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1.  Development and Characterization of a Long-Term Murine Model of Streptococcus pneumoniae Infection of the Lower Airways 
Infection and Immunity  2014;82(8):3289-3298.
Chronic obstructive pulmonary disease (COPD) is characterized by long periods of stable symptoms, but exacerbations occur, which result in a permanent worsening of symptoms. Previous studies have shown a link between bacterial colonization of the lower airways of COPD sufferers and an increase in exacerbation frequency. One of the most frequent bacterial colonizers is Streptococcus pneumoniae. To mimic this aspect of COPD, a murine model of low-level pneumococcal colonization in the lung has been developed, in which S. pneumoniae persisted in the lungs for at least 28 days. From day 14 postinfection, bacterial numbers remained constant until at least 28 days postinfection, and animals showed no outward signs of disease. The bacterial presence correlated with a low-level inflammatory response that was localized to small foci across the left and inferior lobes of the lung. The cellular response was predominantly monocytic, and focal fibroplasia was observed at the airway transitional zones. Physiological changes in the lungs were investigated with a Forced Maneuvers system. This new model provides a means of study of a long-term pulmonary infection with a human pathogen in a rodent system. This is an excellent tool for the development of future models that mimic complex respiratory diseases such as COPD and asthma.
PMCID: PMC4136212  PMID: 24866797
2.  Respiratory Syncytial Virus Increases the Virulence of Streptococcus pneumoniae by Binding to Penicillin Binding Protein 1a. A New Paradigm in Respiratory Infection 
Rationale: Respiratory syncytial virus (RSV) and Streptococcus pneumoniae are major respiratory pathogens. Coinfection with RSV and S. pneumoniae is associated with severe and often fatal pneumonia but the molecular basis for this remains unclear.
Objectives: To determine if interaction between RSV and pneumococci enhances pneumococcal virulence.
Methods: We used confocal microscopy and Western blot to identify the receptors involved in direct binding of RSV and pneumococci, the effects of which were studied in both in vivo and in vitro models of infection. Human ciliated respiratory epithelial cell cultures were infected with RSV for 72 hours and then challenged with pneumococci. Pneumococci were collected after 2 hours exposure and changes in gene expression determined using quantitative real-time polymerase chain reaction.
Measurements and Main Results: Following incubation with RSV or purified G protein, pneumococci demonstrated a significant increase in the inflammatory response and bacterial adherence to human ciliated epithelial cultures and markedly increased virulence in a pneumonia model in mice. This was associated with extensive changes in the pneumococcal transcriptome and significant up-regulation in the expression of key pneumococcal virulence genes, including the gene for the pneumococcal toxin, pneumolysin. We show that mechanistically this is caused by RSV G glycoprotein binding penicillin binding protein 1a.
Conclusions: The direct interaction between a respiratory virus protein and the pneumococcus resulting in increased bacterial virulence and worsening disease outcome is a new paradigm in respiratory infection.
PMCID: PMC4226051  PMID: 24941423
respiratory syncytial virus; pneumococcus; cilia; virulence; G protein
3.  Oleoyl Coenzyme A Regulates Interaction of Transcriptional Regulator RaaS (Rv1219c) with DNA in Mycobacteria* 
The Journal of Biological Chemistry  2014;289(36):25241-25249.
Background: RaaS mediates mycobacterial survival in nonpermissive growth conditions by controlling expression of ATP-dependent efflux pumps.
Results: Oleoyl-CoA regulates binding of the RaaS transcription factor to DNA and thus expression of the RaaS regulon and RaaS-mediated persistence.
Conclusion: The activity of bacterial efflux is regulated by metabolites that are produced during active growth.
Significance: Dysregulation of efflux pumps results in killing of persisting mycobacteria with low metabolic activity.
We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria.
PMCID: PMC4155686  PMID: 25012658
ABC Transporter; Antibiotics; Fatty Acid; Ligand-binding Protein; Mycobacteria; Transcription Repressor
4.  Antimicrobial Treatment Improves Mycobacterial Survival in Nonpermissive Growth Conditions 
Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis.
PMCID: PMC3993263  PMID: 24590482
5.  Septicaemia models using Streptococcus pneumoniae and Listeria monocytogenes: understanding the role of complement properdin 
Medical Microbiology and Immunology  2014;203(4):257-271.
Streptococcus pneumoniae and Listeria monocytogenes, pathogens which can cause severe infectious disease in human, were used to infect properdin-deficient and wildtype mice. The aim was to deduce a role for properdin, positive regulator of the alternative pathway of complement activation, by comparing and contrasting the immune response of the two genotypes in vivo. We show that properdin-deficient and wildtype mice mounted antipneumococcal serotype-specific IgM antibodies, which were protective. Properdin-deficient mice, however, had increased survival in the model of streptococcal pneumonia and sepsis. Low activity of the classical pathway of complement and modulation of FcγR2b expression appear to be pathogenically involved. In listeriosis, however, properdin-deficient mice had reduced survival and a dendritic cell population that was impaired in maturation and activity. In vitro analyses of splenocytes and bone marrow-derived myeloid cells support the view that the opposing outcomes of properdin-deficient and wildtype mice in these two infection models is likely to be due to a skewing of macrophage activity to an M2 phenotype in the properdin-deficient mice. The phenotypes observed thus appear to reflect the extent to which M2- or M1-polarised macrophages are involved in the immune responses to S. pneumoniae and L. monocytogenes. We conclude that properdin controls the strength of immune responses by affecting humoral as well as cellular phenotypes during acute bacterial infection and ensuing inflammation.
Electronic supplementary material
The online version of this article (doi:10.1007/s00430-013-0324-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4118039  PMID: 24728387
Complement; Mouse model; Bacterial infection; Dendritic cells; Macrophages; Fc receptor
6.  The Role of Host and Microbial Factors in the Pathogenesis of Pneumococcal Bacteraemia Arising from a Single Bacterial Cell Bottleneck 
PLoS Pathogens  2014;10(3):e1004026.
The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease.
Author Summary
Decades of research on bacterial sepsis have been devoted to analysing the steps that lead from a local event, either carriage or a localised infection, to systemic disease. Our work analyses in depth the events determining systemic infection by one of the main human pathogens, Streptococcus pneumoniae. Consistent with similar findings on the pathogenesis of bacteraemia due to other commensal pathogens, our results show that after an intravenous inoculum of a million pneumococci, the resulting septicaemia is often founded by a single bacterial cell. Investigation into the nature of this monoclonal infection identified strong within-host selective pressure for metabolic fitness during outgrowth of the bacterial population.
PMCID: PMC3961388  PMID: 24651834
7.  Genetic Factors Regulating Lung Vasculature and Immune Cell Functions Associate with Resistance to Pneumococcal Infection 
PLoS ONE  2014;9(3):e89831.
Streptococcus pneumoniae is an important human pathogen responsible for high mortality and morbidity worldwide. The susceptibility to pneumococcal infections is controlled by as yet unknown genetic factors. To elucidate these factors could help to develop new medical treatments and tools to identify those most at risk. In recent years genome wide association studies (GWAS) in mice and humans have proved successful in identification of causal genes involved in many complex diseases for example diabetes, systemic lupus or cholesterol metabolism. In this study a GWAS approach was used to map genetic loci associated with susceptibility to pneumococcal infection in 26 inbred mouse strains. As a result four candidate QTLs were identified on chromosomes 7, 13, 18 and 19. Interestingly, the QTL on chromosome 7 was located within S. pneumoniae resistance QTL (Spir1) identified previously in a linkage study of BALB/cOlaHsd and CBA/CaOlaHsd F2 intercrosses. We showed that only a limited number of genes encoded within the QTLs carried phenotype-associated polymorphisms (22 genes out of several hundred located within the QTLs). These candidate genes are known to regulate TGFβ signalling, smooth muscle and immune cells functions. Interestingly, our pulmonary histopathology and gene expression data demonstrated, lung vasculature plays an important role in resistance to pneumococcal infection. Therefore we concluded that the cumulative effect of these candidate genes on vasculature and immune cells functions as contributory factors in the observed differences in susceptibility to pneumococcal infection. We also propose that TGFβ-mediated regulation of fibroblast differentiation plays an important role in development of invasive pneumococcal disease. Gene expression data submitted to the NCBI Gene Expression Omnibus Accession No: GSE49533
SNP data submitted to NCBI dbSNP Short Genetic Variation
PMCID: PMC3940657  PMID: 24594938
8.  Recombinant Plants Provide a New Approach to the Production of Bacterial Polysaccharide for Vaccines 
PLoS ONE  2014;9(2):e88144.
Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections.
PMCID: PMC3912152  PMID: 24498433
9.  Correction: Impacts of Ocean Acidification on Early Life-History Stages and Settlement of the Coral-Eating Sea Star Acanthaster planci 
PLoS ONE  2014;9(1):10.1371/annotation/b03dc5d7-0cfd-4182-b39d-fb9299275d5c.
PMCID: PMC3883590
10.  Impacts of Ocean Acidification on Early Life-History Stages and Settlement of the Coral-Eating Sea Star Acanthaster planci 
PLoS ONE  2013;8(12):e82938.
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.
PMCID: PMC3865153  PMID: 24358240
11.  Human L-ficolin, a Recognition Molecule of the Lectin Activation Pathway of Complement, Activates Complement by Binding to Pneumolysin, the Major Toxin of Streptococcus pneumoniae 
PLoS ONE  2013;8(12):e82583.
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.
PMCID: PMC3861440  PMID: 24349316
12.  Serum Phosphate as a Risk Factor for Cardiovascular Events in People with and without Chronic Kidney Disease: A Large Community Based Cohort Study 
PLoS ONE  2013;8(9):e74996.
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.
PMCID: PMC3769279  PMID: 24040373
13.  Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments 
Infection and Immunity  2012;80(12):4333-4343.
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.
PMCID: PMC3497430  PMID: 23027531
14.  Analysis Tool Web Services from the EMBL-EBI 
Nucleic Acids Research  2013;41(Web Server issue):W597-W600.
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 ( 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.
PMCID: PMC3692137  PMID: 23671338
15.  Spir2; a novel QTL on chromosome 4 contributes to susceptibility to pneumococcal infection in mice 
BMC Genomics  2013;14:242.
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.
PMCID: PMC3751763  PMID: 23577770
Streptococcus pneumoniae; Host susceptibility; Host genetics; Quantitative trait loci; Model organism; Mouse; Bacterial infection; Inflammation
16.  Optimization of a direct spectrophotometric method to investigate the kinetics and inhibition of sialidases 
BMC Biochemistry  2012;13:19.
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.
PMCID: PMC3483245  PMID: 23031230
Sialidase; Neuraminidase; Chromogenic sialic acids; Kinetic assay; Streptococcus pneumoniae
17.  Regulation of neuraminidase expression in Streptococcus pneumoniae 
BMC Microbiology  2012;12:200.
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.
PMCID: PMC3509027  PMID: 22963456
Sialic acid; Metabolic regulation; Carbon catabolite repression
18.  ciliaFA: a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software 
Cilia  2012;1:14.
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.
PMCID: PMC3607980  PMID: 23351276
19.  The Lectin Pathway of Complement Activation Is a Critical Component of the Innate Immune Response to Pneumococcal Infection 
PLoS Pathogens  2012;8(7):e1002793.
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.
Author Summary
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.
PMCID: PMC3390405  PMID: 22792067
20.  Two Zinc Uptake Systems Contribute to the Full Virulence of Listeria monocytogenes during Growth In Vitro and In Vivo 
Infection and Immunity  2012;80(1):14-21.
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.
PMCID: PMC3255676  PMID: 22025520
21.  T Regulatory Cells Control Susceptibility to Invasive Pneumococcal Pneumonia in Mice 
PLoS Pathogens  2012;8(4):e1002660.
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.
Author Summary
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.
PMCID: PMC3334885  PMID: 22563306
22.  Invariant NKT cells recognize glycolipids from pathogenic Gram-positive bacteria 
Nature immunology  2011;12(10):966-974.
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.
PMCID: PMC3178673  PMID: 21892173
23.  Treatment of Tuberculosis in a Region with High Drug Resistance: Outcomes, Drug Resistance Amplification and Re-Infection 
PLoS ONE  2011;6(8):e23081.
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.
PMCID: PMC3160294  PMID: 21886778
24.  Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4 
PLoS Pathogens  2010;6(11):e1001191.
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.
Author Summary
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.
PMCID: PMC2978728  PMID: 21085613
25.  Acute exposure of mice to high-dose ultrafine carbon black decreases susceptibility to pneumococcal pneumonia 
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.
PMCID: PMC2976728  PMID: 20958976

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