In this work, we present a simple and rapid method to synthesize red luminescent gold nanoclusters (AuNCs) with high quantum yield (QY, ~16%), excellent photostability and biocompatibility. Next, we fabricated a solid membrane by loading the as-prepared AuNCs in an agar matrix. Different from nanomaterials dispersed in solution, the AuNCs-based solid membrane has distinct advantages including convenience of transportation, while still maintaining strong red luminescence, and relatively long duration storage without aggregation. Taking hydrogen peroxide (H₂O₂) as a typical example, we then employed the AuNCs as a luminescent probe and investigated their sensing performance, either in solution phase or on a solid substrate. The detection of H₂O₂ could be achieved in wide concentration ranges over 805 nM-1.61 mM and 161 μM-19.32 mM in solution and on a solid membrane, respectively, with limits of detection (LOD) of 80 nM and 20 μM. Moreover, the AuNCs-based membrane could also be used for visual detection of H₂O₂ in the range of 0-3.22 mM. In view of the convenient synthesis route and attractive luminescent properties, the AuNCs-based membrane presented in this work is quite promising for applications such as optical sensing, fluorescent imaging, and photovoltaics.

Due to the widespread abusage of antibiotics, antibiotic-resistance in Streptococcus pneumoniae (S. pneumoniae) has been increasing quickly in recent years, and it is obviously urgent to develop new types of antibiotics. Two-component systems (TCSs) are the major signal transduction pathways in bacteria and have emerged as potential targets for antibacterial drugs. Among the 13 pairs of TCSs proteins presenting in S. pneumoniae, VicR/K is the unique one essential for bacterium growth, and block agents to which, if can be found, may be developed as effective antibiotics against S. pneumoniae infection.

Streptococcus pneumoniae is a major pathogen leading to substantial morbidity and mortality in children under 5 years of age. Vaccination is an effective strategy to prevent S. pneumoniae infection. SPY1 is a pneumococcal vaccine candidate strain obtained in our previous study. To improve its stability and immunogencity, in this study, we constructed the SPY1ΔlytA strain that lacks autolysin activity and was coated with an artificial exterior surface calcium phosphate shell by in situ mineralization. The resulting strain SPY1ΔlytACaPi displayed enhanced thermal stability enabling storage at 37°C for 1 week. Furthermore, mucosal and subcutaneous immunization with the SPY1ΔlytACaPi strain induced better protective effects than SPY1ΔlytA in anti-colonization after challenging with 19F and anti-invasion by D39 in mice. Subcutaneous immunization with SPY1ΔlytACaPi elicited higher IgG level while mucosal immunization primarily elicited an immune response which is supposed to be related to Th17 cells. Taken together, the mineralized strain may be a promising candidate for an attenuated S. pneumoniae vaccine.

As a heat shock protein, DnaJ plays an important role in the pathogenesis of pneumococcal infection. However, how the virulence factor-DnaJ elicits host natural immunity still remains unclear. In this study, we investigated the effects of dnaJ deficiency in Streptococcus pneumoniae (S. pneumoniae) on bacterial virulence, and further explored the related molecular mechanisms in vivo and in vitro. By generating dnaJ deficient mutant (ΔdnaJ), the virulence and colonization were detected in murine pneumonia and sepsis models in vivo. Compared with wild-type parent strain, the abilities of rapid colonization and induction of inflammatory responses of ΔdnaJ in mouse lungs were significantly impaired. Simultaneously, recombinant DnaJ purified from E. coli expression system (rDnaJ) induced macrophage strain RAW264.7 to secrete IL-6 by activation of PI3K and JNK signal pathways, which were confirmed by the specific signaling inhibitors. In conclusion, DnaJ, a novel virulence protein, was essential for the virulence and colonization of S. pneumoniae and induced pro-inflammatory cytokine production in macrophages through PI3K/JNK.

Streptococcus pneumoniae is an opportunistic pathogen that can alter its cell surface phenotype in response to the host environment. We demonstrated that the transcriptional regulator FabT is an indirect regulator of capsular polysaccharide, an important virulence factor of Streptococcus pneumoniae. Transcriptome analysis between the wild-type D39s and D39ΔfabT mutant strains unexpectedly identified a differentially expressed gene encoding a site-specific recombinase, PsrA. PsrA catalyzes the inversion of 3 homologous hsdS genes in a type I restriction-modification (RM) system SpnD39III locus and is responsible for the reversible switch of phase variation. Our study demonstrated that upregulation of PsrA in a D39ΔfabT mutant correlated with an increased ratio of transparent (T) phase variants. Inactivation of the invertase PsrA led to uniform opaque (O) variants. Direct quantification of allelic variants of hsdS derivatives and inversions of inverted repeats indicated that the recombinase PsrA fully catalyzes the inversion mediated by IR1 and IR3, and FabT mediated the recombination of the hsdS alleles in PsrA-dependent and PsrA-independent manners. In addition, compared to D39s, the ΔfabT mutant exhibited reduced nasopharyngeal colonization and was more resistant to phagocytosis and less adhesive to epithelial cells. These results indicated that phase variation in the ΔfabT mutant also affects other cell surface components involved in host interactions. IMPORTANCE Streptococcus pneumoniae is a major human pathogen, and its virulence factors and especially the capsular polysaccharide have been extensively studied. In addition to virulence components that are present on its cell surface that directly interact with the host, S. pneumoniae undergoes a spontaneous and reversible phase variation that allows survival in different host environments. This phase variation is manipulated by the recombination of allelic hsdS genes that encode the sequence recognition proteins of the type I RM system SpnD39III locus. The recombination of hsdS alleles is catalyzed by the DNA invertase PsrA. Interestingly, we found the opaque colony morphology can be reversed by inactivation of the transcriptional regulator FabT, which regulates fatty acid biosynthesis. Inactivation of FabT leads to a significant decrease in capsule production and systematic virulence, but these phase variations do not correlate with the capsule production. This phase variation is mediated via the upregulated invertase PsrA in the ΔfabT mutant. These results identify an unexpected link between the specific phase variations and FabT that strongly suggests an underlying mechanism regulating the DNA invertase PsrA.

Global transcriptional regulators are prevalent in gram-positive pathogens. The transcriptional regulators of the Mga/AtxA family regulate target gene expression by directly binding to the promoter regions, that results in the coordinated expression of virulence factors. The spd_1587 gene of Streptococcus pneumoniae strain D39 encodes MgaSpn, which shares sequence similarity with global transcriptional regulators of the Mga/AtxA family. In this study, we demonstrated that MgaSpn regulates the biosynthesis of the capsule and phosphorylcholine, which play key roles in disease severity in S. pneumoniae infections. MgaSpn directly binds to the cps and lic1 promoters and affects the biosynthesis of the capsule and phosphorylcholine. MgaSpn binds to two specific sites on the promoter of cps, one of which contains the -35 box of the promoter, with high affinity. Consistently, low-molecular-weight capsule components were observed in the mgaSpn-null mutant strain. Moreover, we found that phosphorylcholine content was notably increased in the unencapsulated mgaSpn mutant strain. The mgaSpn null mutant caused more severe systemic disease than the parental strain D39. These findings indicate that the pneumococcal MgaSpn protein can inhibit capsule and phosphorylcholine production, thereby affecting the virulence of S. pneumoniae.

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.

Early microbial exposure is associate with protective allergic asthma. We have previously demonstrated that Streptococcus pneumoniae aminopeptidase N (PepN), one of the pneumococcal components, inhibits ovalbumin (OVA) -induced airway inflammation in murine models of allergic asthma, but the underlying mechanism was incompletely determined.

Mast cells participate in immune responses by releasing potent immune system modifiers via degranulation. Due to currently reported controversial roles of mast cells in Streptococcus pneumoniae infections, this study aimed to determine the role and mechanism of mast cells in clearing S. pneumoniae in mice.

Identification of new therapeutic targets for the treatment of sepsis is imperative. We report here that cytokine IL-28 (IFN-λ) levels were elevated in clinical and experimental sepsis. Neutralization of IL-28 protected mice from lethal sepsis induced by cecal ligation and puncture (CLP), which was associated with improved bacterial clearance and enhanced neutrophil infiltration. Conversely, administration of recombinant IL-28 aggravated mortality, facilitated bacterial dissimilation and limited neutrophil recruitment, in the model of sepsis induced by CLP. This study defines IL-28 as a detrimental mediator during sepsis and identifies a potential therapeutic target for the immune therapy in sepsis.

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.

Subunit vaccines that are poorly immunogenic are often combined with adjuvants for immunization. Our previous research identified a pneumolysin variant (ΔA146Ply), a Toll-like receptor 4 agonist, that was an effective adjuvant in the protection of fusion protein DnaJ-ΔA146Ply against mucosal Streptococcus pneumoniae infections. For pneumococcal vaccines, World Health Organization recommend injection as a regular vaccination approach. Subcutaneous immunization is a common and effective method of injection, so we explored the immunity mechanism of subcutaneous immunization with DnaJ-ΔA146Ply. We found that mice immunized subcutaneously with fusion proteins ΔA146Ply-DnaJ and DnaJ-ΔA146Ply produced a higher anti-DnaJ IgG titer than when DnaJ alone was administered. DnaJ-ΔA146Ply induced both B-cell and T-cell-dependent protection against both colonization and lethal pneumococcal infections. Levels of IFN-γ, IL-4, and IL-17A were also elevated in DnaJ-ΔA146Ply immunized mice. However, all these effects were negated in TLR4-/- mice compared to WT mice immunized with DnaJ-ΔA146Ply. B-cell-deficient μMT mice, nude mice, IFN-γ-/-, and IL-4-/- mice immunized with DnaJ-ΔA146Ply could not resist infection with pneumococci. IL-17A-/- and TLR4-/- mice did not benefit from DnaJ-ΔPly immunization in colonization experiments although their survival was not impaired compared with WT mice. Collectively, our data indicated that ΔA146Ply can be a potential subcutaneous adjuvant, and the DnaJ-ΔA146Ply fusion protein induces both humoral and cellular immune response to resist S. pneumoniae infection. The protective effect of colonization also depends on TLR4.

Increasing evidence demonstrates that microorganisms and their products can modulate host responses to cancer therapies and contribute to tumor shrinkage via various mechanisms, including intracellular signaling pathways modulation and immunomodulation. Detoxified pneumolysin derivative ΔA146Ply is a pneumolysin mutant lacking hemolytic activity. To determine the antitumor activity of ΔA146Ply, the combination of ΔA146Ply and berbamine, a well-established antitumor agent, was used for breast cancer therapy, especially for triple-negative breast cancer. The efficacy of the combination therapy was evaluated in vitro using four breast cancer cell lines and in vivo using a synergistic mouse tumor model. We demonstrated that in vitro, the combination therapy significantly inhibited cancer cell proliferation, promoted cancer cell apoptosis, caused cancer cell-cycle arrest, and suppressed cancer cell migration and invasion. In vivo, the combination therapy significantly suppressed tumor growth and prolonged the median survival time of tumor-bearing mice partially through inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and activating systemic antitumor immune responses. The safety analysis demonstrated that the combination therapy showed no obvious liver and kidney toxicity to tumor-bearing mice. Our study provides a new treatment option for breast cancer and lays the experimental basis for the development of ΔA146Ply as an antitumor agent.

Sepsis remains a highly lethal disease, whereas the precise reasons for death remain poorly understood. Prokineticin2 is a secreted protein that regulates diverse biological processes. Whether prokineticin2 is beneficial or deleterious to sepsis and the underlying mechanisms remain unknown.

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.

The initial clinical manifestations and abdominal imaging findings of neonates with necrotising enterocolitis (NEC) and food protein-induced enterocolitis syndrome (FPIES) are sometimes similar; however, their prognosis and therapies are different. We aimed to evaluate the utility of interleukin (IL)-27 as a differentiation marker between NEC and highly suspected early onset (HSEO)-FPIES.

Vaccine effectiveness is mainly determined by the mechanism mediating protection, emphasizing the importance of unraveling the protective mechanism for novel pneumococcal vaccine development. We previously demonstrated that the regulatory T cell (Treg) immune response has a protective effect against pneumococcal infection elicited by the live-attenuated pneumococcal vaccine SPY1. However, the mechanism underlying this protective effect remains unclear. In this study, a short synthetic peptide (P17) was used to downregulate Tregs during immunization and subsequent challenges in a mouse model. In immunized mice, increase in immune cytokines (IL-12p70, IL-4, IL-5, and IL-17A) induced by SPY1 were further upregulated by P17 treatment, whereas the decrease in the infection-associated inflammatory cytokine TNF-α by SPY1 was reversed. P17 also inhibited the increase in the immunosuppressive cytokine IL-10 and inflammatory mediator IL-6 in immunized mice. More severe pulmonary injuries and more dramatic inflammatory responses with worse survival in P17-treated immunized mice indicated the indispensable role of the Treg immune response in protection against pneumococcal infection by maintaining a balance among acquired immune responses stimulated by SPY1. Further studies revealed that the significant elevation of active transforming growth factor β (TGF-β)1 by SPY1 vaccination activated FOXP3, leading to increased frequencies of CD4+CD25+Foxp3+ T cells. Moreover, SPY1 vaccination elevated the levels of Smad2/3 and phosphor-Smad2/3 and downregulated the negative regulatory factor Smad7 in a time-dependent manner during pneumococcal infection, and these changes were reversed by P17 treatment. These results illustrate that SPY1-stimulated TGF-β1 induced the generation of SPY1-specific Tregs via the Smad2/3 signaling pathway. In addition, SPY1-specific Tregs may participate in protection via the enhanced expression of PD-1 and CTLA-4. The data presented here extend our understanding of how the SPY1-induced acquired Treg immune response contributes to protection elicited by live-attenuated vaccines and may be helpful for the evaluation of live vaccines and other mucosal vaccine candidates.

Streptococcus pneumoniae is one of the causative agent of pneumonia, meningitis, otitis media and sepsis. Vaccination is an effective strategy to combat S. pneumoniae invasion. We previously reported that SPY1, a novel attenuated vaccine candidate against S. pneumoniae, induces a protective immune response against pneumococcal infection in mice. However, underlying mechanisms have yet to be fully illustrated. To explore the mechanism of innate and adaptive immunities induced by SPY1. In this study, bone marrow-derived dendritic cells (DCs) of mice were infected with SPY1 and its parental wild-type strain D39, SPY1-infected DCs were co-cultured with homologous CD4+T cells or adoptive transfer to C57BL/6 mice. Results showed that SPY1 promoted DCs maturation with increased levels of surface molecules such as CD40, CD86, and MHC II, and upregulated the expression of proinflammatory cytokines, including TNF-α, IL-6, IL-12p40, IL-12p70 and IL-23. By contrast, D39 did not efficiently induce DCs activation and maturation. SPY1 could also activate MAPK and NF-κB signaling pathways in DC, but D39 unlikely affected this pathways. SPY1 treated DCs also induced Th1 and Th17 responses in vitro and in vivo. Our results supported the potential of SPY1 as a novel attenuated pneumococcus vaccine, because SPY1-activated DCs exhibit fully matured phenotype, initiated an adaptive immune response, and orchestrated Th1 and Th17 responses.

Postinfluenza pneumococcal pneumonia is a common cause of death in humans. However, the role of IL-27 in the pathogenesis of secondary pneumococcal pneumonia after influenza is unknown. We now report that influenza infection induced pulmonary IL-27 production in a type I IFN-α/β receptor (IFNAR) signalling-dependent manner, which sensitized mice to secondary pneumococcal infection downstream of IFNAR pathway. Mice deficient in IL-27 receptor were resistant to secondary pneumococcal infection and generated more IL-17A-producing γδ T cells but not αβ T cells, thereby leading to enhanced neutrophil response during the early phase of host defence. IL-27 treatment could suppress the development of IL-17A-producing γδ T cells activated by Streptococcus pneumoniae and dendritic cells. This suppressive activity of IL-27 on γδ T cells was dependent on transcription factor STAT1. Finally, neutralization of IL-27 or administration of IL-17A restored the role of γδ T cells in combating secondary pneumococcal infection. Our study defines what we believe to be a novel role of IL-27 in impairing host innate immunity against pneumococcal infection.

Necrotizing enterocolitis (NEC) is the leading cause of neonatal gastrointestinal-related death, while the etiology and pathogenesis are poorly understood.