Broad-spectrum in vitro activity of macrophage infectivity potentiator inhibitors against Gram-negative bacteria and Leishmania major.

BACKGROUND: The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip. OBJECTIVES: In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. METHODS: Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. RESULTS: Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. CONCLUSIONS: These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications.

Cystic Fibrosis Clinical Isolates of Aspergillus fumigatus Induce Similar Muco-inflammatory Responses in Primary Airway Epithelial Cells.

Aspergillus is increasingly associated with lung inflammation and mucus plugging in early cystic fibrosis (CF) disease during which conidia burden is low and strains appear to be highly diverse. It is unknown whether clinical Aspergillus strains vary in their capacity to induce epithelial inflammation and mucus production. We tested the hypothesis that individual colonising strains of Aspergillus fumigatus would induce different responses. Ten paediatric CF Aspergillus isolates were compared along with two systemically invasive clinical isolates and an ATCC reference strain. Isolates were first characterised by ITS gene sequencing and screened for antifungal susceptibility. Three clusters (A-C) of Aspergillus isolates were identified by ITS. Antifungal susceptibility was variable, particularly for itraconazole. Submerged CF and non-CF monolayers as well as differentiated primary airway epithelial cell cultures were incubated with conidia for 24 h to allow germination. None of the clinical isolates were found to significantly differ from one another in either IL-6 or IL-8 release or gene expression of secretory mucins. Clinical Aspergillus isolates appear to be largely homogenous in their mucostimulatory and immunostimulatory capacities and, therefore, only the antifungal resistance characteristics are likely to be clinically important.

Linking the westernised oropharyngeal microbiome to the immune response in Chinese immigrants.

BACKGROUND: Human microbiota plays a fundamental role in modulating the immune response. Western environment and lifestyle are envisaged to alter the human microbiota with a new microbiome profile established in Chinese immigrants, which fails to prime the immune system. Here, we investigated how differences in composition of oropharyngeal microbiome may contribute to patterns of interaction between the microbiome and immune system in Chinese immigrants living in Australia. METHODS: We recruited 44 adult Chinese immigrants: newly-arrived (n = 22, living in Australia < 6 months) and long-term Chinese immigrants (n = 22, living in Australia > 5 years), with age and gender matched. Oropharyngeal swabs, serum and whole blood were collected. The 16 s ribosomal RNA gene from the swabs was sequenced on the Illumina MiSeq platform. Innate immune responses were determined by 23 Toll-like receptors (TLR) pathway cytokines, while adaptive immune responses were determined by IgG-associated response to specific microbial/viral pathogens. RESULTS: The relative abundance of the genus Leptotrichia was higher in long-term immigrants as compared to that in newly-arrived Chinese immigrants, while the genus Deinococcus was significantly lower in long-term Chinese immigrants. The genera uncultured Lachnospiraceae, Erysipelotrichaceae UCG-007, Veillonella, and Actinomycetales_ambiguous taxa were negatively correlated with cytokine IL-6 in long-term Chinese immigrants (rho range: - 0.46 ~ - 0.73). With respect to adaptive immunity, several microbial taxa were significantly associated with IgG1 responsiveness to microbial antigens in long-term immigrants, while a significant correlation with IgG1 responsiveness to viral antigens was detected in newly-arrived immigrants. CONCLUSIONS: The composition of the oropharyngeal microbiome varies between newly-arrived and long-term Chinese immigrants. Specific microbial taxa are significantly associated with immunological parameters but with different association patterns between newly-arrived and long-term Chinese immigrants.

A new longitudinal variation in the structure of collagen fibrils and its relationship to locations of mechanical damage susceptibility.

The hierarchical architecture of the collagen fibril is well understood, involving non-integer staggering of collagen molecules which results in a 67 nm periodic molecular density variation termed D-banding. Other than this variation, collagen fibrils are considered to be homogeneous at the micro-scale and beyond. Interestingly, serial kink structures have been shown to form at discrete locations along the length of collagen fibrils from some mechanically overloaded tendons. The formation of these kinks at discrete locations along the length of fibrils (discrete plasticity) may indicate pre-existing structural variations at a length scale greater than that of the D-banding. Using a high velocity nanomechanical mapping technique, 25 tendon collagen fibrils, were mechanically and structurally mapped along 10 µm of their length in dehydrated and hydrated states with resolutions of 20 nm and 8 nm respectively. Analysis of the variation in hydrated indentation modulus along individual collagen fibrils revealed a micro-scale structural variation not observed in the hydrated or dehydrated structural maps. The spacing distribution of this variation was similar to that observed for inter-kink distances seen in SEM images of discrete plasticity type damage. We propose that longitudinal variation in collagen fibril structure leads to localized mechanical susceptibility to damage under overload. Furthermore, we suggest that this variation has its origins in heterogeneous crosslink density along the length of collagen fibrils. The presence of pre-existing sites of mechanical vulnerability along the length of collagen fibrils may be important to biological remodeling of tendon, with mechanically-activated sites having distinct protein binding capabilities and enzyme susceptibility.

Reviewing the Pathogenic Potential of the Otitis-Associated Bacteria Alloiococcus otitidis and Turicella otitidis.

Alloiococcus otitidis and Turicella otitidis are common bacteria of the human ear. They have frequently been isolated from the middle ear of children with otitis media (OM), though their potential role in this disease remains unclear and confounded due to their presence as commensal inhabitants of the external auditory canal. In this review, we summarize the current literature on these organisms with an emphasis on their role in OM. Much of the literature focuses on the presence and abundance of these organisms, and little work has been done to explore their activity in the middle ear. We find there is currently insufficient evidence available to determine whether these organisms are pathogens, commensals or contribute indirectly to the pathogenesis of OM. However, building on the knowledge currently available, we suggest future approaches aimed at providing stronger evidence to determine whether A. otitidis and T. otitidis are involved in the pathogenesis of OM. Such evidence will increase our understanding of the microbial risk factors contributing to OM and may lead to novel treatment approaches for severe and recurrent disease.

Western oropharyngeal and gut microbial profiles are associated with allergic conditions in Chinese immigrant children.

BACKGROUND: The allergy epidemic resulting from western environment/lifestyles is potentially due to modifications of the human microbiome. Therefore, it is of interest to study immigrants living in a western environment as well as their counterparts in the country of origin to understand differences in their microbiomes and health status. METHODS: We investigated 58 Australian Chinese (AC) children from Perth, Western Australia as well as 63 Chinese-born Chinese (CC) children from a city in China. Oropharyngeal (OP) and fecal samples were collected. To assess the microbiomes, 16s ribosomal RNA (rRNA) sequencing for variable regions V3 and V4 was used. Skin prick tests (SPT) were performed to measure the children's atopic status. Information on food allergy and wheezing were acquired from a questionnaire. RESULTS: AC children had more allergic conditions than CC children. The alpha diversity (mean species diversity) of both OP and gut microbiome was lower in AC children compared to CC children for richness estimate (Chao1), while diversity evenness (Shannon index) was higher. The beta diversity (community similarity) displayed a distinct separation of the OP and gut microbiota between AC and CC children. An apparent difference in microbial abundance was observed for many bacteria. In AC children, we sought to establish consistent trends in bacterial relative abundance that are either higher or lower in AC versus CC children and higher or lower in children with allergy versus those without allergy. The majority of OP taxa showed a consistent trend while the majority of fecal taxa showed a contrasting trend. CONCLUSION: Distinct differences in microbiome compositions were found in both oropharyngeal and fecal samples of AC and CC children. The association of the OP microbiome with allergic condition is different from that of the gut microbiome in AC children. The microbiome profiles are changed by the western environment/lifestyle and are associated with allergies in Chinese immigrant children in Australia.

Moraxella catarrhalis Restriction-Modification Systems Are Associated with Phylogenetic Lineage and Disease.

Moraxella catarrhalis is a human-adapted pathogen, and a major cause of otitis media (OM) and exacerbations of chronic obstructive pulmonary disease. The species is comprised of two main phylogenetic lineages, RB1 and RB2/3. Restriction-modification (R-M) systems are among the few lineage-associated genes identified in other bacterial genera and have multiple functions including defense against foreign invading DNA, maintenance of speciation, and epigenetic regulation of gene expression. Here, we define the repertoire of R-M systems in 51 publicly available M. catarrhalis genomes and report their distribution among M. catarrhalis phylogenetic lineages. An association with phylogenetic lineage (RB1 or RB2/3) was observed for six R-M systems, which may contribute to the evolution of the lineages by restricting DNA transformation. In addition, we observed a relationship between a mutually exclusive Type I R-M system and a Type III R-M system at a single locus conserved throughout a geographically and clinically diverse set of M. catarrhalis isolates. The Type III R-M system at this locus contains the phase-variable Type III DNA methyltransferase, modM, which controls a phasevarion (phase-variable regulon). We observed an association between modM presence and OM-associated middle ear isolates, indicating a potential role for ModM-mediated epigenetic regulation in OM pathobiology.

The marsupial trypanosome Trypanosoma copemani is not an obligate intracellular parasite, although it adversely affects cell health.

BACKGROUND: Trypanosoma cruzi invades and replicates inside mammalian cells, which can lead to chronic Chagas disease in humans. Trypanosoma copemani infects Australian marsupials and recent investigations indicate it may be able to invade mammalian cells in vitro, similar to T. cruzi. Here, T. cruzi 10R26 strain (TcIIa) and two strains of T. copemani [genotype 1 (G1) and genotype 2 (G2)] were incubated with marsupial cells in vitro. Live-cell time-lapse and fluorescent microscopy, combined with high-resolution microscopy (transmission and scanning electron microscopy) were used to investigate surface interactions between parasites and mammalian cells. RESULTS: The number of parasites invading cells was significantly higher in T. cruzi compared to either genotype of T. copemani, between which there was no significant difference. While capable of cellular invasion, T. copemani did not multiply in host cells in vitro as there was no increase in intracellular amastigotes over time and no release of new trypomastigotes from host cells, as observed in T. cruzi. Exposure of host cells to G2 trypomastigotes resulted in increased host cell membrane permeability within 24 h of infection, and host cell death/blebbing was also observed. G2 parasites also became embedded in the host cell membrane. CONCLUSIONS: Trypanosoma copemani is unlikely to have an obligate intracellular life-cycle like T. cruzi. However, T. copemani adversely affects cell health in vitro and should be investigated in vivo in infected host tissues to better understand this host-parasite relationship. Future research should focus on increasing understanding of the T. copemani life history and the genetic, physiological and ecological differences between different genotypes.

A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera.

BACKGROUND: Recurrent acute otitis media (rAOM, recurrent ear infection) is a common childhood disease caused by bacteria termed otopathogens, for which current treatments have limited effectiveness. Generic probiotic therapies have shown promise, but seem to lack specificity. We hypothesised that healthy children with no history of AOM carry protective commensal bacteria that could be translated into a specific probiotic therapy to break the cycle of re-infection. We characterised the nasopharyngeal microbiome of these children (controls) in comparison to children with rAOM (cases) to identify potentially protective bacteria. As some children with rAOM do not appear to carry any of the known otopathogens, we also hypothesised that characterisation of the middle ear microbiome could identify novel otopathogens, which may also guide the development of more effective therapies. RESULTS: Middle ear fluids, middle ear rinses and ear canal swabs from the cases and nasopharyngeal swabs from both groups underwent 16S rRNA gene sequencing. The nasopharyngeal microbiomes of cases and controls were distinct. We observed a significantly higher abundance of Corynebacterium and Dolosigranulum in the nasopharynx of controls. Alloiococcus, Staphylococcus and Turicella were abundant in the middle ear and ear canal of cases, but were uncommon in the nasopharynx of both groups. Gemella and Neisseria were characteristic of the case nasopharynx, but were not prevalent in the middle ear. CONCLUSIONS: Corynebacterium and Dolosigranulum are characteristic of a healthy nasopharyngeal microbiome. Alloiococcus, Staphylococcus and Turicella are possible novel otopathogens, though their rarity in the nasopharynx and prevalence in the ear canal means that their role as normal aural flora cannot be ruled out. Gemella and Neisseria are unlikely to be novel otopathogens as they do not appear to colonise the middle ear in children with rAOM.

A comparative molecular and 3-dimensional structural investigation into cross-continental and novel avian Trypanosoma spp. in Australia.

BACKGROUND: Molecular and structural information on avian Trypanosoma spp. throughout Australia is limited despite their intrinsic value in understanding trypanosomatid evolution, diversity, and structural biology. In Western Australia tissue samples (n = 429) extracted from 93 birds in 25 bird species were screened using generic PCR primers to investigate the diversity of Trypanosoma spp. To investigate avian trypanosome structural biology the first 3-dimensional ultrastructural models of a Trypanosoma spp. (Trypanosoma sp. AAT) isolated from a bird (currawong, Strepera spp.) were generated using focussed ion beam milling combined with scanning electron microscopy (FIB-SEM). RESULTS: Here, we confirm four intercontinental species of avian trypanosomes in native Australian birds, and identify a new avian Trypanosoma. Trypanosome infection was identified in 18 birds from 13 different bird species (19%). A single new genotype was isolated and found to be closely related to T. culicavium (Trypanosoma sp. CC2016 B002). Other Trypanosoma spp. identified include T. avium, T. culicavium, T. thomasbancrofti, Trypanosoma sp. TL.AQ.22, Trypanosoma sp. AAT, and an uncharacterised Trypanosoma sp. (group C-III sensu Zidková et al. (Infect Genet Evol 12:102-112, 2012)), all previously identified in Australia or other continents. Serially-sectioning Trypanosoma sp. AAT epimastigotes using FIB-SEM revealed the disc-shaped kinetoplast pocket attached perpendicular to the branching mitochondrion. Additionally, the universal minicircle sequence within the kinetoplast DNA and the associated binding protein were determined in Trypanosoma sp. AAT. CONCLUSIONS: These results indicate that bird trypanosomes are relatively conserved across continents, while being locally diverse, which supports the hypothesis that bird trypanosomes exist as fewer species than described in the literature. Evidence exists that avian Trypanosoma spp. are infecting mammals and could be transmitted by haemadipsid leeches. Trypanosoma sp. AAT is most likely a separate species currently found only in Australia and the first 3-dimentional ultrastructural analysis of an avian trypanosome provides interesting information on their morphology and organelle arrangement.

In vitro drug susceptibility of two strains of the wildlife trypanosome, Trypanosoma copemani: A comparison with Trypanosoma cruzi.

Trypanosomes are blood protozoan parasites that are capable of producing illness in the vertebrate host. Within Australia, several native Trypanosoma species have been described infecting wildlife. However, only Trypanosoma copemani has been associated with pathological lesions in wildlife hosts and more recently has been associated with the drastic decline of the critically endangered woylie (Bettongia penicillata). The impact that some trypanosomes have on the health of the vertebrate host has led to the development of numerous drug compounds that could inhibit the growth or kill the parasite. This study investigated and compared the in vitro susceptibility of two strains of T. copemani (G1 and G2) and one strain of Trypanosoma cruzi (10R26) against drugs that are known to show trypanocidal activity (benznidazole, posaconazole, miltefosine and melarsoprol) and against four lead compounds, two fenarimols and two pyridine derivatives (EPL-BS1937, EPL-BS2391, EPL-BS0967, and EPL-BS1246), that have been developed primarily against T.cruzi. The in vitro cytotoxicity of all drugs against L6 rat myoblast cells was also assessed. Results showed that both strains of T. copemani were more susceptible to all drugs and lead compounds than T. cruzi, with all IC50 values in the low and sub-µM range for both species. Melarsoprol and miltefosine exhibited the highest drug activity against both T. copemani and T. cruzi, but they also showed the highest toxicity in L6 cells. Interestingly, both fenarimol and pyridine derivative compounds were more active against T. copemani and T. cruzi than the reference drugs benznidazole and posaconazole. T. copemani strains exhibited differences in susceptibility to all drugs demonstrating once again considerable differences in their biological behaviour.

Towards a Better Understanding of the Life Cycle of Trypanosoma copemani.

Trypanosoma copemani has been found infecting several threatened/endangered marsupial species within Australia and is thought to be a key player in the rapid decline of the woylie (Bettongia penicillata). To better understand the biology and life cycle of this parasite, the growth requirements, and kinetics of infection of two newly described genotypes, T. copemani G1 and G2, were investigated and compared with the T. cruzi strain-10R26 in vitro. Both G1 and G2 were able to infect all four cell lines tested. The number of infected cells where at least one intracellular amastigote of T. copemani G1 and G2 was seen was below 7% and 15% respectively in most cell lines. However, in VERO cells the rate of infection for T. copemani G2 was 70%-approximately seven and two times higher than for G1 and T. cruzi respectively. Despite the higher infection rate, the number of intracellular forms of T. copemani G2 was lower compared with T. cruzi, and intracellular replicating forms were not observed. The capability of T. copemani G2 to infect cells may have important consequences for pathogenicity and suggests it might employ similar strategies to complete its life cycle in the vertebrate host to those seen in T. cruzi.

A retrospective study of Babesia macropus associated with morbidity and mortality in eastern grey kangaroos (Macropus giganteus) and agile wallabies (Macropus agilis).

This is a retrospective study of 38 cases of infection by Babesia macropus, associated with a syndrome of anaemia and debility in hand-reared or free-ranging juvenile eastern grey kangaroos (Macropus giganteus) from coastal New South Wales and south-eastern Queensland between 1995 and 2013. Infection with B. macropus is recorded for the first time in agile wallabies (Macropus agilis) from far north Queensland. Animals in which B. macropus infection was considered to be the primary cause of morbidity had marked anaemia, lethargy and neurological signs, and often died. In these cases, parasitised erythrocytes were few or undetectable in peripheral blood samples but were sequestered in large numbers within small vessels of visceral organs, particularly in the kidney and brain, associated with distinctive clusters of extraerythrocytic organisms. Initial identification of this piroplasm in peripheral blood smears and in tissue impression smears and histological sections was confirmed using transmission electron microscopy and molecular analysis. Samples of kidney, brain or blood were tested using PCR and DNA sequencing of the 18S ribosomal RNA and heat shock protein 70 gene using primers specific for piroplasms. The piroplasm detected in these samples had 100% sequence identity in the 18S rRNA region with the recently described Babesia macropus in two eastern grey kangaroos from New South Wales and Queensland, and a high degree of similarity to an unnamed Babesia sp. recently detected in three woylies (Bettongia penicillata ogilbyi) in Western Australia.

Comparative analysis of the full genome of Helicobacter pylori isolate Sahul64 identifies genes of high divergence.

Isolates of Helicobacter pylori can be classified phylogeographically. High genetic diversity and rapid microevolution are a hallmark of H. pylori genomes, a phenomenon that is proposed to play a functional role in persistence and colonization of diverse human populations. To provide further genomic evidence in the lineage of H. pylori and to further characterize diverse strains of this pathogen in different human populations, we report the finished genome sequence of Sahul64, an H. pylori strain isolated from an indigenous Australian. Our analysis identified genes that were highly divergent compared to the 38 publically available genomes, which include genes involved in the biosynthesis and modification of lipopolysaccharide, putative prophage genes, restriction modification components, and hypothetical genes. Furthermore, the virulence-associated vacA locus is a pseudogene and the cag pathogenicity island (cagPAI) is not present. However, the genome does contain a gene cluster associated with pathogenicity, including dupA. Our analysis found that with the addition of Sahul64 to the 38 genomes, the core genome content of H. pylori is reduced by approximately 14% (∼170 genes) and the pan-genome has expanded from 2,070 to 2,238 genes. We have identified three putative horizontally acquired regions, including one that is likely to have been acquired from the closely related Helicobacter cetorum prior to speciation. Our results suggest that Sahul64, with the absence of cagPAI, highly divergent cell envelope proteins, and a predicted nontransportable VacA protein, could be more highly adapted to ancient indigenous Australian people but with lower virulence potential compared to other sequenced and cagPAI-positive H. pylori strains.

Attachment and invasion of Neisseria meningitidis to host cells is related to surface hydrophobicity, bacterial cell size and capsule.

We compared exemplar strains from two hypervirulent clonal complexes, strain NMB-CDC from ST-8/11 cc and strain MC58 from ST-32/269 cc, in host cell attachment and invasion. Strain NMB-CDC attached to and invaded host cells at a significantly greater frequency than strain MC58. Type IV pili retained the primary role for initial attachment to host cells for both isolates regardless of pilin class and glycosylation pattern. In strain MC58, the serogroup B capsule was the major inhibitory determinant affecting both bacterial attachment to and invasion of host cells. Removal of terminal sialylation of lipooligosaccharide (LOS) in the presence of capsule did not influence rates of attachment or invasion for strain MC58. However, removal of either serogroup B capsule or LOS sialylation in strain NMB-CDC increased bacterial attachment to host cells to the same extent. Although the level of inhibition of attachment by capsule was different between these strains, the regulation of the capsule synthesis locus by the two-component response regulator MisR, and the level of surface capsule determined by flow cytometry were not significantly different. However, the diplococci of strain NMB-CDC were shown to have a 1.89-fold greater surface area than strain MC58 by flow cytometry. It was proposed that the increase in surface area without changing the amount of anchored glycolipid capsule in the outer membrane would result in a sparser capsule and increase surface hydrophobicity. Strain NMB-CDC was shown to be more hydrophobic than strain MC58 using hydrophobicity interaction chromatography and microbial adhesion-to-solvents assays. In conclusion, improved levels of adherence of strain NMB-CDC to cell lines was associated with increased bacterial cell surface and surface hydrophobicity. This study shows that there is diversity in bacterial cell surface area and surface hydrophobicity within N. meningitidis which influence steps in meningococcal pathogenesis.

Trypanosomes genetic diversity, polyparasitism and the population decline of the critically endangered Australian marsupial, the brush tailed bettong or woylie (Bettongia penicillata).

While much is known of the impact of trypanosomes on human and livestock health, trypanosomes in wildlife, although ubiquitous, have largely been considered to be non-pathogenic. We describe the genetic diversity, tissue tropism and potential pathogenicity of trypanosomes naturally infecting Western Australian marsupials. Blood samples collected from 554 live-animals and 250 tissue samples extracted from 50 carcasses of sick-euthanized or road-killed animals, belonging to 10 species of marsupials, were screened for the presence of trypanosomes using a PCR of the 18S rDNA gene. PCR results revealed a rate of infection of 67% in blood and 60% in tissues. Inferred phylogenetic trees using 18S rDNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) sequences showed the presence of eight genotypes that clustered into three clades: a clade including Trypanosoma copemani, a new clade closely related to Trypanosoma gilletti, and a clade including Trypanosoma H25 from an Australian kangaroo. Trypanosome infections were compared in a declining and in a stable population of the endangered Australian marsupial, the brush tailed bettong or woylie (Bettongia penicillata). This marsupial showed high rates of infection with Clade A genotypes (96%) in the declining population, whereas in the stable population, Clade B genotypes were predominant (89%). Mixed infections were common in woylies from the declining but not from the stable population. Histopathological findings associated with either mixed or single infections involving Clade A genotypes, showed a strong inflammatory process and tissue degeneration predominantly in heart, oesophagus and tongue. Trypanosomes were successfully grown in culture and for the first time we demonstrate that a genotype within Clade A has the capacity to not only colonize different tissues in the host but also to invade cells in vitro. These results provide evidence for the potential role of trypanosomes in the decline of a formerly abundant marsupial that is now critically endangered.

Genetic and functional evidence implicating DLL1 as the gene that influences susceptibility to visceral leishmaniasis at chromosome 6q27.

BACKGROUND: Visceral leishmaniasis (VL) is caused by Leishmania donovani and Leishmania infantum chagasi. Genome-wide linkage studies from Sudan and Brazil identified a putative susceptibility locus on chromosome 6q27. METHODS: Twenty-two single-nucleotide polymorphisms (SNPs) at genes PHF10, C6orf70, DLL1, FAM120B, PSMB1, and TBP were genotyped in 193 VL cases from 85 Sudanese families, and 8 SNPs at genes PHF10, C6orf70, DLL1, PSMB1, and TBP were genotyped in 194 VL cases from 80 Brazilian families. Family-based association, haplotype, and linkage disequilibrium analyses were performed. Multispecies comparative sequence analysis was used to identify conserved noncoding sequences carrying putative regulatory elements. Quantitative reverse-transcription polymerase chain reaction measured expression of candidate genes in splenic aspirates from Indian patients with VL compared with that in the control spleen sample. RESULTS: Positive associations were observed at PHF10, C6orf70, DLL1, PSMB1, and TBP in Sudan, but only at DLL1 in Brazil (combined P = 3 × 10(-4) at DLL1 across Sudan and Brazil). No functional coding region variants were observed in resequencing of 22 Sudanese VL cases. DLL1 expression was significantly (P = 2 × 10(-7)) reduced (mean fold change, 3.5 [SEM, 0.7]) in splenic aspirates from patients with VL, whereas other 6q27 genes showed higher levels (1.27 × 10(-6) < P < .01) than did the control spleen sample. A cluster of conserved noncoding sequences with putative regulatory variants was identified in the distal promoter of DLL1. CONCLUSIONS: DLL1, which encodes Delta-like 1, the ligand for Notch3, is strongly implicated as the chromosome 6q27 VL susceptibility gene.

A genomic-based approach combining in vivo selection in mice to identify a novel virulence gene in Leishmania.

BACKGROUND: Infection with Leishmania results in a broad spectrum of pathologies where L. infantum and L. donovani cause fatal visceral leishmaniasis and L. major causes destructive cutaneous lesions. The identification and characterization of Leishmania virulence genes may define the genetic basis for these different pathologies. METHODS AND FINDINGS: Comparison of the recently completed L. major and L. infantum genomes revealed a relatively small number of genes that are absent or present as pseudogenes in L. major and potentially encode proteins in L. infantum. To investigate the potential role of genetic differences between species in visceral infection, seven genes initially classified as absent in L. major but present in L. infantum were cloned from the closely related L. donovani genome and introduced into L. major. The transgenic L. major expressing the L. donovani genes were then introduced into BALB/c mice to select for parasites with increased virulence in the spleen to determine whether any of the L. donovani genes increased visceral infection levels. During the course of these experiments, one of the selected genes (LinJ32_V3.1040 (Li1040)) was reclassified as also present in the L. major genome. Interestingly, only the Li1040 gene significantly increased visceral infection in the L. major transfectants. The Li1040 gene encodes a protein containing a putative component of an endosomal protein sorting complex involved with protein transport. CONCLUSIONS: These observations demonstrate that the levels of expression and sequence variations in genes ubiquitously shared between Leishmania species have the potential to significantly influence virulence and tissue tropism.

Genetic and epigenetic factors at COL2A1 and ABCA4 influence clinical outcome in congenital toxoplasmosis.

BACKGROUND: Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute. METHODS AND FINDINGS: In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mother's genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting. CONCLUSIONS: These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite.