Brucella is a Gram-negative facultative intracellular pathogen that causes widespread zoonotic infections. Macrophages are crucial immune and antigen-presenting cells that differentiate into proinflammatory M1-type or anti-inflammatory M2-type cells in different microenvironments. The BtpB protein of Brucella is a type IV secretion system effector protein that modulates host inflammatory responses by inhibiting Toll-like receptor signaling and controlling dendritic cell activation. A high expression of intracellular proinflammatory factors is induced in B. suis deficient in BtpB (B. suis mutant strain ΔbtpB). However, the role of BtpB in macrophage polarization triggered by Brucella infection is uncertain. In this study, RAW264.7 macrophages were utilized as a model to investigate the impact of BtpB on macrophage polarization. The cells were infected with wild-type B. suis strain S2, B. suis mutant strain ΔbtpB, or the B. suis complemented strain C-ΔbtpB followed by flow cytometry, reverse transcription quantitative PCR (RT-qPCR), western blotting, immunohistochemistry, and metabolic detection analyses. The expression of BtpB suppressed M1 polarization and promoted M2 macrophage polarization. Moreover, BtpB upregulated the expression of signal transducer and activator of transcription 3 (STAT3) in host cells. Inhibition of STAT3 promoted the expression of nitric oxide synthase (NOS2) in cells infected with either B. suis or C-ΔbtpB, with no observable differences compared to cells infected with ΔbtpB. Conversely, STAT3 overexpression resulted in a downregulation of NOS2 levels in both wild-type and mutant cells. These findings were corroborated by messenger RNA (mRNA) assay and enzyme-linked immunosorbent assay (ELISA) that confirmed the regulatory role of STAT3 in modulating NOS2 expression. Additionally, intracellular proliferation assays under STAT3-modulated conditions indicated that STAT3 suppresses replication of Brucella. In conclusion, the results demonstrate that BtpB inhibits M1 polarization in macrophages by regulating STAT3 expression which provides a strong foundation for improved understanding of Brucella infection mechanisms.
Porcine genital chlamydiosis is considered a potential cause of reproductive failure, with Chlamydia (C.) suis being the most prevalent chlamydial species. To assess its pathogenic relevance, 50 Austrian piglet-producing farms were investigated: 40 farms with reproductive disorders (20 with tetracycline-treatment (tet), 20 without (no-tet)) and 10 clinically unaffected control farms. Samples from 770 sows (tet: n = 333, no-tet: n = 337, control: n =100) including blood, cervical and rectal swabs, dust samples and one pooled nursery fecal sample per farm were analyzed. Thirty-seven reproductive-tracts and six abortion cases were examined. Species-specific real-time PCR detected cervical C. suis shedding in 2.1
Streptococcus suis is a major zoonotic pathogen, and strains originating from clinically healthy pigs pose an increasingly serious threat to human health. This study investigated 315 S. suis isolates collected from clinically healthy pigs in Guangdong and Guangxi provinces between 2024 and 2025. Among these isolates, 34 capsular polysaccharide (cps) types were identified, with 40.63
The bovine respiratory disease complex (BRDC) is a major health and welfare challenge driven by multifactorial interactions among pathogens, the host, the microbiota, and environmental pressures, reflecting the holobiont nature of the calf. Many implicated bacterial pathogens are also respiratory tract commensals, complicating diagnosis, risk assessment, and prevention. Although the potential association of the respiratory and gut microbiotas in BRDC is only beginning to be recognized, their integrated effects have not yet been investigated. Here, we dynamically followed the microbiota, pathogen load, and host response in 30 calves over 147 days under commercial rearing conditions. Nearly half developed BRDC, presenting fever, cough, and abnormal lung sounds, with peak symptoms at day 58 after arrival. Notably, the large majority of these were acute cases, with only a small subset experiencing more than one episode. Pathogen detection in the nasal cavity revealed clear temporal dynamics. Mycoplasma bovis and bovine coronavirus (BCoV) showed their highest loads during the first 2 weeks of life. At 1 month, influenza D virus (IDV) and Histophilus somni peaked, and Pasteurella multocida also reached its maximum abundance and persisted thereafter. Mannheimia haemolytica became prominent later, with a marked increase after 2 months. Importantly, M. haemolytica and P. multocida loads correlated with higher BRDC scores, whereas BCoV was associated with diarrhea. Microbiota analysis showed that nasal beta-diversity diverged between groups at the symptomatic window, and that healthy animals exhibited early life fecal diversity and evenness. Respiratory pathobionts such as Pasteurella and Corynebacterium were enriched in diseased calves, whereas potentially protective families (Lachnospiraceae, Oscillospiraceae) were more abundant in healthy ones. Multivariate analyses further showed that antibiotic treatments and short-chain fatty acids, especially propionic, butyric, iso-valeric, and iso-butyric acids, modulated both fecal and nasal microbiota, with consistently stronger effects in diseased animals. Together, these findings indicate that BRDC outcomes are shaped not by pathogen burden alone but by the interplay among respiratory and digestive microbiotas, pathogens, environment, and management factors. Our study highlights the importance of a holobiont perspective that integrates both gut and respiratory microbiotas to better elucidate the complexity of BRDC. Such an inclusive framework may provide new insights into disease mechanisms and inform the development of innovative therapeutic strategies.
Schistosomiasis remains an important zoonotic parasitic disease, and hepatic fibrosis is a major cause of chronic pathology following Schistosoma japonicum infection. In schistosomiasis, hepatic fibrosis is the major cause of severe pathological damage. However, during the long-term co-evolution and coexistence between Schistosoma parasites and their hosts, the parasite can modulate the host hepatic fibrotic process through extracellular vesicle (EV)-mediated cross-species communication. In this study, we identified an EV-derived microRNA from Schistosoma japonicum, sja-miR-2a-3p, that suppresses hepatic pathology in the host. Using transwell co-culture and EV-isolation experiments, we confirmed that the helminth-specific sja-miR-2a-3p could be transferred to hepatic stellate cells (HSCs) via EVs. Bioinformatic and dual-luciferase assays revealed that host BCL2, a major anti-apoptotic gene, is a direct target of sja-miR-2a-3p. In vitro, sja-miR-2a-3p overexpression suppressed HSC proliferation and migration and induced mitochondrial dysfunction, caspase-3/-7/-9 activation, and apoptosis. In vivo, administration of sja-miR-2a-3p agomir to S. japonicum-infected mice significantly downregulated hepatic BCL2 expression, enhanced HSC apoptosis, alleviated granuloma formation, collagen deposition, and inflammation. Collectively, our findings reveal that schistosome EV-delivered sja-miR-2a-3p attenuates liver fibrosis by promoting mitochondrial apoptosis and functionally suppressing HSC activation. This study supports sja-miR-2a-3p as a candidate antifibrotic molecule.
Fowl adenovirus serotype 4 (FAdV-4) is a nonenveloped double-stranded DNA virus with a 43–45 kb genome, making it a promising viral vector for developing multivalent poultry vaccines. However, limited genomic studies have hindered the design of strategies for generating recombinant FAdV-4 capable of expressing multiple foreign proteins while maintaining high viral titers. In this study, three deletion mutants (rD14AB, rD2042, rD19A) were generated to assess their roles in viral replication and pathogenicity. Only rD2042 showed impaired replication and virulence compared with wild-type FAdV-4. Foreign gene expression assays demonstrated that the D14AB site had a maximum insertion capacity of 2349 bp, and an SV40 poly(A) signal was required for efficient protein expression and viral propagation at this locus. In contrast, the D19A site tolerated insertions of up to 1257 bp in the absence of this signal. Using the insertion strategy optimized for wild-type CH_JS_2017, we modified the previously reported nonpathogenic rON1 strain (harboring the Hexon gene from the attenuated ON1 strain) to co-express mCherry, eGFP, and mTagBFP2 at the two independent sites. SPF chicken trials verified the safety of the engineered virus at immunization age and the genetic stability of exogenous genes. The recombinant virus maintained wild-type-comparable titers while carrying 3606 bp of total foreign sequences, highlighting its dual-site advantage and potential as a multivalent poultry vaccine vector.
Mycoplasma gallisepticum is a major poultry pathogen responsible for chronic respiratory disease and substantial global economic losses. Its ability to establish chronic infections reflects its effective immune evasion strategies, but the mechanisms underlying this remain poorly understood. Some pathogenic mammalian mycoplasmas use the Mycoplasma Immunoglobulin Binding-Protease (MIB-MIP) system to capture and cleave host immunoglobulins (Ig), but the functionality and host-specificity of this system in M. gallisepticum have not been examined. We aimed to functionally characterise all the MIB-MIP homologues in M. gallisepticum and examine their host specificity. Five putative MIB and five putative MIP genes of M. gallisepticum were cloned, expressed as recombinant GST-fusion proteins, and purified for functional analysis. Immunoglobulin-binding assays showed that all MIB proteins bound both avian and mammalian immunoglobulins, forming stable MIB-Ig complexes, with distinct binding capacities. In contrast, proteolytic assays revealed that only three of the five MIP proteins could cleave avian immunoglobulins, when complexed with any of the five MIBs, generating characteristic Ig heavy-chain fragments. Only one MIP protease showed detectable interaction with mammalian immunoglobulins, indicating strong host specificity of these MIPs and functional specialisation for avian immunoglobulin-cleavage. These results revealed that MIB and MIP proteins of M. gallisepticum are adapted to cleavage of avian immunoglobins, thereby interfering with antibody-mediated host immune responses. Bioinformatic analysis suggested that MIB-MIP homologues are widespread among avian mycoplasmas that share similar hosts, tissue tropisms and transmission patterns, and detected evidence of horizontal gene transfer and recombination, indicating that there have been MIB-MIP evolutionary adaptations among the avian mycoplasmas.
Phage therapy typically fails when bacteria rapidly evolve resistance, yet the inevitable fitness costs of escape mutants remain underexploited. In this study, an iterative and targeted phage-cocktail formulation strategy (ITPFS) was presented and developed. A potent lytic phage, PHZ055, was firstly isolated against a colistin-resistant Salmonella Pullorum strain. Co-cultured until resistant mutants emerged, a second lytic phage, PYW047_3, was screened for the resistant variant. The two phages were then combined as a cocktail. Results showed that bacterial growth was strongly suppressed and the emergence of resistance was markedly delayed. Receptor identification revealed that both phages use lipopolysaccharide (LPS) as a receptor, but at distinct sites: PHZ055 targets WaaL, and PYW047_3 targets WaaP. Mutation of WaaL was recently shown to be responsible for the loss of polymerized O-antigen units and thereby exposed the PYW047_3 binding region, indicating that phage cocktails targeting distinct receptor sites can be directionally screened. Moreover, mutants that escaped the two phages not only incurred a growth cost but also exhibited a 32-fold increase in colistin sensitivity. Combining the two phages together with 1/4 MIC colistin almost eradicated all bacteria within 24 h. In all, our study successfully provides a repeatable strategy that incorporates adaptive trade-offs to boost therapeutic efficacy.
Infectious bursal disease virus (IBDV) is an important avian pathogen that damages the bursa of Fabricius and continues to threaten the poultry industry. Pyroptosis is an inflammatory form of programmed cell death, but its contribution to IBDV-induced cellular injury remains incompletely defined. Here, using DF-1 cells, we found that IBDV infection was associated with caspase-3 activation, cleavage of gasdermin E (GSDME), generation of the pore-forming N-terminal fragment (GSDME-N), increased lactate dehydrogenase release, plasma membrane damage, and enhanced inflammatory cytokine-associated readouts, including increased interleukin (IL)-1β secretion and elevated IL-18 messenger RNA (mRNA) expression. IBDV infection also induced mitochondrial dysfunction, as evidenced by loss of mitochondrial membrane potential (ΔΨm), increased reactive oxygen species production, and activation of caspase-9, supporting the involvement of a mitochondria-associated caspase-9/3 pathway. Pharmacological inhibition of caspase-9 or caspase-3 reduced pyroptosis-associated readouts, indicating that mitochondrial damage contributes to IBDV-induced lytic cell death under the tested conditions. Expression of viral protein (VP)2 or VP5 alone caused detectable cytotoxicity but did not robustly reproduce GSDME cleavage, suggesting that viral proteins may contribute to upstream cellular stress that is further amplified during infection. Together, these findings support that IBDV engages a mitochondria-associated caspase-9/3–GSDME axis to promote pyroptosis-like lytic cell death in DF-1 cells.
Type IV interferon (IFN), a recently identified class of IFN, exhibits broad-spectrum antiviral and antibacterial functions across species. In this study, the type IV IFN gene, IFN-υ, and its cognate receptor subunits (IFN-υR1 and IL10RB) were functionally characterized in an avian species, the rock pigeon (Columba livia). Phylogenetic analysis showed that rock pigeon IFN-υ, IFN-υR1, and IL10RB were clustered closely with their respective orthologs in other vertebrates. Expression profiling revealed the constitutive transcription of these genes across multiple tissues. Furthermore, the expression of these genes was significantly upregulated upon stimulation with the viral double-stranded RNA (dsRNA) mimic, poly(I:C). The recombinant rock pigeon IFN-υ activated the canonical Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling pathway, inducing STAT1 phosphorylation and the consequent expression of key interferon-stimulated genes (ISGs) such as OAS1, PKR, and RSAD2. Using overexpression and knockdown assays, the biological activity of IFN-υ was found to signal through a heterodimeric receptor complex comprising IFN-υR1 and IL10RB. Importantly, two conserved tyrosine residues (Tyr334 and Tyr525) within the intracellular domain of IFN-υR1 were found essential for IFN-υ-mediated JAK-STAT pathway activation, ISG induction, and antiviral activity. These findings elucidate the conserved functional architecture and signaling mechanism of type IV IFN in vertebrates.
Accumulating data support the role of exosomes from virus-infected cells (especially RNA viruses) in the selective encapsulation of viral proteins, genomic material, and even whole virions, thereby mediating intercellular communication events and contributing to viral transmission. Tembusu virus (TMUV), a single-stranded RNA virus, has posed a threat to global public health since its emergence in 2010. However, whether exosomes can mediate TMUV intercellular transmission remains obscure. Here, we show that exosomes derived from TMUV-infected cells (TMUV-exosomes) contained viral genomic RNA and partial viral proteins (C, prM, E, NS1, NS2B, NS4B, and NS5). TMUV-exosomes enter HEK293 cells primarily via a dynamin-dependent, cholesterol-sensitive caveolae-mediated (CavME) endocytosis pathway, thereby suppressing the subsequent expression of key antiviral immune genes to create a proviral microenvironment that supports productive TMUV infection. Remarkably, central to this exosomal hijacking mechanism, the viral NS4A protein and host Rab27a protein form a critical regulatory axis. Specifically, NS4A upregulates Rab27a expression to potentiate the release of exosomes encapsulating viral nucleic acids and proteomic components. Overall, our study provides a mechanistic model for understanding the role of exosomes in TMUV intercellular transmission and identifies the NS4A–Rab27a axis as a critical regulatory node. Disrupting this interaction may represent a potential therapeutic strategy for TMUV infection, warranting further investigation.
Hemotropic mycoplasmas (hemoplasmas) infecting cattle were first detected in France in 2019 during a clinical outbreak, but their prevalence both at the species and strain levels remains unknown. This study therefore aimed to determine the prevalence of hemoplasmas and their coinfections, and to assess the within and between herd diversity in five fully sampled dairy herds. Detection was performed using 16S rRNA quantitative polymerase chain reaction (qPCR), as well as three strain-specific rnpB qPCR assays targeting Candidatus Mycoplasma haematobovis (CMh, formerly Ca. M. haemobos), Mycoplasma wenyonii strain Mexico (Mex), and Mycoplasma wenyonii strain Massachusetts (Mass). A total of 1011 cattle were sampled, of which 88.7
Porcine reproductive and respiratory syndrome is one of the most devastating diseases affecting global swine production. Viral nonstructural protein 12 (nsp12) is a key factor in viral replication during viral subgenomic RNA synthesis. We identified the host E3 ubiquitin ligase RING finger protein 187 (RNF187) as a novel anti-porcine reproductive and respiratory syndrome virus (PRRSV) host-restriction factor. RNF187 directly interacts with nsp12, and the overexpression of RNF187 significantly inhibits the expression of the viral nucleocapsid (N) protein and the titer of viruses, whereas the knockdown of RNF187 promotes viral replication. Mechanistic studies showed that RNF187 mediates the degradation of nsp12 in a dose-dependent manner. This degradation process can be blocked by the autophagy inhibitor, 3-methyladenine (3-MA). These results indicate that RNF187 degrades nsp12 via the autophagy pathway rather than the proteasome pathway. Mechanistically, RNF187 acts as a scaffold protein to recruit the autophagic cargo receptor NDP52 and promotes the K63-linked polyubiquitination at lysine 130 (K130) of nsp12, thereby targeting nsp12 for autophagy-lysosomal degradation. This study revealed a novel host defense mechanism mediated by the RNF187-NDP52 axis, which restricts PRRSV infection by targeting nsp12, thereby providing a potential target for the development of antiviral strategies.
The emergence of highly lethal genotype I/II recombinant African swine fever virus (ASFV) strains in China has rendered existing genotype II-based live attenuated vaccines ineffective, underscoring the urgent need for novel vaccine candidates. Although single-gene deletions of A137R or I226R have shown promise against genotype II strains, their efficacy and safety in the context of recombinant strains remain unexplored. Using homologous recombination, we constructed a single gene-deleted mutant (JX23-02ΔI226R) and a double gene-deleted mutant (JX23-02ΔI226RΔA137R) from the genotype I/II recombinant ASFV strain JX23-02. The replication kinetics, pathogenicity, immunogenicity, and protective efficacy of these mutants were evaluated in vitro and in pigs. Both deletion mutants exhibited significantly reduced replication in porcine alveolar macrophages. Immunization with JX23-02ΔI226R resulted in 40
The first epidemic of lumpy skin disease (LSD) in France was detected in June 2025, with a total of 117 outbreaks recorded by the end of the year. This fast-spreading vector-borne disease of cattle prompted the implementation of strict control measures, including total depopulation of affected herds, resulting in the culling of more than 3500 cattle. Over the course of the epidemic, this measure became increasingly unacceptable, leading to major protests. To contribute to this veterinary public health debate, we present a mathematical model, accounting for both cattle and vector populations, that compares within-herd transmission dynamics under selective or total depopulation strategies, and different vector control scenarios. The selective depopulation strategies are modelled based on a bi-daily test-and-cull approach implemented upon detection of the first clinical case in an unvaccinated herd, with imperfect diagnostic tests capable of detecting infection in asymptomatic cattle under different assumptions of test sensitivity and of time from infection to detectability. Our model shows that the selective culling strategy is insufficient to control the spread of the disease in the absence of vector population control, with the entire herd eventually becoming infected. In the best-case scenario with highly sensitive and timely diagnostic tests and an 80
The porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious pathogen. Viral infections often enhance their replication by modulating the structure and expression of host genes. However, it remains unclear whether PRRSV employs a similar mechanism to achieve self-replication. To address this question, the current study combined assay for transposase accessible chromatin sequencing (ATAC-seq) and ribonucleic acid (RNA) sequencing (RNA-seq) to identify accessible chromatin regions and key host genes associated with PRRSV infection. By comparing the PRRSV-infected group with the control group, we initially detected 8664 differentially accessible chromatin regions and 4037 differentially expressed genes. Motif analysis of these differential chromatin regions revealed several potential cis-regulatory elements containing binding sites for transcription factors. Further integration of ATAC-seq and RNA-seq results identified 1352 overlapping genes between the PRRSV-infected and control groups. A significant positive correlation between differential gene expression and chromatin accessibility signals suggests that chromatin remodeling may drive transcriptional changes during infection. Protein–protein interaction (PPI) network analysis highlighted candidate genes potentially associated with PRRSV infection in hosts, such as IL1B, CCL20, CXCL10, CSF3, etc. Given their potential association with the infection mechanism, these genes could serve as candidate targets for the future development of prophylactic vaccines and therapeutic strategies. Additionally, several signaling pathways that may regulate immune and inflammatory responses were significantly enriched in our ATAC-seq and RNA-seq analyses. These findings provide valuable insights into the molecular mechanisms underlying PRRSV infection and pave the way for developing more effective preventive and treatment measures.
Although previous studies have suggested a role for GAPLINC in regulating influenza A virus (IAV) infection, the functional involvement of GAPLINC in IAV infection in vitro and in vivo remains largely unknown. Here, we found that expression of lncRNA GAPLINC is significantly downregulated by infections with several strains of IAV, including PR8, WSN, H3N2, and H9N2. Interestingly, infections with several other viruses, such as pseudorabies virus (PRV), Sendai virus (SeV), and Herpes simplex virus (HSV), also result in a significant reduction in GAPLINC expression. During IAV infection, activation of NF-κB and the downstream IL-6/STAT3 signaling pathway contribute, at least in part, to the downregulation of GAPLINC expression. Knockdown of GAPLINC in host cells impairs the viral replication, whereas overexpression of GAPLINC increases the viral titers. Both heterozygous GAPLINC knockout (KO) mice (GAPLINC+/-) and homozygous GAPLINC KO mice (GAPLINC⁻/⁻) were further employed to determine its function in vivo. GAPLINC knockout renders mice more resistant to IAV infection than wild-type counterparts, as evidenced by lower viral load and lung injury, slower body weight loss, and improved survival. We confirmed that GAPLINC obviously suppresses the IRF3 activation in IAV-infected cells. Moreover, we noticed that inhibition of ATG7-involved autophagy weakens the pro-viral activity of GAPLINC. Together, the results support the conclusion that GAPLINC plays a critical role in enhancing the pathogenesis of IAV, at least by targeting the IRF3 and ATG7-mediated autophagy pathway.
The prevalence of porcine rotavirus A (PoRVA) in China has increased significantly, threatening the swine industry. Pigs serve as “mixing vessels” for rotaviruses, facilitating reassortment between human and animal strains, leading to the emergence of zoonotic variants. In this study, two PoRVA strains, XXW2023 (G9P[7]) and HD2023 (G1P[7]), were isolated from diarrheic piglets in Guangdong Province, China. Genomic analysis revealed that both strains were human-porcine reassortants, with VP1, VP3, and NSP1 genes closely related to human rotaviruses. Intragenic recombination was identified in the VP4 and VP6 genes. Pathogenicity was evaluated in 7-day-old mice and 1-day-old piglets. Both strains caused persistent diarrhea in mice and severe watery diarrhea, intestinal lesions, and death within 48 h in piglets. Systemic infection was confirmed, with viral replication detected in the lungs. Infectious virus titers, VP6 antigen, and NSP4 were detected in lung tissues, providing evidence of active replication in the respiratory tract. The strains exhibited distinct tissue tropism, with XXW2023 being enterotropic and HD2023 showing pulmonary tropism. Viral RNA and antigen levels in the lungs of HD2023-infected piglets exceeded those in their intestines, and the infectious virus titer in their lungs was significantly higher than that in the lungs of XXW2023-infected piglets. These findings demonstrate that reassortant rotavirus strains infect the respiratory tract, extending the conventional view of rotavirus as a strictly enteric pathogen. The emergence of these highly pathogenic, phenotypically divergent human-porcine reassortants underscores their zoonotic risk, highlighting the need for enhanced surveillance and reconsideration of vaccine coverage.
Canine distemper virus (CDV) can cause fatal viral infection in domestic and wild animals globally. Several lineages are known, originating from distinct geographical regions and hosts, and can spread naturally or through human intervention into new geographic areas. The Arctic lineage was first described in carnivores of the Arctic ecosystems and subsequently reported in several European and Asian countries, yet its origin, evolution, and ecology remain partially unresolved. In this study, we generated genome sequence data of (n = 16) CDV strains of Arctic lineage collected from dogs in Italy over a nearly 15-year period, providing an extensive dataset to investigate the evolution of this particular lineage. We also generated genome data of seven Europe strains of another major lineage collected during the same period from red foxes (n = 3) and dogs (n = 4). Inter-lineage recombination events were identified in two CDV sequences. Sequence 2008 of the European lineage acquired a fragment from an Arctic lineage virus between the N and P genes. Sequence 2015 of the Arctic lineage displayed a more complex recombination pattern with fragments from Europe, America-2, and Rockborn lineages across multiple genes and hosts. Phylogenetic tree showed that the oldest Italian Arctic lineage from 2006 was more similar to the oldest Arctic CDV isolates, whilst a well-defined sub-cluster circulated from 2009 onwards in domestic and wild carnivores. These results provide novel insights into CDV evolution in Europe and emphasize the importance of ongoing genomic monitoring.
Brucella melitensis, a facultative intracellular pathogen, relies on membrane integrity and homeostasis to resist host defenses and establish infection. The plsC gene encodes 1-acyl-sn-glycerol-3-phosphate acyltransferase, a key enzyme in the glycerophospholipid pathway that catalyzes the synthesis of phosphatidic acid, an essential precursor for membrane lipid formation. However, its role in B. melitensis virulence remains poorly understood. Here, we constructed a plsC deletion mutant (ΔplsC) and a complemented strain (ΔplsC-Com) in B. melitensis strain M5 and characterized their phenotypes. Deletion of plsC impaired bacterial growth in nutrient-limited media, reduced tolerance to hydrogen peroxide and polymyxin B, and decreased lipid synthesis while increasing outer membrane permeability. Ultrastructural analysis revealed surface roughness, cytoplasmic voids, and nucleoid condensation in the mutant. Although ΔplsC retained normal adhesion and invasion capabilities in RAW264.7 macrophages and HeLa cells, its intracellular survival was specifically attenuated in macrophages at 48 h post-infection. In a mouse model, ΔplsC showed significantly reduced colonization of the spleen and liver and induced fewer and smaller liver granulomas as compared with the parental and complemented strains. These results demonstrate that PlsC is essential for maintaining membrane homeostasis and stress resistance in Brucella, which in turn supports its survival within professional phagocytes and full virulence in vivo. Our study suggests a critical link between phospholipid metabolism and Brucella pathogenicity.
