In this study, we identify a major spore surface protein, BclA, and provide evidence that this protein is glycosylated. Following extraction of the spore surface, solubilized proteins were separated by one-dimensional PAGE and stained with glycostain to reveal a reactive high-molecular-mass region of approximately 600 kDa. Tandem mass spectrometry analysis of in-gel digests showed this band to contain peptides corresponding to a putative exosporangial glycoprotein (BclA3) and identified a number of glycopeptides modified with multiple N-acetyl hexosamine moieties and, in some cases, capped with novel glycans. In addition, we demonstrate that the glycosyltransferase gene sgtA (gene CD3350 in strain 630 and CDR3194 in strain R20291), which is located immediately upstream of the bclA3 homolog, is involved in the glycosylation of the spore surface, and is cotranscribed with bclA3. The presence of anti-β-O-GlcNAc-reactive material was demonstrated on the surface of spores by immunofluorescence and in surface extracts by Western blotting, although each strain produced a distinct pattern of reactivity. Reactivity of the spore surface with the anti-β-O-GlcNAc antibody was abolished in the 630 and R20291 glycosyltransferase mutant strains, while complementation with a wild-type copy of the gene restored the β-O-GlcNAc reactivity. Phenotypic testing of R20291 glycosyltransferase mutant spores revealed no significant change in sensitivity to ethanol or lysozyme. However, a change in the resistance to heat of R20291 glycosyltransferase mutant spores compared to R20291 spores was observed, as was the ability to adhere to and be internalized by macrophages.
Telomere length has a biological link to cancer, with excessive telomere shortening leading to genetic instability and resultant malignant transformation. Telomere length is heritable and genetic variants determining telomere length have been identified. Telomere biology has been implicated in the development of hematological malignancies (HMs), therefore, closer examination of telomere length in HMs may provide further insight into genetic etiology of disease development and support for telomere length as a prognostic factor in HMs. We retrospectively examined mean relative telomere length in the Tasmanian Familial Hematological Malignancies Study using a quantitative PCR method on genomic DNA from peripheral blood samples. Fifty-five familial HM cases, 191 unaffected relatives of familial HM cases and 75 non-familial HM cases were compared with 758 population controls. Variance components modeling was employed to identify factors influencing variation in telomere length. Overall, HM cases had shorter mean relative telomere length (P=2.9×10−6) and this was observed across both familial and non-familial HM cases (P=2.2×10−4 and 2.2×10−5, respectively) as well as additional subgroupings of HM cases according to broad subtypes. Mean relative telomere length was also significantly heritable (62.6%; P=4.7×10−5) in the HM families in the present study. We present new evidence of significantly shorter mean relative telomere length in both familial and non-familial HM cases from the same population adding further support to the potential use of telomere length as a prognostic factor in HMs. Whether telomere shortening is the cause of or the result of HMs is yet to be determined, but as telomere length was found to be highly heritable in our HM families this suggests that genetics driving the variation in telomere length is related to HM disease risk.
telomere length; hematological malignancies; familial cancer
Planococcus halocryophilus strain Or1, isolated from high Arctic permafrost, grows and divides at −15 °C, the lowest temperature demonstrated to date, and is metabolically active at −25 °C in frozen permafrost microcosms. To understand how P. halocryophilus Or1 remains active under the subzero and osmotically dynamic conditions that characterize its native permafrost habitat, we investigated the genome, cell physiology and transcriptomes of growth at −15 °C and 18% NaCl compared with optimal (25 °C) temperatures. Subzero growth coincides with unusual cell envelope features of encrustations surrounding cells, while the cytoplasmic membrane is significantly remodeled favouring a higher ratio of saturated to branched fatty acids. Analyses of the 3.4 Mbp genome revealed that a suite of cold and osmotic-specific adaptive mechanisms are present as well as an amino acid distribution favouring increased flexibility of proteins. Genomic redundancy within 17% of the genome could enable P. halocryophilus Or1 to exploit isozyme exchange to maintain growth under stress, including multiple copies of osmolyte uptake genes (Opu and Pro genes). Isozyme exchange was observed between the transcriptome data sets, with selective upregulation of multi-copy genes involved in cell division, fatty acid synthesis, solute binding, oxidative stress response and transcriptional regulation. The combination of protein flexibility, resource efficiency, genomic plasticity and synergistic adaptation likely compensate against osmotic and cold stresses. These results suggest that non-spore forming P. halocryophilus Or1 is specifically suited for active growth in its Arctic permafrost habitat (ambient temp. ∼−16 °C), indicating that such cryoenvironments harbor a more active microbial ecosystem than previously thought.
cryophile; permafrost; cold-active; cold/osmotic adaptation; subzero environments
The human erythrocyte contains an abundance of the thiol-dependant peroxidase Peroxiredoxin-2 (Prx2), which protects the cell from the pro-oxidant environment it encounters during its 120 days of life in the blood stream. In malarial infections, the Plasmodium parasite invades red cells and imports Prx2 during intraerythrocytic development, presumably to supplement in its own degradation of peroxides generated during cell metabolism, especially hemoglobin (Hb) digestion. Here we demonstrate that an irreversible Prx2 inhibitor, Conoidin A (2,3-bis(bromomethyl)-1,4-dioxide-quinoxaline; BBMQ), has potent cytocidal activity against cultured P. falciparum. Parasite growth was also inhibited in red cells that were treated with BBMQ and then washed prior to parasite infection. These cells remained susceptible to merozoite invasion, but failed to support normal intraerythrocytic development. In addition the potency of chloroquine (CQ), an antimalarial drug that prevents the detoxification of Hb-derived heme, was significantly enhanced in the presence of BBMQ. CQ IC50 values decreased an order of magnitude when parasites were either co-incubated with BBMQ, or introduced into BBMQ-pretreated cells; these effects were equivalent for both drug-resistant and drug-sensitive parasite lines. Together these results indicate that treatment of red cells with BBMQ renders them incapable of supporting parasite growth and increases parasite sensitivity to CQ. We also propose that molecules such as BBMQ that target host cell proteins may constitute a novel host-directed therapeutic approach for treating malaria.
Malaria treatments are becoming less effective due to the rapid spread of drug resistant parasites. Increased understanding of the host/parasite interaction is crucial in order to develop treatments that will be less prone to resistance. Parasite invasion of the red blood cell (RBC) is a critical aspect of the parasite life cycle and is, therefore, a promising target for the development of malaria treatments. Assays for analysing parasite invasion in vitro have been developed, but no equivalent assays exist for in vivo studies. This article describes a novel flow cytometric in vivo parasite invasion assay.
Experiments were conducted with mice infected with erythrocytic stages of Plasmodium chabaudi adami strain DS. Exogenously labelled blood cells were transfused into infected mice at schizogony, and collected blood samples stained and analysed using flow cytometry to specifically detect and measure proportions of labelled RBC containing newly invaded parasites. A combination of antibodies (CD45 and CD71) and fluorescent dyes, Hoechst (DNA) and JC-1 (mitochondrial membrane potential), were used to differentiate parasitized RBCs from uninfected cells, RBCs containing Howell-Jolly bodies, leukocytes and RBC progenitors. Blood cells were treated ex vivo with proteases to examine the effects on in vivo parasite invasion.
The staining and flow cytometry analysis method was accurate in determining the parasitaemia down to 0.013% with the limit of detection at 0.007%. Transfused labelled blood supported normal rates of parasite invasion. Protease-treated red cells resulted in 35% decrease in the rate of parasite invasion within 30 minutes of introduction into the bloodstream of infected mice.
The invasion assay presented here is a versatile method for the study of in vivo red cell invasion efficiency of Plasmodium parasites in mice, and allows direct comparison of invasion in red cells derived from two different populations. The method also serves as an accurate alternative method of estimating blood parasitaemia.
Malaria; Plasmodium chabaudi; Plasmodium berghei; Flow cytometry; Parasitaemia; Merozoite; Invasion; In vivo. JC-1
Platelets, as well as regulating blood hemostasis, are an important component of the body’s defense against invading microbial pathogens. We previously reported that platelets protect during malaria infection by binding Plasmodium-infected erythrocytes (IE) and killing the parasite within. More recent studies have now revealed the platelet plasmocidal factor, platelet factor 4 (PF4) and the red cell-expressed Duffy-antigen molecule as the central players in the parasite killing activity of platelets.
Platelet; malaria; Plasmodium falciparum; platelet factor 4; Duffy-antigen; innate immunity
We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile.
Mouse models of allergic asthma are characterized by airway hyperreactivity (AHR), Th2-driven eosinophilic airway inflammation, high allergen-specific IgE (anti-OVA IgE) levels in serum, and airway remodeling. Because asthma susceptibility has a strong genetic component, we aimed to identify new asthma susceptibility genes in the mouse by analyzing the asthma phenotypes of the Leishmania major resistant (lmr) recombinant congenic (RC) strains. The lmr RC strains are derived from C57BL/6 and BALB/c intercrosses and carry congenic loci on chromosome 17 (lmr1) and 9 (lmr2) in both backgrounds. Whereas the lmr2 locus on chromosome 9 contributes to a small background-specific effect on anti-OVA IgE and AHR, the lmr1 locus on chromosome 17 mediates a strong effect on Th2-driven eosinophilic airway inflammation and background-specific effects on anti-OVA IgE and AHR. The lmr1 locus contains almost 600 polymorphic genes. To narrow down this number of candidate genes, we performed genome-wide transcriptional profiling on lung tissue from C.lmr1 RC mice and BALB/c control mice. We identified a small number of differentially expressed genes located within the congenic fragment, including a number of Mhc genes, polymorphic between BALB/c and C57Bl/6. The analysis of asthma phenotypes in the C.B10-H2b RC strain, carrying the C57Bl/6 haplotype of the Mhc locus in a BALB/c genetic background, reveals a strikingly similar asthma phenotype compared with C.lmr1, indicating that the differentially expressed genes located within the C.B10-H2b congenic fragment are the most likely candidate genes to contribute to the reduced asthma phenotypes associated with the C57Bl/6 allele of lmr1.
allergic asthma; quantitative trait locus; recombinant congenic mice; chromosome 17; mouse model
The blood stage of the plasmodium parasite life cycle is responsible for the clinical symptoms of malaria. Epidemiological studies have identified coincidental malarial endemicity and multiple red blood cell (RBC) disorders. Many RBC disorders result from mutations in genes encoding cytoskeletal proteins and these are associated with increased protection against malarial infections. However the mechanisms underpinning these genetic, host responses remain obscure. We have performed an N-ethyl-N-nitrosourea (ENU) mutagenesis screen and have identified a novel dominant (haploinsufficient) mutation in the Ank-1 gene (Ank1MRI23420) of mice displaying hereditary spherocytosis (HS). Female mice, heterozygous for the Ank-1 mutation showed increased survival to infection by Plasmodium chabaudi adami DS with a concomitant 30% decrease in parasitemia compared to wild-type, isogenic mice (wt). A comparative in vivo red cell invasion and parasite growth assay showed a RBC-autonomous effect characterised by decreased proportion of infected heterozygous RBCs. Within approximately 6–8 hours post-invasion, TUNEL staining of intraerythrocytic parasites, showed a significant increase in dead parasites in heterozygotes. This was especially notable at the ring and trophozoite stages in the blood of infected heterozygous mutant mice compared to wt (p<0.05). We conclude that increased malaria resistance due to ankyrin-1 deficiency is caused by the intraerythrocytic death of P. chabaudi parasites.
Multiple sclerosis (OMIM 126200) is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability.1 Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals;2,3 and systematic attempts to identify linkage in multiplex families have confirmed that variation within the Major Histocompatibility Complex (MHC) exerts the greatest individual effect on risk.4 Modestly powered Genome-Wide Association Studies (GWAS)5-10 have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects play a key role in disease susceptibility.11 Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the Class I region. Immunologically relevant genes are significantly over-represented amongst those mapping close to the identified loci and particularly implicate T helper cell differentiation in the pathogenesis of multiple sclerosis.
multiple sclerosis; GWAS; genetics
Genetic linkage studies of the host response to Leishmania major, the causative agent of cutaneous leishmaniasis, have identified significant genetic complexity in humans and mice. In the mouse model, multiple loci have been implicated in susceptibility to infection, but to date, the genes underlying these loci have not been identified. We now describe the contribution of a novel candidate gene, Fli1, to both L. major resistance and enhanced wound healing. We have previously mapped the L. major response locus, lmr2, to proximal chromosome 9 in a genetic cross between the resistant C57BL/6 strain and the susceptible BALB/c strain. We now show that the presence of the resistant C57BL/6 lmr2 allele in susceptible BALB/c mice confers an enhanced L. major resistance and wound healing phenotype. Fine mapping of the lmr2 locus permitted the localization of the lmr2 quantitative trait locus to a 5-Mb interval comprising 21 genes, of which microarray analysis was able to identify differential expression in 1 gene—Fli1. Analysis of Fli1 expression in wounded and L. major-infected skin and naïve and infected lymph nodes validated the importance of Fli1 in lesion resolution and wound healing and identified 3 polymorphisms in the Fli1 promoter, among which a GA repeat element may be the important contributor.
We conducted an association study across the human leukocyte antigen (HLA) complex to identify loci associated with multiple sclerosis (MS). Comparing 1927 SNPs in 1618 MS cases and 3413 controls of European ancestry, we identified seven SNPs that were independently associated with MS conditional on the others (each ). All associations were significant in an independent replication cohort of 2212 cases and 2251 controls () and were highly significant in the combined dataset (). The associated SNPs included proxies for HLA-DRB1*15:01 and HLA-DRB1*03:01, and SNPs in moderate linkage disequilibrium (LD) with HLA-A*02:01, HLA-DRB1*04:01 and HLA-DRB1*13:03. We also found a strong association with rs9277535 in the class II gene HLA-DPB1 (discovery set , replication set , combined ). HLA-DPB1 is located centromeric of the more commonly typed class II genes HLA-DRB1, -DQA1 and -DQB1. It is separated from these genes by a recombination hotspot, and the association is not affected by conditioning on genotypes at DRB1, DQA1 and DQB1. Hence rs9277535 represents an independent MS-susceptibility locus of genome-wide significance. It is correlated with the HLA-DPB1*03:01 allele, which has been implicated previously in MS in smaller studies. Further genotyping in large datasets is required to confirm and resolve this association.
Recent association studies in multiple sclerosis (MS) have identified and replicated several single nucleotide polymorphism (SNP) susceptibility loci including CLEC16A, IL2RA, IL7R, RPL5, CD58, CD40 and chromosome 12q13–14 in addition to the well established allele HLA-DR15. There is potential that these genetic susceptibility factors could also modulate MS disease severity, as demonstrated previously for the MS risk allele HLA-DR15. We investigated this hypothesis in a cohort of 1006 well characterised MS patients from South-Eastern Australia. We tested the MS-associated SNPs for association with five measures of disease severity incorporating disability, age of onset, cognition and brain atrophy. We observed trends towards association between the RPL5 risk SNP and time between first demyelinating event and relapse, and between the CD40 risk SNP and symbol digit test score. No associations were significant after correction for multiple testing. We found no evidence for the hypothesis that these new MS disease risk-associated SNPs influence disease severity.
Genetic heterogeneity is a difficulty frequently encountered in the search for genes conferring susceptibility to prostate cancer. To circumvent this issue, we selected a large prostate cancer pedigree for genome-wide linkage analysis from a population that is genetically homogeneous. Selected cases and first-degree relatives were genotyped with Affymetrix 10K SNP arrays, identifying a 14 Mb haplotype on chromosome 5 (5p13–q12) inherited identical-by-descent (IBD) by multiple cases. Microsatellite genotyping of additional deceased case samples confirmed that a total of eight cases inherited the common haplotype (P=0.0017). Re-sequencing of eight prioritised candidate genes in the region in six selected individuals identified 15 SNPs segregating with the IBD haplotype, located within the ITGA2 gene. Three of these polymorphisms were selected for genotyping in an independent Tasmanian data set comprising 127 cases with familial prostate cancer, 412 sporadic cases and 319 unaffected controls. Two were associated with prostate cancer risk: rs3212649 (OR=1.67 (1.07–2.6), P=0.0009) and rs1126643 (OR=1.52 (1.01–2.28), P=0.0088). Significant association was observed in both familial and sporadic prostate cancer. Although the functional SNP remains to be identified, considerable circumstantial evidence, provided by in vivo and in vitro studies, supports a role for ITGA2 in tumour development.
genetic; susceptibility; prostate; cancer; risk
Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is a highly contagious respiratory pathogen that causes severe losses to the swine industry worldwide. Current commercially-available vaccines are of limited value because they do not induce cross-serovar immunity and do not prevent development of the carrier state. Microarray-based comparative genomic hybridizations (M-CGH) were used to estimate whole genomic diversity of representative Actinobacillus pleuropneumoniae strains. Our goal was to identify conserved genes, especially those predicted to encode outer membrane proteins and lipoproteins because of their potential for the development of more effective vaccines.
Using hierarchical clustering, our M-CGH results showed that the majority of the genes in the genome of the serovar 5 A. pleuropneumoniae L20 strain were conserved in the reference strains of all 15 serovars and in representative field isolates. Fifty-eight conserved genes predicted to encode for outer membrane proteins or lipoproteins were identified. As well, there were several clusters of diverged or absent genes including those associated with capsule biosynthesis, toxin production as well as genes typically associated with mobile elements.
Although A. pleuropneumoniae strains are essentially clonal, M-CGH analysis of the reference strains of the fifteen serovars and representative field isolates revealed several classes of genes that were divergent or absent. Not surprisingly, these included genes associated with capsule biosynthesis as the capsule is associated with sero-specificity. Several of the conserved genes were identified as candidates for vaccine development, and we conclude that M-CGH is a valuable tool for reverse vaccinology.
There are 16 capsule-based serotypes of Actinobacillus pleuropneumoniae, all of which are capable of causing disease in pigs. Here we report the finished and annotated genome sequence of the reference serotype 5b strain L20. This strain has a rough appearance and readily forms biofilms, as is typical for most field isolates (6).
Animals congenic for the char2 host response locus to the murine malarial parasite Plasmodium chabaudi have been bred, and they demonstrated a phenotypic difference from the parental lines. These congenic lines have been crossed back to the parental line to generate recombinants across the congenic intervals. The recombinants were inbred, and the subcongenic intervals were fixed. These lines were then challenged with parasites and assessed as being either resistant or susceptible. From the analysis of many subcongenic lines, it has become obvious that there are at least two loci underlying the char2 locus and that both of these mediate resistance when the haplotype derives from the resistant C57BL/6 strain.
Aeromonas salmonicida has been isolated from numerous fish species and shows wide variation in virulence and pathogenicity. As part of a larger research program to identify virulence genes and candidates for vaccine development, a DNA microarray was constructed using a subset of 2024 genes from the draft genome sequence of A. salmonicida subsp. salmonicida strain A449. The microarray included genes encoding known virulence-associated factors in A. salmonicida and homologs of virulence genes of other pathogens. We used microarray-based comparative genomic hybridizations (M-CGH) to compare selected A. salmonicida sub-species and other Aeromonas species from different hosts and geographic locations.
Results showed variable carriage of virulence-associated genes and generally increased variation in gene content across sub-species and species boundaries. The greatest variation was observed among genes associated with plasmids and transposons. There was little correlation between geographic region and degree of variation for all isolates tested.
We have used the M-CGH technique to identify subsets of conserved genes from amongst this set of A. salmonicida virulence genes for further investigation as potential vaccine candidates. Unlike other bacterial characterization methods that use a small number of gene or DNA-based functions, M-CGH examines thousands of genes and/or whole genomes and thus is a more comprehensive analytical tool for veterinary or even human health research.
A major advance has been made towards the positional cloning of char2 (a quantitative trait locus encoding resistance to Plasmodium chabaudi malaria). Mice congenic for the locus have been used to fine map the gene and to prove that char2 plays a significant role in the outcome of malarial infection, independently of other resistance loci.
In Leishmaniasis, as in many infectious diseases, clinical manifestations are determined by the interaction between the genetics of the host and of the parasite. Here we describe studies mapping two loci controlling resistance to murine cutaneous leishmaniasis. Mice infected with L. major show marked genetic differences in disease manifestations: BALB/c mice are susceptible, exhibiting enlarging lesions that progress to systemic disease and death, whereas C57BL/6 are resistant, developing small, self-healing lesions. F2 animals from a C57BL/6 × BALB/c cross showed a continuous distribution of lesion score. Quantitative trait loci (QTL) have been mapped after a non-parametric QTL analysis on a genome-wide scan on 199 animals. QTLs identified were confirmed in a second cross of 271 animals. Linkage was confirmed to a chromosome 9 locus (D9Mit67–D9Mit71) and to a region including the H2 locus on chromosome 17. These have been named lmr2 and lmr1, respectively.