USA300, a dominant clone of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), is circulating globally and can cause necrotizing pneumonia with high morbidity and mortality. To further reveal the host anti-MRSA infection immune response, we established a mouse model of acute primary MRSA pneumonia challenged with aerosols of the USA300 clone. A time-course transcriptome analysis of the lungs collected at 0, 12, 24, 48 and 96 h post-infection (hpi) was conducted using RNA sequencing (RNA-seq) and multiple bioinformatic analysis methods. The change trend of histopathology and five innate immune cell (neutrophils, mononuclear cells, eosinophils, macrophages, DC cells) proportions in the lungs after infection was also examined. We observed a distinct acute pulmonary recovery process. A rapid initiation period of inflammation was present at 12 hpi, during which the IL-17 pathway dominantly mediated inflammation and immune defense. The main stages of host inflammatory response occurred at 24 and 48 hpi, and the regulation of interferon activation and macrophage polarization played an important role in the control of inflammatory balance at this stage. At 96 hpi, cellular proliferation processes associated with host repair were observed, as well as adaptive immunity and complement system responses involving C1q molecules. More importantly, the data provide new insight into and identify potential functional genes involved in the checks and balances occurring between host anti-inflammatory and proinflammatory responses. To the best of our knowledge, this is the first study to investigate transcriptional responses throughout the inflammatory recovery process in the lungs after MRSA infection. Our study uncovers valuable research targets for key regulatory mechanisms underlying the pathogenesis of MRSA lung infections, which may help to develop novel treatment strategies for MRSA pneumonia.

Background: Outer Membrane Vesicles (OMVs), nanosized particles derived from Gram-negative bacteria, are promising vaccine carriers due to innate immunogenicity and self-adjuvant properties. Yet the systematic evaluations of OMV-associated toxicity remain limited. Methods: We developed a CRISPR/Cas9-engineered Salmonella enterica serovar Typhimurium ΔmsbB mutant (Mut4_STM) to produce detoxified OMVs (Mut4_OMVs) with enhanced yield. Subcutaneous immunization of BALB/c mice with Mut4_OMVs to evaluate safety, and the adjuvant efficacy was also determined by injecting Mut4_OMVs with Yersinia pestis F1Vmut or Bacillus anthracis PA_D4 antigens, mixing formulation, respectively. Results: Mut4_OMVs showed a 9-fold protein yield increase over wild-type OMVs. While all mice injected with wild-type OMVs died, 100% survival was observed in those receiving Mut4_OMVs. However, dose-dependent pathological alterations became evident in specific organs as the administration dose escalated, such as induced splenic extramedullary hematopoiesis and renal edema. Despite residual toxicity, 2–3 doses of 10 μg Mut4_OMVs elicited antigen-specific antibody titers comparable to aluminum adjuvant controls and superior T-cell immune responses. Conclusion: While Mut4_OMVs retain potent adjuvant activity, their residual toxicity necessitates further biocompatibility optimization for safe vaccine applications.

Background: An emerging trend of mutual convergence between drug-resistant and highly virulent strains of K. pneumoniae has been identified, highlighting the urgent need for the development of novel vaccines. Methods: To delete the target genes and eliminate the plasmids carrying antibiotic resistance genes, CRISPR-Cas9 technology was employed to perform genome editing on a clinically isolated O2 serotype of K. pneumoniae. Subsequently, this strain was utilized as a host to express genes associated with the synthesis of O1 serotype LPSs to construct the recombinant strain capable of simultaneously expressing LPSs of both O1 and O2 serotypes. This recombinant strain was then used as the production strain for the preparation of vaccines based on GMMAs (Generalized Modules for Membrane Antigens), and its biological characteristics were characterized. Finally, the safety and immunogenicity of the vaccine were evaluated using mice as the model animals. Result: a GMMA vaccine characterized by a high yield and low toxicity was gained. Importantly, the lipopolysaccharides (LPSs) of both O1 and O2 serotypes of K. pneumoniae were successfully expressed on the surface of the outer membrane vesicles. Following immunization with the GMMA vaccine, mice were capable of producing antibodies against the GMMA and demonstrated resistance to the invasion of both serotypes of clinically isolated K. pneumoniae. Conclusions: The GMMA vaccine showed significant promise as a bivalent vaccine against K. pneumoniae.

Mycoplasma pneumoniae pneumonia (MPP) is a common respiratory infection characterized by significant inflammatory responses and lung tissue injury. However, the precise immunological mechanisms and temporal dynamics of key cytokines driving pulmonary inflammation in MPP are still unclear. This study aimed to investigate the underlying immunological mechanisms and cytokine dynamics in MPP. We established an acute MPP murine model via intranasal administration of M. pneumoniae. This model recapitulates key features of human MPP, such as robust airway inflammation and cytokine production. Comprehensive analyses were conducted, including histopathology, flow cytometry, and cytokine profiling. Results showed severe inflammatory responses with prominent infiltration of neutrophils and macrophages in lung tissue, whereas monocyte populations were significantly reduced, indicating a shift towards myeloid cell predominance. Notably, 36 cytokines, including pro-inflammatory interleukins (IL-1β, IL-6, IL-17A) and chemokines, were statistically significantly upregulated in bronchoalveolar lavage fluid compared to the normal group, highlighting a cytokine storm associated with lung inflammation and tissue damage. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis further revealed enriched pathways related to cytokine-cytokine receptor interactions and IL-17 signaling, suggesting potential therapeutic targets. In conclusion, this study preclinical provides insights into the innate immune response and cytokine-driven pathology in acute MPP, underscoring the pivotal roles of myeloid cells and pro-inflammatory cytokines. Future research should focus on clinical validation of these findings to assess their translational potential and the exploration of immunomodulatory strategies informed by this model to mitigate MPP severity.