Streptococcus pneumoniae endopeptidase O induces trained immunity and confers protection against various pathogenic infections.

Antibiotic resistance and the surge of infectious diseases during the pandemic present significant threats to human health. Trained immunity emerges as a promising and innovative approach to address these infections. Synthetic or natural fungal, parasitic and viral components have been reported to induce trained immunity. However, it is not clear whether bacterial virulence proteins can induce protective trained immunity. Our research demonstrates Streptococcus pneumoniae virulence protein PepO, is a highly potent trained immunity inducer for combating broad-spectrum infection. Our findings showcase that rPepO training confers robust protection to mice against various pathogenic infections by enhancing macrophage functionality. rPepO effectively re-programs macrophages, re-configures their epigenetic modifications and bolsters their immunological responses, which is independent of T or B lymphocytes. In vivo and in vitro experiments confirm that trained macrophage-secreted complement C3 activates peritoneal B lymphocyte and enhances its bactericidal capacity. In addition, we provide the first evidence that granulocyte colony-stimulating factor (G-CSF) derived from trained macrophages plays a pivotal role in shaping central-trained immunity. In summation, our research demonstrates the capability of rPepO to induce both peripheral and central trained immunity in mice, underscoring its potential application in broad-spectrum anti-infection therapy. Our research provides a new molecule and some new target options for infectious disease prevention.

Identification of cis-acting elements upstream of regR gene in streptococcus pneumoniae.

The identification and characterization of functional cis-acting elements is of fundamental importance for comprehending the regulatory mechanisms of gene transcription and bacterial pathogenesis. The transcription factor RegR has been demonstrated to control both competence and virulence in Streptococcus pneumoniae. Despite the clear contribution of RegR to these pathways, the mechanisms underlying its transcriptional regulation remain poorly understood. In this study, we conducted mutational analysis, gene dissection and luciferase activity assays to characterize the cis-elements situated upstream of the regR gene. Our findings revealed that a 311 bp 3'-terminal DNA sequence of the spd0300 gene represents a central region of the upstream cis-acting element of regR. Further investigations identified two structurally similar enhancer-like sequences within this region which feature prominently in the regulation of regR transcription. Furthermore, employing DNA pull-down assays allowed us to enrich the trans-acting factors with the potential to interact with these cis-acting elements. Notably, we found that the competence regulator ComE was implicated in the regulation of regR transcription, a finding which was corroborated by electrophoretic mobility shift assays (EMSA) and quantitative real-time PCR analyses (qRT-PCR). Taken together, our data thus provide fresh insight into the transcriptional regulation of regR.

A Streptococcus pneumoniae endolysin mutant protein ΔA146Ply elicits rapid broad-spectrum mucosal protection in mice via upregulation of GPX4 through TLR4/IRG1/NRF2 to alleviate macrophage ferroptosis.

Innovative solutions for rapid protection against broad-spectrum infections are very important in dealing with complex infection environments. We utilized a functionally inactive mutated endolysin protein of Streptococcus pneumoniae (ΔA146Ply) to immunize mice against pneumonic infections by multidrug-resistant bacteria, Candida albicans and influenza virus type A. ΔA146Ply protection relied on both immunized tissue-resident and monocyte-derived alveolar macrophages and inhibited infection induced ferroptosis that upregulated expression of GPX4 (glutathione peroxidase) in alveolar macrophages. Ferroptosis resistance endowed macrophages with enhanced phagocytosis by inhibiting lipid peroxidation during infection. Moreover, we demonstrated ΔA146Ply upregulated GPX4 through the TLR4/IRG1/NRF2 pathway. ΔA146Ply also induced ferroptosis inhibition and phagocytosis improvement in human monocytes. This mode of action is a novel and potentially prophylactic and rapid broad-spectrum anti-infection mechanism. Our study provides new insights into protective interventions that act by regulating ferroptosis to improve multiple pathogen resistance via GPX4 targeting.

MiR-181b targets Six2 and inhibits the proliferation of metanephric mesenchymal cells in vitro.

MicroRNAs (miRNAs) are small non-coding RNAs that down-regulate gene expression by binding to target mRNA for cleavage or translational repression, and play important regulatory roles in renal development. Despite increasing genes have been predicted to be miRNA targets by bioinformatic analysis during kidney development, few of them have been verified by experiment. The objective of our study is to identify the miRNAs targeting Six2, a critical transcription factor that maintains the mesenchymal progenitor pool via self-renewal (proliferation) during renal development. We initially analyzed the 3'UTR of Six2 and found 37 binding sites targeted by 50 putative miRNAs in the 3'UTR of Six2. Among the 50 miRNAs, miR-181b is the miRNAs predicted by the three used websites. In our study, the results of luciferase reporter assay, realtime-PCR and Western blot demonstrated that miR-181b directly targeted on the 3'UTR of Six2 and down-regulate the expression of Six2 at mRNA and protein levels. Furthermore, EdU proliferation assay along with the Six2 rescue strategy showed that miR-181b suppresses the proliferation of metanephric mesenchymal by targeting Six2 in part. In our research, we concluded that by targeting the transcription factor gene Six2, miR-181b inhibits the proliferation of metanephric mesenchymal cells in vitro and might play an important role in the formation of nephrons.

The Streptococcus virulence protein PepO triggers anti-tumor immune responses by reprograming tumor-associated macrophages in a mouse triple negative breast cancer model.

BACKGROUND: The efficacy of current surgery and chemotherapy for triple negative breast cancer (TNBC) is limited due to heterogenous and immunosuppressive tumor microenvironment (TME). Tumor associated macrophages (TAMs), which are regarded as an M2 tumor-promoting phenotype, are crucial in the development of the immunosuppressive TME. Targeting TAM reprograming is a promising strategy in anti-tumor therapy since reprogramming techniques provide the opportunity to actively enhance the antitumor immunological activity of TAM in addition to eliminating their tumor-supportive roles, which is rarely applied in TNBC clinically. However, how to drive M2 macrophages reprogramming into M1 with high potency remains a challenge and the molecular mechanisms how M2 macrophages polarized into M1 are poorly understood. Here, we identified a new immunoregulatory molecular PepO that was served as an immunoregulatory molecule governed the transformation of tumor-promoting M2 to tumor-inhibitory M1 cells and represented an effective anti-tumor property. RESULTS: At the present study, we identified a new immunoregulatory molecular PepO, as a harmless immunoregulatory molecule, governed the transformation of tumor-promoting M2 to tumor-inhibitory M1 cells efficiently. PepO-primed M2 macrophages decreased the expression of tumor-supportive molecules like Arg-1, Tgfb, Vegfa and IL-10, and increased the expression of iNOS, Cxcl9, Cxcl10, TNF-α and IL-6 to inhibit TNBC growth. Moreover, PepO enhanced the functions of macrophages related to cell killing, phagocytosis and nitric oxide biosynthetic process, thereby inhibiting the development of tumors in vivo and in vitro. Mechanistically, PepO reprogramed TAMs toward M1 by activating PI3K-AKT-mTOR pathway via TLR4 and suppressed the function of M2 by inhibiting JAK2-STAT3 pathway via TLR2. The PI3K inhibitor LY294002 abrogated the role of PepO in switching M2 macrophages into M1 and in inhibiting TNBC growth in vivo. And PepO failed to govern the M2 macrophages to reprogram into M1 macrophages and inhibit TNBC when TLR2 or TLR4 was deficient. Moreover, PepO enhanced the antitumor activity of doxorubicin and the combination exerted a synergistic effect on TNBC suppression. CONCLUSIONS: Our research identified a possible macrophage-based TNBC immunotherapeutic approach and suggested a novel anticancer immunoregulatory molecular called PepO.

comCDE (Competence) Operon Is Regulated by CcpA in Streptococcus pneumoniae D39.

Natural transformation plays an important role in the formation of drug-resistant bacteria. Exploring the regulatory mechanism of natural transformation can aid the discovery of new antibacterial targets and reduce the emergence of drug-resistant bacteria. Competence is a prerequisite of natural transformation in Streptococcus pneumoniae, in which comCDE operon is the core regulator of competence. To date, only ComE has been shown to directly regulate comCDE transcription. In this study, a transcriptional regulator, the catabolite control protein A (CcpA), was identified that directly regulated comCDE transcription. We confirmed that CcpA binds to the cis-acting catabolite response elements (cre) in the comCDE promoter region to regulate comCDE transcription and transformation. Moreover, CcpA can coregulate comCDE transcription with phosphorylated and dephosphorylated ComE. Regulation of comCDE transcription and transformation by CcpA was also affected by carbon source signals. Together, these insights demonstrate the versatility of CcpA and provide a theoretical basis for reducing the emergence of drug-resistant bacteria. IMPORTANCE Streptococcus pneumoniae is a major cause of bacterial infections in humans, such as pneumonia, bacteremia, meningitis, otitis media, and sinusitis. Like most streptococci, S. pneumoniae is naturally competent and employs this ability to augment its adaptive evolution. The current study illustrates CcpA, a carbon catabolite regulator, can participate in the competence process by regulating comCDE transcription, and this process is regulated by different carbon source signals. These hidden abilities are likely critical for adaptation and colonization in the environment.

ΔA146Ply-HA stem protein immunization protects mice against influenza A virus infection and co-infection with Streptococcus pneumoniae.

Influenza virus (IV) is a common pathogen affecting the upper respiratory tract, that causes various diseases. Secondary bacterial pneumonia is a common complication and a major cause of death in influenza patients. Streptococcus pneumoniae (S. pneumoniae) is the predominant co-infected bacteria in the pandemic, which colonizes healthy people but can cause diseases in immunocompromised individuals. Vaccination is a crucial strategy for avoiding infection, however, no universal influenza vaccine (UIV) that is resistant to multiple influenza viruses is available. Despite its limited immunogenicity, the hemagglutinin (HA) stem is a candidate peptide for UIV. ΔA146Ply (pneumolysin with a single deletion of A146) not only retains the Toll-like receptor 4 agonist effect but also is a potential vaccine adjuvant and a candidate protein for the S. pneumoniae vaccine. We constructed the fusion protein ΔA146Ply-HA stem and studied its immunoprotective effect in mice infection models. The results showed that intramuscular immunization of ΔA146Ply-HA stem without adjuvant could induce specific antibodies against HA stem and specific CD4+ T and CD8+ T cellular immunity in BALB/c and C57BL/6 mice, which could improve the survival rate of mice infected with IAV and co-infected with S. pneumoniae, but the protective effect on BALB/c mice was better than that on C57BL/6 mice. ΔA146Ply-HA stem serum antibody could protect BALB/c and C57BL/6 mice from IAV, and recognized HA polypeptides of H3N2, H5N1, H7N9, and H9N2 viruses. Moreover, ΔA146Ply-HA stem intramuscular immunization had a high safety profile with no obvious toxic side effects. The results indicated that coupling ΔA146Ply with influenza protein as a vaccine was a safe and effective strategy against the IV and secondary S. pneumoniae infection.

rM2e-ΔPly protein immunization induces protection against influenza viruses and its co-infection with Streptococcus pneumoniae in mice.

Current seasonal influenza A virus (IAV) vaccines only protect against specific virus and require annual reconstitution to accommodate the viral mutations. A universal influenza vaccination that protects against all IAV strains is urgently needed. The influenza matrix protein 2 ectodomain (M2e) is a potential universal IAV vaccine candidate, but it has a low immunogenicity. ΔA146Ply was proved to be an effective protein adjuvant. Therefore, ΔA146Ply was used as an adjuvant of M2e in this study to evaluate its protective effect against IAV-related infection. Herein, a novel rM2e protein containing multiple M2e originated from different species of IAV was constructed and expressed in Escherichia coli (E. coli). Meanwhile, we also constructed and expressed the rM2e-ΔPly protein in E. coli. These proteins were administered intramuscularly to BALB/c mice. rM2e-ΔPly protein induces higher levels of humoral and cellular responses compared with their comprising protein mixture or rM2e alone. rM2e-ΔPly protein enhances the survival rate, reduces viral loads and inflammatory response in lung challenged with PR8. The serum induced by rM2e-ΔPly protein can protect against PR8 by passive immunity and cross-react with multiple M2e peptides derived from different IAV subtypes. After IAV and Streptococcus pneumoniae (S. pneumoniae) co-infection, rM2e-Ply protein can improve survival, lower viral and bacterial burdens, and diminish inflammatory response in the lungs. These results demonstrate that rM2e-ΔPly protein can significantly protect against influenza virus, IAV and S. pneumoniae co-infection, indicating that rM2e-ΔPly protein has the potential to become a universal influenza vaccine.

IL-4 plays an essential role in DnaJ-ΔA146Ply-mediated immunoprotection against Streptococcus pneumoniae in mice.

The fusion protein DnaJ-ΔA146Ply is protective against pneumococcal infections in mice. However, we found that immunized IL-4-/- mice showed significant lower survival rates and higher bacterial loads than did wild-type (WT) mice after being challenged. We explored the role of IL-4 in the protective immunity conferred by DnaJ-ΔA146Ply. Our results showed that there were no significant differences in antibody titers between immunized WT mice and IL-4-/- mice. The bacterial loads of passively immunized IL-4-/- mice were significantly higher than those of WT mice, while mice immunized with anti-DnaJ-ΔA146Ply serum from WT and IL-4-/- mice showed similar capacity for bacterial clearance. DnaJ-ΔA146Ply-dependent phagocytosis of IL-4-/- neutrophils was significant decreased compared with that of WT neutrophils. The levels of Syk and phosphor-Syk in IL-4-/- neutrophils were decreased compared with those in WT neutrophils. Additionally, Splenocytes in IL-4-/- mice triggered significantly higher levels of IFN-γ and IL-17A than did splenocytes in WT mice. Taken together, our findings illustrate that IL-4 deficiency does not influence the antibody production or antibody effect, but change the cellular immune response induced by DnaJ-ΔA146Ply. Additionally, IL-4 can enhance the antibody-dependent phagocytosis of neutrophils partially by activating Syk and participate in the protective immunity induced by DnaJ-ΔA146Ply.

Biomineralization improves the stability of a Streptococcus pneumoniae protein vaccine at high temperatures.

Aim: Protein vaccines have been the focus of research for vaccine development due to their safety record and facile production. Improving the stability of proteins is of great significance to the application of protein vaccines. Materials & methods: Based on the proteins pneumolysin and DnaJ of Streptococcus pneumoniae, biomineralization was carried out to prepare protein nanoparticles, and their thermal stability was tested both in vivo and in vitro. Results: Mineralized nanoparticles were formed successfully and these calcium phosphate-encapsulated proteins were resistant to proteinase K degradation and were thermally stable at high temperatures. The mineralized proteins retained the immunoreactivity of the original proteins. Conclusion: Mineralization technology is an effective means to stabilize protein vaccines, presenting a safe and economical method for vaccine administration.

Streptococcus pneumoniae Endopeptidase O Promotes the Clearance of Staphylococcus aureus and Streptococcus pneumoniae via SH2 Domain-Containing Inositol Phosphatase 1-Mediated Complement Receptor 3 Upregulation.

Increasing evidences demonstrate that microorganism and their products protect against bacterial and viral pathogens through various mechanisms including immunomodulation. Streptococcus pneumoniae endopeptidase O (PepO), a pneumococcal virulence protein, has been proven to enhance the phagocytosis of Staphylococcus aureus and Streptococcus pneumoniae by macrophages in our previous study, where we detected the down regulation of SH2 domain-containing inositol phosphatase 1 (SHIP1) and the up regulation of complement receptor 3 (CR3) in PepO-stimulated macrophages. In the present study, using SHIP1 over-expression plasmid and CR3 siRNA, we proved that the down regulation of SHIP1 and the up regulation of CR3 mediate the enhanced phagocytosis of S. aureus and S. pneumoniae by PepO-stimulated macrophages. The down regulation of SHIP1 also mediates the up regulation of CR3. To further determine whether PepO protects against respiratory pathogens, we constructed a mouse model with intranasal infection of S. aureus or S. pneumoniae and found that PepO significantly promoted their clearance. The down regulation of SHIP1 and the up regulation of CR3 also play a role in this process. This study provides a new preventive and therapeutic option for respiratory infectious diseases and lays the theoretical basis for the development of PepO as an immunomodulation agent.

IL-6 During Influenza-Streptococcus pneumoniae Co-Infected Pneumonia-A Protector.

Understanding of pathogenesis and protection mechanisms underlying influenza-Streptococcus pneumoniae co-infection may provide potential strategies for decreasing its high morbidity and mortality. Interleukin-6 (IL-6) is an important cytokine that acts to limit infection-related inflammation; however, its role in co-infected pneumonia remains unclear. Here we show that the clinically relevant co-infected mice displayed dramatically elevated IL-6 levels; which was also observed in patients with co-infected pneumonia. IL-6-/- mice presented with increased bacterial burden, early dissemination of bacteria to extrapulmonary sites accompanied by aggravated pulmonary lesions and high mortality when co-infection. This protective function of IL-6 is associated with cellular death and macrophage function. Importantly, therapeutic administration of recombinant IL-6 protein reduced cells death in BALF, and enhanced macrophage phagocytosis through increased MARCO expression. This protective immune mechanism furthers our understanding of the potential impact of immune components during infection and provides potential therapeutic avenues for influenza-Streptococcus pneumoniae co-infected pneumonia.