Lf Roddam

Genetic characterization of pilin glycosylation and phase variation 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 either an O‐linked trisaccharide, Gal (β1‐4) Gal (α1‐3) 2,4‐diacetamido‐2,4,6‐trideoxyhexose or an O‐linked disaccharide Gal (α1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pglB/B2, pglF, pglG and pglH. A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pglB2 polymorphisms were not found in strain C311♯3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311♯3, a strain with well‐defined lipopolysaccharide (LPS) and pilin‐linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating truncation of the trisaccharide structure. Antisera specific for the C311♯3 trisaccharide failed to react with pilin from these pglE and pglF mutants. GC‐MS analysis of the sugar composition of the pglE mutant showed a reduction in galactose compared with C311♯3 wild type. Analysis of amino acid sequence homologies has suggested specific roles for pglE and pglF in the biosynthesis of the trisaccharide structure. Further, we present evidence that pglE, which contains heptanucleotide repeats, is responsible for the phase variation between trisaccharide and disaccharide structures in strain C311♯3 and other strains. We also present evidence that pglG, pglH and pglB2 are potentially phase variable.

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.

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.

Pseudomonas aeruginosa Uses Multiple Pathways To Acquire Iron during Chronic Infection in Cystic Fibrosis Lungs

ABSTRACT 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.

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.

Virulence gene distribution in clinical, nosocomial and environmental isolates of Pseudomonas aeruginosa.

The virulence factor genotypes of a large cohort of clinical, nosocomial environment and community environment isolates (184 in total) of Pseudomonas aeruginosa from Tasmania, Australia, were determined by PCR. The virulence factor genotype of the majority of isolates was highly conserved, with the exception of the virulence gene exoU, which demonstrated low prevalence (33 isolates; 18 %) in the population tested. Isolates collected from the environment of intensive therapy wards (intensive care unit and neurosurgical units) of the major tertiary referral hospital in Tasmania were found to be more likely (P<0.001 and P<0.05, respectively) to possess the virulence factor gene exoU than all other isolates. Adult cystic fibrosis isolates showed a decreased prevalence of the exoU gene (P<0.01) when compared to other clinical isolates (P<0.01), which may indicate decreased virulence. No specific virulence factor genotype was associated with the cystic fibrosis epidemic strains tested.

Biosignificance of bacterial cyanogenesis in the CF lung

Two recent studies have demonstrated the presence of biologically significant amounts of cyanide within the airways of cystic fibrosis (CF) patients infected with Pseudomonas aeruginosa. Whilst environmental strains of P. aeruginosa are known to synthesise cyanide, there has been a relative lack of investigation into bacterial cyanogenesis from a medical viewpoint, despite the role P. aeruginosa plays in many serious infection settings and especially in CF lung disease. This review discusses the implications of cyanogenesis in the CF airway in terms of bacterial ecology, host immune response, progression of lung disease and potential treatment options.

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 (3OC12HSL) 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 3OC12HSL 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 3OC12HSL 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.

Molecular analysis of changes in Pseudomonas aeruginosa load during treatment of a pulmonary exacerbation in cystic fibrosis

BACKGROUND Intravenous antibiotics for pulmonary exacerbations (PEs) of cystic fibrosis (CF) usually target Pseudomonas aeruginosa. Insights into the CF lung microbiome have questioned this approach. We used RT-qPCR to determine whether intravenous antibiotics reduced P. aeruginosa numbers and whether this correlated with improved lung function. We also investigated antibiotic effects on other common respiratory pathogens in CF. METHODS Sputa were collected from patients when stable and again during a PE. Sputa were expectorated into a RNA preservation buffer for RNA extraction and preparation of cDNA. qPCR was used to enumerate viable P. aeruginosa as well as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Burkholderia cepacia complex and Aspergillus fumigatus. RESULTS Fifteen CF patients were followed through 21 PEs. A complete set of serial sputum samples was unavailable for two patients (three separate PEs). P. aeruginosa numbers did not increase immediately prior to a PE, but numbers during intravenous antibiotic treatment were reduced ≥4-log in 6/18 and ≥1-log in 4/18 PEs. In 7/18 PEs, P. aeruginosa numbers changed very little with intravenous antibiotics and one patient demonstrated a ≥2-log increase in P. aeruginosa load. H. influenzae and S. pneumoniae were detected in ten and five PEs respectively, but with antibiotic treatment these bacteria rapidly became undetectable in 6/10 and 4/5 PEs, respectively. There was a negative correlation between P. aeruginosa numbers and FEV1 during stable phase (r(s)=0.75, p<0.05), and reductions in P. aeruginosa load with intravenous antibiotic treatment correlated with improved FEV1 (r(s)=0.52, p<0.05). CONCLUSIONS Exacerbations are not due to increased P. aeruginosa numbers in CF adults. However, lung function improvements correlate with reduced P. aeruginosa burden suggesting that current antibiotic treatment strategies remain appropriate in most patients. Improved understanding of PE characterised by unchanged P. aeruginosa numbers and minimal lung function improvement following treatment may allow better targeted therapies.

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-β+ CD4+ cells. We observed higher proportions of Treg, IL-10+ and TGF-β+ CD4+ cells in CF adults (≥ 18 years old), but not children/adolescents, compared with controls. Within the CF group, high TGF-β+% 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.