 unique secreted proteins of Mmm, and 4 homologous proteins. In the M. bovis secretome, 19 proteins were predicted to be virulence factors, while 4 putative virulence factors were identified in the Mmm secretome. In addition, five unique secreted proteins of Mmm were expressed and purified, and their antigenicity was confirmed by Western blotting assay and indirect ELISA. Among them, Ts1133 and Ts0085 were verified as potential candidates for distinguishing Mmm infection from M. bovis infection.


Introduction
Mycoplasmas are minimal and wall-less bacteria that include many pathogenic species that cause respiratory diseases, arthritis, and urogenital tract diseases in humans and animals [1].Mycoplasma bovis (M.bovis) and Mycoplasma mycoides subsp.mycoides (Mmm) are two pathogenic species in bovines.Mmm is a well-known pathogen causing contagious bovine pleuropneumonia (CBPP), a notifiable disease listed in the World Organization for Animal Health (WOAH) characterized by pneumonia, pleurisy, and a high mortality rate of up to 50% without treatment [2].Mmm has been eradicated in many parts of the world, including Europe (Portugal, Spain, France, and Italy) [3], Australia [4], and China [5], however, it is still the most serious animal disease endemic in vast areas of sub-Saharan Africa [6].On the other hand, M. bovis is a common cause of subacute and chronic pneumonia in feedlot cattle and dairy calves throughout the world [7].Due to the poor outcome of antibiotic treatment and the unsatisfactory efficacy of vaccines against CBPP and the lack of commercial vaccines available for M. bovis, both pathogens have gained significant economic importance for the worldwide cattle industry.The main reason for this status is attributed to a poor understanding of the pathogenesis and immune responses of both mycoplasma species [8].
Secreted proteins of bacteria are generally considered virulence factors because they usually function as toxins, adhesins, and virulence-determining enzymes, thereby mediating bacterial adhesion, invasion, propagation, and suppressing host defense [9].Accordingly, several secreted proteins of mycoplasmas have been identified as virulence factors, including P102 of M. hyopneumoniae [10], Mpn491 and the community-acquired respiratory distress syndrome (CARDS) toxin of M. pneumoniae [11,12], and MBOV_RS02825 and MbovP280 of M. bovis [13,14].Also, identifying novel immunogenic proteins for improved diagnostics and vaccines for Mmm and M. bovis has been confirmed [15,16].Further, the secretome and extracellular vesicles of several mycoplasma species have been identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), isobaric tags for relative and absolute quantitation (iTRACK), and label-free proteomic analyses [17][18][19][20].In addition, comparative proteomics has been used to analyze the whole-cell proteomes of two Mycoplasma hyopneumoniae strains [21], the whole-cell proteomes of the virulent M. hyopneumoniae strain and its attenuated strain [22], and the secretomes of Mycoplasma hyopneumoniae and Mycoplasma flocculare [19].However, since mycoplasmas are fastidious species that require their media to contain sera, which could change the mycoplasmas protein pattern [23] and contaminate the extracted secreted proteins, the above studies could not exclude the interference of the foreign proteins from the sera of the medium.Therefore, serum-free media would be an ideal solution to avoid contamination of secreted proteomics of mycoplasmas by sera.
In the present study, a semi-defined medium without serum was used to culture M. bovis strain HB0801 and Mmm strain Afadé for the extraction of the soluble secreted proteins in the culture supernatant, and LC-MS/MS was used to identify secreted proteomics.The further comparative analysis was performed to first reveal homologous and unique secreted proteins between M. bovis and Mmm.The findings are significant to further investigate the virulence or immunological properties of related secreted proteins for elucidating the pathogenesis and immune response of both mycoplasmas.

Bacterial Strains and Culture
Mycoplasma mycoides subsp.mycoides strain Afadé (GenBank accession no.GCA _000959065.1)originates from Northern Cameroon and was isolated at the Farcha laboratories in Tchad in 1965 [2].M. bovis strain HB0801 (GenBank accession no.NC_018077.1)was isolated and characterized by the National Key Laboratory of Agricultural Microbiology, HZAU, China [7].Mmm was grown in the semi-defined medium without serum [24], while M. bovis was grown in the same semi-defined medium without serum but supplemented with sodium pyruvate (0.01 mol/L).The semi-defined medium was produced without animal serum or bovine serum albumin, as previously described [24].In order to observe the growth of mycoplasmas, their growth curves were determined with the conventional colony counting method.

Enrichment of Proteins from Culture Supernatant of Mycoplasma
For enrichment of the soluble secreted proteins of M. bovis and Mmm from their culture supernatants, both were cultured in 500 mL semi-defined medium for 48 h at 37 • C. The bacteria were pelleted by centrifugation at 3500 g for 30 min, and culture supernatants were filtered through a 0.22 µm filter membrane.Then, the filtrates were centrifuged at 12,000 g for 30 min, filtered (0.22 µm), and dialyzed against sodium acetate at 5 mM and pH 5.0 using the SnakeSkin ® Dialysis Tubing (Thermo Scientific, IL, USA), with a molecular weight cut-off of 3.5 kDa.The final filtrates were freeze-dried and resolved in 10 mL PBS (pH 7.4).

Identification of Soluble Secreted Proteins by LC-MS/MS
A total of 100 µg of the secretome of M. bovis and Mmm was resolved with 12% SDS-PAGE.The gel samples for the secretomes of M. bovis and Mmm were prepared in parallel.Each sample was digested with 5 µL of 2.5-10 ng/µL trypsin solution (Promega, USA) at 37 • C for 20 h.Then, the solution was transferred into a new tube, and 100 µL of supersaturated alpha-cyano-4-hydroxycinnamic acid matrix solution (the solvent is 50% CAN and 0.1% TFA) were added, and the solvent was freeze-dried.In total, 60 µL of ddH 2 O (containing 0.1% formaldehyde) was used to resuspend the freeze-dried sample, and LC-MS/MS was performed by a previously described method [20].Mascot 2.2 software was used for LC-MS/MS queries from NCBI (NCBI RefSeq assembly: M. bovis HB0801 (GCF_000270525.1),Mmm Afade (GCF_000959065.1)).

In Silico Comparative and Functional Analysis
In silico functional analysis of the M. bovis and Mmm secretomes was performed according to the previous description with minor modifications.Subcellular protein localization was predicted using PSORTb version 3.0.2(https://www.psort.org/psortb/accessed on 1 September 2022).Classically secreted proteins that carry signal sequences and are, therefore, secreted by classical pathways were predicted by the SignalP 5.0 server (http://www.cbs.dtu.dk/services/SignalP/accessed on 1 September 2022).No signal peptide-triggered protein secretion was predicted by the use of the SecretomeP 2.0 server (http://www.cbs.dtu.dk/services/SecretomeP/accessed on 1 September 2022).Prediction of virulence factors for secreted proteins was performed using VFDB (http://www.mgc.ac.cn/VFs/ accessed on 1 September 2022).After obtaining amino sequences from the NCBI, each protein was aligned separately against the VFDB full dataset by the BLAST algorithm.A matrix was created by VFDB output consisting of the BLAST score and E-value for each input protein.The data were screened on the basis of a BLAST score of ≥80.In addition, COG functional annotation for the proteins identified was acquired using the EggNOG database version 5.0 (http://eggnog5.embl.de/#/app/seqscanaccessed on 1 September 2022).Furthermore, MolliGen 3.0 (http://services.cbib.u-bordeaux.fr/molligen/accessed on 1 September 2022) was applied online to analyze the homologous proteins between M. bovis HB0801 and Mmm Afadé.The homologous proteins of Ts0085 and Ts1133 were identified from NCBI, then the multiple sequence alignment was performed by ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript/index.php accessed on 1 September 2022).

Gene Cloning and Expression of Mmm-Secreted Proteins
The sequences of genes were site-direct edited by replacing the TGA codon with TGG to ensure that Mycoplasma tryptophan was correctly translated in Escherichia coli (E.coli).The sequence-encoded Ts0029, Ts0085, Ts0484, Ts0707, and Ts1133 were synthesized by Beijing Tianyi Huiyuan Bioscience & Technology Inc. (Wuhan, China) and ligated into the pET-30a vector (Novagen, Darmstadt, Germany).E. coli strain BL21 (TransGen, Beijing, China) was then transformed with each of the constructed recombinant plasmids individually, and the recombinant proteins were expressed after the E. coli cells were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.8 mM).The proteins were purified with nickel affinity chromatography (GE Healthcare, NJ, USA), as described previously [14].

Characterization of the Antigenicity of Mmm-Secreted Proteins
Then, 1 µg of each purified recombinant protein (Ts1133, Ts0029, Ts0707, Ts0085, and Ts0484) was separated by 12% SDS-PAGE and transferred onto PVDF membranes (Immun-Blot ® , USA).After blocking, recombinant proteins were probed with five serum samples of calves naturally infected with Mmm, kindly provided by Prof. Jiuqing Xin from China National CBPP Reference Laboratory, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences [25], whereas sera from non-infected calves were used as the negative control.Then, the Western blotting assay was developed with HRP-conjugated goat anti-bovine IgG (Southern Biotech Co., USA) for 1 h and finally visualized with WesternBright™ ECL (Advansta, CA, USA) [26].
In addition, the five recombinant proteins were characterized using indirect ELISA.In brief, 96-well microtitre plates were coated overnight at 4 • C with 200 ng of each purified recombinant protein diluted in 100 µL sodium carbonate buffer (pH 9.6) and washed with PBS containing 0.05% Tween 20 (PBST).After blocking, the plates were probed for 1 h at 37 • C with sera collected from M. bovis and Mmm naturally infected calves [25,27].After washing with PBST, the plates were incubated for 1 h at 37 • C with goat anti-bovine IgG-HRP (1:5000) (Southern Biotech Co., USA) and washed with PBST, followed by the addition of tetramethylbenzidine (TMB)/H 2 O 2 (Wuhan Keqian Biological Co., Ltd., China) as a substrate.The reaction was stopped after 5 min, and OD values at 630 nm (OD 630 ) were obtained with a microtiter plate reader (BioTek, USA).

Statistical Analysis
The data were expressed as means ± standard error mean (SEM).Samples are normally distributed.A Student's t-test was used for a single comparison with the GraphPad Prism version 5 software (GraphPad Software, La Jolla, CA, USA).* p < 0.05, ** p < 0.01, *** p < 0.001 indicate statistically significant differences, while ns indicates no difference.

Soluble Secreted Proteins of M. bovis and Mmm
The growth curves showed that both M. bovis and Mmm could grow well in the semi-defined medium (Figure 1A,B).Then, the soluble secreted proteins were extracted from culture supernatants and resolved with SDS-PAGE.The results revealed that similar SDS-PAGE protein profiles were observed for the cultural supernatants of M. bovis, Mmm, or medium (Figure 1C).PAGE protein profiles were observed for the cultural supernatants of M. bovis, Mmm, or medium (Figure 1C).

Identification of Proteins of M. bovis and Mmm in the Culture Supernatant
In order to identify as many secreted proteins as possible, the LC-MS/MS analysis was performed twice, and all identified proteins were selected for analysis.As a result, 48 Mmm and 59 M. bovis proteins were identified from culture supernatants (Tables S1 and  S2).Further, MolliGen 3.0 (http://services.cbib.u-bordeaux.fr/molligen/)was applied to analyze online the homologous proteins between M. bovis HB0801 and Mmm Afadé.Although 454 homologous proteins were identified (Table S3), after comparing with the secreted proteins obtained, only 4 homologous secreted proteins were shared between M. bovis and Mmm, including hypothetical protein (Mbov_0154/TS60_0462), ribosomal protein S12 (Mbov_0678/TS60_0185), heat shock protein (Mbov_0817/TS60_0689), and ATP synthase subunit alpha (Mbov_0440/TS60_0995).Moreover, M. bovis and Mmm have 55 and 44 unique secreted proteins, respectively (Figure 2A).

Identification of Proteins of M. bovis and Mmm in the Culture Supernatant
In order to identify as many secreted proteins as possible, the LC-MS/MS analysis was performed twice, and all identified proteins were selected for analysis.As a result, 48 Mmm and 59 M. bovis proteins were identified from culture supernatants (Tables S1 and S2).Further, MolliGen 3.0 (http://services.cbib.u-bordeaux.fr/molligen/accessed on 1 September 2022) was applied to analyze online the homologous proteins between M. bovis HB0801 and Mmm Afadé.Although 454 homologous proteins were identified (Table S3), after comparing with the secreted proteins obtained, only 4 homologous secreted proteins were shared between M. bovis and Mmm, including hypothetical protein (Mbov_0154/TS60_0462), ribosomal protein S12 (Mbov_0678/TS60_0185), heat shock protein (Mbov_0817/TS60_0689), and ATP synthase subunit alpha (Mbov_0440/TS60_0995).Moreover, M. bovis and Mmm have 55 and 44 unique secreted proteins, respectively (Figure 2A).

Prediction of Secreted Pathways, Locations, and Functional Analysis
Extracellular proteins are a subject of extreme interest because of their pivotal roles in bacterial lifestyles [28].Proteins identified within the sets of M. bovis and Mmm secreted proteins were classified according to predicted subcellular protein localization using PSORTb version 3.0.2(https://www.psort.org/psortb/accessed on 1 September 2022) and secretion pathways.For M. bovis, 25 proteins (42.4%) were described as located in the cytoplasm, 8 proteins (13.6%) as cell membrane proteins, 2 (3.4%) as extracellular, and 24 (40.6%) as unknown proteins (Figure 2D).Regarding secreted pathways, 19 proteins (32.2%) were predicted to be secreted by the classical pathway, while 18 proteins (30.5%) were predicted to be non-classically secreted, and 22 proteins (37.3%) were assigned to an undefined type of secretion (Figure 2C).For Mmm, 24 proteins (50%) were predicted to be localized in the cytoplasm, 2 (4.2%) as extracellular, and 22 (45.8%) as unknown (Figure 2D).In this Mmm secretome, classical secretion was predicted for 13 proteins (27.1%), non-classical secretion was predicted for 13 proteins (27.1%), and 22 proteins (45.8%) were assigned to an undefined secretion type (Figure 2C).These proportions of location distribution for the secreted proteins of both strains are similar, but no cell membrane-localized proteins were identified in Mmm.In addition, the classically secreted proteins and non-classically secreted proteins in M. bovis and Mmm accounted for 62.7% and 54.2%, respectively.

Prediction of Secreted Pathways, Locations, and Functional Analysis
Extracellular proteins are a subject of extreme interest because of their pivotal roles in bacterial lifestyles [28].Proteins identified within the sets of M. bovis and Mmm secreted proteins were classified according to predicted subcellular protein localization using PSORTb version 3.0.2(https://www.psort.org/psortb/accessed on 1 September 2022) and secretion pathways.For M. bovis, 25 proteins (42.4%) were described as located in the cytoplasm, 8 proteins (13.6%) as cell membrane proteins, 2 (3.4%) as extracellular, and 24 (40.6%) as unknown proteins (Figure 2D).Regarding secreted pathways, 19 proteins (32.2%) were predicted to be secreted by the classical pathway, while 18 proteins (30.5%) were predicted to be non-classically secreted, and 22 proteins (37.3%) were assigned to an undefined type of secretion (Figure 2C).For Mmm, 24 proteins (50%) were predicted to be localized in the cytoplasm, 2 (4.2%) as extracellular, and 22 (45.8%) as unknown (Figure 2D).In this Mmm secretome, classical secretion was predicted for 13 proteins (27.1%), nonclassical secretion was predicted for 13 proteins (27.1%), and 22 proteins (45.8%) were assigned to an undefined secretion type (Figure 2C).These proportions of location distribution for the secreted proteins of both strains are similar, but no cell membrane-localized proteins were identified in Mmm.In addition, the classically secreted proteins and nonclassically secreted proteins in M. bovis and Mmm accounted for 62.7% and 54.2%, respectively.
The secreted proteins of M. bovis and Mmm were further categorized according to COG, and the functional prediction was summarized in Figure 2B for both M. bovis and Mmm.The identified secreted proteins were mostly related to the "function unknown" (S; 22% and 29%, respectively).In addition, the most occupied categories were cell wall/mem- The secreted proteins of M. bovis and Mmm were further categorized according to COG, and the functional prediction was summarized in Figure 2B for both M. bovis and Mmm.The identified secreted proteins were mostly related to the "function unknown" (S; 22% and 29%, respectively).In addition, the most occupied categories were cell wall/membrane/envelope biogenesis (M; 13.6% and 8.3%, respectively), replication, recombination, and repair (L; 11.9% and 8.3%, respectively), and translation, ribosomal structure, and biogenesis (J; 8.5% and 16.7%, respectively).Metabolism-related categories were also well represented for both species, comprising 25.4% of M. bovis identified proteins (divided in G, F, E, C, and P) and 14.6% of Mmm identified proteins (divided in F, E, C, P, and H).Three functional categories related to carbohydrate transport and metabolism (five proteins), defense mechanisms (three proteins), and intracellular trafficking, secretion, and vesicular transport (one protein) were found only in M. bovis, while the coenzyme transport and metabolism (two proteins) category was found only in Mmm.

The Evaluation of Differential Diagnostic Biomarkers
In order to discover the potential secreted biomarkers for distinguishing Mmm infection from M. bovis infection, we compared 48 secreted proteins of Mmm in our study with previously reported proteins of EV-membranes from Mmm [17].The results indicated eight common proteins, and six of them were predicted as secreted proteins by bioinformatic tools (Table 3).We further expressed and purified five secreted proteins from the eight common proteins successfully (Figure 3A).Then, the five proteins were resolved with SDS-PAGE, transferred onto membranes, and incubated with antisera from five calves infected with Mmm.The results indicated Ts1133 and Ts0085 displayed signals in response to five antisera, respectively, but Ts0707, Ts0484, and Ts0029 did not (Figure 3B-G).iELISA was also used to demonstrate the antigenicity of recombinant proteins.In contrast to the Western blotting assay, iELISA revealed all proteins generated antibody responses with antiserum from calves infected with Mmm (n = 5), but Ts0085 and Ts1133 showed a stronger reaction than other proteins.In addition, the proteins in iELISA did not react with the serum infected with M. bovis (n = 10) or serum from the uninfected animals (n = 11).The ratio of OD values of Mmm-positive serum to -negative serum OD and M. bovis-positive serum was calculated and evaluated for the potential proteins as diagnosis targets (Figure 4).Furthermore, multiple sequence alignments revealed Ts0085 and Ts1133 were highly conserved proteins among 18 strains of Mmm isolated worldwide (Figures S1 and S2).

M. bovis and Mmm Secretomes Are Greatly Affected by Essential Medium Components
The secretome profiles extracted from mycoplasmal culture supernatant remain extremely challenging due to the relative underrepresentation of secreted proteins in comparison to the complex background of overrepresented serum proteins [30].Several studies attempted to identify secreted proteins in the secretomes and extracellular vesicles of several Mycoplasma species by proteomic approaches, such as two-dimensional electrophoresis, liquid chromatography-tandem mass spectrometry, and label-free proteomic analyses [17][18][19][20].Our study used a semi-defined medium without serum to remove interference and facilitate the identification of secreted proteins.This approach first made the mycoplasmas adapt to the new medium shown by the normal growth curves, which,

M. bovis and Mmm Secretomes Are Greatly Affected by Essential Medium Components
The secretome profiles extracted from mycoplasmal culture supernatant remain extremely challenging due to the relative underrepresentation of secreted proteins in comparison to the complex background of overrepresented serum proteins [30].Several studies attempted to identify secreted proteins in the secretomes and extracellular vesicles of several Mycoplasma species by proteomic approaches, such as two-dimensional electrophoresis, liq-uid chromatography-tandem mass spectrometry, and label-free proteomic analyses [17][18][19][20].Our study used a semi-defined medium without serum to remove interference and facilitate the identification of secreted proteins.This approach first made the mycoplasmas adapt to the new medium shown by the normal growth curves, which, therefore, decreased the nutritional and environmental stress on the mycoplasmas to the maximum and obtained secretomes closer to those generated by mycoplasmas under the natural state compared to the previous studies [31].However, compared to the previous findings, much less secreted proteins were obtained in this study (59 for M. bovis, 48 for Mmm), demonstrating that the secretomes greatly varied with the inclusion of medium serum, probably through disturbance of cell metabolism, protein expression, and secretion [32].Among our secreted proteins, 25 of the M. bovis secreted proteins were previously identified, and 24 were predicted to be secreted by the classical pathway or non-classical pathway, indicating the current results are reliable (Table 4).Meanwhile, we also identified 48 secreted proteins of Mmm, and 8 of them were identified as proteins of EV-membranes, indicating that some secreted proteins of Mmm may be secreted as EV proteins.Putative lipoprotein Classical a : the overlapped secreted proteins of M. bovis between our data and published secretomes.
On the other hand, although we successfully excluded serum interruption of the mycoplasma secretomes, the possible contamination with intracellular proteins in the supernatant could not be completely excluded.Therefore, these secretomes should be further verified with the mycoplasmas or their extracted secretomes by some other methods, such as Western blotting assays, ELISA, and in situ localization [14].

Identification of Virulence-Associated Secreted Proteins
In order to identify the virulence-associated secreted proteins, all identified proteins in the secretome were analyzed using VFDB [33,34].As a result, there was a big difference in the number of unique virulence-associated secreted proteins: 19 for M. bovis and 4 for Mmm.
The reason remains to be investigated.Among these proteins, the MbovP016 encoded by the Mbov_0016 gene has already been identified as a virulence-related factor that was able to induce apoptosis of host cells through the endoplasmic reticulum stress-dependent signaling pathway [35]; the peptide of VSP could suppress the concanavalin A-induced proliferation of bovine lymphocytes [36].The EF-Tu encoded by the TS60_0188 gene was previously identified as an adhesin in M. hyopneumoniae [37].In addition, MbovP274 was confirmed to be a secreted protein and could significantly increase the production of IL-8, IL-12, and IFN-γ [38].Considering the limited knowledge on secreted proteins of Mmm and M. bovis, our findings provided significant reference for further identifying potential virulence-associated factors to elucidate mycoplasmal pathogenicity.

Potential Diagnostic Targets for Differentiation of Mmm from M. bovis
Although China and some other countries have already eradicated CBPP, its reemergence would be possible because the exchange activities across countries free of CBPP and African countries with epidemics of CBPP are extensively performed under the conditions of economic globalization and international trade.Therefore, the development of a rapid and accurate method to rapidly differentiate Mmm from M. bovis infection is of the utmost importance.From the expressed five Mmm unique proteins predicted as secreted, we showed that TS0085 and Ts1133 can react solely with Mmm-positive cattle sera rather than M. bovis-positive cattle sera and negative cattle sera.Previous studies revealed MSC519 (Ts0484) and MSC636 were the best targets to identify infected animals [39][40][41].But, our results indicated Ts0085 and Ts1133 had higher sensitivity to diagnose Mmm infection than Ts0484.In addition, both Ts0085 and Ts1133 were highly conserved proteins between different known strains.Although we need to test more positive sera, especially Mmm-positive sera, to confirm the current findings, Ts0085 and Ts1133 already show a promising application prospect as potential biomarkers for differential diagnosis of Mmm and M. bovis infection, with greater promise than other conventional diagnostic methods such as PCR.

Conclusions
This study identified 59 secreted proteins of M. bovis and 48 secreted proteins of Mmm grown in serum-free medium.Among them, the unique Mmm-secreted proteins, Ts0085 and Ts1133, might serve as biomarker candidates for diagnosis of Mmm infection and differentiation of Mmm from M. bovis infection.

Figure 1 .
Figure 1.The growth of M. bovis and Mmm in the semi-defined medium without serum and their secreted proteins (three replicates each).(A) The growth curve of M. bovis in the semi-defined medium supplemented with sodium pyruvate (0.01 mol/L).(B) The growth curve of Mmm in the semidefined medium.(C) The resolution of soluble secreted proteins of M. bovis or Mmm extracted from the culture supernatants with SDS-PAGE.The proteins extracted from the medium served as a control.The original gel figure can be viewed in Figure S3.

Figure 1 .
Figure 1.The growth of M. bovis and Mmm in the semi-defined medium without serum and their secreted proteins (three replicates each).(A) The growth curve of M. bovis in the semi-defined medium supplemented with sodium pyruvate (0.01 mol/L).(B) The growth curve of Mmm in the semi-defined medium.(C) The resolution of soluble secreted proteins of M. bovis or Mmm extracted from the culture supernatants with SDS-PAGE.The proteins extracted from the medium served as a control.The original gel figure can be viewed in Figure S3.

14 Figure 2 .
Figure 2. In silico functional prediction for M. bovis and Mmm secreted proteins.(A) The Venn diagram was created from the secreted proteins of M. bovis and Mmm.(B) The COG functional distribution of secreted proteins of M. bovis and Mmm.(C) The type of protein secretion was predicted by SignalP 5.0 and SecretomeP 2.0 servers.(D) The subcellular localization of secreted proteins was predicted by PSORTb version 3.0.2.

Figure 2 .
Figure 2. In silico functional prediction for M. bovis and Mmm secreted proteins.(A) The Venn diagram was created from the secreted proteins of M. bovis and Mmm.(B) The COG functional distribution of secreted proteins of M. bovis and Mmm.(C) The type of protein secretion was predicted by SignalP 5.0 and SecretomeP 2.0 servers.(D) The subcellular localization of secreted proteins was predicted by PSORTb version 3.0.2.

Figure 4 .
Figure 4.The antigenicity of 5 unique secreted proteins of Mmm was detected by indirect ELISA.Ts0484 (A), Ts0707 (B), Ts1133 (C), Ts0029 (D), or Ts0085 (E) were used to generate antibody responses with antiserum.In total, 200 ng of each protein was coated in each well overnight and incubated with antiserums of Mmm infection (n = 5), and M. bovis infection (n = 10), and negative serum (n = 11) at a dilution of 1:100 for 1 h at 37 °C.The OD630nm value represented antibody responses.The ratio of Mmm-positive serum OD to negative serum OD or M. bovis-positive serum OD was calculated to evaluate the ideal protein for diagnosis.** p < 0.01, *** p < 0.001 indicate statistically significant differences.

Figure 4 .
Figure 4.The antigenicity of 5 unique secreted proteins of Mmm was detected by indirect ELISA.Ts0484 (A), Ts0707 (B), Ts1133 (C), Ts0029 (D), or Ts0085 (E) were used to generate antibody responses with antiserum.In total, 200 ng of each protein was coated in each well overnight and incubated with antiserums of Mmm infection (n = 5), and M. bovis infection (n = 10), and negative serum (n = 11) at a dilution of 1:100 for 1 h at 37 • C. The OD 630 nm value represented antibody responses.The ratio of Mmm-positive serum OD to negative serum OD or M. bovis-positive serum OD was calculated to evaluate the ideal protein for diagnosis.** p < 0.01, *** p < 0.001 indicate statistically significant differences.

Table 1 .
Virulence-related factor identification in M. bovis using the VFDB.

Table 2 .
Virulence-related factor identification in Mmm using the VFDB.

Table 3 .
[17]lapped secreted proteins of Mmm between our data and published data[17].: the overlapped secreted proteins of Mmm between our data and secreted proteins in the EV. a