Streptococcus pneumoniae is a conditionally pathogenic bacteria that colonizes the nasopharynx of 27% to 65% of children and 10% of adults. Capsular polysaccharides are the most critical virulence factor of S. pneumoniae, and nonencapsulated strains are usually non-pathogenic. Previous studies have shown that glucose regulates capsule synthesis. To investigate the mechanism of carbon metabolism regulatory factors CcpA and HPr regulating capsule synthesis in the presence of glucose as the sole carbon source, we constructed deletion mutants (D39ΔccpA and ΔptsH) and complemented strains (D39ΔccpA::ccpA and ΔptsH::ptsH). In this study, we found that the promoting effect of capsule synthesis by glucose disappeared after the deletion of ccpA and ptsH, and demonstrated that the protein CcpA regulates capsule synthesis by binding to the cps promoter and altering the transcription level of the cps gene cluster. Increased glucose concentration up-regulated the level of HPr-Ser46~P, which enhanced the binding ability of CcpA to the DNA sequence of the cps promoter, thus promoting capsule synthesis. HPr also has a regulatory effect on capsule synthesis. These insights reveal a new synthesis mechanism of capsular polysaccharide and provide a new strategy of antibacterial drugs for S. pneumoniae.

Uracil metabolism is an important step in the growth and metabolism of Streptococcus pneumoniae, and pyrimidine nucleotides play an important role in the expression and production of S. pneumoniae capsules. MgaSpn(spd_1587),as a transcriptional ragulator of host environment adaptation, regulates the biosynthesis of the capsules and phosphorylcholine. However, the underlying regulation mechanism between uracil metabolism and biosynthesis of capsules remains incompletely understood. Here, we first described the relationship between uracil metabolism and capsule expression via the pyrR gene(spd_1134) in S. pneumoniae. Electrophoretic mobility-shift assays (EMSAs) and DNase I footprinting assays showed a direct interaction between MgaSpn and the pyrR promoter (PpyrR) at two specific binding sites. MgaSpn negatively regulated capsule production through pyrR as confirmed by complementing pyrR expression in D39ΔmgaSpnΔpyrR (mgaSpn and pyrR double-defective strain). Virulence experiments showed that the MgaSpn-pyrR interaction was necessary for both pneumococcal colonization and invasive infection. For the first time, the present study demonstrated that the de novo synthesis gene pyrR of S. pneumoniae is regulated by the MgaSpn transcriptional regulator.Taken together,these results provide an insight into the regulation of capsule production mediated by uracil metabolism and its important roles in pneumococcal pathogenesis.

Secondary bacterial pneumonia is a common cause of influenza infection associated deaths. This study investigated the potential therapeutic role of inhibiting toll-like receptor 7 (TLR7) in influenza-associated Staphylococcus aureus (S.aureus) coinfection. Both TLR7 deficiency and TLR7 antagonist IRS661 treatment significantly improved survival in mice coinfected with influenza virus (PR8) and S.aureus while significantly reducing pulmonary damage as evidenced by decreased total protein (TP) and lactate dehydrogenase (LDH) levels in bronchoalveolar lavage fluid (BALF) and serum alanine aminotransferase (ALT) and urea nitrogen (UREA) levels. Notably, both interventions significantly maintained the accumulated inflammatory infiltration observed within 6 h post-coinfection, and up to 48 h. Mechanistically, IRS661 enhanced macrophage phagocytosis, bactericidal activity, and reactive oxygen species (ROS) production ex vivo and in vivo. This effect was correlated with reduced c-Jun N-terminal kinase (JNK) phosphorylation. Furthermore, during early infection, this modulation was accompanied by significantly reduced levels of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, but a concomitant increase in IFN-γ. Transcriptomic analysis revealed IRS661 modulated pathways involved in cell adhesion, metabolism, and oxidative stress response. These findings suggested that TLR7 antagonism, specifically through IRS661, represented a promising therapeutic strategy for influenza-associated secondary bacterial infections, paving further investigations into its clinical translation potential.

OBJECTIVE:Pneumococcal meningitis is a serious infectious disease with a high mortality rate and a global presence, and survivors have different degrees of neurological sequelae as a consequence of the host response to the infection. Progranulin (PGRN) is a multifunctional autocrine growth factor that is also a major immunoregulator. We want to investigate the role for PGRN in Pneumococcal meningitis in vivo and in vitro. METHOD:Mouse and cell models were established to explore the protective effect and mechanism of PGRN against pneumococcal meningitis. RESULTS:Progranulin plays a protective role in pneumococcal meningitis by inhibiting pyroptosis. Pyroptosis resulted from exposure of BV-2 cells to the bacterium and this was confirmed in the in vivo model. Administration of the NLRP3 inflammasome inhibitor MCC950 to mice prior to infection inhibited pyroptosis and protected PGRN -/- mice and BV-2 cell model from meningitis. CONCLUSION:This study implicates a protective role for PGRN in pneumococcal meningitis by inhibiting pyroptosis, indicating that PGRN may have therapeutic potential.

Streptococcus pneumoniae capsular polysaccharide (CPS) is a crucial virulence factor for this pathogenic bacterium and is partially under transcriptional control. In this study, we used electrophoretic mobility shift assays and DNA enzyme footprinting to identified the hypothetical protein SPD_0410 as a negative regulator of cps locus. Our results showed that the D39Δspd0410 mutant strain exhibited significantly elevated CPS levels compared to the parental strain D39s. SPD_0410 directly binds at two specific sites on the cps promoter. The regulatory effect of SPD_0410 on CPS was weakened after the mutation of specific binding sites in the promoter. RNAseq analysis revealed that the deletion of spd0410 led to alterations in glucose metabolism. However, the altered glucose levels appeared to eliminate the regulation of CPS synthesis by SPD_0410. Deleting the spd0410 gene resulted in higher invasion and phagocytic resistance of bacteria and in vivo mouse experiments confirmed that D39Δspd0410 caused more severe systemic disease than the parental strain D39s. Our results indicated that SPD_0410 negatively regulates the synthesis of S. pneumoniae capsules and can directly alter pneumococcal virulence.

AIMS:Group 2 innate lymphoid cells (ILC2s) play key roles in allergic asthma development. We have previously discovered that CD5 antigen-like protein (CD5L) can inhibit allergic airway inflammation. In this study, we investigate the effect of CD5L on ILC2s and the underlying mechanism. RESULTS:Our findings demonstrated that CD5L suppresses allergic airway inflammation by inhibiting ILC2s. CD5L inhibited NF-κB, MAPK, and PI3K-AKT pathways in ILC2s, thus reducing interleukin (IL)-5 and IL-13 production. CD5L increased the level of lysophosphatidylcholine (lysoPC) in ILC2s through the transforming growth factor beta (TGF-β) signaling pathway. The elevated lysoPC further induced reactive oxygen species (ROS) production in ILC2s, and the increased ROS fed back to increase the level of lysoPC. The accumulated ROS induced ILC2 apoptosis. The scavenger receptor CD36 mediated the inhibitory effect of CD5L on ILC2s and allergic airway inflammation. Finally, CD5L was shown to be potential therapeutic for allergic asthma. INNOVATION:This study is the first to demonstrate that CD5L suppresses allergic airway inflammation by inhibiting ILC2 responses. It is the initial discovery that CD5L promotes ILC2 apoptosis, whereas CD5L was previously recognized as an apoptosis inhibitor. The regulation of TGF-β signaling pathway on lysoPC is demonstrated for the first time. CONCLUSION:This study demonstrated that CD5L inhibits ILC2 activation and induces ILC2 apoptosis, thereby suppressing allergic airway inflammation. CD5L can serve as a novel therapeutic strategy for allergic asthma. Antioxid. Redox Signal. 44, 393-409.

The global transcriptional regulator MgaSpn is a significant virulence factor of Streptococcus pneumoniae. In our previous study, we found that MgaSpn is a regulator of bacterial virulence by modulating the levels of phosphorylcholine (ChoP) and capsular polysaccharides (CPS) on the surface of S. pneumoniae. Here, we report for the first time that pcpA expression was significantly increased in mgaSpn deletion strains and significantly decreased when mgaSpn was overexpressed. Electrophoretic mobility-shift and DNase I footprinting assays confirmed that MgaSpn interacts with the pcpA promoter (PpcpA) at two specific binding sites. Virulence experiments demonstrated that the interaction between MgaSpn and PcpA is necessary for pneumococcal colonization and invasive infection. Western blot analysis indicated that iron concentration can influences the regulation of PcpA expression via MgaSpn. In summary, these results revealed that MgaSpn regulates PcpA and plays a significant role in pneumococcal pathogenesis.

The persistent challenge of sepsis-related mortality underscores the necessity for deeper insights. Our multicenter, cross-age cohort study identified insulin-like growth factor binding protein 6 (IGFBP6) as a critical regulator in sepsis diagnosis, prognosis, and mortality risk evaluation. Mechanistically, IGFBP6 engages in IGF-independent binding to prohibitin2 (PHB2) on epithelial cells, driving PHB2 tyrosine phosphorylation during sepsis. This process disrupts STAT1 phosphorylation, nuclear translocation, and its recruitment to the CCL2 promoter, ultimately impairing CCL2 transcription and macrophage chemotaxis. Crucially, PHB2 silencing via siPHB2 and STAT1 activation using 2-NP restored CCL2 expression in vitro and in vivo, improving bacterial clearance and survival in septic mice. Concurrently, IGFBP6 compromised macrophage bactericidal activity by inhibiting Akt phosphorylation, reducing ROS/IL-1β production and phagocytic capacity - defects reversible by Akt agonist SC79. Collectively, IGFBP6 emerges as an endogenous driver of sepsis pathogenesis, positioning it as a dual diagnostic biomarker and therapeutic target. Intervention strategies targeting IGFBP6-mediated signaling may offer transformative approaches for sepsis management.

Neonatal necrotizing enterocolitis (NEC) is a common intestinal disease that threatens the lives of newborns and is characterized by ischemic necrosis of the small intestine and colon. As early diagnosis of NEC improves prognosis, the identification of new or complementary biomarkers is of great importance. In this study, we evaluate the diagnostic value of CCL3 in NEC and compare its effectiveness with other commonly used biomarkers, such as procalcitonin (PCT) and C-reactive protein (CRP). Serum samples were collected from 64 patients with NEC and 38 jaundice neonatal controls. Before initiating therapy, blood samples for the whole blood count, CRP, PCT and CCL3 were obtained from all neonates. Receiver operating characteristic (ROC) curve and multivariate logistic regression analyses were performed. Department of Clinical Laboratory, Children’s Hospital of Chongqing Medical University. The serum CCL3 level of the NEC group was significantly higher than that of the Control group. The ROC area under the curve (AUC) was 0.8614 (95

Background. Sepsis is characterized by a life-threatening syndrome caused by an unbalanced host response to infection. Fibroblast growth factor 8 (FGF8) has been newly identified to play important roles in inflammation and innate immunity, but its role in host response to sepsis is undefined. Methods. A cecal ligation and puncture (CLP)-induced mouse sepsis model was established to evaluate the immunomodulatory function of FGF8 during sepsis. The underlying molecular mechanisms were elucidated by cell models using relevant molecular biology experiments. The clinical value of FGF8 in the adjuvant diagnosis of sepsis was evaluated using clinical samples. Results. FGF8 protein concentrations were elevated in CLP-induced septic mice compared to controls. In vivo, FGF8 blockade using anti-FGF8 antibody significantly increased mortality and bacterial burden and was paralleled by significantly aggravated tissue injury after CLP. Therapeutic administration of recombinant FGF8 (rFGF8) improved the bacterial clearance and mortality of septic mice in a FGFR1-dependent manner. In vitro, FGF8 directly enhanced bacterial phagocytosis and killing of macrophages by enhancing the phosphorylation of the ERK1/2 signaling pathway, which could be abrogated with the ERK1/2 pathway inhibitor U0126. Clinically, serum FGF8 levels in both adult and pediatric patients with sepsis in an intensive care unit were significantly higher than those in healthy controls. Conclusions. These results present a previously unrecognized role of FGF8 in improving survival of sepsis by enhancing host immune defense. Therefore, targeting FGF8 may provide new strategies for the diagnosis and immunotherapy of sepsis.

Candidemia is a severe disease with high mortality in both intensive care unit (ICU) and non-ICU settings. Considering that progranulin (PGRN) is a potential therapeutic target for the candidemia caused by C. albicans, we determined the serum level of PGRN after candidemia and evaluated its association with mortality. A retrospective discovery cohort (62 patients) and a validation cohort (70 patients) were enrolled. Blood was collected on day of first blood culture positivity for C. albicans, and serum PGRN levels were then measured. In the discovery cohort, all serum PGRN studied were expressed at higher levels in candidemia patients than in bacteremia patients and healthy volunteers, non-survivors presented with significantly higher serum PGRN concentrations when compared with survivors. Serum PGRN concentration was associated with 30-day mortality and patients at a higher risk of death showed higher serum PGRN levels. These results were confirmed in the independent validation cohort. Interestingly, in vitro study demonstrated that macrophages, neutrophils and lymphocytes may be the major source of PGRN production after C. albicans infection instead of epithelial cells. Our findings highlight that serum PGRN appears as a biomarker in candidemia patients and as a promising tool for mortality risk stratification in managing candidemia.

Defining next-generation immune therapeutics for the treatment of sepsis will involve biomarker-based therapeutic decision-making. Bone morphogenetic protein 9 (BMP9) is a cytokine in the transforming growth factor–β superfamily. Here, circulating BMP9 concentrations were quantified in two independent cohorts of patients with sepsis. Decreased concentrations of serum BMP9 were observed in the patients with sepsis at the time of admission as compared with healthy controls. Concentrations of BMP9 at the time of admission were also associated with 28-day mortality, because patients with sepsis at a higher risk of death had lower BMP9 concentrations. The mechanism driving the contribution of BMP9 to host immunity was further investigated using in vivo murine sepsis models and in vitro cell models. We found that BMP9 treatment improved outcome in mice with experimental sepsis. BMP9-treated mice exhibited increased macrophage influx into the peritoneal cavity and more efficient bacterial clearance than untreated mice. In vitro, BMP9 promoted macrophage recruitment, phagocytosis, and subsequent bacterial killing. We further found that deletion of the type 1 BMP receptor ALK1 in macrophages abolished BMP9-mediated protection against polymicrobial sepsis in vivo. Further experiments indicated that the regulation of macrophage activation by the BMP9-ALK1 axis was mainly mediated through the suppressor of mother against decapentaplegic 1/5 signaling pathway. Together, these results suggest that BMP9 can both serve as a biomarker for patient stratification with an independent prognostic value and be developed as a host-directed therapy for sepsis.

Current pneumococcal vaccines, including the pneumococcal polysaccharide (PPV23) and conjugate (PCV13) vaccines, offer protection against specific serotypes but pose risks of serotype replacement that can alter the composition of the nasopharyngeal microbiota. To address this challenge, a novel strategy has been proposed to provide effective protection without disrupting the colonization of other bacterial populations. In our study, we found that subcutaneous immunization with recombinant peptidoglycan N-acetylglucosamine deacetylase A (rPgdA) elicited robust humoral and cellular immune responses, significantly reducing the invasion of Streptococcus pneumoniae in the lungs without affecting nasopharyngeal carriage. Furthermore, rPgdA antisera were shown to diminish bacterial invasion of lung epithelial cells in vitro. Notably, sera from patients with invasive pneumococcal infections exhibited higher levels of antibodies against the PgdA protein compared to sera from healthy adults, suggesting that a natural immune response to this protein occurs during infection. These results suggest a promising new target for the development of pneumococcal vaccines.

The detoxified pneumolysin derivative ΔA146Ply has been proven to have a direct anti-triple negative breast cancer effect by our group, but its work model remains unclear. In this study, we focused on its ability to inhibit triple-negative breast cancer metastasis. We found that ΔA146Ply suppressed the migration and invasion of triple-negative breast cancer cells by activating mannose receptor and toll-like receptor 4. Their activation triggers the activation of the mammalian target of rapamycin signalling, sequentially leading to autophagy, transforming growth factor-β1, and epithelial-mesenchymal transition inhibition. Furthermore, the combination of doxorubicin and ΔA146Ply significantly inhibited triple-negative breast cancer progression and prolonged survival in tumour-bearing mice. Taken together, our study provides an alternative microbiome-based mannose receptor-targeted therapy for triple-negative breast cancer and a novel theoretical and experimental basis for the downstream signalling pathway of the mannose receptor.

Competence development is essential for bacterial transformation since it enables bacteria to take up free DNA from the surrounding environment. The regulation of teichoic acid biosynthesis is tightly controlled during pneumococcal competence; however, the mechanism governing this regulation and its impact on transformation remains poorly understood. We demonstrated that a defect in lipoteichoic acid ligase (TacL)-mediated lipoteichoic acids (LTAs) biosynthesis was associated with impaired pneumococcal transformation. Using a fragment of tacL regulatory probe as bait in a DNA pulldown assay, we successfully identified several regulatory proteins, including ComE. Electrophoretic mobility shift assays revealed that phosphomimetic ComE, but not wild-type ComE, exhibited specific binding to the probe. DNase I footprinting assays revealed the specific binding sequences encompassing around 30 base pairs located 31 base pairs upstream from the start codon of tacL. Expression of tacL was found to be upregulated in the ΔcomE strain, and the addition of exogenous competence-stimulating peptide repressed the tacL transcription in the wild-type strain but not the ΔcomE mutant, indicating that ComE exerted a negative regulatory effect on the transcription of tacL. Mutation in the JH2 region of tacL upstream regulatory sequence led to increased LTAs abundance and displayed higher transformation efficiency. Collectively, our work identified the regulatory mechanisms that control LTAs biosynthesis during competence and thereby unveiled a repression mechanism underlying pneumococcal transformation.

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 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. 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. Our research identified a possible macrophage-based TNBC immunotherapeutic approach and suggested a novel anticancer immunoregulatory molecular called PepO.

Cytokines play a critical role in the development of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Here we investigated whether IL-27 was elevated in patients with ALI/ARDS and its potential clinical significance. Bronchoalveolar lavage (BAL) and serum samples were obtained from 58 ALI/ARDS patients, and 25 control healthy volunteers. IL-27 and other inflammatory mediators were measured in BAL and serum by ELISA. Besides, a mouse model of cecal ligation and puncture (CLP)-induced lung inflammation/injury was established, and serum, BAL fluid and tissues were collected for analyses in the presence or absence of IL-27 neutralizing antibodies. BAL IL-27 was found to be significantly higher in patients with ALI/ARDS than that in controls, particularly of pulmonary origin; serum IL-27 was also significantly higher. Increased IL-27 was associated with markers of inflammation, and correlated with disease severity of patients in ALI/ARDS. In a mouse model of CLP-induced lung inflammation/injury, elevated IL-27 levels were observed in the lung, serum, and BAL fluids. IL-27 neutralizing antibody treatment reduced pulmonary inflammation and lung injury and improved mouse survival in response to CLP. Therefore, IL-27 is a critical cytokine in ALI/ARDS and inhibition of IL-27 may open a promising approach for ALI/ARDS patients.

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.