M Thillai, C Eberhardt, AM Lewin, L Potiphar, S Hingley-Wilson, S Sridhar, J Macintyre, OM Kon, M Wickremasinghe, Athol Wells, ME Weeks, D Mitchell, A Lalvani (2012)Sarcoidosis and tuberculosis cytokine profiles: indistinguishable in bronchoalveolar lavage but different in blood., In: PLoS One7(7)pp. e38083-?
The clinical, radiological and pathological similarities between sarcoidosis and tuberculosis can make disease differentiation challenging. A complicating factor is that some cases of sarcoidosis may be initiated by mycobacteria. We hypothesised that immunological profiling might provide insight into a possible relationship between the diseases or allow us to distinguish between them.
Suzanne M. Hingley-Wilson, Nan Ma, Yin Hu, Rosalyn Casey, Anders Bramming, Richard J. Curry, Hongying Lilian Tang, Huihai Wu, Rachel E. Butler, William R. Jacobs, Andrea Rocco, Johnjoe McFadden (2020)Loss of phenotypic inheritance associated with ydcI mutation leads to increased frequency of small, slow persisters in Escherichia coli, In: Proceedings of the National Academy of Sciences117(8)pp. 4152-4157
National Academy of Sciences
Persistence, the phenomenon whereby a small subpopulation of bacterial cells survive sterilization, prolongs antibiotic treatment and contributes to the development of genetic antimicrobial drug resistance (AMR). In this study we performed single-cell tracking of wild-type and high-persister mutant strains of Escherichia coli to identify factors that correlate with persistence. We found, as expected, persistence correlated with slow growth, but also with small birth size. We investigated intergenerational (mother–daughter) and intragenerational (sister–sister) phenotypic inheritance of growth parameters and discovered the mutant phenotype was associated with lower levels of phenotypic inheritance and identified the gene responsible, the transcription factor ydcI. Targeting pathways involved in persistence could reveal approaches to impeding persistence and the development of AMR.
Cell growth experiments with a microfluidic device produce large scale time-lapse image data, which contain important information on cell growth and patterns in their genealogy. To extract such information, we propose a scheme to segment and track bacterial cells automatically. In contrast to most published approaches, which often split segmentation and tracking into two independent procedures, we focus on designing an algorithm that describes cell properties evolving between consecutive frames by feeding segmentation and tracking results from one frame to the next one. The cell boundaries are extracted by minimising the Distance Regularised Level Set Evolution model. Each individual cell was identified and tracked by identifying cell septum and membrane as well as developing a trajectory energy minimisation function along time-lapse series. Experiments show that by applying this scheme, cell growth and division can be measured automatically. The results show the efficiency of the approach when testing on different datasets while comparing with other existing algorithms. The proposed approach demonstrates great potential for large scale bacterial cell growth analysis.
A Allmendinger, J Spavieri, M Kaiser, R Casey, S Hingley-Wilson, A Lalvani, M Guiry, G Blunden, D Tasdemir (2010)Antiprotozoal, Antimycobacterial and Cytotoxic Potential of Twenty-Three British and Irish Red Algae, In: PHYTOTHERAPY RESEARCH24(7)pp. 1099-1103
JOHN WILEY & SONS LTD
Suzie Hingley-Wilson, Nan Ma, Yin Hu, Rosalyn Casey, Anders Bramming, Richard J. Curry, Hongying Lilian Tang, Huihai Wu, Rachel Butler, William R. Jacobs Jr., Andrea Rocco, Johnjoe McFadden (2020)Loss of phenotypic inheritance associated with ydcI mutation leads to increased frequency of small, slow persisters in Escherichia coli, In: Proceedings of the National Academy of Sciences
National Academy of Sciences
Whenever a genetically homogenous population of bacterial cells is exposed to antibiotics, a tiny fraction of cells survives the treatment,the phenomenon known as bacterial persistence [G.L. Hobby et al., Exp. Biol. Med. 50, 281–285 (1942); J. Bigger, The Lancet 244, 497– 500 (1944)]. Despite its biomedical relevance, the origin of the phenomenon is still unknown, and as a rare, phenotypically resistant subpopulation, persisters are notoriously hard to study and define. Using computerized tracking we show that persisters are small at birth and slowly replicating. We also determine that the highpersister mutant strain of Escherichia coli, HipQ, is associated with the phenotype of reduced phenotypic inheritance (RPI). We identify the gene responsible for RPI, ydcI, which encodes a transcription factor, and propose a mechanism whereby loss of phenotypic inheritance causes increased frequency of persisters. These results provide insight into the generation and maintenance of phenotypic variation and provide potential targets for the development of therapeutic strategies that tackle persistence in bacterial infections.
A biocoating confines non-growing, metabolically-active bacteria within a synthetic colloidal polymer (i.e. latex) film. Bacteria encapsulated inside biocoatings can perform useful functions, such as a biocatalyst in wastewater treatment. A biocoating needs to have high a permeability to allow a high rate of mass transfer for rehydration and the transport of both nutrients and metabolic products. It therefore requires an interconnected porous structure. Tuning the porosity architecture is a challenge. Here, we exploited rigid tubular nanoclays (halloysite) and non-toxic latex particles (with a relatively high glass transition temperature) as the colloidal “building blocks” to tailor the porosity inside biocoatings containing Escherichia coli bacteria as a model organism. Electron microscope images revealed inefficient packing of the rigid nanotubes and proved the existence of nanovoids along the halloysite/polymer interfaces. Single-cell observations using confocal laser scanning microscopy provided evidence for metabolic activity of the E. coli within the biocoatings through the expression of yellow fluorescent protein. A custom-built apparatus was used to measure the permeability of a fluorescein sodium salt in the biocoatings. Whereas there was no measurable permeability in a coating made from only latex particles, the permeability coefficient of the composite biocoatings increased with increasing halloysite content up to a value of 110-4 m h-1. The effects of this increase in permeability was demonstrated through a specially-developed resazurin reduction assay. Bacteria encapsulated in halloysite composite biocoatings had statistically significant higher metabolic activities in comparison to bacteria encapsulated in a non-optimized coating made from latex particles alone.
J Spavieri, A Allmendinger, M Kaiser, R Casey, S Hingley-Wilson, A Lalvani, MD Guiry, G Blunden, D Tasdemir (2010)Antimycobacterial, Antiprotozoal and Cytotoxic Potential of Twenty-one Brown Algae (Phaeophyceae) from British and Irish Waters, In: PHYTOTHERAPY RESEARCH24(11)pp. 1724-1729
JOHN WILEY & SONS LTD
T Hsu, Suzie Hingley-Wilson, B Chen, M Chen, AZ Dai, PM Morin, CB Marks, J Padiyar, C Goulding, M Gingery, D Eisenberg, RG Russell, SC Derrick, FM Collins, SL Morris, CH King, WR Jr Jacobs The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue, In: Proceedings of the National Academy of Sciences of the United States of America100(21)pp. 12420-12425
National Academy of Sciences
Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette–Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting ΔRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis ΔRD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the ΔRD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette–Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.
With the completion of genome sequencing of Mycobacterium tuberculosis and upsurge in the incidence of M. tuberculosis infection worldwide partly as a result of HIV pandemic, there is need for rationale approach to vaccine and chemotherapy discoveries for M. tuberculosis. The homologue of mig gene of Mycobacterium avium was searched for in the M. tuberculosis database at The Institute of Genomic Research (TIGR), USA and The Sanger Institute, UK. Homologue of the gene was found and comprehensively analysed. Reverse transcription PCR (RT-PCR) was carried out on the mig (fadD19) gene homologue and echA19 gene. The result of the RT-PCR showed that the mig gene was at least 2-fold upregulated during intracellular infection of macrophage compared to the broth grown bacilli as opposed to the demonstrated specific expression of mig gene in M. avium infected macrophage. The echA19 gene was also found to be upregulated. . © Ibadan Biomedical Communications Group.
Mcl-1 protein expression was found to be up-regulated during infection with virulent Mycobacterium tuberculosis strain H37Rv. Mcl-1 induction in THP-1 cells was optimal at a multiplicity of infection of 0.8–1.2 bacilli per macrophage and was independent of opsonin coating of the bacteria. Mcl-1 expression was elevated as early as 4 h, peaked at 5.8-fold above control cells at 24 h, and remained elevated at 48 h after infection. In THP-1 cells, mMcl-1 mRNA was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. In THP-1 cells and monocyte-derived macrophages (MDMs), Mcl-1 protein was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. Treatment of uninfected, H37Ra-infected, and H37Rv-infected THP-1 cells and MDMs with antisense oligonucleotides to mcl-1 reduced Mcl-1 expression by >84%. This resulted in an increase in apoptosis of both MDMs and THP-1 cells that were infected with H37Rv, but not cells that were uninfected or infected with H37Ra. Increased apoptosis correlated with a decrease in M. tuberculosis CFUs recovered from antisense-treated, H37Rv-infected cells at 4 and 7 days after infection. In contrast, CFU recoveries from sense-treated, H37Rv-infected cells or from antisense- or sense-treated, H37Ra-infected cells were unchanged from controls. Thus, the antiapoptotic effect of the induction of Mcl-1 expression in H37Rv-infected macrophages promotes the survival of virulent M. tuberculosis.
N Upton, DJ Jackson, AA Nikonova, Suzie Hingley-Wilson, M Khaitov, A Del Rosario, S Traub, MB Trujillo-Torralbo, M Habibi, SL Elkin, OM Kon, MR Edwards, P Mallia, J Footitt, J Macintyre, LA Stanciu, SL Johnston, A Sykes (2017)Rhinovirus induction of fractalkine (CX3CL1) in airway and peripheral blood mononuclear cells in asthma, In: PLoS One12(8)e0183864
Public Library of Science
Rhinovirus infection is associated with the majority of asthma exacerbations. The role of fractalkine in anti-viral (type 1) and pathogenic (type 2) responses to rhinovirus infection in allergic asthma is unknown. To determine whether (1) fractalkine is produced in airway cells and in peripheral blood leucocytes, (2) rhinovirus infection increases production of fractalkine and (3) levels of fractalkine differ in asthmatic compared to non-asthmatic subjects. Fractalkine protein and mRNA levels were measured in bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs) from non-asthmatic controls (n = 15) and mild allergic asthmatic (n = 15) subjects. Protein levels of fractalkine were also measured in macrophages polarised ex vivo to give M1 (type 1) and M2 (type 2) macrophages and in BAL fluid obtained from mild (n = 11) and moderate (n = 14) allergic asthmatic and non-asthmatic control (n = 10) subjects pre and post in vivo rhinovirus infection. BAL cells produced significantly greater levels of fractalkine than PBMCs. Rhinovirus infection increased production of fractalkine by BAL cells from non-asthmatic controls (P<0.01) and in M1-polarised macrophages (P<0.05), but not in BAL cells from mild asthmatics or in M2 polarised macrophages. Rhinovirus induced fractalkine in PBMCs from asthmatic (P<0.001) and healthy control subjects (P<0.05). Trends towards induction of fractalkine in moderate asthmatic subjects during in vivo rhinovirus infection failed to reach statistical significance. Fractalkine may be involved in both immunopathological and anti-viral immune responses to rhinovirus infection. Further investigation into how fractalkine is regulated across different cell types and into the effect of stimulation including rhinovirus infection is warranted to better understand the precise role of this unique dual adhesion factor and chemokine in immune cell recruitment.
D Montamat-Sicotte, K Millington, CR Willcox, S Hingley-Wilson, S Hackforth, J Innes, OM Kon, DE Minnikin, GS Besra, BE Willcox, A Lalvani (2010)Mycolic acid-specific T-cells in human tuberculosis are dynamically related to antigen load and exhibit memory expansion after cure, In: IMMUNOLOGY131pp. 171-171 SM Hingley-Wilson, R Casey, D Connell, S Bremang, JT Evans, PM Hawkey, GE Smith, A Jepson, S Philip, OM Kon, A Lalvani (2013)Undetected multidrug-resistant tuberculosis amplified by first-line therapy in mixed infection., In: Emerg Infect Dis19(7)pp. 1138-1141
Infections with >1 Mycobacterium tuberculosis strain(s) are underrecognized. We show, in vitro and in vivo, how first-line treatment conferred a competitive growth advantage to amplify a multidrug-resistant M. tuberculosis strain in a patient with mixed infection. Diagnostic techniques that identify mixed tubercle bacilli populations are needed to curb the spread of multidrug resistance.
Studying defined mutants of Mycobacterium tuberculosis in the mouse model of infection has led to the discovery of attenuated mutants that fall into several phenotypic classes. These mutants are categorized by their growth characteristics compared with those of wild-type M. tuberculosis, and include severe growth in vivo mutants, growth in vivo mutants, persistence mutants, pathology mutants and dissemination mutants. Here, examples of each of these mutant phenotypes are described and classified accordingly. Defining the importance of mycobacterial gene products responsible for in vivo growth, persistence and the induction of immunopathology will lead to a greater understanding of the host-pathogen interaction and potentially to new antimycobacterial treatment options.
SM Hingley-Wilson, R Casey, D Connell, S Bremang, JT Evans, PM Hawkey, GE Smith, A Jepson, S Philip, OM Kon, A Lalvani (2013)Undetected Multidrug-Resistant Tuberculosis Amplified by First-line Therapy in Mixed Infection, In: EMERGING INFECTIOUS DISEASES19(7)pp. 1138-1141
CENTERS DISEASE CONTROL
The 6-kDa early secretory antigenic target of Mycobacterium tuberculosis (ESAT-6) and the 10-kDa culture filtrate antigen (CFP-10), encoded in region of difference 1 (RD1) and secreted by the ESAT-6 system 1 (Esx-1) secretion system, are the most immunodominant and highly M. tuberculosis (MTB)-specific antigens. These attributes are responsible for their primary importance in tuberculosis (TB) immunodiagnosis and vaccine development. Rv3615c [Esx-1 substrate protein C (EspC)], encoded outside RD1, is similar in size and sequence homology to CFP-10 and ESAT-6, suggesting it might be a target of cellular immunity in TB. Using ex vivo enzyme-linked immunospot- and flow cytometry-based cytokine-secretion assay, we comprehensively assessed cellular immune responses to EspC in patients with active TB, latently infected persons, and uninfected bacillus Calmette-Guérin (BCG)-vaccinated controls. EspC was at least as immunodominant as ESAT-6 and CFP-10 in both active and latent TB infection. EspC contained broadly recognized CD4(+) and CD8(+) epitopes, inducing a predominantly CD4(+) T-cell response that comprised functional T-cell subsets secreting both IFN-γ and IL-2 as well as functional T-cell subsets secreting only IFN-γ. Surprisingly, T-cell responses to EspC were as highly specific (93%) for MTB infection as responses to ESAT-6 and CFP-10, with only 2 of 27 BCG-vaccinated controls responding to each antigen. Using quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains, we identified the mechanism of the specificity of anti-EspC immunity as the Esx-1 dependence of EspC secretion. The high immunodominance of EspC, equivalent to that of ESAT-6 and CFP-10, makes it a TB vaccine candidate, and its high specificity confers strong potential for T-cell-based immunodiagnosis.
It is thought that during latent infection, Mycobacterium tuberculosis bacilli are retained within granulomas in a low-oxygen environment. The dormancy survival (Dos) regulon, regulated by the response regulator DosR, appears to be essential for hypoxic survival in M. tuberculosis, but it is not known how the regulon promotes survival. Here we report that mycobacteria, in contrast to enteric bacteria, do not form higher-order structures (e.g. ribosomal dimers) upon entry into stasis. Instead, ribosomes are stabilized in the associated form (70S). Using a strategy incorporating microfluidic, proteomic, and ribosomal profiling techniques to elucidate the fate of mycobacterial ribosomes during hypoxic stasis, we show that the dormancy regulator DosR is required for optimal ribosome stabilization. We present evidence that the majority of this effect is mediated by the DosR-regulated protein MSMEG_3935 (a S30AE domain protein), which is associated with the ribosome under hypoxic conditions. A Δ3935 mutant phenocopies the ΔdosR mutant during hypoxia, and complementation of ΔdosR with the MSMEG_3935 gene leads to complete recovery of dosR mutant phenotypes during hypoxia. We suggest that this protein is named ribosome-associated factor under hypoxia (RafH) and that it is the major factor responsible for DosR-mediated hypoxic survival in mycobacteria.
SM Hingley-Wilson, D Connell, K Pollock, L Grass, L Potiphar, S Bremang, A Lalvani, T Hsu, WR Jacobs Jr, E Tchilian, A Sykes, OM Kon (2014)ESX1-dependent fractalkine mediates chemotaxis and Mycobacterium tuberculosis infection in humans, In: Tuberculosis94(3)pp. 262-270
Mycobacterium tuberculosis-induced cellular aggregation is essential for granuloma formation and may assist establishment and early spread of M. tuberculosis infection. The M. tuberculosis ESX1 mutant, which has a non-functional type VII secretion system, induced significantly less production of the host macrophage-derived chemokine fractalkine (CX3CL1). Upon infection of human macrophages ESX1-dependent fractalkine production mediated selective recruitment of CD11b+ monocytic cells and increased infection of neighbouring cells consistent with early local spread of infection. Fractalkine levels were raised in vivo at tuberculous disease sites in humans and were significantly associated with increased CD11b+ monocytic cellular recruitment and extent of granulomatous disease. These findings suggest a novel fractalkine-dependent ESX1-mediated mechanism in early tuberculous disease pathogenesis in humans. Modulation of M. tuberculosis-mediated fractalkine induction may represent a potential treatment option in the future, perhaps allowing us to switch off a key mechanism required by the pathogen to spread between cells. © 2014 The Authors.
M Pareek, J Evans, J Innes, G Smith, S Hingley-Wilson, KE Lougheed, S Sridhar, M Dedicoat, P Hawkey, A Lalvani (2013)Ethnicity and mycobacterial lineage as determinants of tuberculosis disease phenotype., In: Thorax68(3)pp. 221-229
BACKGROUND: Emerging evidence suggests that Mycobacterium tuberculosis (Mtb) lineage and host ethnicity can determine tuberculosis (TB) clinical disease patterns but their relative importance and interaction are unknown. METHODS: We evaluated prospectively collected TB surveillance and Mtb strain typing data in an ethnically heterogeneous UK population. Lineage assignment was denoted using 15-loci mycobacterial interspersed repetitive units containing variable numbers of tandem repeats (MIRU-VNTR) and MIRU-VNTRplus. Geographical and ethnic associations of the six global Mtb lineages were identified and the influence of lineage and demographic factors on clinical phenotype were assessed using multivariate logistic regression. RESULTS: Data were available for 1070 individuals with active TB which was pulmonary only, extrapulmonary only and concurrent pulmonary-extrapulmonary in 52.1%, 36.9% and 11.0% respectively. The most prevalent lineages were Euro-American (43.7%), East African Indian (30.2%), Indo-Oceanic (13.6%) and East Asian (12.2%) and were geo-ethnically restricted with, for example, Indian subcontinent ethnicity inversely associated with Euro-American lineage (OR 0.23; 95% CI 0.14 to 0.39) and positively associated with the East African-Indian lineage (OR 4.04; 95% CI 2.19 to 7.45). Disease phenotype was most strongly associated with ethnicity (OR for extrathoracic disease 21.14 (95% CI 6.08 to 73.48) for Indian subcontinent and 14.05 (3.97 to 49.65) for Afro-Caribbean), after adjusting for lineage. With East Asian lineage as the reference category, the Euro-American (OR 0.54; 95% CI 0.32 to 0.91) and East-African Indian (OR 0.50; 95% CI 0.29 to 0.86) lineages were negatively associated with extrathoracic disease, compared with pulmonary disease, after adjusting for ethnicity. CONCLUSIONS: Ethnicity is a powerful determinant of clinical TB phenotype independently of mycobacterial lineage and the role of ethnicity-associated factors in pathogenesis warrants investigation.
DJ Montamat-Sicotte, KA Millington, CR Willcox, S Hingley-Wilson, S Hackforth, J Innes, OM Kon, DA Lammas, DE Minnikin, GS Besra, BE Willcox, A Lalvani (2011)A mycolic acid-specific CD1-restricted T cell population contributes to acute and memory immune responses in human tuberculosis infection., In: J Clin Invest121(6)pp. 2493-2503
Current tuberculosis (TB) vaccine strategies are largely aimed at activating conventional T cell responses to mycobacterial protein antigens. However, the lipid-rich cell wall of Mycobacterium tuberculosis (M. tuberculosis) is essential for pathogenicity and provides targets for unconventional T cell recognition. Group 1 CD1-restricted T cells recognize mycobacterial lipids, but their function in human TB is unclear and their ability to establish memory is unknown. Here, we characterized T cells specific for mycolic acid (MA), the predominant mycobacterial cell wall lipid and key virulence factor, in patients with active TB infection. MA-specific T cells were predominant in TB patients at diagnosis, but were absent in uninfected bacillus Calmette-Guérin-vaccinated (BCG-vaccinated) controls. These T cells were CD1b restricted, detectable in blood and disease sites, produced both IFN-γ and IL-2, and exhibited effector and central memory phenotypes. MA-specific responses contracted markedly with declining pathogen burden and, in patients followed longitudinally, exhibited recall expansion upon antigen reencounter in vitro long after successful treatment, indicative of lipid-specific immunological memory. T cell recognition of MA is therefore a significant component of the acute adaptive and memory immune response in TB, suggesting that mycobacterial lipids may be promising targets for improved TB vaccines.
B Broniatowska, A Allmendinger, D Tasdemir, M Kaiser, D Montamat-Sicotte, S Hingley-Wilson, A Lalvani, M Guiry, G Blunden (2011)Antiprotozoal, antitubercular and cytotoxic potential of cyanobacterial (blue-green algal) extracts from Ireland, In: Natural Product Communications6(5)pp. 689-694
Cyanobacteria (= blue-green algae) are prolific producers of structurally distinct and biologically active metabolites. In the continuation of our search for new sources of anti-infective natural products, we have assessed the in vitro antiprotozoal (Plasmodium falciparum, Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani) and antitubercular (Mycobacterium tuberculosis) potential of samples of two terrestrial cyanobacteria, Nostoc commune (collected when desiccated and wet) and Rivularia biasolettiana. The cytotoxic potential of the extracts was also evaluated against primary L6 cells. Except for T. cruzi and M. tuberculosis, the crude extracts were active against all the organisms tested and showed no toxicity. The crude extracts were then partitioned between n-hexane, chloroform and aqueous methanol and retested against the same panel of pathogens. The chloroform sub-extracts of both N. commune samples showed significant activity against T. b. rhodesiense (IC values 2.0 and 3.5 μg/mL) and P. falciparum (IC s 7.4 and 5.8 μg/mL), with low toxicity. This trend was also true for R. biasolettiana extracts, and its chloroform sub-extract showed notable activity against all parasitic protozoa. There were differences in the biological activity profiles of extracts derived from desiccated and hydrated forms of N. commune. To our knowledge, this is the first study assessing the anti-infective activity of desiccated and hydrated forms of N. commune, as well as R. biasolettiana. Furthermore, the present work reports such biological activity in terrestrial cyanobacteria from Ireland for the first time. These results warrant the further study of Irish terrestrial cyanobacteria as a valuable source of new natural product leads for the treatment of parasitic protozoal infections.
Neisseria meningitidis causes meningococcal disease, a global life threatening illness with annual incidences of between 1 and 1000 per 100,000 population. Humans are the only known host with approximately 10% of people having asymptomatic nasopharyngeal carriage at any one time. Thus, the ability of meningococci to attach, invade, and grow in the epithelium is crucial for both its commensal and pathogenic properties. In the rare event that meningococci cross the epithelium into the bloodstream, disease may occur. In order to better understand the mechanisms of meningococcal pathogenesis, transposon mutagenesis was used to identify bacterial genes involved in epithelial cell adherence and internalization as well as traversal of the epithelial barrier. Three epithelial cell lines of respiratory origin, A549 cells, 16HBE14o- cells and Detroit 562 cells were used to examine N. meningitidis L91543 (C:2a:P1.2, ST-11; ET-37) pathogenesis. First, adhesion, invasion and traversal assays were optimized for bacterial uptake to enable the maximum number of mutants to be tested and to avoid stochastic loss from the transposon library. Since the highest level of meningococci adherence and invasion was observed using 16HBE14o- cells, this cell line was chosen for subsequent traversal assays, where an intact epithelial barrier was established on Transwell® membrane inserts. Epithelial barrier integrity was assessed by measuring transepithelial electrical resistance (TEER), permeability of the marker protein, 70 kDa Dextran, and by examining the distribution of the tight junction proteins, occludin and ZO-1, by immunofluorescence. Next, transposon mutagenesis libraries comprising of approx. 14,500 N. meningitidis L91543 mutants, were used to probe meningococcal interactions with 16HBE14o- epithelial cells. Illumina sequencing of amplified transposon junctions was performed on DNA extracted from both input and output pools obtained from the various assays. Comparative analysis of input/output pools showed reduced fitness, not only of genes associated with type IV pili, but mainly of genes involved in metabolism especially nucleotide and amino acid metabolism. Genes involved in membrane transport, regulatory functions and cellular processes also showed reduced fitness. The function of putative genes of interest was validated by generating insertion knockout mutants and testing them independently for their ability to alter meningococcal-epithelial cell interactions. The knockout mutant assays showed 67-100% agreement to the Tn-Seq analysis prediction. Based on the knock out mutant assays as well the Tn-Seq prediction we can conclude that type IV pili, nucleotide biosynthesis, glucose and amino acid metabolism, as well as resistance to antimicrobial peptide are critical for meningococcal interaction with epithelial cells.