Pro-inflammatory interactions of streptolysin O toxin with human neutrophils in vitro.

The recent global resurgence of severe infections caused by the Group A streptococcus (GAS) pathogen, Streptococcus pyogenes, has focused attention on this microbial pathogen, which produces an array of virulence factors, such as the pore-forming toxin, streptolysin O (SOT). Importantly, the interactions of SOT with human neutrophils (PMN), are not well understood. The current study was designed to investigate the effects of pretreatment of isolated human PMN with purified SOT on several pro-inflammatory activities, including generation of reactive oxygen species (ROS), degranulation (elastase release), influx of extracellular calcium (Ca2+) and release of extracellular DNA (NETosis), using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of PMN to SOT alone caused modest production of ROS and elastase release, while pretreatment with the toxin caused significant augmentation of chemoattractant (fMLP)-activated ROS generation and release of elastase by activated PMN. These effects of treatment of PMN with SOT were associated with both a marked and sustained elevation of cytosolic Ca2+concentrations and significant increases in the concentrations of extracellular DNA, indicative of NETosis. The current study has identified a potential role for SOT in augmenting the Ca2+-dependent pro-inflammatory interactions of PMN, which, if operative in a clinical setting, may contribute to hyper-activation of PMN and GAS-mediated tissue injury.

Pneumococcal sialidase promotes bacterial survival by fine-tuning of pneumolysin-mediated membrane disruption.

Pneumolysin (Ply) is an indispensable cholesterol-dependent cytolysin for pneumococcal infection. Although Ply-induced disruption of pneumococci-containing endosomal vesicles is a prerequisite for the evasion of endolysosomal bacterial clearance, its potent activity can be a double-edged sword, having a detrimental effect on bacterial survivability by inducing severe endosomal disruption, bactericidal autophagy, and scaffold epithelial cell death. Thus, Ply activity must be maintained at optimal levels. We develop a highly sensitive assay to monitor endosomal disruption using NanoBiT-Nanobody, which shows that the pneumococcal sialidase NanA can fine-tune Ply activity by trimming sialic acid from cell-membrane-bound glycans. In addition, oseltamivir, an influenza A virus sialidase inhibitor, promotes Ply-induced endosomal disruption and cytotoxicity by inhibiting NanA activity in vitro and greater tissue damage and bacterial clearance in vivo. Our findings provide a foundation for innovative therapeutic strategies for severe pneumococcal infections by exploiting the duality of Ply activity.

Streptolysin S is required for Streptococcus pyogenes nasopharyngeal and skin infection in HLA-transgenic mice.

Streptococcus pyogenes is a human-specific pathogen that commonly colonizes the upper respiratory tract and skin, causing a wide variety of diseases ranging from pharyngitis to necrotizing fasciitis and toxic shock syndrome. S. pyogenes has a repertoire of secreted virulence factors that promote infection and evasion of the host immune system including the cytolysins streptolysin O (SLO) and streptolysin S (SLS). S. pyogenes does not naturally infect the upper respiratory tract of mice although mice transgenic for MHC class II human leukocyte antigens (HLA) become highly susceptible. Here we used HLA-transgenic mice to assess the role of both SLO and SLS during both nasopharyngeal and skin infection. Using S. pyogenes MGAS8232 as a model strain, we found that an SLS-deficient strain exhibited a 100-fold reduction in bacterial recovery from the nasopharynx and a 10-fold reduction in bacterial burden in the skin, whereas an SLO-deficient strain did not exhibit any infection defects in these models. Furthermore, depletion of neutrophils significantly restored the bacterial burden of the SLS-deficient bacteria in skin, but not in the nasopharynx. In mice nasally infected with the wildtype S. pyogenes, there was a marked change in localization of the tight junction protein ZO-1 at the site of infection, demonstrating damage to the nasal epithelia that was absent in mice infected with the SLS-deficient strain. Overall, we conclude that SLS is required for the establishment of nasopharyngeal infection and skin infection in HLA-transgenic mice by S. pyogenes MGAS8232 and provide evidence that SLS contributes to nasopharyngeal infection through the localized destruction of nasal epithelia.

Estimates of differential toxin expression governing heterogeneous intracellular lifespans of Streptococcus pneumoniae.

Following invasion of the host cell, pore-forming toxins secreted by pathogens compromise vacuole integrity and expose the microbe to diverse intracellular defence mechanisms. However, the quantitative correlation between toxin expression levels and consequent pore dynamics, fostering the intracellular life of pathogens, remains largely unexplored. In this study, using Streptococcus pneumoniae and its secreted pore-forming toxin pneumolysin (Ply) as a model system, we explored various facets of host-pathogen interactions in the host cytosol. Using time-lapse fluorescence imaging, we monitored pore formation dynamics and lifespans of different pneumococcal subpopulations inside host cells. Based on experimental histograms of various event timescales such as pore formation time, vacuolar death or cytosolic escape time and total degradation time, we developed a mathematical model based on first-passage processes that could correlate the event timescales to intravacuolar toxin accumulation. This allowed us to estimate Ply production rate, burst size and threshold Ply quantities that trigger these outcomes. Collectively, we present a general method that illustrates a correlation between toxin expression levels and pore dynamics, dictating intracellular lifespans of pathogens.

Development and characterization of a hemolysis inhibition assay to determine functionality of anti-Streptolysin O antibodies in human sera.

The high burden of disease and the long-lasting sequelae following Streptococcus pyogenes (Strep A) infections make the development of an effective vaccine a global health priority. Streptolysin O (SLO), is a key toxin in the complex pathogenesis of Strep A infection. Antibodies are elicited against SLO after natural exposure and represent a key target for vaccine-induced immunity. Here we present the setup and characterization of a hemolysis assay to measure functionality of anti-SLO antibodies in human sera. Assay specificity, precision, linearity, reproducibility, and repeatability were determined. The assay was demonstrated to be highly sensitive, specific, reproducible, linear and performed well in assessing functionality of anti-SLO antibodies induced by exposed individuals. Moreover, different sources of critical reagents, in particular red- blood cells, have been compared and had minimal impact on assay performance. The assay presented here has throughput suitable for evaluating sera in vaccine clinical trials and sero-epidemiological studies to gain further insights into the functionality of infection- and vaccine-induced antibodies.

hsdSA regulated extracellular vesicle-associated PLY to protect Streptococcus pneumoniae from macrophage killing via LAPosomes.

IMPORTANCE: S. pneumoniae is a major human pathogen that undergoes a spontaneous and reversible phase variation that allows it to survive in different host environments. Interestingly, we found hsdSA , a gene that manipulated the phase variation, promoted the survival and replication of S. pneumoniae in macrophages by regulating EV production and EV-associated PLY. More importantly, here we provided the first evidence that higher EV-associated PLY (produced by D39) could form LAPosomes that were single membrane compartments containing S. pneumoniae, which are induced by integrin ß1/NOX2/ROS pathway. At the same time, EV-associated PLY increased the permeability of lysosome membrane and induced an insufficient acidification to escape the host killing, and ultimately prolonged the survival of S. pneumoniae in macrophages. In contrast, lower EV-associated PLY (produced by D39ΔhsdSA ) activated ULK1 recruitment to form double-layered autophagosomes to eliminate bacteria.

Impact of Endogenous Pneumococcal Hydrogen Peroxide on the Activity and Release of Pneumolysin.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. The pore-forming cholesterol-dependent cytolysin (CDC) pneumolysin (PLY) and the physiological metabolite hydrogen peroxide (H2O2) can greatly increase the virulence of pneumococci. Although most studies have focused on the contribution of both virulence factors to the course of pneumococcal infection, it is unknown whether or how H2O2 can affect PLY activity. Of note, S. pneumoniae exploits endogenous H2O2 as an intracellular signalling molecule to modulate the activity of several proteins. Here, we demonstrate that H2O2 negatively affects the haemolytic activity of PLY in a concentration-dependent manner. Prevention of cysteine-dependent sulfenylation upon substitution of the unique and highly conserved cysteine residue to serine in PLY significantly reduces the toxin's susceptibility to H2O2 treatment and completely abolishes the ability of DTT to activate PLY. We also detect a clear gradual correlation between endogenous H2O2 generation and PLY release, with decreased H2O2 production causing a decline in the release of PLY. Comparative transcriptome sequencing analysis of the wild-type S. pneumoniae strain and three mutants impaired in H2O2 production indicates enhanced expression of several genes involved in peptidoglycan (PG) synthesis and in the production of choline-binding proteins (CPBs). One explanation for the impact of H2O2 on PLY release is the observed upregulation of the PG bridge formation alanyltransferases MurM and MurN, which evidentially negatively affect the PLY release. Our findings shed light on the significance of endogenous pneumococcal H2O2 in controlling PLY activity and release.

Isorhamnetin as a novel inhibitor of pneumolysin against Streptococcus pneumoniae infection in vivo/in vitro.

The increasing incidence of Streptococcus pneumoniae (S. pneumoniae) infection severely threatened the global public heath, causing a significant fatality in immunocompromised hosts. Notably, pneumolysin (PLY) as a pore-forming cytolysin plays a crucial role in the pathogenesis of pneumococcal pneumonia and lung injury. In this study, a natural flavonoid isorhamnetin was identified as a PLY inhibition to suppress PLY-induced hemolysis by engaging the predicted residues and attenuate cytolysin PLY-mediated A549 cells injury. Underlying mechanisms revealed that PLY inhibitor isorhamnetin further contributed to decrease the formation of bacterial biofilms without affecting the expression of PLY. In vivo S. pneumoniae infection confirmed that the pathological injury of lung tissue evoked by S. pneumoniae was ameliorated by isorhamnetin treatment. Collectively, these results presented that isorhamnetin could inhibit the biological activity of PLY, thus reducing the pathogenicity of S. pneumoniae. In summary, our study laid a foundation for the feasible anti-virulence strategy targeting PLY, and provided a promising PLY inhibitor for the treatment of S. pneumoniae infection.

Preparation a Recombinant Form of Pneumolysin Protein from Streptococcus pneumoniae.

A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.

Pneumolysin as a target for new therapies against pneumococcal infections: A systematic review.

BACKGROUND: This systematic review evaluates pneumolysin (PLY) as a target for new treatments against pneumococcal infections. Pneumolysin is one of the main virulence factors produced by all types of pneumococci. This toxin (53 kDa) is a highly conserved protein that binds to cholesterol in eukaryotic cells, forming pores that lead to cell destruction. METHODS: The databases consulted were MEDLINE, Web of Science, and Scopus. Articles were independently screened by title, abstract, and full text by two researchers, and using consensus to resolve any disagreements that occurred. Articles in other languages different from English, patents, cases report, notes, chapter books and reviews were excluded. Searches were restricted to the years 2000 to 2021. Methodological quality was evaluated using OHAT framework. RESULTS: Forty-one articles describing the effects of different molecules that inhibit PLY were reviewed. Briefly, the inhibitory molecules found were classified into three main groups: those exerting a direct effect by binding and/or blocking PLY, those acting indirectly by preventing its effects on host cells, and those whose mechanisms are unknown. Although many molecules are proposed as toxin blockers, only some of them, such as antibiotics, peptides, sterols, and statins, have the probability of being implemented as clinical treatment. In contrast, for other molecules, there are limited studies that demonstrate efficacy in animal models with sufficient reliability. DISCUSSION: Most of the studies reviewed has a good level of confidence. However, one of the limitations of this systematic review is the lack of homogeneity of the studies, what prevented to carry out a statistical comparison of the results or meta-analysis. CONCLUSION: A panel of molecules blocking PLY activity are associated with the improvement of the inflammatory process triggered by the pneumococcal infection. Some molecules have already been used in humans for other purposes, so they could be safe for use in patients with pneumococcal infections. These patients might benefit from a second line treatment during the initial stages of the infection preventing acute respiratory distress syndrome and invasive pneumococcal diseases. Additional research using the presented set of compounds might further improve the clinical management of these patients.

Naturally-occurring serotype 3 Streptococcus pneumoniae strains that lack functional pneumolysin and autolysin have attenuated virulence but induce localized protective immune responses.

Streptococcus pneumoniae is an important cause of fatal pneumonia in humans. These bacteria express virulence factors, such as the toxins pneumolysin and autolysin, that drive host inflammatory responses. In this study we confirm loss of pneumolysin and autolysin function in a group of clonal pneumococci that have a chromosomal deletion resulting in a pneumolysin-autolysin fusion gene Δ(lytA'-ply')593. The Δ(lytA'-ply')593 pneumococci strains naturally occur in horses and infection is associated with mild clinical signs. Here we use immortalized and primary macrophage in vitro models, which include pattern recognition receptor knock-out cells, and a murine acute pneumonia model to show that a Δ(lytA'-ply')593 strain induces cytokine production by cultured macrophages, however, unlike the serotype-matched ply+lytA+ strain, it induces less tumour necrosis factor α (TNFα) and no interleukin-1ß production. The TNFα induced by the Δ(lytA'-ply')593 strain requires MyD88 but, in contrast to the ply+lytA+ strain, is not reduced in cells lacking TLR2, 4 or 9. In comparison to the ply+lytA+ strain in a mouse model of acute pneumonia, infection with the Δ(lytA'-ply')593 strain resulted in less severe lung pathology, comparable levels of interleukin-1α, but minimal release of other pro-inflammatory cytokines, including interferon-γ, interleukin-6 and TNFα. These results suggest a mechanism by which a naturally occurring Δ(lytA'-ply')593 mutant strain of S. pneumoniae that resides in a non-human host has reduced inflammatory and invasive capacity compared to a human S. pneumoniae strain. These data probably explain the relatively mild clinical disease in response to S. pneumoniae infection seen in horses in comparison to humans.

Streptolysin O Deficiency in Streptococcus pyogenes M1T1 covR/S Mutant Strain Attenuates Virulence in In Vitro and In Vivo Infection Models.

Mutation within the Streptococcus pyogenes (Streptococcus group A; Strep A) covR/S regulatory system has been associated with a hypervirulent phenotype resulting from the upregulation of several virulence factors, including the pore-forming toxin, streptolysin O (SLO). In this study, we utilized a range of covR/S mutants, including M1T1 clonal strains (5448 and a covS mutant generated through mouse passage designated 5448AP), to investigate the contribution of SLO to the pathogenesis of covR/S mutant Strep A disease. Up-regulation of slo in 5448AP resulted in increased SLO-mediated hemolysis, decreased dendritic cell (DC) viability post coculture with Strep A, and increased production of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1) by DCs. Mouse passage of an isogenic 5448 slo-deletion mutant resulted in recovery of several covR/S mutants within the 5448Δslo background. Passage also introduced mutations in non-covR/S genes, but these were considered to have no impact on virulence. Although slo-deficient mutants exhibited the characteristic covR/S-controlled virulence factor upregulation, these mutants caused increased DC viability with reduced inflammatory cytokine production by infected DCs. In vivo, slo expression correlated with decreased DC numbers in infected murine skin and significant bacteremia by 3 days postinfection, with severe pathology at the infection site. Conversely, the absence of slo in the infecting strain (covR/S mutant or wild-type) resulted in detection of DCs in the skin and attenuated virulence in a murine model of pyoderma. slo-sufficient and -deficient covR/S mutants were susceptible to immune clearance mediated by a combination vaccine consisting of a conserved M protein peptide and a peptide from the CXC chemokine protease SpyCEP. IMPORTANCE Streptococcus pyogenes is responsible for significant numbers of invasive and noninvasive infections which cause significant morbidity and mortality globally. Strep A isolates with mutations in the covR/S system display greater propensity to cause severe invasive diseases, which are responsible for more than 163,000 deaths each year. This is due to the upregulation of virulence factors, including the pore-forming toxin streptolysin O. Utilizing covR/S and slo-knockout mutants, we investigated the role of SLO in virulence. We found that SLO alters interactions with host cell populations and increases Strep A viability at sterile sites of the host, such as the blood, and that its absence results in significantly less virulence. This work underscores the importance of SLO in Strep A virulence while highlighting the complex nature of Strep A pathogenesis. This improved insight into host-pathogen interactions will enable a better understanding of host immune evasion mechanisms and inform streptococcal vaccine development programs.

Structural basis underlying the synergism of NADase and SLO during group A Streptococcus infection.

Group A Streptococcus (GAS) is a strict human pathogen possessing a unique pathogenic trait that utilizes the cooperative activity of NAD+-glycohydrolase (NADase) and Streptolysin O (SLO) to enhance its virulence. How NADase interacts with SLO to synergistically promote GAS cytotoxicity and intracellular survival is a long-standing question. Here, the structure and dynamic nature of the NADase/SLO complex are elucidated by X-ray crystallography and small-angle scattering, illustrating atomic details of the complex interface and functionally relevant conformations. Structure-guided studies reveal a salt-bridge interaction between NADase and SLO is important to cytotoxicity and resistance to phagocytic killing during GAS infection. Furthermore, the biological significance of the NADase/SLO complex in GAS virulence is demonstrated in a murine infection model. Overall, this work delivers the structure-functional relationship of the NADase/SLO complex and pinpoints the key interacting residues that are central to the coordinated actions of NADase and SLO in the pathogenesis of GAS infection.

Visualizing the Domino-Like Prepore-to-Pore Transition of Streptolysin O by High-Speed AFM.

Pore-forming proteins (PFPs) are produced by various organisms, including pathogenic bacteria, and form pores within the target cell membrane. Streptolysin O (SLO) is a PFP produced by Streptococcus pyogenes and forms high-order oligomers on the membrane surface. In this prepore state, multiple α-helices in domain 3 of each subunit exist as unfolded structures and transiently interact with each other. They subsequently transition into transmembrane ß-hairpins (TMHs) and form pores with diameters of 20-30 nm. However, in this pore formation process, the trigger of the transition in a subunit and collaboration between subunits remains elusive. Here, I observed the dynamic pore formation process using high-speed atomic force microscopy. During the oligomer transition process, each subunit was sequentially inserted into the membrane, propagating along the oligomer in a domino-like fashion (chain reaction). This process also occurred on hybrid oligomers containing wildtype and mutant subunits, which cannot insert into the membrane because of an introduced disulfide bond. Furthermore, propagation still occurred when an excessive force was added to hybrid oligomers in the prepore state. Based on the observed chain reactions, I estimate the free energies and forces that trigger the transition in a subunit. Furthermore, I hypothesize that the collaboration between subunits is related to the structure of their TMH regions and interactions between TMH-TMH and TMH-lipid molecules.

Human serum albumin stabilizes streptolysin S activity secreted in the extracellular milieu by streptolysin S-producing streptococci.

Anginosus group streptococci (AGS) are opportunistic pathogens of the human oral cavity; however, their pathogenicity has not been discussed in detail. Oral streptococci live in the gingival sulcus, from where they can easily translocate into the bloodstream due to periodontal diseases and dental treatment and cause hazardous effects on the host through their virulence factors. Streptolysin S (SLS), a pathogenic factor produced by ß-hemolytic species/strains belonging to AGS, plays an important role in damaging host cells. Therefore, we investigated the SLS-dependent cytotoxicity of ß-hemolytic Streptococcus anginosus subsp. anginosus (SAA), focusing on different growth conditions such as in the bloodstream. Consequently, SLS-dependent hemolytic activity/cytotoxicity in the culture supernatant of ß-hemolytic SAA was stabilized by blood components, particularly human serum albumin (HSA). The present study suggests that the secreted SLS, not only from ß-hemolytic SAA, but also from other SLS-producing streptococci, is stabilized by HSA. As HSA is the most abundant protein in human plasma, the results of this study provide new insights into the risk of SLS-producing streptococci which can translocate into the bloodstream.

Recombinant Pneumolysin of Pneumococci Induces TLR4 Expression and Maturation of Dendritic Cells In Vitro.

Pneumolysin (Ply) is a target for the development of serotype-independent pneumococcal vaccines, an important condition for the efficacy of which is their ability to activate innate immunity with the subsequent formation of adaptive immunity. In this study, the ability of recombinant full-length Ply (rPly) of pneumococci to induce TLR expression and maturation of dendritic cells generated from mouse bone marrow was evaluated. It was shown that rPly in vitro increased the number of dendritic cells expressing Toll-like receptor 4 (TLR4) on the membrane. rPly caused maturation of dendritic cells generated from mouse bone marrow, which manifested in a decrease in the number of progenitor cells (CD34), an increase in the number of cells expressing the adhesion molecule CD38, costimulatory molecules CD80 and CD86, molecules of terminal differentiation of dendritic cells CD83, as well as molecules of antigenic presentation of the major histocompatibility complex class II.

Streptolysin S targets the sodium-bicarbonate cotransporter NBCn1 to induce inflammation and cytotoxicity in human keratinocytes during Group A Streptococcal infection.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that employs several secreted and surface-bound virulence factors to manipulate its environment, allowing it to cause a variety of disease outcomes. One such virulence factor is Streptolysin S (SLS), a ribosomally-produced peptide toxin that undergoes extensive post-translational modifications. The activity of SLS has been studied for over 100 years owing to its rapid and potent ability to lyse red blood cells, and the toxin has been shown to play a major role in GAS virulence in vivo. We have previously demonstrated that SLS induces hemolysis by targeting the chloride-bicarbonate exchanger Band 3 in erythrocytes, indicating that SLS is capable of targeting host proteins to promote cell lysis. However, the possibility that SLS has additional protein targets in other cell types, such as keratinocytes, has not been explored. Here, we use bioinformatics analysis and chemical inhibition studies to demonstrate that SLS targets the electroneutral sodium-bicarbonate cotransporter NBCn1 in keratinocytes during GAS infection. SLS induces NF-κB activation and host cytotoxicity in human keratinocytes, and these processes can be mitigated by treating keratinocytes with the sodium-bicarbonate cotransport inhibitor S0859. Furthermore, treating keratinocytes with SLS disrupts the ability of host cells to regulate their intracellular pH, and this can be monitored in real time using the pH-sensitive dye pHrodo Red AM in live imaging studies. These results demonstrate that SLS is a multifunctional bacterial toxin that GAS uses in numerous context-dependent ways to promote host cell cytotoxicity and increase disease severity. Studies to elucidate additional host targets of SLS have the potential to impact the development of therapeutics for severe GAS infections.

Shionone-Targeted Pneumolysin to Ameliorate Acute Lung Injury Induced by Streptococcus pneumoniae In Vivo and In Vitro.

Streptococcus pneumoniae (S. pneumoniae), as a Gram-positive bacterium, can cause severe bacterial pneumonia, and result in high morbidity and mortality in infected people. Meanwhile, isolated drug-resistant S. pneumoniae is growing, which raises concerns about strategies for combatting S. pneumoniae infection. To disturb S. pneumoniae pathogenicity and its drug-resistance, developing novel anti-infective strategies or compounds is urgent. In this study, the anti-infective effect of shionone was explored. A minimum inhibitory concentration (MIC) assay and growth curve determination were performed to evaluate the effect of the tetracyclic triterpenoid compound shionone against S. pneumoniae. Hemolysis tests, western blotting, oligomerization inhibition assays, and molecular docking were carried out to explore the anti-infective mechanism of shionone. Moreover, the protective effect of shionone was also confirmed in a mousepneumonia model. The results showed that the excellent hemolytic inhibitory activity of shionone was observed at less than 8 µg/mL. Meanwhile, shionone could disturb the oligomerization of pneumolysin (PLY) but did not interfere with PLY expression at less than 4 µg/mL. Molecular docking suggested that shionone targeted the ASP-59, ILE-60, THR-57, PHE-344, and ASN-346 amino acid sites to reduce S. pneumoniae pathogenicity. Furthermore, shionone alleviated lung histopathologic injury and decreased lung bacterial colonization in vivo. The above results showed that shionone could bind to the PLY active pocket under the concentrations of 8 µg/mL and neutralize PLY hemolysis activity to reduce S. pneumoniae pathogenicity in vitro and in vivo.

Negative Cross-Talk between TLR2/4-Independent AMPKα1 and TLR2/4-Dependent JNK Regulates S. pneumoniae-Induced Mucosal Innate Immune Response.

Streptococcus pneumoniae is major cause of otitis media (OM) and life-threatening pneumonia. Overproduction of mucin, the major component of mucus, plays a critical role in the pathogenesis of both OM and pneumonia. However, the molecular mechanisms underlying the tight regulation of mucin upregulation in the mucosal epithelium by S. pneumoniae infection remain largely unknown. In this study, we show that S. pneumoniae pneumolysin (PLY) activates AMP-activated protein kinase α1 (AMPKα1), the master regulator of energy homeostasis, which is required for S. pneumoniae-induced mucin MUC5AC upregulation in vitro and in vivo. Moreover, we found that PLY activates AMPKα1 via cholesterol-dependent membrane binding of PLY and subsequent activation of the Ca2+- Ca2+/calmodulin-dependent kinase kinase ß (CaMKKß) and Cdc42-mixed-lineage protein kinase 3 (MLK3) signaling axis in a TLR2/4-independent manner. AMPKα1 positively regulates PLY-induced MUC5AC expression via negative cross-talk with TLR2/4-dependent activation of MAPK JNK, the negative regulator of MUC5AC expression. Moreover, pharmacological inhibition of AMPKα1 suppressed MUC5AC induction in the S. pneumoniae-induced OM mouse model, thereby demonstrating its therapeutic potential in suppressing mucus overproduction in OM. Taken together, our data unveil a novel mechanism by which negative cross-talk between TLR2/4-independent activation of AMPKα1 and TLR2/4-dependent activation of JNK tightly regulates the S. pneumoniae PLY-induced host mucosal innate immune response.

Electrochemical Detection for Isothermal Loop-Mediated Amplification of Pneumolysin Gene of Streptococcus pneumoniae Based on the Oxidation of Phenol Red Indicator.

A highly sensitive electrochemical methodology for end-point detection of loop-mediated isothermal nucleic acid amplification reactions was developed. It is based on the oxidation process of phenol red (PR), commonly used as a visual indicator. The dependence of its redox process on pH, which changes during amplification, allows performing quantitative measurements. Thus, the change in the oxidation potential of PR during the amplification is used, for the first time, as the analytical signal that correlates with the number of initial DNA copies. As a proof-of-concept, the amplification of the pneumolysin gene from Streptococcus pneumoniae, one of the main pathogens causing community-acquired pneumonia, is performed. Combination of isothermal amplification with electrochemical detection, performed on small-size flexible electrodes, allows easy decentralization. Adaptation to the detection of other pathogens causing infectious diseases would be very useful in the prevention of future epidemics.