Pathogenicity of Misfolded and Dimeric HLA-B27 Molecules

The association between HLA-B27 and the group of autoimmune inflammatory arthritic diseases, the spondyloarthropathies (SpAs) which include ankylosing spondylitis (AS) and Reactive Arthritis (ReA), has been well established and remains the strongest association between any HLA molecule and autoimmune disease. The mechanism behind this striking association remains elusive; however animal model and biochemical data suggest that HLA-B27 misfolding may be key to understanding its association with the SpAs. Recent investigations have focused on the unusual biochemical structures of HLA-B27 and their potential role in SpA pathogenesis. Here we discuss how these unusual biochemical structures may participate in cellular events leading to chronic inflammation and thus disease progression

Annotation of two large contiguous regions from the Haemonchus contortus genome using RNA-seq and comparative analysis with Caenorhabditis elegans

The genomes of numerous parasitic nematodes are currently being sequenced, but their complexity and size, together with high levels of intra-specific sequence variation and a lack of reference genomes, makes their assembly and annotation a challenging task. Haemonchus contortus is an economically significant parasite of livestock that is widely used for basic research as well as for vaccine development and drug discovery. It is one of many medically and economically important parasites within the strongylid nematode group. This group of parasites has the closest phylogenetic relationship with the model organism Caenorhabditis elegans, making comparative analysis a potentially powerful tool for genome annotation and functional studies. To investigate this hypothesis, we sequenced two contiguous fragments from the H. contortus genome and undertook detailed annotation and comparative analysis with C. elegans. The adult H. contortus transcriptome was sequenced using an Illumina platform and RNA-seq was used to annotate a 409 kb overlapping BAC tiling path relating to the X chromosome and a 181 kb BAC insert relating to chromosome I. In total, 40 genes and 12 putative transposable elements were identified. 97.5% of the annotated genes had detectable homologues in C. elegans of which 60% had putative orthologues, significantly higher than previous analyses based on EST analysis. Gene density appears to be less in H. contortus than in C. elegans, with annotated H. contortus genes being an average of two-to-three times larger than their putative C. elegans orthologues due to a greater intron number and size. Synteny appears high but gene order is generally poorly conserved, although areas of conserved microsynteny are apparent. C. elegans operons appear to be partially conserved in H. contortus. Our findings suggest that a combination of RNA-seq and comparative analysis with C. elegans is a powerful approach for the annotation and analysis of strongylid nematode genomes

Streptococcal necrotising fasciitis from diverse strains of Streptococcus pyogenes in tropical northern Australia: case series and comparison with the literature

BACKGROUND: Since the mid-1980's there has been a worldwide resurgence of severe disease from group A streptococcus (GAS), with clonal clusters implicated in Europe and the United States. However GAS associated sepsis and rheumatic fever have always remained at high levels in many less developed countries. In this context we aimed to study GAS necrotising fasciitis (NF) in a region where there are high background rates of GAS carriage and disease. METHODS: We describe the epidemiology, clinical and laboratory features of 14 consecutive cases of GAS NF treated over a seven year period from tropical northern Australia. RESULTS: Incidence rates of GAS NF in the Aboriginal population were up to five times those previously published from other countries. Clinical features were similar to those described elsewhere, with 7/14 (50%) bacteremic and 9/14 (64%) having associated streptococcal toxic shock syndrome. 11/14 (79%) had underlying chronic illnesses, including all four fatalities (29% mortality overall). Important laboratory differences from other series were that leukocytosis was absent in 9/14 (64%) but all had substantial lymphopenia. Sequence typing of the 14 NF-associated GAS isolates showed no clonality, with only one emm type 1 and two emm type 3 strains. CONCLUSIONS: While NF clusters can occur from a single emergent GAS clone, this was not evident in our tropical region, where high rates of NF parallel high overall rates of GAS infection from a wide diversity of strains. The specific virulence factors of GAS strains which do cause NF and the basis of the inadequate host response in those patients who develop NF on infection with these GAS require further elucidation

Brugia malayi Gene Expression in Response to the Targeting of the Wolbachia Endosymbiont by Tetracycline Treatment

Filarial parasites afflict hundreds of millions of individuals worldwide, and cause significant public health problems in many of the poorest countries in the world. Most of the human filarial parasite species, including Brugia malayi, harbor endosymbiotic bacteria of the genus Wolbachia. Elimination of the endosymbiont leads to sterilization of the adult female worm. The need exists for the development of new chemotherapeutic approaches that can practically exploit the vulnerability of the filaria to the loss of the Wolbachia. In this study we performed ultrastructural and microarray analyses of female worms collected from infected jirds treated with tetracycline. Results suggest that the endosymbiotic bacteria were specifically affected by the antibiotic. Furthermore, in response to the targeting of the endosymbiont, the parasites modulated expression of their genes. When exposed to tetracycline, the parasites over-expressed genes involved in protein synthesis. Expression of genes involved in cuticle biosynthesis and energy metabolism was, on the other hand, limited

Development of an In Vivo RNAi Protocol to Investigate Gene Function in the Filarial Nematode, Brugia malayi

Our ability to control diseases caused by parasitic nematodes is constrained by a limited portfolio of effective drugs and a paucity of robust tools to investigate parasitic nematode biology. RNA interference (RNAi) is a reverse-genetics tool with great potential to identify novel drug targets and interrogate parasite gene function, but present RNAi protocols for parasitic nematodes, which remove the parasite from the host and execute RNAi in vitro, are unreliable and inconsistent. We have established an alternative in vivo RNAi protocol targeting the filarial nematode Brugia malayi as it develops in an intermediate host, the mosquito Aedes aegypti. Injection of worm-derived short interfering RNA (siRNA) and double stranded RNA (dsRNA) into parasitized mosquitoes elicits suppression of B. malayi target gene transcript abundance in a concentration-dependent fashion. The suppression of this gene, a cathepsin L-like cysteine protease (Bm-cpl-1) is specific and profound, both injection of siRNA and dsRNA reduce transcript abundance by 83%. In vivo Bm-cpl-1 suppression results in multiple aberrant phenotypes; worm motility is inhibited by up to 69% and parasites exhibit slow-moving, kinked and partial-paralysis postures. Bm-cpl-1 suppression also retards worm growth by 48%. Bm-cpl-1 suppression ultimately prevents parasite development within the mosquito and effectively abolishes transmission potential because parasites do not migrate to the head and proboscis. Finally, Bm-cpl-1 suppression decreases parasite burden and increases mosquito survival. This is the first demonstration of in vivo RNAi in animal parasitic nematodes and results indicate this protocol is more effective than existing in vitro RNAi methods. The potential of this new protocol to investigate parasitic nematode biology and to identify and validate novel anthelmintic drug targets is discussed

Gender-Associated Genes in Filarial Nematodes Are Important for Reproduction and Potential Intervention Targets

Lymphatic filariasis is a neglected tropical disease that is caused by thread-like parasitic worms that live and reproduce in lymphatic vessels of the human host. There are no vaccines to prevent filariasis, and available drugs are not effective against all stages of the parasite. In addition, recent reports suggest that the filarial nematodes may be developing resistance to key medications. Therefore, there is an urgent need to identify new drug targets in filarial worms. The purpose of this study was to perform a genome-wide analysis of gender-associated gene transcription to improve understanding of key reproductive processes in filarial nematodes. Our results indicate that thousands of genes are differentially expressed in male and female adult worms. Many of those genes are involved in specific reproductive processes such as embryogenesis and spermatogenesis. In addition, expression of some of those genes is suppressed by tetracycline, a drug that leads to sterilization of adult female worms in many filarial species. Thus, gender-associated genes represent priority targets for design of vaccines and drugs that interfere with reproduction of filarial nematodes. Additional work with this type of integrated systems biology approach should lead to important new tools for controlling filarial diseases

Functional Analysis of the Cathepsin-Like Cysteine Protease Genes in Adult Brugia malayi Using RNA Interference

Filarial nematodes are an important group of human pathogens, causing lymphatic filariasis and onchocerciasis, and infecting around 150 million people throughout the tropics with more than 1.5 billion at risk of infection. Control of filariasis currently relies on mass drug administration (MDA) programs using drugs which principally target the microfilarial life-cycle stage. These control programs are facing major challenges, including the absence of a drug with macrofilaricidal or permanent sterilizing activity, and the possibility of the development of drug-resistance against the drugs available. Cysteine proteases are essential enzymes which play important roles in a wide range of cellular processes, and the cathepsin-like cysteine proteases have been identified as potential targets for drug or vaccine development in many parasites. Here we have studied the function of several of the cathepsin-like enzymes in the filarial nematode, B. malayi, and demonstrate that these cysteine proteases are involved in the development of embryos, show similar functions to their counterparts in C. elegans, and therefore, provide an important target for future drug development targeted to eliminate filariasis

Brugia malayi Excreted/Secreted Proteins at the Host/Parasite Interface: Stage- and Gender-Specific Proteomic Profiling

Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES) products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf), L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs) in the available databases. Moreover, this analysis was able to confirm the presence of 274 “hypothetical” proteins inferred from gene prediction algorithms applied to the B. malayi (Bm) genome. Not surprisingly, the majority (160/274) of these “hypothetical” proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase), MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females) compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host–parasite interaction