Complete Genome Sequences of Clinical Pandoraea fibrosis Isolates.

Pandoraea fibrosis is a newly identified Gram-negative bacterial species that was isolated from the respiratory tract of an Australian cystic fibrosis patient. The complete assembled genome sequences of two consecutive isolates (second isolate collected 11 months after antibiotic treatment) from the same individual are presented here.

Pandoraea fibrosis sp. nov., a novel Pandoraea species isolated from clinical respiratory samples.

Pandoraea species have been isolated from diverse environmental samples and are emerging important respiratory pathogens, particularly in people with cystic fibrosis (CF). In the present study, two bacterial isolates initially recovered from consecutive sputum samples collected from a CF patient and identified as Pandoraea pnomenusa underwent a polyphasic taxonomic analysis. The isolates were found to be Gram-negative, facultative anaerobic motile bacilli and subsequently designated as strains 6399T (=LMG29626T=DSM103228T) and 7641 (=LMG29627=DSM103229), respectively. Phylogenetic analysis based on 16S rRNA and gyrB gene sequences revealed that 6399T and 7641 formed a distinct phylogenetic lineage within the genus Pandoraea. Genome sequence comparison analysis indicated that strains 6399T and 7641 are clonal and share 100 % similarity, however, similarity to other type strains (ANIb 73.2-88.8 %, ANIm 83.5-89.9 % and OrthoANI 83.2-89.3 %) indicates that 6399T and 7641 do not belong to any of the reported type species. The major cellular fatty acids of 6399T were C16 : 0 (32.1 %) C17 : 0cyclo (18.7 %) and C18 : 1ω7c (14.5 %), while Q-8 was the only respiratory quinone detected. The major polar lipids identified were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G+C content of 6399T was 62.9 (mol%). Strain 6399T can be differentiated from other members of Pandoraea by the absence of C19 : 0ω8c cyclo and by the presence of C17 : 0ω8c cyclo. Together our data show that the bacterial strains 6399T and 7641 represent a novel species of the genus Pandoraea, for which the name Pandoraea fibrosis sp. nov. is proposed (type strain 6399T).

Widespread alterations in the peripheral blood innate immune cell profile in cystic fibrosis reflect lung pathology.

Cystic fibrosis (CF) is caused by mutations to the CF transmembrane conductance regulator (CFTR) gene. CFTR is known to be expressed on multiple immune cell subtypes, dendritic cells, monocytes/macrophages, neutrophils and lymphocytes. We hypothesized that the lack of CFTR expression on peripheral blood innate immune cells would result in an altered cell profile in the periphery and that this profile would reflect lung pathology. We performed a flow cytometric phenotypic investigation of innate immune cell proportions in peripheral blood collected from 17 CF patients and 15 age-matched healthy controls. We observed significant differences between CF patients and controls in the relative proportions of natural killer (NK) cells, monocytes and their subsets, with significant correlations observed between proportions of NK and monocyte cell subsets and lung function (forced expiratory volume in 1 sec, % predicted; FEV1% predicted) in CF patients. This study demonstrates the widespread nature of immune dysregulation in CF and provides a basis for identification of potential therapeutic targets. Modulation of the distinct CF-related immune cell phenotype identified could also be an important biomarker for evaluating CFTR-targeted drug efficacy.

Differentially expressed genes from RNA-Seq and functional enrichment results are affected by the choice of single-end versus paired-end reads and stranded versus non-stranded protocols.

BACKGROUND: RNA-Seq is now widely used as a research tool. Choices must be made whether to use paired-end (PE) or single-end (SE) sequencing, and whether to use strand-specific or non-specific (NS) library preparation kits. To date there has been no analysis of the effect of these choices on identifying differentially expressed genes (DEGs) between controls and treated samples and on downstream functional analysis. RESULTS: We undertook four mammalian transcriptomics experiments to compare the effect of SE and PE protocols on read mapping, feature counting, identification of DEGs and functional analysis. For three of these experiments we also compared a non-stranded (NS) and a strand-specific approach to mapping the paired-end data. SE mapping resulted in a reduced number of reads mapped to features, in all four experiments, and lower read count per gene. Up to 4.3% of genes in the SE data and up to 12.3% of genes in the NS data had read counts which were significantly different compared to the PE data. Comparison of DEGs showed the presence of false positives (average 5%, using voom) and false negatives (average 5%, using voom) using the SE reads. These increased further, by one or two percentage points, with the NS data. Gene ontology functional enrichment (GO) of the DEGs arising from SE or NS approaches, revealed striking differences in the top 20 GO terms, with as little as 40% concordance with PE results. Caution is therefore advised in the interpretation of such results. By comparison, there was overall consistency in gene set enrichment analysis results. CONCLUSIONS: A strand-specific protocol should be used in library preparation to generate the most reliable and accurate profile of expression. Ideally PE reads are also recommended particularly for transcriptome assembly. Whilst SE reads produce a DEG list with around 5% of false positives and false negatives, this method can substantially reduce sequencing cost and this saving could be used to increase the number of biological replicates thereby increasing the power of the experiment. As SE reads, when used in association with gene set enrichment, can generate accurate biological results, this may be a desirable trade-off.

Pandoraea pnomenusa Isolated from an Australian Patient with Cystic Fibrosis.

Pandoraea species are considered as emerging pathogens in people with cystic fibrosis (CF). The contribution of these organisms to disease progression in CF patients is not fully understood owing in large measure to the scant reports in clinical and research literature describing their colonization of CF patients and their associated virulence determinants. In an effort to increase awareness and evidence for Pandoraea spp. infection in people with CF, and to stimulate research aimed at unraveling the pathogenic properties of Pandoraea, we report a case of a 26-year-old Australian (Tasmanian) man with CF who was chronically infected with Pandoraea pnomenusa for at least one year prior to his death from respiratory failure. In addition, we describe for the first time evidence suggesting that this bacterium is a facultative anaerobe and report on the availability of a whole genome sequence for this organism. To the best of our knowledge, this report represents only the second clinical case study of P. pnomenusa infection in the world, and the first in an Australian CF patient.

Functionalized polyanilines disrupt Pseudomonas aeruginosa and Staphylococcus aureus biofilms.

The purpose of the present study was to investigate the antimicrobial effects of functionalized polyanilines (fPANIs) against stationary phase cells and biofilms of Pseudomonas aeruginosa and Staphylococcus aureus using homopolymer of sulfanilic acid (poly-SO3H) as a model. The chemically synthesized poly-SO3H was characterized using Fourier Transform Infra-Red (FTIR) and Ultraviolet-Visible (UV-Vis) spectroscopies. The molecular weight (Mw) and elemental analysis of homopolymer poly-SO3H were also examined. We found that poly-SO3H was bactericidal against stationary phase cells of P. aeruginosa and S. aureus at a concentration of 20 mgml(-1). Surprisingly, we discovered that the same concentration (20 mgml(-1)) of poly-SO3H significantly disrupted and killed bacterial cells present in pre-established forty-eight hour static biofilms of these organisms, as shown by crystal violet and bacterial live/dead fluorescence staining assays. In support of these data, poly-SO3H extensively diminished the expression of bacterial genes related to biofilm formation in stationary phase cells of P. aeruginosa, and seemed to greatly reduce the amount of the quorum sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) able to be recovered from biofilms of this organism. Furthermore, we found that poly-SO3H was able to effectively penetrate and kill cells in biofilms formed by the P. aeruginosa (AESIII) isolate derived from the sputum of a cystic fibrosis patient. Taken together, the results of the present study emphasise the broad antimicrobial activities of fPANI, and suggest that they could be developed further and used in some novel ways to construct medical devices and/or industrial equipment that are refractory to colonization by biofilm-forming bacteria.

High peripheral blood th17 percent associated with poor lung function in cystic fibrosis.

People with cystic fibrosis (CF) have been reported to make lung T cell responses that are biased towards T helper (Th) 2 or Th17. We hypothesized that CF-related T cell regulatory defects could be detected by analyzing CD4+ lymphocyte subsets in peripheral blood. Peripheral blood mononuclear cells from 42 CF patients (6 months-53 years old) and 78 healthy controls (2-61 years old) were analyzed for Th1 (IFN-γ+), Th2 (IL-4+), Th17 (IL-17+), Treg (FOXP3+), IL-10+ and TGF-ß+ CD4+ cells. We observed higher proportions of Treg, IL-10+ and TGF-ß+ CD4+ cells in CF adults (≥ 18 years old), but not children/adolescents, compared with controls. Within the CF group, high TGF-ß+% was associated with chronic Pseudomonas aeruginosa lung infection (p < 0.006). We observed no significant differences between control and CF groups in the proportions of Th1, Th2 or Th17 cells, and no association within the CF group of any subset with sex, CFTR genotype, or clinical exacerbation. However, high Th17% was strongly associated with poor lung function (FEV1 % predicted) (p = 0.0008), and this association was strongest when both lung function testing and blood sampling were performed within one week. Our results are consistent with reports of CF as a Th17 disease and suggest that peripheral blood Th17 levels may be a surrogate marker of lung function in CF.

Pseudomonas aeruginosa uses multiple pathways to acquire iron during chronic infection in cystic fibrosis lungs.

Pseudomonas aeruginosa chronically infects the lungs of more than 80% of adult patients with cystic fibrosis (CF) and is a major contributor to the progression of disease pathology. P. aeruginosa requires iron for growth and has multiple iron uptake systems that have been studied in bacteria grown in laboratory culture. The purpose of this research was to determine which of these are active during infection in CF. RNA was extracted from 149 sputum samples obtained from 23 CF patients. Reverse transcription-quantitative real-time PCR (RT-qPCR) was used to measure the expression of P. aeruginosa genes encoding transport systems for the siderophores pyoverdine and pyochelin, for heme, and for ferrous ions. Expression of P. aeruginosa genes could be quantified in 89% of the sputum samples. Expression of genes associated with siderophore-mediated iron uptake was detected in most samples but was at low levels in some samples, indicating that other iron uptake mechanisms are active. Expression of genes encoding heme transport systems was also detected in most samples, indicating that heme uptake occurs during infection in CF. feoB expression was detected in all sputum samples, implying an important role for ferrous ion uptake by P. aeruginosa in CF. Our data show that multiple P. aeruginosa iron uptake mechanisms are active in chronic CF infection and that RT-qPCR of RNA extracted from sputum provides a powerful tool for investigating bacterial physiology during infection in CF.

Expression of PPARγ and paraoxonase 2 correlated with Pseudomonas aeruginosa infection in cystic fibrosis.

The Pseudomonas aeruginosa quorum sensing signal molecule N-3-oxododecanoyl-l-homoserine lactone (3OC(12)HSL) can inhibit function of the mammalian anti-inflammatory transcription factor peroxisome proliferator activated receptor (PPAR)γ, and can be degraded by human paraoxonase (PON)2. Because 3OC(12)HSL is detected in lungs of cystic fibrosis (CF) patients infected with P. aeruginosa, we investigated the relationship between P. aeruginosa infection and gene expression of PPARγ and PON2 in bronchoalveolar lavage fluid (BALF) of children with CF. Total RNA was extracted from cell pellets of BALF from 43 children aged 6 months-5 years and analyzed by reverse transcription-quantitative real time PCR for gene expression of PPARγ, PON2, and P. aeruginosa lasI, the 3OC(12)HSL synthase. Patients with culture-confirmed P. aeruginosa infection had significantly lower gene expression of PPARγ and PON2 than patients without P. aeruginosa infection. All samples that were culture-positive for P. aeruginosa were also positive for lasI expression. There was no significant difference in PPARγ or PON2 expression between patients without culture-detectable infection and those with non-Pseudomonal bacterial infection, so reduced expression was specifically associated with P. aeruginosa infection. Expression of both PPARγ and PON2 was inversely correlated with neutrophil counts in BALF, but showed no correlation with other variables evaluated. Thus, lower PPARγ and PON2 gene expression in the BALF of children with CF is associated specifically with P. aeruginosa infection and neutrophilia. We cannot differentiate whether this is a cause or the effect of P. aeruginosa infection, but propose that the level of expression of these genes may be a marker for susceptibility to early acquisition of P. aeruginosa in children with CF.

Neisseria gonorrhoeae pilin glycan contributes to CR3 activation during challenge of primary cervical epithelial cells.

Expression of type IV pili by Neisseria gonorrhoeae plays a critical role in mediating adherence to human epithelial cells. Gonococcal pilin is modified with an O-linked glycan, which may be present as a di- or monosaccharide because of phase variation of select pilin glycosylation genes. It is accepted that bacterial proteins may be glycosylated; less clear is how the protein glycan may mediate virulence. Using primary, human, cervical epithelial (i.e. pex) cells, we now provide evidence to indicate that the pilin glycan mediates productive cervical infection. In this regard, pilin glycan-deficient mutant gonococci exhibited an early hyper-adhesive phenotype but were attenuated in their ability to invade pex cells. Our data further indicate that the pilin glycan was required for gonococci to bind to the I-domain region of complement receptor 3, which is naturally expressed by pex cells. Comparative, quantitative, infection assays revealed that mutant gonococci lacking the pilin glycan did not bind to the I-domain when it is in a closed, low-affinity conformation and cannot induce an active conformation to complement receptor 3 during pex cell challenge. To our knowledge, these are the first data to directly demonstrate how a protein-associated bacterial glycan may contribute to pathogenesis.

Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions.

The success of Pseudomonas aeruginosa in cystic fibrosis (CF) and other chronic infections is largely attributed to its ability to grow in antibiotic-resistant biofilm communities. This study investigated the effects of limiting iron levels as a strategy for preventing/disrupting P. aeruginosa biofilms. A range of synthetic and naturally occurring iron-chelating agents were examined. Biofilm development by P. aeruginosa strain PAO1 and CF sputum isolates from chronically infected individuals was significantly decreased by iron removal under aerobic atmospheres. CF strains formed poor biofilms under anaerobic conditions. Strain PAO1 was also tested under anaerobic conditions. Biofilm formation by this model strain was almost totally prevented by several of the chelators tested. The ability of synthetic chelators to impair biofilm formation could be reversed by iron addition to cultures, providing evidence that these effective chelating compounds functioned by directly reducing availability of iron to P. aeruginosa. In contrast, the biological chelator lactoferrin demonstrated enhanced anti-biofilm effects as iron supplementation increased. Hence biofilm inhibition by lactoferrin appeared to occur through more complex mechanisms to those of the synthetic chelators. Overall, our results demonstrate the importance of iron availability to biofilms and that iron chelators have potential as adjunct therapies for preventing biofilm development, especially under low oxygen conditions such as encountered in the chronically infected CF lung.

Recombinant probiotics for treatment and prevention of enterotoxigenic Escherichia coli diarrhea.

BACKGROUND & AIMS: We have developed a therapeutic strategy for gastrointestinal infections that is based on molecular mimicry of host receptors for bacterial toxins on the surface of harmless gut bacteria. The aim of this study was to apply this to the development of a recombinant probiotic for treatment and prevention of diarrheal disease caused by enterotoxigenic Escherichia coli strains that produce heat-labile enterotoxin. METHODS: This was achieved by expressing glycosyltransferase genes from Neisseria meningitidis or Campylobacter jejuni in a harmless Escherichia coli strain (CWG308), resulting in the production of a chimeric lipopolysaccharide capable of binding heat-labile enterotoxin with high avidity. RESULTS: The strongest heat-labile enterotoxin binding was achieved with a construct (CWG308:pLNT) that expresses a mimic of lacto-N-neotetraose, which neutralized > or = 93.8% of the heat-labile enterotoxin activity in culture lysates of diverse enterotoxigenic Escherichia coli strains of both human and porcine origin. When tested with purified heat-labile enterotoxin, it was capable of adsorbing approximately 5% of its own weight of toxin. Weaker toxin neutralization was achieved with a construct that mimicked the ganglioside GM2. Preabsorption with, or coadministration of, CWG308:pLNT also resulted in significant in vivo protection from heat-labile enterotoxin-induced fluid secretion in rabbit ligated ileal loops. CONCLUSIONS: Toxin-binding probiotics such as those described here have considerable potential for prophylaxis and treatment of enterotoxigenic Escherichia coli-induced travelers' diarrhea.

Analysis of the role of pglI in pilin glycosylation of Neisseria meningitidis.

Pilin is the major subunit of the essential virulence factor pili and is glycosylated at Ser63. In this study we investigated the gene pglI to determine whether it is involved in the biosynthesis of the pilin-linked glycan of Neisseria meningitidis strain C311#3. A N. meningitidis C311#3pglI mutant resulted in a change of apparent molecular weight in SDS-PAGE and altered binding of antisera, consistent with a role in the biosynthesis of the pilin-linked glycan. These data, in conjunction with homology with well-characterised acyltransferases suggests a specific role for pglI in the biosynthesis of the basal 2,4-diacetamido-2,4,6-trideoxyhexose residue of the pilin-linked glycan.

Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis.

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translationally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria.

Two novel chromosomal loci influence cultivar-specific nodulation failure in the interaction between strain ANU794 and subterranean clover cv. Woogenellup.

The nodulation failure resulting from the interaction between Rhizobium leguminosarum biovar trifolii strain ANU794 and the Trifolium subterraneum cv. Woogenellup was examined by transposon mutagenesis to resolve whether multiple determinants were involved in cultivar-specificity. Three new transposon-induced mutants of ANU794 (W72, W78 and W710) with significantly enhanced nodulation ability on cv. Woogenellup were identified. The W72 and W78 mutations are chromosomally-located, whereas the W710 mutation isplasmid-located. The ethylene synthesis inhibitor, aminoethoxyvinylglycine, fails to enhance the nodulation ability of ANU794, ANU7943 (csn1::Tn5) and W78 on cv. Woogenellup, but enhances the nodulation ability of W72,W710 and ANU7941 (nodM::Tn5). DNA sequencing of the W78 locus reveals strong homology to an unknown Mycobacterium open reading frame, and to several bacterial non-haem chloroperoxidases. The previously identified csn1 locus showed homology to the 50S ribosomal protein, L9, with the Tn5 insertion being located in the 5'-untranslated region. The results suggest that cultivar-specificity is mediated by at least two independent mechanisms or determinants, and not by a simple gene-for-gene interaction. The role of ethylene in cultivar specificity is discussed. Cultivar-specific interactions may prove useful in identifying pathways involved in efficient nodule formation and plant-microbe interactions.

Erratum to: Two novel chromosomal loci influence cultivar-specific nodulation failure in the interaction between strain ANU794 and subterranean clover cv. Woogenellup.

The nodulation failure resulting from the interaction between Rhizobium leguminosarum biovar trifolii strain ANU794 and the Trifolium subterraneum cv. Woogenellup was examined by transposon mutagenesis to resolve whether multiple determinants were involved in cultivar-specificity. Three new transposon-induced mutants of ANU794 (W72, W78 and W710) with significantly enhanced nodulation ability on cv. Woogenellup were identified. The W72 and W78 mutations are chromosomally-located, whereas the W710 mutation isplasmid-located. The ethylene synthesis inhibitor, aminoethoxyvinylglycine, fails to enhance the nodulation ability of ANU794, ANU7943 (csn1::Tn5) and W78 on cv. Woogenellup, but enhances the nodulation ability of W72,W710 and ANU7941 (nodM::Tn5). DNA sequencing of the W78 locus reveals strong homology to an unknown Mycobacterium open reading frame, and to several bacterial non-haem chloroperoxidases. The previously identified csn1 locus showed homology to the 50S ribosomal protein, L9, with the Tn5 insertion being located in the 5'-untranslated region. The results suggest that cultivar-specificity is mediated by at least two independent mechanisms or determinants, and not by a simple gene-for-gene interaction. The role of ethylene in cultivar specificity is discussed. Cultivar-specific interactions may prove useful in identifying pathways involved in efficient nodule formation and plant-microbe interactions.

Genetic characterization of pilin glycosylation in Neisseria meningitidis.

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of an O-linked trisaccharide, Gal(beta1-4)Gal(alpha1-3)2,4-diacetimido-2,4,6-trideoxyhexose++ +. In a previous study the authors identified and characterized a gene, pglA, encoding a galactosyltransferase involved in pilin glycosylation. In this study a set of random genomic sequences from N. meningitidis strain MC58 was used to search for further genes involved in pilin glycosylation. Initially, an open reading frame was identified, and designated pglD (pilin glycosylation gene D), which was homologous to genes involved in polysaccharide biosynthesis. The region adjacent to this gene was cloned and nucleotide sequence analysis revealed two further genes, pglB and pglC, which were also homologous with genes involved in polysaccharide biosynthesis. Insertional mutations were constructed in pglB, pglC and pglD in N. meningitidis C311#3, a strain with well-defined LPS and pilin-linked glycan structures, to determine whether these genes had a role in the biosynthesis of either of these molecules. Analysis of these mutants revealed that there was no alteration in the phenotype of LPS in any of the mutant strains as judged by SDS-PAGE gel migration. In contrast, increased gel migration of the pilin subunit molecules of pglB, pglC and pglD mutants by Western analysis was observed. Pilin from each of the pglB, pglC and pglD mutants did not react with a terminal-galactose-specific stain, confirming that the gel migration differences were due to the alteration or absence of the pilin-linked trisaccharide structure in these mutants. In addition, antisera specific for the C311#3 trisaccharide failed to react with pilin from the pglB, pglC, pglD and galE mutants. Analysis of nucleotide sequence homologies has suggested specific roles for pglB, pglC and pglD in the biosynthesis of the 2,4-diacetimido-2,4,6-trideoxyhexose structure.