AnimalMetaOmics: a multi-omics data resources for exploring animal microbial genomes and microbiomes.

The Animal Meta-omics landscape database (AnimalMetaOmics, https://yanglab.hzau.edu.cn/animalmetaomics#/) is a comprehensive and freely available resource that includes metagenomic, metatranscriptomic, and metaproteomic data from various non-human animal species and provides abundant information on animal microbiomes, including cluster analysis of microbial cognate genes, functional gene annotations, active microbiota composition, gene expression abundance, and microbial protein identification. In this work, 55 898 microbial genomes were annotated from 581 animal species, including 42 924 bacterial genomes, 12 336 virus genomes, 496 archaea genomes and 142 fungi genomes. Moreover, 321 metatranscriptomic datasets were analyzed from 31 animal species and 326 metaproteomic datasets from four animal species, as well as the pan-genomic dynamics and compositional characteristics of 679 bacterial species and 13 archaea species from animal hosts. Researchers can efficiently access and acquire the information of cross-host microbiota through a user-friendly interface, such as species, genomes, activity levels, expressed protein sequences and functions, and pan-genome composition. These valuable resources provide an important reference for better exploring the classification, functional diversity, biological process diversity and functional genes of animal microbiota.

Anaplasma phagocytophilum Ats-1 enhances exosome secretion through Syntenin-1.

Anaplasma phagocytophilum is an intracellular obligate parasite that causes granulocytic anaplasmosis. Effector Ats-1 is an important virulence factor of A. phagocytophilum. Multiomics screening and validation has been used to determine that Ats-1 regulates host cell apoptosis and energy metabolism through the respiratory chain mPTP axis. In this study, a total of 19 potential binding proteins of Ats-1 in host cells were preliminarily screened using a yeast two-hybrid assay, and the interaction between syntenin-1 (SDCBP) and Ats-1 was identified through immunoprecipitation. Bioinformatics analysis showed that SDCBP interacted with SDC1, SDC2, and SDC4 and participated in the host exosome secretion pathway. Further studies confirmed that Ats-1 induced the expression of SDC1, SDC2, and SDC4 in HEK293T cells through SDCBP and increased the exosome secretion of these cells. This indicated that SDCBP played an important role in Ats-1 regulating the exosome secretion of the host cells. These findings expand our understanding of the intracellular regulatory mechanism of A. phagocytophilum, which may enhance its own infection and proliferation by regulating host exosome pathways.

A Novel Calibration Method of Line Structured Light Plane Using Spatial Geometry.

The line structured light plane calibration method using a plane target cannot produce satisfactory calibration results due to inaccurate positioning of the calibrated points. Field of view noise and sensor noise affect the target light stripe extraction and camera parameter calculation during the calibration process. These factors will cause the calculation of the coordinates of the calibrated point to deviate, and thus affect the light plane calibration. To solve this problem, we propose a new method to calculate the calibrated point based on spatial geometry. Firstly, for the projection line corresponding to the feature point on the light stripe and the corresponding line on the target, a common perpendicular of these two lines above is established, and since the sum of the squares of the distances from the midpoint to the two straight lines is the smallest, the midpoint of the common perpendicular is taken as the calibrated point. Secondly, the target is moved to different positions, and the non-collinear calibrated points are calculated. Finally, the parameters of the light plane are obtained by fitting these calibrated points. This method requires only a checkerboard target, and has a simple calibration process. The experimental results show that the average error of the calibration method proposed in this paper is 0.011 mm, which is less than the 0.031 mm of the calibration method based on the plane target with cross-ratio invariant.

Scanning iron response regulator binding sites using Dap-seq in the Brucella genome.

Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Previous studies suggested that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, in the present study, a Dap-seq approach was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes was identified using qRT-PCR. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), ftrA (BME_RS14525), cation-transporting P-type ATPase (zntA) (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. Furthermore, the iron utilization and growth assay experiments confirmed that rirA was involve in iron metabolism and growth of Brucella. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism, and the rirA was identified as a regulon of Irr and it also plays a role in iron metabolism of Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism.

Exosomes released by Brucella-infected macrophages inhibit the intracellular survival of Brucella by promoting the polarization of M1 macrophages.

Exosomes, membrane vesicles released extracellularly from cells, contain nucleic acids, proteins, lipids and other components, allowing the transfer of material information between cells. Recent studies reported the role of exosomes in pathogenic microbial infection and host immune mechanisms. Brucella-invasive bodies can survive in host cells for a long time and cause chronic infection, which causes tissue damage. Whether exosomes are involved in host anti-Brucella congenital immune responses has not been reported. Here, we extracted and identified exosomes secreted by Brucella melitensis M5 (Exo-M5)-infected macrophages, and performed in vivo and in vitro studies to examine the effects of exosomes carrying antigen on the polarization of macrophages and immune activation. Exo-M5 promoted the polarization of M1 macrophages, which induced the significant secretion of M1 cytokines (tumour necrosis factor-α and interferon-γ) through NF-κB signalling pathways and inhibited the secretion of M2 cytokines (IL-10), thereby inhibiting the intracellular survival of Brucella. Exo-M5 activated innate immunity and promoted the release of IgG2a antibodies that protected mice from Brucella infection and reduced the parasitaemia of Brucella in the spleen. Furthermore, Exo-M5 contained Brucella antigen components, including Omp31 and OmpA. These results demonstrated that exosomes have an important role in immune responses against Brucella, which might help elucidate the mechanisms of host immunity against Brucella infection and aid the search for Brucella biomarkers and the development of new vaccine candidates.

Multiomics analyses reveals Anaplasma phagocytophilum Ats-1 induces anti-apoptosis and energy metabolism by upregulating the respiratory chain-mPTP axis in eukaryotic mitochondria.

BACKGROUND: Anaplasma translocated substrate 1 (Ats-1) is an effector of type 4 secretory systems (T4SS) and the main virulence factor of Anaplasma phagocytophilum. Ats-1 is involved in the regulation of host cell biological processes, but the specific molecular mechanism of its action is unclear. RESULTS: In this study, we identified Ats-1 as involved in mitochondrial respiratory regulation of HEK293T cells by multi-omics analysis. After intracellular expression of Ats-1, adenosine triphosphate levels and the proliferation of HEK293T cells were both up-regulated, while HEK293T cells apoptosis was inhibited. Ats-1 targeted translocation to the mitochondria where it up-regulated the expression of NDUFB5, NDUFB3, NDUFS7, COX6C, and SLC25A5, thereby enhancing energy production and inhibiting HEK293T cells apoptosis while enhancing HEK293T cells proliferation, and ultimately facilitating Anaplasma phagocytophilum replication in HEK293T cells. CONCLUSIONS: This study demonstrated that Anaplasma phagocytophilum Ats-1 induces anti-apoptosis and energy metabolism by upregulating the respiratory chain-mPTP axis in eukaryotic mitochondria. These results provide a better understanding of the pathogenic mechanism of Anaplasma phagocytophilum within host cells.

Using a Relative Quantitative Proteomic Method to Identify Differentially Abundant Proteins in Brucella melitensis Biovar 3 and Brucella melitensis M5-90.

Brucellosis, caused by Brucella spp., is one of the most widespread bacterial zoonoses worldwide. Vaccination is still considered the best way to control brucellosis. An investigation into the differential proteome expression patterns of wild and vaccine strains may help researchers and clinicians differentiate between the strains to diagnose and better understand the mechanism(s) underlying differences in virulence. In the present study, a mass spectrometry-based, label-free relative quantitative proteomics approach was used to investigate the proteins expressed by the wild strain, B. melitensis biovar 3 and compare it with those expressed by B. melitensis M5-90. The higher level of virulence for B. melitensis biovar 3 compared to B. melitensis M5-90 was validated in vitro and in vivo. A total of 2133 proteins, encompassing 68% of the theoretical proteome, were identified and quantified by proteomic analysis, resulting in broad coverage of the B. melitensis proteome. A total of 147 proteins were identified as differentially expressed (DE) between these two strains. In addition, 9 proteins and 30 proteins were identified as unique to B. melitensis M5-90 and B. melitensis biovar 3, respectively. Pathway analysis revealed that the majority of the DE proteins were involved in iron uptake, quorum sensing, pyrimidine metabolism, glycine betaine biosynthetic and metabolic processes, thiamine-containing compound metabolism and ABC transporters. The expression of BtpA and VjbR proteins (two well-known virulence factors) in B. melitensis biovar 3 was 8-fold and 2-fold higher than in B. melitensis M5-90. In summary, our results identified many unique proteins that could be selected as candidate markers for differentiating vaccinated animals from animals with wild-type infections. BtpA and VjbR proteins might be responsible for the residual virulence of B. melitensis M5-90, while ABC transporters and thiamine metabolism associated proteins may be newly identified Brucella virulence factors. All of the identified DE proteins provide valuable information for the development of vaccines and the discovery of novel therapeutic targets.

Crosstalk of Brucella abortus nucleomodulin BspG and host DNA replication process/mitochondrial respiratory pathway promote anti-apoptosis and infection.

The secretory proteins of Brucella mediate the expression of the bacterium in the host, thereby facilitating intracellular parasitism. With the exception of the recently reported BspJ, the Brucella nucleomodulin has not yet been characterized. We defined the Brucella nucleomodulin BspG and verified six proteins (PCBP1, KMT5C, NDUFS6, PCNA, CIAO2B, and SDHB) that interacted with BspG using a yeast two-hybrid assay and co-immunoprecipitation (CO-IP) screening. The deletion of BspG decreased the intracellular proliferation of B. abortus in both in vivo and in vitro experiments. The analysis found that these interacting proteins were related to energy generation, gene expression, and apoptosis of host cells. The crosstalk between B. abortus nucleomodulin BspG and host DNA replication/mitochondrial respiratory pathways promotes anti-apoptosis and infection, but the mechanism needs additional study.

Brucella induces M1 to M2 polarization of macrophages through STAT6 signaling pathway to promote bacterial intracellular survival.

Brucella are serious intracellular pathogens that parasitize macrophages and cause persistent infection in humans and animals. Although macrophages are an important bridge between natural and acquired immunity, their role in Brucella infection is not completely clear. Recently, studies have reported that Brucella can induce macrophage polarization, although the specific molecular mechanism involved is not known. Therefore, in the current study the replication ability of Brucella melitensis strain M5 (Brucella M5) was examined as well as its macrophage polarization and cytokine production, in a host. The role of Signal transducers and activators of transcription 6 (STAT6) in macrophage polarization induced by Brucella infection was also investigated. The results showed that Brucella M5 survived in vivo for a prolonged period of time and caused damage to the spleen and uterus tissues. The expression of type M2 cytokines was induced after Brucella M5 infection. Immunohistochemistry showed that STAT6 was upregulated in spleen and uterus tissues. At the cellular level, Brucella M5 induced macrophagetransformation from M1 to M2-type during the later stage of infection. When STAT6 was silenced, the polarization of M2-type was inhibited, and the intracellular survival rate of Brucella decreased significantly. In conclusion, these findings demonstrate that STAT6 is the key factor regulates M2 polarization of macrophages and promotes the intracellular survival of Brucella in the late stage of infection and provides an explanation of the mechanism responsible for persistent Brucella infection.

A SARS-CoV-2 nanobody that can bind to the RBD region may be used for treatment in COVID-19 in animals.

Coronavirus disease 2019 (COVID-19) caused by an infectious virus, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), poses a threat to the world. The suitable treatments must be identified for this disease in animals. Nanobody have therapeutic potential in the COVID-19. In this study, SARS-CoV-2 Spike RBD protein was used to make the nanobody. Nanobodies binding to the SARS-CoV-2 Spike RBD protein was obtained. Interestingly, the nanobody could bind to SARS-CoV-2 Spike S protein and RBD protein at the same time. Nanobodies were validated with a neutralizing antibody detection kit. The use of pseudoviruses confirmed that nanobodies could prevent pseudoviruses from infecting cells. We believe the nanobody are very valuable and could be used in the treatment of COVID-19. SARS-CoV-2 nanobodies can be rapidly mass-produced from microorganisms to block SARS-CoV-2 infection in vitro and in vivo with preventive and therapeutic effects.

Nucleomodulin BspJ as an effector promotes the colonization of Brucella abortus in the host.

BACKGROUND: Brucella infection induces brucellosis, a zoonotic disease. The intracellular circulation process and virulence of Brucella mainly depend on its type IV secretion system (T4SS) expressing secretory effectors. Secreted protein BspJ is a nucleomodulin of Brucella that invades the host cell nucleus. BspJ mediates host energy synthesis and apoptosis through interaction with proteins. However, the mechanism of BspJ as it affects the intracellular survival of Brucella remains to be clarified. OBJECTIVES: To verify the functions of nucleomodulin BspJ in Brucella's intracellular infection cycles. METHODS: Constructed Brucella abortus BspJ gene deletion strain (B. abortus ΔBspJ) and complement strain (B. abortus pBspJ) and studied their roles in the proliferation of Brucella both in vivo and in vitro. RESULTS: BspJ gene deletion reduced the survival and intracellular proliferation of Brucella at the replicating Brucella-containing vacuoles (rBCV) stage. Compared with the parent strain, the colonization ability of the bacteria in mice was significantly reduced, causing less inflammatory infiltration and pathological damage. We also found that the knockout of BspJ altered the secretion of cytokines (interleukin [IL]-6, IL-1ß, IL-10, tumor necrosis factor-α, interferon-γ) in host cells and in mice to affect the intracellular survival of Brucella. CONCLUSIONS: BspJ is extremely important for the circulatory proliferation of Brucella in the host, and it may be involved in a previously unknown mechanism of Brucella's intracellular survival.

The Recombinant Expression Proteins FnBP and ClfA From Staphylococcus aureus in Addition to GapC and Sip From Streptococcus agalactiae Can Protect BALB/c Mice From Bacterial Infection.

Dairy cow mastitis is a serious disease that is mainly caused by intramammary infection with Staphylococcus aureus and Streptococcus agalactiae [group B streptococcus (GBS)]. FnBP and ClfA are the virulence factors of S. aureus, while GapC is the respective factor for S. agalactiae. Sip is a highly immunogenic protein, and it is conserved in all GBS serotypes. In this study, we analyzed the abovementioned four genes prepared a FnBP+ClfA chimeric protein (FC), a GapC+Sip chimeric protein (GS), and a FnBP+ClfA+GapC+Sip chimeric protein (FCGS) based on the antigenic sites to evaluate their use in vaccine development. After expression and purification of the recombinant proteins in Escherichia coli, BALB/c mice were immunized with them to examine resistance effects. The total lethal and half lethal doses of S. aureus and S. agalactiae were then measured, and the immunoprotective effects of the fusion proteins were evaluated. The FC and FCGS chimeric proteins could induce mice to produce high levels of antibodies, and bacterial loads were significantly reduced in the spleens and livers after challenge. After immunization with FCGS, the recipients resisted the attacks of both S. aureus and S. agalactiae, indicating the potential of the fusion protein as a mastitis vaccine.

Anaplasma phagocytophilum AptA enhances the UPS, autophagy, and anti-apoptosis of host cells by PSMG3.

Anaplasma phagocytophilum is an obligate intracellular bacterium and a common tick-borne infectious pathogen that can cause human granulocytic anaplasmosis (HGA). Effector proteins play an important role in the pathogenic mechanism of A. phagocytophilum, but the specifics of the disease mechanism are unclear. We studied the effector protein AptA (A. phagocytophilum toxin A) using yeast two hybrid assays to screen its interacting protein proteasome assembly chaperone 3 (PSMG3, PAC3), and identified new mechanisms for the pathogenicity of A. phagocytophilum in HEK293T cells. After AptA enters the host cell, it interacts with PSMG3 to enhance the activity of the proteasome, causing ubiquitination and autophagy in the host cell and thereby increasing cross-talk between the ubiquitination-proteasome system (UPS) and autophagy. AptA also reduces the apoptotic efficiency of the host cells. These results offer new clues as to the pathogenic mechanism of A. phagocytophilum and support the hypothesis that AptA interacts with host PSMG3.

Interferon-Inducible Transmembrane Protein 3-Containing Exosome as a New Carrier for the Cell-to-Cell Transmission of Anti-Brucella Activity.

Exosomes are small extracellular vesicles that are released from cells and that function in intercellular communication. Recently, interferon-inducible transmembrane protein 3 (IFITM3) has been identified as a highly effective anti-intracellular pathogen protein that can inhibit the invasion of a wide range of pathogenic microorganisms. However, whether Brucella infection induces secretion of exosomes and whether these exosomes contain IFITM3 protein remain unknown. Here, we focused on the immune function of extracellular IFITM3 protein in the process of Brucella infection. This study is the first to show that Brucella melitensis strain M5 (Brucella M5) can stimulate macrophages to secrete large amounts of exosomes. Most importantly, we identified exosomes from Brucella M5-infected cells that were rich in molecules of IFITM3, and these exosomes could transmit the IFITM3 from one cell to another, thereby effectively inhibiting the intracellular survival of Brucella. Moreover, immunization with exosomes carrying IFITM3 decreased mouse spleen tissue damage and spleen colony forming unit (CFU), leading to the establishment of an anti-Brucella state in mice. In conclusion, our findings provide new insights into the anti-Brucella mechanism of IFITM3-containg exosomes, thus providing a theoretical foundation for systematic elaboration of the mechanisms of Brucella infection and host immunity. The results provide new ideas for the development of candidate vaccines for Brucella.

Small ubiquitin-related modifier 2 affects the intracellular survival of Brucella abortus 2308 by regulating activation of the NF-κB pathway.

Brucella is a genus of Gram-negative intracellular pathogens that cause animal and human diseases. Brucella survival and replication inside immune cells is critical for the establishment of chronic infections. Protein modifications by small ubiquitin-related modifier proteins and the NF-κB pathway are involved in many cellular activities, playing major roles in regulating protein function that is essential for pathogenic bacteria during infection. However, the relationship between them in the intracellular survival of Brucella is still largely unknown. We demonstrated that Brucella abortus 2308 infection can activate the expression of small ubiquitin-related modifier-2 proteins in a time-dependent manner. We found the production of Th1 cytokines (IFN-γ and TNF-α) and the transcription of NF-κB/p65 were promoted by overexpression and inhibited by interference of small ubiquitin-related modifier-2. In addition, we showed that small ubiquitin-related modifier-2 can inhibit intracellular survival of Brucella abortus 2308 by regulating activation of the NF-κB pathway. Taken together, this work shows that small ubiquitin-related modifier-2 modification of NF-κB2/p65 is essential for the survival of Brucella abortus 2308 inside macrophages. This work may help to unravel the pathogenic mechanisms of Brucella infections.

Brucella abortus BspJ Is a Nucleomodulin That Inhibits Macrophage Apoptosis and Promotes Intracellular Survival of Brucella.

To date, a variety of Brucella effector proteins have been found to mediate host cell secretion, autophagy, inflammation, and other signal pathways, but nuclear effector proteins have not yet been reported. We identified the first Brucella nucleomodulin, BspJ, and we screened out the BspJ interaction host proteins NME/NM23 nucleoside diphosphate kinase 2 (NME2) and creatine kinase B (CKB) through yeast two-hybrid and co-immunoprecipitation assays. These proteins are related to the host cell energy synthesis, metabolism, and apoptosis pathways. Brucella nucleomodulin BspJ will decrease the expression level of NME2 and CKB. In addition, BspJ gene deletion strains promoted the apoptosis of macrophages and reduced the intracellular survival of Brucella in host cells. In short, we found nucleomodulin BspJ may directly or indirectly regulate host cell apoptosis through the interaction with NME2 and CKB by mediating energy metabolism pathways in response to the intracellular circulation of Brucella infection, but the mechanism needs further study.

Screening and evaluation of Mycobacterium tuberculosis diagnostic antigens.

In recent years, the prevalence of tuberculosis worldwide has increased, and with it, the number of drug-resistant tuberculosis strains. This has brought new challenges towards prevention and control of the disease. Therefore, it is urgent to find reliable and rapid diagnostic methods for tuberculosis in general, and for the drug-resistant forms of the disease. To this aim, we assessed 17 tuberculosis-specific protein candidates for the detection of tuberculosis-specific antibodies. First, we established an indirect ELISA method to detect anti-Mycobacterium tuberculosis IgM and IgG. We tested 453 sera and analyzed the efficacy of the protein candidates for diagnosis of tuberculosis. Next, we screened antigens rich in T cell epitopes for their ability to induce high levels of IFN-γ, in order to define their suitability does develop detection tests based on IFN-γ release assay (IGRAs). The antigens CFP-10, PPE57, 38kDa, and Rv3807 showed higher diagnostic potential for the detection of anti-tuberculosis IgM, whereas PPE57, Ag85B, CFP-10, Rv0220, and 38kDa antigens performed better for anti-tuberculosis IgG detection. Worth noting is that CFP-10, 38kDa, and PPE57 detected efficiently both IgM and IgG. Rv1987, Rv3807, PPE57, Rv0220, and MPT64 proteins alone and combinations of Rv1987 + Rv3807, 16kDa + Rv0220, and MPT64 + Rv1986 tested in IGRAs displayed a good correlation with the positive control constituted by a cocktail of ESAT-6 + CFP-10 + TB7.7 (ECT), known for their stimulating properties (r > 0.5, p < 0.01). Among these antigen candidates, Rv0220 and Rv1987 + Rv3807 were the most potent. We discovered CFP-10, 38kDa, and PPE57 for the detection of anti-M. tuberculosis IgM and IgG, and Rv0220 alone or the combination Rv1987 + Rv3807 as the strongest stimulators in IGRAs. These antigens provide new references for the screening of tuberculosis-specific antibodies and effective stimulation in IGRAs.