In Situ Characterization of Splenic Brucella melitensis Reservoir Cells during the Chronic Phase of Infection in Susceptible Mice

Brucella are facultative intracellular Gram-negative coccobacilli that chronically infect humans as well as domestic and wild-type mammals, and cause brucellosis. Alternatively activated macrophages (M2a) induced by IL-4/IL-13 via STAT6 signaling pathways have been frequently described as a favorable niche for long-term persistence of intracellular pathogens. Based on the observation that M2a-like macrophages are induced in the spleen during the chronic phase of B. abortus infection in mice and are strongly infected in vitro, it has been suggested that M2a macrophages could be a potential in vivo niche for Brucella. In order to test this hypothesis, we used a model in which infected cells can be observed directly in situ and where the differentiation of M2a macrophages is favored by the absence of an IL-12-dependent Th1 response. We performed an in situ analysis by fluorescent microscopy of the phenotype of B. melitensis infected spleen cells from intranasally infected IL-12p40^-/- BALB/c mice and the impact of STAT6 deficiency on this phenotype. Most of the infected spleen cells contained high levels of lipids and expressed CD11c and CD205 dendritic cell markers and Arginase1, but were negative for the M2a markers Fizz1 or CD301. Furthermore, STAT6 deficiency had no effect on bacterial growth or the reservoir cell phenotype in vivo, leading us to conclude that, in our model, the infected cells were not Th2-induced M2a macrophages. This characterization of B. melitensis reservoir cells could provide a better understanding of Brucella persistence in the host and lead to the design of more efficient therapeutic strategies.     

Beegle: from literature mining to disease-gene discovery

Disease-gene identification is a challenging process that has multiple applications within functional genomics and personalized medicine. Typically, this process involves both finding genes known to be associated with the disease (through literature search) and carrying out preliminary experiments or screens (e.g. linkage or association studies, copy number analyses, expression profiling) to determine a set of promising candidates for experimental validation. This requires extensive time and monetary resources. We describe Beegle, an online search and discovery engine that attempts to simplify this process by automating the typical approaches. It starts by mining the literature to quickly extract a set of genes known to be linked with a given query, then it integrates the learning methodology of Endeavour (a gene prioritization tool) to train a genomic model and rank a set of candidate genes to generate novel hypotheses. In a realistic evaluation setup, Beegle has an average recall of 84% in the top 100 returned genes as a search engine, which improves the discovery engine by 12.6% in the top 5% prioritized genes. Beegle is publicly available at http://beegle.esat.kuleuven.be/.     

APOE ɛ4 allele and TOMM40‐APOC1 variants jointly contribute to survival to older ages

Age‐related diseases characteristic of post‐reproductive life, aging, and life span are the examples of polygenic non‐Mendelian traits with intricate genetic architectures. Polygenicity of these traits implies that multiple variants can impact their risks independently or jointly as combinations of specific variants. Here, we examined chances to live to older ages, 85 years and older, for carriers of compound genotypes comprised of combinations of genotypes of rs429358 (APOE ɛ4 encoding polymorphism), rs2075650 (TOMM40), and rs12721046 (APOC1) polymorphisms using data from four human studies. The choice of these polymorphisms was motivated by our prior results showing that the ɛ4 carriers having minor alleles of the other two polymorphisms were at exceptionally high risk of Alzheimer''s disease (AD), compared with non‐carriers of the minor alleles. Consistent with our prior findings for AD, we show here that the adverse effect of the ɛ4 allele on survival to older ages is significantly higher in carriers of minor alleles of rs2075650 and/or rs12721046 polymorphisms compared with their non‐carriers. The exclusion of AD cases made this effect stronger. Our results provide compelling evidence that AD does not mediate the associations of the same compound genotypes with chances to survive until older ages, indicating the existence of genetically heterogeneous mechanisms. The survival chances can be mainly associated with lipid‐ and immunity‐related mechanisms, whereas the AD risk, can be driven by the AD‐biomarker‐related mechanism, among others. Targeting heterogeneous polygenic profiles of individuals at high risks of complex traits is promising for the translation of genetic discoveries to health care.     

Structure and Function of Glycosylated Tandem Repeats from Candida albicans Als Adhesins

Tandem repeat (TR) regions are common in yeast adhesins, but their structures are unknown, and their activities are poorly understood. TR regions in Candida albicans Als proteins are conserved glycosylated 36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R. De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke, Eukaryot. Cell 5:1664–1673, 2006). Ab initio modeling with either Rosetta or LINUS generated consistent structures of three-stranded antiparallel β-sheet domains, whereas randomly shuffled sequences with the same composition generated various structures with consistently higher energies. O- and N-glycosylation patterns showed that each TR domain had exposed hydrophobic surfaces surrounded by glycosylation sites. These structures are consistent with domain dimensions and stability measurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A. Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677–1682, 2009) and with circular dichroism determination of secondary structure and thermal stability. Functional assays showed that the hydrophobic surfaces of TR domains supported binding to polystyrene surfaces and other TR domains, leading to nonsaturable homophilic binding. The domain structures are like “classic” subunit interaction surfaces and can explain previously observed patterns of promiscuous interactions between TR domains in any Als proteins or between TR domains and surfaces of other proteins. Together, the modeling techniques and the supporting data lead to an approach that relates structure and function in many kinds of repeat domains in fungal adhesins.Yeast adhesins are a diverse set of cell adhesion proteins that mediate adhesion to host cells, environmental substrates, other fungi, and coinfecting bacteria (6, 8, 20, 21, 23, 29). The adhesins share common features, including compact N-terminal domains similar to Ig or lectin domains, Thr-rich midpieces, often in tandem repeats, and long highly glycosylated Ser/Thr-rich C-terminal regions that extend the functional domains out from the cell surface. No structures for the Thr-rich midpieces are known, but they can mediate aggregation of fungal cells (33, 35, 47). The prevalence and conservation of such repeats argue that they are functionally important, despite limited data on their structure and function.In Candida albicans, the Als adhesins are homologous proteins, products of 8 loci that encode numerous alleles of cell surface adhesins (16). In each mature Als protein, there are, from the N terminus, three tandem Ig-like domains, a β-sheet-rich conserved 127-residue amyloid-forming T region, a variable number of 36-residue tandem repeats (TRs), and a highly glycosylated stalk region that extends the N-terminal domains away from the cell surface (Fig. 1) (16, 33, 41). The C termini of these and other wall-associated adhesins are covalently cross-linked into the cell wall through transglycosylation of a modified glycosylphosphatidylinositol (GPI) anchor (18, 25). This modular design, including tandem repeats, is typical of fungal adhesins (8).Open in a separate windowFig. 1.Schematic diagram of the sequence of Als5p. The regions are named above, and the number of amino acid residues in each region is shown below. The modeled sequences are in the TR region.The Als protein Ig-like region, T region, and TR region all have protein-protein interaction activities (26, 33, 35). The Ig-like regions can interact with diverse mammalian proteins, presumably in a way analogous to antibody-antigen binding, as has been shown in the homologous protein α-agglutinin from Saccharomyces cerevisiae (8, 24, 26, 35). The T regions interact through formation of amyloid-like structures both in vivo and in vitro (33, 34a, 36). An insight into the function of the tandem repeats followed from observations that Als proteins initiate and maintain cell-to-cell aggregations, either spontaneously (“autoaggregation”) or following adhesion to a bead-bound defined ligand (10, 11, 36). Aggregation is more extensive for Als proteins with more tandem repeats (26, 35). This result suggested that the tandem repeats are uniquely structured to facilitate or mediate the aggregative function. Circular dichroism spectroscopy of the TR region of Als5p shows a β-sheet-rich structure in the soluble protein (35).In support of their direct involvement in aggregation, the repeat region of the C. albicans adhesin Als5p mediates cell-cell aggregation in the absence of the Ig-like and T domains (35). Moreover, the repeats can also potentiate binding of Als5p to fibronectin (35). Thus, the TR domains mediate cellular aggregation and increased binding to fibronectin. In addition, TR domains and their amino acid sequences are highly conserved across several Candida species (3). These properties need to be explained by their three-dimensional structure.Because there are no homologous structures known, we modeled by two independent ab initio methods. Rosetta assembles structures by combining short peptide structures extracted from the protein structural database PDB (38), then combines structures in a Monte Carlo approach, and assesses energetics of assembled structures. Rosetta has recently been shown to generate accurate models for protein-sized domains (40). We also predicted structures with LINUS, which generates randomized structures and rapidly estimates energetics to choose low-energy models (45). The models were supported by structural analyses with atomic force microscopy and circular dichroism spectroscopy. Functional assays showed that the TR domains can mediate binding activities predicted from the calculated structures.     

Haemophilus Responses to Nutritional Immunity: Epigenetic and Morphological Contribution to Biofilm Architecture,Invasion, Persistence and Disease Severity

In an effort to suppress microbial outgrowth, the host sequesters essential nutrients in a process termed nutritional immunity. However, inflammatory responses to bacterial insult can restore nutritional resources. Given that nutrient availability modulates virulence factor production and biofilm formation by other bacterial species, we hypothesized that fluctuations in heme-iron availability, particularly at privileged sites, would similarly influence Haemophilus biofilm formation and pathogenesis. Thus, we cultured Haemophilus through sequential heme-iron deplete and heme-iron replete media to determine the effect of transient depletion of internal stores of heme-iron on multiple pathogenic phenotypes. We observed that prior heme-iron restriction potentiates biofilm changes for at least 72 hours that include increased peak height and architectural complexity as compared to biofilms initiated from heme-iron replete bacteria, suggesting a mechanism for epigenetic responses that participate in the changes observed. Additionally, in a co-infection model for human otitis media, heme-iron restricted Haemophilus, although accounting for only 10% of the inoculum (90% heme-iron replete), represented up to 99% of the organisms recovered at 4 days. These data indicate that fluctuations in heme-iron availability promote a survival advantage during disease. Filamentation mediated by a SulA-related ortholog was required for optimal biofilm peak height and persistence during experimental otitis media. Moreover, severity of disease in response to heme-iron restricted Haemophilus was reduced as evidenced by lack of mucosal destruction, decreased erythema, hemorrhagic foci and vasodilatation. Transient restriction of heme-iron also promoted productive invasion events leading to the development of intracellular bacterial communities. Taken together, these data suggest that nutritional immunity, may, in fact, foster long-term phenotypic changes that better equip bacteria for survival at infectious sites.     

In Vivo Adaptation and Persistence of Neisseria meningitidis within the Nasopharyngeal Mucosa

Neisseria meningitidis (Nme) asymptomatically colonizes the human nasopharynx, yet can initiate rapidly-progressing sepsis and meningitis in rare instances. Understanding the meningococcal lifestyle within the nasopharyngeal mucosa, a phase of infection that is prerequisite for disease, has been hampered by the lack of animal models. Herein, we compare mice expressing the four different human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) that can bind the neisserial Opa protein adhesins, and find that expression of human CEACAM1 is necessary and sufficient to establish intranasal colonization. During infection, in vivo selection for phase variants expressing CEACAM1-specific Opa proteins occurs, allowing mucosal attachment and entry into the subepithelial space. Consistent with an essential role for Opa proteins in this process, Opa-deficient meningococci were unable to colonize the CEACAM1-humanized mice. While simple Opa-mediated attachment triggered an innate response regardless of meningococcal viability within the inoculum, persistence of viable Opa-expressing bacteria within the CEACAM1-humanized mice was required for a protective memory response to be achieved. Parenteral immunization with a capsule-based conjugate vaccine led to the accumulation of protective levels of Nme-specific IgG within the nasal mucus, yet the sterilizing immunity afforded by natural colonization was instead conferred by Nme-specific IgA without detectable IgG. Considered together, this study establishes that the availability of CEACAM1 helps define the exquisite host specificity of this human-restricted pathogen, displays a striking example of in vivo selection for the expression of desirable Opa variants, and provides a novel model in which to consider meningococcal infection and immunity within the nasopharyngeal mucosa.     

MLVA Genotyping of Brucella melitensis and Brucella abortus Isolates from Different Animal Species and Humans and Identification of Brucella suis Vaccine Strain S2 from Cattle in China

In China, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep, cattle and swine. Brucella melitensis (biovars 1 and 3) is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1 and 3, and B. suis biovars 1 and 3 are also associated with sporadic human brucellosis. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by multi-locus variable-number tandem-repeat analysis (MLVA). Among the B. melitensis isolates, the majority (74/82) belonged to MLVA8 genotype 42, clustering in the ‘East Mediterranean’ group. Two B. melitensis biovar 1 genotype 47 isolates, belonging to the ‘Americas’ group, were recovered; both were from the Himalayan blue sheep (Pseudois nayaur, a wild animal). The majority of B. abortus isolates (51/70) were biovar 3, genotype 36. Ten B. suis biovar 1 field isolates, including seven outbreak isolates recovered from a cattle farm in Inner Mongolia, were genetically indistinguishable from the vaccine strain S2, based on MLVA cluster analysis. MLVA analysis provided important information for epidemiological trace-back. To the best of our knowledge, this is the first report to associate Brucella cross-infection with the vaccine strain S2 based on molecular comparison of recovered isolates to the vaccine strain. MLVA typing could be an essential assay to improve brucellosis surveillance and control programs.     

Global Positioning System Data-Loggers: A Tool to Quantify Fine-Scale Movement of Domestic Animals to Evaluate Potential for Zoonotic Transmission to an Endangered Wildlife Population

Domesticated animals are an important source of pathogens to endangered wildlife populations, especially when anthropogenic activities increase their overlap with humans and wildlife. Recent work in Tanzania reports the introduction of Cryptosporidium into wild chimpanzee populations and the increased risk of ape mortality associated with SIVcpz-Cryptosporidium co-infection. Here we describe the application of novel GPS technology to track the mobility of domesticated animals (27 goats, 2 sheep and 8 dogs) with the goal of identifying potential routes for Cryptosporidium introduction into Gombe National Park. Only goats (5/27) and sheep (2/2) were positive for Cryptosporidium. Analysis of GPS tracks indicated that a crop field frequented by both chimpanzees and domesticated animals was a potential hotspot for Cryptosporidium transmission. This study demonstrates the applicability of GPS data-loggers in studies of fine-scale mobility of animals and suggests that domesticated animal–wildlife overlap should be considered beyond protected boundaries for long-term conservation strategies.     

Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.     

Toxoplasma gondii Infection in Alpine Red Deer (Cervus elaphus): Its Spread and Effects on Fertility

In contrast to the depth of knowledge on the pathological effects of parasitism in domestic animals, the impact of the vast majority of parasites on wildlife hosts is poorly understood and, besides, information from domestics is rarely usable to disclose the parasites’ impact on free-ranging populations’ dynamics. Here we use Toxoplasmosis as a study model since, until now, the infection process and the protozoan’s effects in natural conditions has received little attention. We analysed 81 sera from red deer (Cervus elaphus) sampled in Italian Alps and through generalized linear models we evaluated (1) the epidemiological factors influencing T. gondii infection dynamics; (2) its impact on female fertility. High seroprevalence of T. gondii infection was recorded in yearling (1 year-old; prevalence = 52.4%) and adult (>2 year-old; prevalence = 51.3%) red deer, while calves (<1 year-old) did not contract the infection suggesting horizontal transmission as the main route of infection. The stable prevalence between yearlings and adults and the higher serological titres of younger individuals lead to two alternative infection processes suggesting a difference between age classes or in acquiring the infection or in responding to the pathogen. No associations between T. gondii serological titres and pregnancy status was observed indicating no direct effect on the probability of being pregnant; nevertheless a relation between females’ higher serological titres and lower foetal development emerged, suggesting potential effects of the parasite infection on deer reproduction. The results demonstrate high seroprevalence of T. gondii infection in free-ranging red deer and, furthermore, the effect on foetal development suggests the potential impact of the parasite on red deer fertility and thus on its population dynamics.     

Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts

The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT) for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT), and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents); Kenya (rodents and shrews); Mwogolo (rodents); Lora (rodents); Qunjian (rodent); serogroup Grippotyphosa (cattle); and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9% in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries.     

Serosurvey of Smooth Brucella,Leptospira spp. and Toxoplasma gondii in Free-Ranging Jaguars (Panthera onca) and Domestic Animals from Brazil

This study investigated the exposure of jaguar populations and domestic animals to smooth Brucella, Leptospira spp. and Toxoplasma gondii in the Cerrado, Pantanal and Amazon biomes of Brazil. Between February 2000 and January 2010, serum samples from 31 jaguars (Panthera onca), 1,245 cattle (Bos taurus), 168 domestic dogs (Canis lupus familiaris) and 29 domestic cats (Felis catus) were collected and analysed by rose bengal test for smooth Brucella, microscopic agglutination test for Leptospira spp. and modified agglutination test for T. gondii. Cattle populations from all sites (9.88%) were exposed to smooth Brucella, but only one jaguar from Cerrado was exposed to this agent. Jaguars captured in the Cerrado (60.0%) and in the Pantanal (45.5%) were seropositive for different serovars of Leptospira spp., cattle (72.18%) and domestic dogs (13.1%) from the three sites and one domestic cat from Pantanal were also seropositive for the agent. The most prevalent serotype of Leptospira spp. identified in jaguars from the Cerrado (Grippotyphosa) and the Pantanal (Pomona) biomes were distinct from those found in the domestic animals sampled. Jaguars (100%), domestic dogs (38.28%) and domestic cats (82.76%) from the three areas were exposed to T. gondii. Our results show that brucellosis and leptospirosis could have been transmitted to jaguars by domestic animals; and jaguars probably play an important role in the maintenance of T. gondii in nature.     

DNA Sequences from Formalin-Fixed Nematodes: Integrating Molecular and Morphological Approaches to Taxonomy

To effectively integrate DNA sequence analysis and classical nematode taxonomy, we must be able to obtain DNA sequences from formalin-fixed specimens. Microdissected sections of nematodes were removed from specimens fixed in formalin, using standard protocols and without destroying morphological features. The fixed sections provided sufficient template for multiple polymerase chain reaction-based DNA sequence analyses.