Robert A. Hirst

The role of pneumolysin in pneumococcal pneumonia and meningitis

Diseases caused by Streptococcus pneumoniae include pneumonia, septicaemia and meningitis. All these are associated with high morbidity and mortality. The pneumococcus can colonize the nasopharynx, and this can be a prelude to bronchopneumonia and invasion of the vasculature space. Proliferation in the blood can result in a breach of the blood–brain barrier and entry into the cerebrospinal fluid (CSF) where the bacteria cause inflammation of the meningeal membranes resulting in meningitis. The infected host may develop septicaemia and/or meningitis secondary to bronchopneumonia. Also septicaemia is a common precursor of meningitis. The mechanisms surrounding the sequence of infection are unknown, but will be dependent on the properties of both the host and bacterium. Treatment of these diseases with antibiotics leads to clearance of the bacteria from the infected tissues, but the bacteriolytic nature of antibiotics leads to an acute release of bacterial toxins and thus after antibiotic therapy the patients can be left with organ‐specific deficits. One of the main toxins released from pneumococci is the membrane pore forming toxin pneumolysin. Here we review the extensive studies on the role of pneumolysin in the pathogenesis of pneumococcal diseases.

Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia

Primary ciliary dyskinesia most often arises from loss of the dynein motors that power ciliary beating. Here we show that DNAAF3 (also known as PF22), a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes before their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in individuals from families with situs inversus and defects in the assembly of inner and outer dynein arms. Knockdown of dnaaf3 in zebrafish likewise disrupts dynein arm assembly and ciliary motility, causing primary ciliary dyskinesia phenotypes that include hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a PF22-null mutant cannot assemble any outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests that DNAAF3 (PF22) acts at a similar stage as other preassembly proteins, for example, DNAAF2 (also known as PF13 or KTU) and DNAAF1 (also known as ODA7 or LRRC50), in the dynein preassembly pathway. These results support the existence of a conserved, multistep pathway for the cytoplasmic formation of assembly competent ciliary dynein complexes.

Centriolar satellites are assembly points for proteins implicated in human ciliopathies, including oral-facial-digital syndrome 1

Ciliopathies are caused by mutations in genes encoding proteins required for cilia organization or function. We show through colocalization with PCM-1, that OFD1 (the product of the gene mutated in oral-facial-digital syndrome 1) as well as BBS4 and CEP290 (proteins encoded by other ciliopathy genes) are primarily components of centriolar satellites, the particles surrounding centrosomes and basal bodies. RNA interference experiments reveal that satellite integrity is mutually dependent upon each of these proteins. Upon satellite dispersal, through mitosis or forced microtubule depolymerization, OFD1 and CEP290 remain centrosomal, whereas BBS4 and PCM-1 do not. OFD1 interacts via its fifth coiled-coil motif with the N-terminal coiled-coil domain of PCM-1, which itself interacts via its C-terminal non-coiled-coil region with BBS4. OFD1 localization to satellites requires its N-terminal region, encompassing the LisH motif, whereas expression of OFD1 C-terminal constructs causes PCM-1 and CEP290 mislocalization. Moreover, in embryonic zebrafish, OFD1 and BBS4 functionally synergize, determining morphogenesis. Our observation that satellites are assembly points for several mutually dependent ciliopathy proteins provides a further possible explanation as to why the clinical spectrum of OFD1, Bardet–Biedl and Joubert syndromes overlap. Furthermore, definition of how OFD1 and PCM-1 interact helps explain why different OFD1 mutations lead to clinically variable phenotypes.

European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia

The diagnosis of primary ciliary dyskinesia is often confirmed with standard, albeit complex and expensive, tests. In many cases, however, the diagnosis remains difficult despite the array of sophisticated diagnostic tests. There is no “gold standard” reference test. Hence, a Task Force supported by the European Respiratory Society has developed this guideline to provide evidence-based recommendations on diagnostic testing, especially in light of new developments in such tests, and the need for robust diagnoses of patients who might enter randomised controlled trials of treatments. The guideline is based on pre-defined questions relevant for clinical care, a systematic review of the literature, and assessment of the evidence using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach. It focuses on clinical presentation, nasal nitric oxide, analysis of ciliary beat frequency and pattern by high-speed video-microscopy analysis, transmission electron microscopy, genotyping and immunofluorescence. It then used a modified Delphi survey to develop an algorithm for the use of diagnostic tests to definitively confirm and exclude the diagnosis of primary ciliary dyskinesia; and to provide advice when the diagnosis was not conclusive. Finally, this guideline proposes a set of quality criteria for future research on the validity of diagnostic methods for primary ciliary dyskinesia. International ERS guidelines recommend a combination of tests to diagnose primary ciliary dyskinesia http://ow.ly/sJhH304InBN

Streptococcus pneumoniae Deficient in Pneumolysin or Autolysin Has Reduced Virulence in Meningitis

BACKGROUND The role played by pneumolysin and autolysin in pneumococcal meningitis is poorly understood. METHODS A rat model was used to investigate the disease, in which surgical implantation of a cisternal catheter allowed bacterial instillation and cerebrospinal fluid (CSF) sampling. RESULTS CSF infection of rats with wild-type pneumococci caused meningitis within 26 h, whereas isogenic mutants that do not express pneumolysin (DeltaPly) or autolysin (LytA(-)) caused very mild or no disease. Wild-type infections resulted in pneumococci in the CSF and cortical homogenates, but a minority of the rats infected with DeltaPly or LytA(-) had bacteria in these locations at 26 h. Leukocyte numbers in the CSF were similar after infection with all pneumococci; however, neutrophils and monocytes predominated after wild-type infection, whereas lymphocytes and atypical lymphocytes predominated after infection with the mutants. Wild-type pneumococci caused disruption to the ependyma, but this was not observed in rats infected with DeltaPly or LytA(-). Cells surrounding the ventricles in wild type-infected animals expressed caspase 3, and astrocytes had hypertrophy; both findings were absent in rats infected with the mutants. CONCLUSIONS This study provides strong in vivo evidence that pneumolysin and autolysin play crucial roles in the pathogenesis of pneumococcal meningitis.

Relative Roles of Pneumolysin and Hydrogen Peroxide from Streptococcus pneumoniae in Inhibition of Ependymal Ciliary Beat Frequency

ABSTRACT Ciliated ependymal cells line the ventricular system of the brain and the cerebral aqueducts. This study characterizes the relative roles of pneumolysin and hydrogen peroxide (H2O2) in pneumococcal meningitis, using the in vitro ependymal ciliary beat frequency (CBF) as an indicator of toxicity. We have developed an ex vivo model to examine the ependymal surface of the brain slices cut from the fourth ventricle. The ependymal cells had cilia beating at a frequency of between 38 and 44Hz. D39 (wild-type) and PLN-A (pneumolysin-negative) pneumococci at 108 CFU/ml both caused ciliary slowing. Catalase protected against PLN-A-induced ciliary slowing but afforded little protection from D39. Lysed PLN-A did not reduce CBF, whereas lysed D39 caused rapid ciliary stasis. There was no effect of catalase, penicillin, or catalase plus penicillin on the CBF. H2O2 at a concentration as low as 100 μM caused ciliary stasis, and this effect was abolished by coincubation with catalase. An additive inhibition of CBF was demonstrated using a combination of both toxins. A significant inhibition of CBF at between 30 and 120 min was demonstrated with both toxins compared with either H2O2 (10 μM) or pneumolysin (1 HU/ml) alone. D39 released equivalent levels of H2O2 to those released by PLN-A, and these concentrations were sufficient to cause ciliary stasis. The brain slices did not produce H2O2, and in the presence of 108 CFU of D39 or PLN-A per ml there was no detectable bacterially induced increase of H2O2release from the brain slice. Coincubation with catalase converted the H2O2 produced by the pneumococci to H2O. Penicillin-induced lysis of bacteria dramatically reduced H2O2 production. The hemolytic activity released from D39 was sufficient to cause rapid ciliary stasis, and there was no detectable release of hemolytic activity from the pneumolysin-negative PLN-A. These data demonstrate that D39 bacteria released pneumolysin, which caused rapid ciliary stasis. D39 also released H2O2, which contributed to the toxicity, but this was masked by the more severe effects of pneumolysin. H2O2 released from intact PLN-A was sufficient to cause rapid ciliary stasis, and catalase protected against H2O2-induced cell toxicity, indicating a role for H2O2 in the response. There is also a slight additive effect of pneumolysin and H2O2 on ependymal toxicity; however, the precise mechanism of action and the role of these toxins in pathogenesis remain unclear.

Comparison of the effects of [Phe1Ψ(CH2‐NH)Gly2]Nociceptin (1–13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors

Nociceptin(NC) is the endogenous ligand for the opioid receptor like‐1 receptor (NC‐receptor). [Phe1ΨC(CH2‐NH)Gly2]Nociceptin(1–13)NH2 ([F/G]NC(1–13)NH2) has been reported to antagonize NC actions in peripheral guinea‐pig and mouse tissues. In this study, we investigated the effects of a range of NC C‐terminal truncated fragments and [F/G]NC(1–13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHONCR) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]‐Tyr14‐NC binding in membranes from rCX and CHONCR cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1–13)NH2 was as potent as NC(1–13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHONCR cells was NCNH2NC=NC(1–13)NH2>NC(1–12)NH2>>NC(1–11)NH2. [F/G]NC(1–13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1–13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5±4.9% and 7.39, 58.9±6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1–13)NH2, displayed a small (instrinsic activity α=0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1–13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1–13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms.

MCIDAS mutations result in a mucociliary clearance disorder with reduced generation of multiple motile cilia

Reduced generation of multiple motile cilia (RGMC) is a rare mucociliary clearance disorder. Affected persons suffer from recurrent infections of upper and lower airways because of highly reduced numbers of multiple motile respiratory cilia. Here we report recessive loss-of-function and missense mutations in MCIDAS-encoding Multicilin, which was shown to promote the early steps of multiciliated cell differentiation in Xenopus. MCIDAS mutant respiratory epithelial cells carry only one or two cilia per cell, which lack ciliary motility-related proteins (DNAH5; CCDC39) as seen in primary ciliary dyskinesia. Consistent with this finding, FOXJ1-regulating axonemal motor protein expression is absent in respiratory cells of MCIDAS mutant individuals. CCNO, when mutated known to cause RGMC, is also absent in MCIDAS mutant respiratory cells, consistent with its downstream activity. Thus, our findings identify Multicilin as a key regulator of CCNO/FOXJ1 for human multiciliated cell differentiation, and highlight the 5q11 region containing CCNO and MCIDAS as a locus underlying RGMC.

Ciliated air-liquid cultures as an aid to diagnostic testing of primary ciliary dyskinesia.

BACKGROUND The diagnosis of primary ciliary dyskinesia (PCD) can prove difficult because of secondary damage of ciliated tissue. METHODS Here we audit culturing cells, obtained by nasal brushing, to a ciliated phenotype using an air-liquid interface method to determine if the effects of secondary damage on cilia were reduced following culture. RESULTS Of 231 patients consecutively referred for diagnostic testing, culture was attempted in 187, with 101 (54%) becoming ciliated. Of the 90 brush biopsy samples with a low dyskinesia score (< 40%), 71 grew cilia after culture (79% success). Significant secondary damage (> 40% dyskinesia) was present in 69 (43%) of the initial brush biopsy samples, and of these, 18 (26%) became ciliated after culture. In these samples, ciliary dyskinesia was significantly (P < .001) reduced (64% ± 6.8% before culture, 31% ± 4.5% after culture). Ciliary beat frequency (CBF) after cell culture was similar to CBF before culture. Cell culture helped to exclude PCD in eight patients for whom ciliary dyskinesia was present in > 70% of the initial brush biopsy sample, a level at which a rebiopsy would normally be requested. In six patients in whom no cilia were found in the initial brush biopsy samples, ciliated cell culture was successful and excluded the diagnosis. PCD was diagnosed in 28 patients and ciliated cell culture was successful in 12 (43%) showing identical ciliary beat pattern and electron microscopy findings. CONCLUSIONS Ciliary dyskinesia was reduced following cell culture to a ciliated phenotype compared with the initial brush biopsy sample. The specific PCD phenotype was maintained after culture.

Inner dynein arm defects causing primary ciliary dyskinesia: repeat testing required

Primary ciliary dyskinesia (PCD) results in chronic nasal symptoms and chest disease leading to bronchiectasis. We noted a number of patients referred for diagnostic testing whose initial results suggested PCD due to an inner dynein arm or radial spoke defect but in whom no abnormality was found on retesting. The present study was an audit of all patients referred for PCD diagnostic testing over a 3-yr period whose initial electron microscopy (EM) and beat pattern analysis suggested an inner dynein arm or radial spoke defect. 21 patients referred for diagnostic testing for PCD suspected of an inner dynein arm defect and six suspected of a radial spoke defect on initial EM and beat pattern analysis had repeat testing performed. On repeat testing, five patients initially suspected of an inner dynein arm defect and one with a radial spoke defect had normal EM and beat pattern, leading to the initial diagnosis being questioned. Patients suspected of PCD due to an inner dynein arm defect or radial spoke defect should have the diagnosis reassessed if it has been based on only one diagnostic sample.