Coral reefs are marine biodiversity hotspots, but their existence is threatened by global change and local pressures such as land-runoff and overfishing. Population explosions of coral-eating crown of thorns sea stars (COTS) are a major contributor to recent decline in coral cover on the Great Barrier Reef. Here, we investigate how projected near-future ocean acidification (OA) conditions can affect early life history stages of COTS, by investigating important milestones including sperm motility, fertilisation rates, and larval development and settlement. OA (increased pCO2 to 900–1200 µatm pCO2) significantly reduced sperm motility and, to a lesser extent, velocity, which strongly reduced fertilization rates at environmentally relevant sperm concentrations. Normal development of 10 d old larvae was significantly lower under elevated pCO2 but larval size was not significantly different between treatments. Settlement of COTS larvae was significantly reduced on crustose coralline algae (known settlement inducers of COTS) that had been exposed to OA conditions for 85 d prior to settlement assays. Effect size analyses illustrated that reduced settlement may be the largest bottleneck for overall juvenile production. Results indicate that reductions in fertilisation and settlement success alone would reduce COTS population replenishment by over 50%. However, it is unlikely that this effect is sufficient to provide respite for corals from other negative anthropogenic impacts and direct stress from OA and warming on corals.
The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q−/− mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.
Serum phosphate is a known risk factor for cardiovascular events and mortality in people with chronic kidney disease (CKD), however data on the association of these outcomes with serum phosphate in the general population are scarce. We investigate this relationship in people with and without CKD in a large community-based population.
Three groups from an adult cohort of the Quality Improvement in Chronic Kidney Disease (QICKD) cluster randomised trial (ISRCTN56023731) were followed over a period of 2.5 years: people with normal renal function (N = 24,184), people with CKD stages 1–2 (N = 20,356), and people with CKD stages 3–5 (N = 13,292). We used a multilevel logistic regression model to determine the association between serum phosphate, in these groups, and a composite outcome of all-cause mortality, cardiovascular events, and advanced coronary artery disease. We adjusted for known cardiovascular risk factors.
Higher phosphate levels were found to correlate with increased cardiovascular risk. In people with normal renal function and CKD stages 1–2, Phosphate levels between 1.25 and 1.50 mmol/l were associated with increased cardiovascular events; odds ratio (OR) 1.36 (95% CI 1.06–1.74; p = 0.016) in people with normal renal function and OR 1.40 (95% CI 1.09–1.81; p = 0.010) in people with CKD stages 1–2. Hypophosphatemia (<0.75 mmol/l) was associated with fewer cardiovascular events in people with normal renal function; OR 0.59 (95% CI 0.36–0.97; p = 0.049). In people with CKD stages 3–5, hyperphosphatemia (>1.50 mmol/l) was associated with increased cardiovascular risk; OR 2.34 (95% CI 1.64–3.32; p<0.001). Other phosphate ranges were not found to have a significant impact on cardiovascular events in people with CKD stages 3–5.
Serum phosphate is associated with cardiovascular events in people with and without CKD. Further research is required to determine the mechanisms underlying these associations.
Streptococcus pneumoniae is an aerotolerant Gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth, S. pneumoniae produces high levels of H2O2. Since S. pneumoniae lacks catalase, the question of how it controls H2O2 levels is of critical importance. The psa locus encodes an ABC Mn2+-permease complex (psaBCA) and a putative thiol peroxidase, tpxD. This study shows that tpxD encodes a functional thiol peroxidase involved in the adjustment of H2O2 homeostasis in the cell. Kinetic experiments showed that recombinant TpxD removed H2O2 efficiently. However, in vivo experiments revealed that TpxD detoxifies only a fraction of the H2O2 generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD Cys58 undergoes selective oxidation in vivo, under conditions where H2O2 is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesis in vitro were significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H2O2 resulted in tpxD upregulation, while psaBCA expression was oppositely affected. However, the challenge of ΔtpxD mutants with H2O2 did not affect psaBCA, implying that TpxD is involved in the regulation of the psa operon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39ΔtpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H2O2.
Since 2004 the European Bioinformatics Institute (EMBL-EBI) has provided access to a wide range of databases and analysis tools via Web Services interfaces. This comprises services to search across the databases available from the EMBL-EBI and to explore the network of cross-references present in the data (e.g. EB-eye), services to retrieve entry data in various data formats and to access the data in specific fields (e.g. dbfetch), and analysis tool services, for example, sequence similarity search (e.g. FASTA and NCBI BLAST), multiple sequence alignment (e.g. Clustal Omega and MUSCLE), pairwise sequence alignment and protein functional analysis (e.g. InterProScan and Phobius). The REST/SOAP Web Services (http://www.ebi.ac.uk/Tools/webservices/) interfaces to these databases and tools allow their integration into other tools, applications, web sites, pipeline processes and analytical workflows. To get users started using the Web Services, sample clients are provided covering a range of programming languages and popular Web Service tool kits, and a brief guide to Web Services technologies, including a set of tutorials, is available for those wishing to learn more and develop their own clients. Users of the Web Services are informed of improvements and updates via a range of methods.
Streptococcus pneumoniae causes over one million deaths worldwide annually, despite recent developments in vaccine and antibiotic therapy. Host susceptibility to pneumococcal infection and disease is controlled by a combination of genetic and environmental influences, but current knowledge remains limited.
In order to identify novel host genetic variants as predictive risk factors or as potential targets for prophylaxis, we have looked for quantitative trait loci in a mouse model of invasive pneumococcal disease. We describe a novel locus, called Streptococcus pneumoniae infection resistance 2 (Spir2) on Chr4, which influences time to morbidity and the development of bacteraemia post-infection.
The two quantitative trait loci we have identified (Spir1 and Spir2) are linked significantly to both bacteraemia and survival time. This may mean that the principle cause of death, in our model of pneumonia, is bacteraemia and the downstream inflammatory effects it precipitates in the host.
Streptococcus pneumoniae; Host susceptibility; Host genetics; Quantitative trait loci; Model organism; Mouse; Bacterial infection; Inflammation
Streptococcus pneumoniae expresses three distinct sialidases, NanA, NanB, and NanC, that are believed to be key virulence factors and thus, potential important drug targets. We previously reported that the three enzymes release different products from sialosides, but could share a common catalytic mechanism before the final step of product formation. However, the kinetic investigations of the three sialidases have not been systematically done thus far, due to the lack of an easy and steady measurement of sialidase reaction rate.
In this work, we present further kinetic characterization of pneumococcal sialidases by using a direct spectrophotometric method with the chromogenic substrate p-nitrophenyl-N-acetylneuraminic acid (p-NP-Neu5Ac). Using our assay, the measured kinetic parameters of the three purified pneumococcal sialidase, NanA, NanB and NanC, were obtained and were in perfect agreement with the previously published data. The major advantage of this alternative method resides in the direct measurement of the released product, allowing to readily determine of initial reaction rates and record complete hydrolysis time courses.
We developed an accurate, fast and sensitive spectrophotometric method to investigate the kinetics of sialidase-catalyzed reactions. This fast, sensitive, inexpensive and accurate method could benefit the study of the kinetics and inhibition of sialidases in general.
Sialidase; Neuraminidase; Chromogenic sialic acids; Kinetic assay; Streptococcus pneumoniae
Sialic acid (N-acetylneuraminic acid; NeuNAc) is one of the most important carbohydrates for Streptococcus pneumoniae due of its role as a carbon and energy source, receptor for adhesion and invasion and molecular signal for promotion of biofilm formation, nasopharyngeal carriage and invasion of the lung.
In this work, NeuNAc and its metabolic derivative N-acetyl mannosamine (ManNAc) were used to analyze regulatory mechanisms of the neuraminidase locus expression. Genomic and metabolic comparison to Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii and Streptococcus sanguinis elucidates the metabolic association of the two amino sugars to different parts of the locus coding for the two main pneumococcal neuraminidases and confirms the substrate specificity of the respective ABC transporters. Quantitative gene expression analysis shows repression of the locus by glucose and induction of all predicted transcriptional units by ManNAc and NeuNAc, each inducing with higher efficiency the operon encoding for the transporter with higher specificity for the respective amino sugar. Cytofluorimetric analysis demonstrated enhanced surface exposure of NanA on pneumococci grown in NeuNAc and ManNAc and an activity assay allowed to quantify approximately twelve times as much neuraminidase activity on induced cells as opposed to glucose grown cells.
The present data increase the understanding of metabolic regulation of the nanAB locus and indicate that experiments aimed at the elucidation of the relevance of neuraminidases in pneumococcal virulence should possibly not be carried out on bacteria grown in glucose containing media.
Sialic acid; Metabolic regulation; Carbon catabolite repression
Analysis of ciliary function for assessment of patients suspected of primary ciliary dyskinesia (PCD) and for research studies of respiratory and ependymal cilia requires assessment of both ciliary beat pattern and beat frequency. While direct measurement of beat frequency from high-speed video recordings is the most accurate and reproducible technique it is extremely time consuming. The aim of this study was to develop a freely available automated method of ciliary beat frequency analysis from digital video (AVI) files that runs on open-source software (ImageJ) coupled to Microsoft Excel, and to validate this by comparison to the direct measuring high-speed video recordings of respiratory and ependymal cilia. These models allowed comparison to cilia beating between 3 and 52 Hz.
Digital video files of motile ciliated ependymal (frequency range 34 to 52 Hz) and respiratory epithelial cells (frequency 3 to 18 Hz) were captured using a high-speed digital video recorder. To cover the range above between 18 and 37 Hz the frequency of ependymal cilia were slowed by the addition of the pneumococcal toxin pneumolysin. Measurements made directly by timing a given number of individual ciliary beat cycles were compared with those obtained using the automated ciliaFA system.
The overall mean difference (± SD) between the ciliaFA and direct measurement high-speed digital imaging methods was −0.05 ± 1.25 Hz, the correlation coefficient was shown to be 0.991 and the Bland-Altman limits of agreement were from −1.99 to 1.49 Hz for respiratory and from −2.55 to 3.25 Hz for ependymal cilia.
A plugin for ImageJ was developed that extracts pixel intensities and performs fast Fourier transformation (FFT) using Microsoft Excel. The ciliaFA software allowed automated, high throughput measurement of respiratory and ependymal ciliary beat frequency (range 3 to 52 Hz) and avoids operator error due to selection bias. We have included free access to the ciliaFA plugin and installation instructions in Additional file 1 accompanying this manuscript that other researchers may use.
The complement system plays a key role in host defense against pneumococcal infection. Three different pathways, the classical, alternative and lectin pathways, mediate complement activation. While there is limited information available on the roles of the classical and the alternative activation pathways of complement in fighting streptococcal infection, little is known about the role of the lectin pathway, mainly due to the lack of appropriate experimental models of lectin pathway deficiency. We have recently established a mouse strain deficient of the lectin pathway effector enzyme mannan-binding lectin associated serine protease-2 (MASP-2) and shown that this mouse strain is unable to form the lectin pathway specific C3 and C5 convertases. Here we report that MASP-2 deficient mice (which can still activate complement via the classical pathway and the alternative pathway) are highly susceptible to pneumococcal infection and fail to opsonize Streptococcus pneumoniae in the none-immune host. This defect in complement opsonisation severely compromises pathogen clearance in the lectin pathway deficient host. Using sera from mice and humans with defined complement deficiencies, we demonstrate that mouse ficolin A, human L-ficolin, and collectin 11 in both species, but not mannan-binding lectin (MBL), are the pattern recognition molecules that drive lectin pathway activation on the surface of S. pneumoniae. We further show that pneumococcal opsonisation via the lectin pathway can proceed in the absence of C4. This study corroborates the essential function of MASP-2 in the lectin pathway and highlights the importance of MBL-independent lectin pathway activation in the host defense against pneumococci.
Streptococcus pneumoniae is a major human pathogen that causes pneumonia, septicemia and meningitis. The host defense against pneumococci is largely dependent on complement, a system of blood proteins which, when activated, attach to bacteria, targeting them for clearance by phagocytes. There are three routes of complement activation: The classical, lectin and alternative pathways. Limited information is available on the roles of the classical and alternative pathways in fighting pneumococci; the role of the lectin pathway has escaped the attention of previous research. This work demonstrates that the lectin pathway is critical in fighting pneumococcal infection. Of the five different lectin pathway recognition molecules in human serum, only L-ficolin and collectin 11 activate complement on pneumococci. Human mannose-binding lectin (MBL), the best-known lectin pathway pattern recognition molecule, has no role whatsoever in fighting pneumococci. Similarly, in mouse serum, only ficolin A and collectin 11 drive complement activation on S. pneumoniae. Hence, MBL deficient mice are not compromised in pneumococcal infection, while ficolin A deficient mice and mice deficient of the key lectin pathway enzyme MBL-associated serine protease-2 (MASP-2) are exquisitely susceptible to infection. This work explains why MBL deficiency, the most frequent hereditary immune deficiency, does not predispose to pneumococcal disease.
We report here the identification and characterization of two zinc uptake systems, ZurAM and ZinABC, in the intracellular pathogen Listeria monocytogenes. Transcription of both operons was zinc responsive and regulated by the zinc-sensing repressor Zur. Deletion of either zurAM or zinA had no detectable effect on growth in defined media, but a double zurAM zinA mutant was unable to grow in the absence of zinc supplementation. Deletion of zinA had no detectable effect on intracellular growth in HeLa epithelial cells. In contrast, growth of the zurAM mutant was significantly impaired in these cells, indicating the importance of the ZurAM system during intracellular growth. Notably, the deletion of both zinA and zurAM severely attenuated intracellular growth, with the double mutant being defective in actin-based motility and unable to spread from cell to cell. Deletion of either zurAM or zinA had a significant effect on virulence in an oral mouse model, indicating that both zinc uptake systems are important in vivo and establishing the importance of zinc acquisition during infection by L. monocytogenes. The presence of two zinc uptake systems may offer a mechanism by which L. monocytogenes can respond to zinc deficiency within a variety of environments and during different stages of infection, with each system making distinct contributions under different stress conditions.
Streptococcus pneumoniae is an important human pathogen responsible for a spectrum of diseases including pneumonia. Immunological and pro-inflammatory processes induced in the lung during pneumococcal infection are well documented, but little is known about the role played by immunoregulatory cells and cytokines in the control of such responses. We demonstrate considerable differences in the immunomodulatory cytokine transforming growth factor (TGF)-β between the pneumococcal pneumonia resistant BALB/c and susceptible CBA/Ca mouse strains. Immunohistochemistry and flow cytometry reveal higher levels of TGF-β protein in BALB/c lungs during pneumococcal pneumonia that correlates with a rapid rise in lung Foxp3+Helios+ T regulatory cells. These cells have protective functions during pneumococcal pneumonia, because blocking their induction with an inhibitor of TGF-β impairs BALB/c resistance to infection and aids bacterial dissemination from lungs. Conversely, adoptive transfer of T regulatory cells to CBA/Ca mice, prior to infection, prolongs survival and decreases bacterial dissemination from lungs to blood. Importantly, strong T regulatory cell responses also correlate with disease-resistance in outbred MF1 mice, confirming the importance of immunoregulatory cells in controlling protective responses to the pneumococcus. This study provides exciting new evidence for the importance of immunomodulation during pulmonary pneumococcal infection and suggests that TGF-β signalling is a potential target for immunotherapy or drug design.
Streptococcus pneumoniae is a major human bacterial pathogen that causes a wide range of diseases including pneumonia, meningitis, sepsis and ear infections. The bacterium is responsible for around 1.2 million deaths per year, mostly in high-risk groups such as children, the elderly and those with a weakened immune system. Infection with the pneumococcus can induce a wide-variety of immune responses and disease symptoms and it is not known why some people are more resistant to infection than others. Here, we identify an important role in natural resistance against pneumococcal pneumonia for a group of cells – known as T regulatory cells – that control the immune response to pneumococcal infection. In mice, strong T regulatory cell responses correlate with resistance to invasive pneumococcal pneumonia. Disease-resistance can be boosted by administering T regulatory cells to highly susceptible mice or inhibited by blocking the activity of these cells in resistant mice. These results advance our understanding of the host immunity differences that underpin resistance to pneumococcal pneumonia and offer hope that in the future we might boost resistance in susceptible individuals through modulation of their immune system.
Natural killer T (NKT) cells recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell receptor (TCR), but the forces driving TCR conservation have remained uncertain. Here we show that NKT cells recognize diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells are required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is found at a low level in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR, and most important, they extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.
Emerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries.
We combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment.
At baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome.
In Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals.
Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.
Streptococcus pneumoniae (pneumococcus) is a pathogen of global significance, causing diseases including pneumonia, meningitis and septicaemia. In order to develop improved pneumococcal vaccines it is essential to understand how the bacterium interacts with the host immune system. Pneumococci produce a range of pathogenicity factors, among which the toxin pneumolysin plays a central role and has potential as a vaccine candidate. Here, we demonstrate that pneumolysin can directly activate innate immune cells and dramatically amplify the production of pro-inflammatory cytokines. These enhancing effects of the toxin do not require Toll-like receptor (TLR)4. In particular, the toxin exerts a potent effect on interleukin (IL)-1, which is an endogenous pyrogen and powerful activator of IL-17A production. This effect results from activation of the NLRP3 inflammasome complex and NLRP3 is required for protection against the pathogen in vivo. To induce protective immunity against pneumococci, IFN-γ and IL-17A are thought to be essential. We show that pneumolysin plays a key role in promoting these cytokines both in vitro and in vivo during respiratory infection. The results add significantly to our understanding of the interactions between pneumococci and the immune system and support investigations into the inclusion of pneumolysin or its derivatives in novel pneumococcal vaccines.
Epidemiological studies suggest that inhalation of carbonaceous particulate matter from biomass combustion increases susceptibility to bacterial pneumonia. In vitro studies report that phagocytosis of carbon black by alveolar macrophages (AM) impairs killing of Streptococcus pneumoniae. We have previously reported high levels of black carbon in AM from biomass smoke-exposed children and adults. We therefore aimed to use a mouse model to test the hypothesis that high levels of carbon loading of AM in vivo increases susceptibility to pneumococcal pneumonia.
Female outbred mice were treated with either intranasal phosphate buffered saline (PBS) or ultrafine carbon black (UF-CB in PBS; 500 μg on day 1 and day 4), and then infected with S. pneumoniae strain D39 on day 5. Survival was assessed over 72 h. The effect of UF-CB on AM carbon loading, airway inflammation, and a urinary marker of pulmonary oxidative stress was assessed in uninfected animals.
Instillation of UF-CB in mice resulted a pattern of AM carbon loading similar to that of biomass-smoke exposed humans. In uninfected animals, UF-CB treated animals had increased urinary 8-oxodG (P = 0.055), and an increased airway neutrophil differential count (P < 0.01). All PBS-treated mice died within 72 h after infection with S. pneumoniae, whereas morbidity and mortality after infection was reduced in UF-CB treated animals (median survival 48 h vs. 30 h, P < 0.001). At 24 hr post-infection, UF-CB treated mice had lower lung and the blood S. pneumoniae colony forming unit counts, and lower airway levels of keratinocyte-derived chemokine/growth-related oncogene (KC/GRO), and interferon gamma.
Acute high level loading of AM with ultrafine carbon black particles per se does not increase the susceptibility of mice to pneumococcal infection in vivo.
Ciliated ependymal cells line the cerebral ventricles and aqueducts separating the infected CSF from the brain parenchyma in meningitis.
Investigation of the interaction of Listeria monocytogenes with cultured rat brain ependymal cells showed that certain strains reduced the beat frequency of the cilia but all the strains studied significantly reduced the ciliary beat amplitude (the linear distance travelled by the tip of each cilium per beat cycle).
The presence of the ependyma caused aggregation of some listeria strains and in some cases extracellular material also was seen in association with bacterial aggregates. These observations were dependent on the expression of genes required for invasion, intracellular survival and listerial cell to cell spread that are regulated by the transcriptional activator, positive regulatory factor A (PrfA).
Streptococcus pneumoniae resides in the oxygen-rich environment of the upper respiratory tract, and therefore the ability to survive in the presence of oxygen is an important aspect of its in vivo survival. To investigate how S. pneumoniae adapts to oxygen, we determined the global gene expression profile of the micro-organism in aerobiosis and anaerobiosis. It was found that exposure to aerobiosis elevated the expression of 54 genes, while the expression of 15 genes was downregulated. Notably there were significant changes in putative genome plasticity and hypothetical genes. In addition, increased expression of rgg, a putative transcriptional regulator, was detected. To test the role of Rgg in the pneumococcal oxidative stress response, an isogenic mutant was constructed. It was found that the mutant was sensitive to oxygen and paraquat, but not to H2O2. In addition, the absence of Rgg strongly reduced the biofilm-forming ability of an unencapsulated pneumococcus. Virulence studies showed that the median survival time of mice infected intranasally with the rgg mutant was significantly longer than that of the wild-type-infected group, and the animals infected with the mutant developed septicaemia later than those infected intranasally with the wild-type.
The pneumococcus obtains its energy from the metabolism of host glycosides. Therefore, efficient degradation of host glycoproteins is integral to pneumococcal virulence. In search of novel pneumococcal glycosidases, we characterized the Streptococcus pneumoniae strain D39 protein encoded by SPD_0065 and found that this gene encodes a β-galactosidase. The SPD_0065 recombinant protein released galactose from desialylated fetuin, which was used here as a model of glycoproteins found in vivo. A pneumococcal mutant with a mutation in SPD_0065 showed diminished β-galactosidase activity, exhibited an extended lag period in mucin-containing defined medium, and cleaved significantly less galactose than the parental strain during growth on mucin. As pneumococcal β-galactosidase activity had been previously attributed solely to SPD_0562 (bgaA), we evaluated the contribution of SPD_0065 and SPD_0562 to total β-galactosidase activity. Mutation of either gene significantly reduced enzymatic activity, but β-galactosidase activity in the double mutant, although significantly less than that in either of the single mutants, was not completely abolished. The expression of SPD_0065 in S. pneumoniae grown in mucin-containing medium or tissues harvested from infected animals was significantly upregulated compared to that in pneumococci from glucose-containing medium. The SPD_0065 mutant strain was found to be attenuated in virulence in a manner specific to the host tissue.
Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pflA genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.
Streptococcus pneumoniae is a human commensal and pathogen able to cause a variety of diseases that annually result in over a million deaths worldwide. The S. pneumoniaeSpain23F sequence type 81 lineage was among the first recognized pandemic clones and was responsible for almost 40% of penicillin-resistant pneumococcal infections in the United States in the late 1990s. Analysis of the chromosome sequence of a representative strain, and comparison with other available genomes, indicates roles for integrative and conjugative elements in the evolution of pneumococci and, more particularly, the emergence of the multidrug-resistant Spain 23F ST81 lineage. A number of recently acquired loci within the chromosome appear to encode proteins involved in the production of, or immunity to, antimicrobial compounds, which may contribute to the proficiency of this strain at nasopharyngeal colonization. However, further sequencing of other pandemic clones will be required to establish whether there are any general attributes shared by these strains that are responsible for their international success.
Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.
The structure of a catalytically active subdomain of the NanA sialidase from S. pneumoniae is reported to a resolution of 2.5 Å. The complex with the inhibitor Neu5Ac2en identifies the key catalytic residues and provides a platform for structure-based development of specific inhibitors.
Streptococcus pneumoniae genomes encode three sialidases, NanA, NanB and NanC, which are key virulence factors that remove sialic acids from various glycoconjugates. The enzymes have potential as drug targets and also as vaccine candidates. The 115 kDa NanA is the largest of the three sialidases and is anchored to the bacterial membrane. Although recombinantly expressed full-length NanA was soluble, it failed to crystallize; therefore, a 56.5 kDa domain that retained full enzyme activity was subcloned. The purified enzyme was crystallized in 0.1 M MES pH 6.5, 30%(w/v) PEG 4000 using the sitting-drop vapour-diffusion method. Data were collected at 100 K to 2.5 Å resolution from a crystal grown in the presence of the inhibitor 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid. The crystal belongs to space group P212121, with unit-cell parameters a = 49.2, b = 95.6, c = 226.6 Å. The structure was solved by molecular replacement and refined to final R and R
free factors of 0.246 and 0.298, respectively.
NanA; sialidases; Streptococcus pneumoniae
The ability of fluorescence resonance energy transfer, molecular-beacon, and TaqMan probes to detect single nucleotide polymorphism (SNP) in the presence of a wild-type allele was evaluated using drug resistance-conferring SNPs in mixed Mycobacterium tuberculosis DNA. It was found that both the absolute quantity and the ratio of alleles determine the detection sensitivity of the probe systems.
We examined the role of the neuraminidases NanA and NanB in colonization and infection in the upper and lower respiratory tract by Streptococcus pneumoniae, as well as the role of these neuraminidases in the onset and development of septicemia following both intranasal and intravenous infection. We demonstrated for the first time using outbred MF1 mouse models of infection that both NanA and NanB were essential for the successful colonization and infection of the upper and lower respiratory tract, respectively, as well as pneumococcal survival in nonmucosal sites, such as the blood. Our studies have shown that in vivo a neuraminidase A mutant is cleared from the nasopharynx, trachea, and lungs within 12 h postinfection, while a neuraminidase B mutant persists but does not increase in either the nasopharynx, trachea, or lungs. We also demonstrated both neuraminidase mutants were unable to cause sepsis following intranasal infections. When administered intravenously, however, both mutants survived initially but were unable to persist in the blood beyond 48 h postinfection and were progressively cleared. The work presented here demonstrates the importance of pneumococcal neuraminidase A and for the first time neuraminidase B in the development of upper and lower respiratory tract infection and sepsis.