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 and suggests a critical link between phospholipid metabolism and Brucella pathogenicity.
Abstract
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.
Brucellosis caused by Brucella melitensis is a worldwide zoonotic disease, yet the role of the c-di-GMP phosphodiesterase BpdB in virulence remains incompletely understood. This study aimed to investigate the function of BpdB in B. melitensis pathogenicity and its involvement in host STING signaling. A bpdB deletion strain (ΔbpdB) and a complemented strain (CbpdB) were constructed in B. melitensis M5. Bacterial growth, stress tolerance, intracellular survival in macrophages, cytokine expression, and mouse virulence were evaluated. Deletion of bpdB did not affect in vitro growth but significantly attenuated virulence in BALB/c mice, reducing splenomegaly, splenic bacterial load, and hepatic granuloma formation. The ΔbpdB strain exhibited enhanced sensitivity to oxidative stress, whereas resistance to acid, polymyxin B, and SDS remained unchanged. Intracellular survival of ΔbpdB in RAW264.7 macrophages was reduced at 72 h post infection, a defect that was completely abrogated in STING-knockout RAW264.7 cells. ΔbpdB infection induced higher transcriptional levels of IFN-β and IL-1β, with IFN-β induction strictly dependent on STING. In C57BL/6J mice, the virulence attenuation of ΔbpdB was partially STING-dependent, as the reduction in splenic bacterial load was smaller in STING-knockout than in wild-type mice. These findings demonstrate that BpdB contributes to B. melitensis virulence by enhancing oxidative stress resistance and dampening STING-dependent host responses, providing new insights into c-di-GMP-mediated host–pathogen interactions.
Na Li, Qiumei Shi, Simin Chen et al.· International Journal of Mol...· 0 citations
ABSTRACT LiaS is a histidine kinase receptor in two-component systems that senses environmental stress signals. This study investigated the roles of LiaS in Listeria monocytogenes stress resistance and virulence. Phenotypic assays showed that deletion of liaS (ΔliaS) resulted in significantly impaired growth under acidic, alkaline, and osmotic stresses, as well as reduced survival under strong acidic conditions. These defects were partially restored in the complemented strain (CΔliaS). The growth defects were also observed in the ΔliaS under Cu2+ or H2O2 exposure. Furthermore, the ΔliaS strain exhibited impaired invasion and intracellular migration in host cells, along with attenuated colonization in the liver and spleen at 24 h post-infection. Bacterial loads in the spleen of infected mice remained lower at 48 h. The mortality was also delayed in ΔliaS‑infected mice. Transcriptional analysis revealed that compared to neutral conditions, osmotic stress-related genes (except opucB and gbuC) were significantly upregulated in the ΔliaS strain under acidic conditions, indicating that LiaS modulates acid tolerance through transcriptional regulation. Collectively, LiaS is essential for the adaptation of Listeria monocytogenes to acidic, osmotic, and oxidative stresses. Its absence attenuates host cell invasion, intracellular motility, and organ-specific colonization, highlighting its dual role in environmental resilience and pathogenicity. These findings provide a theoretical basis for understanding host–pathogen interactions and offer new strategies against antimicrobial-resistant pathogens.
Yongshu Wu, Jiali Xu, Yifan Wang et al.· Virulence· 0 citations
Brucella spp. is a bacterium that can survive under conditions of nutrient starvation and is responsible for foodborne illnesses. The OmpR-type transcriptional regulator is characterized by an N-terminal receiver domain and a C-terminal domain, and it plays a regulatory role in diverse physiological processes, notably nitrogen and carbon metabolism. The genome of Brucella melitensis 16M contains genes for multiple OmpR-family regulators. However, the genetic program associated with nitrogen metabolism remains elusive. Herein, it was demonstrated that the B. melitensis 16M ΔftcR mutant, which lacks the OmpR-type regulator FtcR, displayed compromised viability upon recovery in a nitrogen- and carbon-free medium. Chromatin immunoprecipitation and next-generation sequencing were used to characterize the DNA-binding sites of FtcR. Our genome-wide analysis revealed extensive FtcR-binding sites throughout the B. melitensis 16M genome, including the identification of glnB as a novel target. As glnB encodes a P-II nitrogen regulator, this suggests FtcR plays a direct role in modulating nitrogen, carbon, and energy metabolism under prolonged nutrient starvation. In summary, our findings not only advance our understanding of the transcriptional regulation of nitrogen metabolism but also highlight its critical role in brucellosis pathogenesis, thereby paving the way for the development of novel therapeutics.
Yidan Zhang, Yu Zhang, Shengnan Song et al.· Microorganisms· 0 citations
M. tuberculosis, an intracellular pathogen, survives within the membrane-bound vacuole, the phagosome with acidic pH and limited access to nutrients. To survive and replicate within human host, M. tuberculosis must adapt and fulfil its nutritional requirements. To investigate how the intracellular bacillus scavenges nutrients from its host, we studied mycobacterial acquisition of host-derived amino acids, the preferred nitrogen sources. We discovered that PhoP, a key determinant of mycobacterial adaptation to phagosomal acidification, controls expression of AnsP1 and AnsP2 to facilitate acquisition of host aspartate and asparagine, respectively. Thus, macrophage-infected WT-H37Rv showed a significantly higher level of intra-bacterial Asn compared to the phoPR-KO mutant and a complemented mutant could restore Asn level to that of WT-H37Rv. Under acidic conditions, elevated DNA binding of PhoP within the promoters lead to direct activation of ansP1 and ansP2. Consistently, phoPR-KO is unable to utilize Asn under acidic condition, and over-expression of ansP1 or ansP2 in the mutant restored intracellular growth defect of the mutant. These findings uncover the regulatory network allowing utilization of organic nitrogen sources by the pathogen during infection.
Bhanwar Bamniya, Kajal, Khushboo Mehta et al.· Journal of Biological Chemis...· 0 citations
ABSTRACT ClpX functions as a component of the ClpXP protease, a conserved intracellular protease that regulates protein turnover, stress responses, and virulence in multiple bacterial species. Our lab has established that clpX is necessary for resistance to cell envelope targeting antibiotics, such as penicillin and daptomycin in Bacillus anthracis Sterne. Previous microarray data identified the msrA/B gene encoding a bifunctional methionine sulfoxide reductase as upregulated in the ΔclpX mutant. Methionine sulfoxide reductases (Msr) repair oxidatively damaged proteins by reducing methionine sulfoxide residues back to methionine. While Msr enzymes are primarily associated with oxidative stress, cell wall antibiotics induce expression of msrA1 and msrB in S. aureus. Here, we investigated the role of MsrA/B in oxidative and cell envelope stress. Our results show that although hydrogen peroxide and paraquat induce msrA/B expression, the ΔmsrA/B strain was not susceptible to either oxidant, whereas the ΔclpX strain was sensitive to both. We also found that loss of msrA/B conferred penicillin‐specific sensitivity, but, unlike ΔclpX, increased sensitivity was not seen with other cell wall or cell membrane targeting antibiotics. Inactivation of the catalytic cysteine of either Msr domain of MsrA/B failed to complement, suggesting that the reducing activity of MsrA/B is required for penicillin resistance. These findings indicate that while MsrA/B contributes to penicillin resistance, other proteins in the ClpXP modulon must also play a role in oxidative and cell envelope stress.
Aeron B Pennington, Salina Hona, Josey I Austin et al.· MicrobiologyOpen· 0 citations
Campylobacter jejuni is the leading cause of bacterial foodborne diarrheal disease worldwide. Despite its microaerophilic nature, C. jejuni is ubiquitous in aerobic environments and must possess specific adaptation mechanisms against oxidative stress. Here, we identified a novel role for FlhF, a GTPase essential for proper flagellar assembly, in promoting resistance to hydrogen peroxide (H2O2). Comparative transcriptomic analysis under H2O2 stress revealed that deletion of flhF leads to significant downregulation of oxidative stress-related genes. FlhF directly interacts with TonB2, an iron transport-associated protein, via its B and N domains. Codeletion of flhF and tonB2 leads to increased sensitivity to H2O2, suggesting a synergistic interaction. Moreover, the FlhF-TonB2 interaction promotes H2O2 detoxification, potentially by modulating intracellular iron homeostasis and influencing redox processes. Together, these findings reveal a novel function of FlhF in the oxidative stress response of C. jejuni, offering new insights into flagella-associated defense mechanisms in this pathogen.