Helminth pathogen cathepsin proteases:it's a family affair

Helminth pathogens express papain-like cysteine peptidases, termed cathepsins, which have important roles in virulence, including host entry, tissue migration and the suppression of host immune responses. The liver fluke Fasciola hepatica, an emerging human pathogen, expresses the largest cathepsin L cysteine protease family yet described. Recent phylogenetic, biochemical and structural studies indicate that this family contains five separate clades, which exhibit overlapping but distinct substrate specificities created by a process of gene duplication followed by subtle residue divergence within the protease active site. The developmentally regulated expression of these proteases correlates with the passage of the parasite through host tissues and its encounters with different host macromolecules.

Nontypeable Haemophilus influenzae clearance by alveolar macrophages is impaired by exposure to cigarette smoke

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-alpha) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-alpha secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-alpha secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.

MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae

Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis.

Respiratory syncytial virus interaction with human airway epithelium

Although respiratory syncytial virus (RSV) is a major human respiratory pathogen, our knowledge of how it causes disease in humans is limited. Airway epithelial cells are the primary targets of RSV infection in vivo, so the generation and exploitation of RSV infection models based on morphologically and physiologically authentic well-differentiated primary human airway epithelial cells cultured at an air-liquid interface (WD-PAECs) provide timely developments that will help to bridge this gap. Here we review the interaction of RSV with WD-PAEC cultures, the authenticity of the RSV-WD-PAEC models relative to RSV infection of human airway epithelium in vivo, and future directions for their exploitation in our quest to understand RSV pathogenesis in humans.

The Opportunistic Pathogen Propionibacterium acnes: Insights into Typing, Human Disease, Clonal Diversification and CAMP Factor Evolution

We previously described a Multilocus Sequence Typing (MLST) scheme based on eight genes that facilitates population genetic and evolutionary analysis of P. acnes. While MLST is a portable method for unambiguous typing of bacteria, it is expensive and labour intensive. Against this background, we now describe a refined version of this scheme based on two housekeeping (aroE; guaA) and two putative virulence (tly; camp2) genes (MLST) that correctly predicted the phylogroup (IA, IA, IB, IC, II, III), clonal complex (CC) and sequence type (ST) (novel or described) status for 91% isolates (n = 372) via cross-referencing of the four gene allelic profiles to the full eight gene versions available in the MLST database (http://pubmlst.org/pacnes/). Even in the small number of cases where specific STs were not completely resolved, the MLST method still correctly determined phylogroup and CC membership. Examination of nucleotide changes within all the MLST loci provides evidence that point mutations generate new alleles approximately 1.5 times as frequently as recombination; although the latter still plays an important role in the bacterium's evolution. The secreted/cell-associated 'virulence' factors tly and camp2 show no clear evidence of episodic or pervasive positive selection and have diversified at a rate similar to housekeeping loci. The co-evolution of these genes with the core genome might also indicate a role in commensal/normal existence constraining their diversity and preventing their loss from the P. acnes population. The possibility that members of the expanded CAMP factor protein family, including camp2, may have been lost from other propionibacteria, but not P. acnes, would further argue for a possible role in niche/host adaption leading to their retention within the genome. These evolutionary insights may prove important for discussions surrounding camp2 as an immunotherapy target for acne, and the effect such treatments may have on commensal lineages. © 2013 McDowell et al.

The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense

Toll-like receptors (TLRs) are crucial in the innate immune response to pathogens, in that they recognize and respond to pathogen associated molecular patterns, which leads to activation of intracellular signaling pathways and altered gene expression. Vaccinia virus (VV), the poxvirus used to vaccinate against smallpox, encodes proteins that antagonize important components of host antiviral defense. Here we show that the VV protein A52R blocks the activation of the transcription factor nuclear factor kappa B (NF-kappa B) by multiple TLRs, including TLR3, a recently identified receptor for viral RNA. A52R associates with both interleukin 1 receptor-associated kinase 2 (IRAK2) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two key proteins important in TLR signal transduction. Further, A52R could disrupt signaling complexes containing these proteins. A virus deletion mutant lacking the A52R gene was attenuated compared with wild-type and revertant controls in a murine intranasal model of infection. This study reveals a novel mechanism used by VV to suppress the host immunity. We demonstrate viral disabling of TLRs, providing further evidence for an important role for this family of receptors in the antiviral response.

Detectability of bovine TB using the tuberculin skin test does not vary significantly according to pathogen genotype within Northern Ireland

Strains of many infectious diseases differ in parameters that influence epidemic spread, for example virulence, transmissibility, detectability and host specificity. Knowledge of inter-strain variation can be exploited to improve management and decrease disease incidence. Bovine tuberculosis (bTB) is increasingly prevalent among farmed cattle in the UK, exerting a heavy economic burden on the farming industry and government. We aimed to determine whether strains of Mycobacterium bovis (the causative agent of bTB) identified and classified using genetic markers (spoligotyping and multi-locus VNTR analysis) varied in response to the tuberculin skin test; this being the primary method of bTB detection used in the UK. Inter-strain variation in detectability of M. bovis could have important implications for disease control. The skin test is based on a differential delayed type hypersensitivity (DTH) response to intradermal injections of purified protein derivative (PPD) from M. bovis (PPD-B) and Mycobacterium avium (PPD-A). We searched for an association between skin test response (PPD-B skin rise minus PPD-A skin rise) and M. bovis genotype at the disclosing test in culture-confirmed cases using a field dataset consisting of 21,000 isolates belonging to 63 genotypes of M. bovis from cattle in Northern Ireland. We found no substantial variation among genotypes (estimated responses clustered tightly around the mean) controlling for animal sex, breed and test effects. We also estimated the ratio of skin test detected to undetected cases (i.e. cases only detected at abattoir). The skin test detection ratio varied among abattoirs with some detecting a greater proportion of cases than others but this variation was unrelated to the community composition of genotypes within each abattoir catchment. These two lines of evidence indicate that M. bovis genotypes in Northern Ireland have similar detectability using the skin test.

Diagnostic accuracy of pulmonary host inflammatory mediators in the exclusion of ventilator-acquired pneumonia

Background: Excessive use of empirical antibiotics is common in critically ill patients. Rapid biomarker-based exclusion of infection may improve antibiotic stewardship in ventilator-acquired pneumonia (VAP). However, successful validation of the usefulness of potential markers in this setting is exceptionally rare.

Objectives: We sought to validate the capacity for specific host inflammatory mediators to exclude pneumonia in patients with suspected VAP. 

Methods: A prospective, multicentre, validation study of patients with suspected VAP was conducted in 12 intensive care units. VAP was confirmed following bronchoscopy by culture of a potential pathogen in bronchoalveolar lavage fluid (BALF) at >10⁴ colony forming units per millilitre (cfu/mL). Interleukin-1 beta (IL-1β), IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were quantified in BALF. Diagnostic utility was determined for biomarkers individually and in combination. 

Results: Paired BALF culture and biomarker results were available for 150 patients. 53 patients (35%) had VAP and 97 (65%) patients formed the non-VAP group. All biomarkers were significantly higher in the VAP group (p<0.001). The area under the receiver operator characteristic curve for IL-1β was 0.81; IL-8, 0.74; MMP-8, 0.76; MMP-9, 0.79 and HNE, 0.78. A combination of IL-1β and IL-8, at the optimal cut-point, excluded VAP with a sensitivity of 100%, a specificity of 44.3% and a post-test probability of 0% (95% CI 0% to 9.2%). 

Conclusions: Low BALF IL-1β in combination with IL-8 confidently excludes VAP and could form a rapid biomarker-based rule-out test, with the potential to improve antibiotic stewardship.

On the alignment of debris discs and their host stars' rotation axis - implications for spin-orbit misalignment in exoplanetary systems

It has been widely thought that measuring the misalignment angle between the orbital plane of a transiting exoplanet and the spin of its host star was a good discriminator between different migration processes for hot-Jupiters. Specifically, well-aligned hot-Jupiter systems (as measured by the Rossiter-McLaughlin effect) were thought to have formed via migration through interaction with a viscous disc, while misaligned systems were thought to have undergone a more violent dynamical history. These conclusions were based on the assumption that the planet-forming disc was well-aligned with the host star. Recent work by a number of authors has challenged this assumption by proposing mechanisms that act to drive the star-disc interaction out of alignment during the pre-main-sequence phase. We have estimated the stellar rotation axis of a sample of stars which host spatially resolved debris discs. Comparison of our derived stellar rotation axis inclination angles with the geometrically measured debris-disc inclinations shows no evidence for a misalignment between the two.

The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution

BACKGROUND: The liver fluke Fasciola hepatica is a major pathogen of livestock worldwide, causing huge economic losses to agriculture, as well as 2.4 million human infections annually.

RESULTS: Here we provide a draft genome for F. hepatica, which we find to be among the largest known pathogen genomes at 1.3 Gb. This size cannot be explained by genome duplication or expansion of a single repeat element, and remains a paradox given the burden it may impose on egg production necessary to transmit infection. Despite the potential for inbreeding by facultative self-fertilisation, substantial levels of polymorphism were found, which highlights the evolutionary potential for rapid adaptation to changes in host availability, climate change or to drug or vaccine interventions. Non-synonymous polymorphisms were elevated in genes shared with parasitic taxa, which may be particularly relevant for the ability of the parasite to adapt to a broad range of definitive mammalian and intermediate molluscan hosts. Large-scale transcriptional changes, particularly within expanded protease and tubulin families, were found as the parasite migrated from the gut, across the peritoneum and through the liver to mature in the bile ducts. We identify novel members of anti-oxidant and detoxification pathways and defined their differential expression through infection, which may explain the stage-specific efficacy of different anthelmintic drugs.

CONCLUSIONS: The genome analysis described here provides new insights into the evolution of this important pathogen, its adaptation to the host environment and external selection pressures. This analysis also provides a platform for research into novel drugs and vaccines.

Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

PS1-10bzj: A Fast, Hydrogen-poor Superluminous Supernova in a Metal-poor Host Galaxy

We present observations and analysis of PS1-10bzj, a superluminous supernova (SLSN) discovered in the Pan-STARRS Medium Deep Survey at a redshift z = 0.650. Spectroscopically, PS1-10bzj is similar to the hydrogen-poor SLSNe 2005ap and SCP 06F6, though with a steeper rise and lower peak luminosity (M bol ~= -21.4 mag) than previous events. We construct a bolometric light curve, and show that while PS1-10bzj's energetics were less extreme than previous events, its luminosity still cannot be explained by radioactive nickel decay alone. We explore both a magnetar spin-down and circumstellar interaction scenario and find that either can fit the data. PS1-10bzj is located in the Extended Chandra Deep Field South and the host galaxy is imaged in a number of surveys, including with the Hubble Space Telescope. The host is a compact dwarf galaxy (MB ≈ -18 mag, diameter

The host galaxy and late-time evolution of the superluminous supernova PTF12dam

Superluminous supernovae (SLSNe) of Type Ic have a tendency to occur in faint host galaxies which are likely to have low mass and low metallicity. PTF12dam is one of the closest and best-studied superluminous explosions that has a broad and slowly fading light curve similar to SN 2007bi. Here we present new photometry and spectroscopy for PTF12dam from 200-500 d (rest frame) after peak and a detailed analysis of the host galaxy (SDSS J142446.21+461348.6 at z = 0.107). Using deep templates and image subtraction we show that the light curve can be fit with a magnetar model if escape of high-energy gamma rays is taken into account. The full bolometric light curve from -53 to +399 d (with respect to peak) cannot be fit satisfactorily with the pair-instability models. An alternative model of interaction with a dense circumstellar material (CSM) produces a good fit to the data although this requires a very large mass (˜13 M⊙) of hydrogen-free CSM. The host galaxy is a compact dwarf (physical size ˜1.9 kpc) and with Mg = -19.33 ± 0.10, it is the brightest nearby SLSN Ic host discovered so far. The host is a low-mass system (2.8 × 108 M⊙) with a star formation rate (5.0 M⊙ yr-1), which implies a very high specific star formation rate (17.9 Gyr-1). The remarkably strong nebular emission provide detections of the [O III] λ4363 and [O II] λλ7320, 7330auroral lines and an accurate oxygen abundance of 12 + log (O/H) = 8.05 ± 0.09. We show here that they are at the extreme end of the metallicity distribution of dwarf galaxies and propose that low metallicity is a requirement to produce these rare and peculiar SNe.

The extracellular vesicles of the helminth pathogen, Fasciola hepatica: biogenesis pathways and cargo molecules involved in parasite pathogenesis.

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterisation of the total secretome of this zoonotic parasite. Fasciola secretes at least two sub-populations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialised cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic datasets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular machinery required for EV biogenesis is constitutively expressed across the intra-mammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.

Function of host defence peptide LL-37 in human periodontal disease.

Objectives: Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods: Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results: At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL-37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion: The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.