Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains

Background

Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity.

Methodology and Findings

We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer.

Conclusions

These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers {"type":"entrez-nucleotide","attrs":{"text":"CP001809","term_id":"340539261","term_text":"CP001809"}}CP001809 and {"type":"entrez-nucleotide","attrs":{"text":"CP001829","term_id":"302205157","term_text":"CP001829"}}CP001829.     

Geographical Information Systems risk assessment models for zoonotic fascioliasis in the South American Andes region

The WHO recognises Fasciola hepatica to be an important human health problem. The Andean countries of Peru, Bolivia and Chile are those most severely affected by this distomatosis, though areas of Ecuador, Colombia and Venezuela are also affected. As part of a multidisciplinary project, we present results of use of a Geographical Information Systems (GIS) forecast model to conduct an epidemiological analysis of human and animal fasciolosis in the central part of the Andes mountains. The GIS approach enabled us to develop a spatial and temporal epidemiological model to map the disease in the areas studied and to classify transmission risk into low, moderate and high risk areas so that areas requiring the implementation of control activities can be identified. Current results are available on a local scale for: (1) the northern Bolivian Altiplano, (2) Puno in the Peruvian Altiplano, (3) the Cajamarca and Mantaro Peruvian valleys, and (4) the Ecuadorian provinces of Azuay, Cotopaxi and Imbabura. Analysis of results demonstrated the validity of a forecast model that combines use of climatic data to calculate of forecast indices with remote sensing data, through the classification of Normalized Difference Vegetation Index (NDVI) maps.     

PI3Kgamma inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease generated by deregulation of T cell-mediated B-cell activation, which results in glomerulonephritis and renal failure. Disease is treated with immunosuppressants and cytostatic agents that have numerous side effects. Here we examine the use of inhibitors of phosphoinositide 3-kinase (PI3K) gamma, a lipid kinase that regulates inflammation, in the MRL-lpr mouse model of SLE. Treatment reduced glomerulonephritis and prolonged lifespan, suggesting that P13Kgamma may be a useful target in the treatment of chronic inflammation.     

Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis

Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kgamma (encoded by Pik3cg). We show that Pik3cg(-/-) mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kgamma as a therapeutic target. We also describe that oral treatment with a PI3Kgamma inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg(-/-) mice. Our results identify selective PI3Kgamma inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.     

Dynamic shear stress in parallel-plate flow chambers

An in vitro model using a parallel-plate fluid flow chamber is supposed to simulate in vivo fluid shear stresses on various cell types exposed to dynamic fluid flow in their physiological environment. The metabolic response of cells in vitro is associated with the wall shear stress. However, parallel-plate flow chambers have not been characterized for dynamic fluid flow experiments. We use a dimensionless ratio h / lambda(v), in determining the exact magnitude of the dynamic wall shear stress, with its oscillating components scaled by a shear factor T. It is shown that, in order to expose cells to predictable levels of dynamic fluid shear stress, two conditions have to be met: (1) h / lambda(v) < 2, where h is the distance between the plates and lambda(v) is the viscous penetration depth; and (2) f(0) < f(c) / m, where the critical frequency f(c) is the upper threshold for this flow regime, m is the highest harmonic mode of the flow, and f(0) is the fundamental frequency of fluid flow.     

The DRY motif as a molecular switch of the human oxytocin receptor

The human oxytocin receptor is known to exhibit promiscuous activity by coupling to both Galpha(q) and Galpha(i) G proteins to activate distinct signaling pathways. A single-amino acid substitution within the highly conserved E/DRY motif at the cytosolic extension of helix 3 [i.e., D136(3.49)N] increased the rate of both basal and agonist-stimulated inositol phosphate (IP(3)) accumulation of the receptor. Furthermore, like for a typical constitutively active receptor, the partial agonist arginine vasopressin behaved as a full agonist for the D136(3.49)N mutant. Subsequently, both oxytocin and arginine vasopressin showed an increased potency in stimulating IP3 accumulation as compared to the wild-type receptor. Very interestingly, our experiments provide strong evidence that the D136(3.49)N mutant inhibits receptor signaling via Galpha(i)-mediated pathways while increasing the activity through the Galpha(q)-mediated pathways. Molecular simulations of the free and OT-bound forms of wild-type OTR and of the D136(3.49)N constitutively active mutant suggest that the receptor portions close to the E/DRY and NPxxY motifs are particularly susceptible to undergoing structural modification in response to activating mutations and agonist binding. Furthermore, computational modeling suggests that the OT-bound form of wild-type OTR is able to explore more states than the OT-bound form of the D136(3.49)N constitutively active mutant, consistent with its G protein promiscuity. Taken together, these observations emphasize the important role of the E/DRY motif not only in receptor activation but also in the promiscuity of G protein coupling. Knowledge of the mechanism of selective G protein coupling could aid drug discovery efforts to identify signaling specific therapies.     

Involvement of phosphoinositide 3-kinase gamma, Rac, and PAK signaling in chemokine-induced macrophage migration

In macrophages, chemotactic stimuli cause the activation of Rac and PAK, but little is known about the signaling pathways involved and their role in chemotactic gradient sensing. Herein, we report that in macrophages, the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 activates the small GTPase Rac and its downstream target PAK2 within seconds. This response depends on Gi activation and largely on the subsequent triggering of phosphoinositide 3-kinase gamma (PI3Kgamma) and Rac. Retroviral transduction of tagged Rac1 and -2 indicates that RANTES/CCL5-mediated activation of PI3Kgamma triggers Rac1 but not Rac2. In agreement, silencing of Rac1 by shRNA blocks PAK2 activity and inhibits RANTES/CCL5-induced macrophage polarization and directional migration. On the other hand, the tyrosine kinase receptor agonist CSF-1 activates PAK2 independently of PI3Kgamma and Rac. Our results thus demonstrate a chemokine-specific signaling pathway in which Gi and PI3Kgamma coordinate to drive Rac1 and PAK2 activation that eventually controls the chemotactic response.     

Precise excision of the large pathogenicity island, SPI7, in Salmonella enterica serovar Typhi

The large pathogenicity island (SPI7) of Salmonella enterica serovar Typhi is a 133,477-bp segment of DNA flanked by two 52-bp direct repeats overlapping the pheU (phenylalanyl-tRNA) gene, contains 151 potential open reading frames, and includes the viaB operon involved in the synthesis of Vi antigen. Some clinical isolates of S. enterica serovar Typhi are missing the entire SPI7, due to its precise excision; these strains have lost the ability to produce Vi antigen, are resistant to phage Vi-II, and invade a human epithelial cell line more rapidly. Excision of SPI7 occurs spontaneously in a clinical isolate of S. enterica serovar Typhi when it is grown in the laboratory, leaves an intact copy of the pheU gene at its novel join point, and results in the same three phenotypic consequences. SPI7 is an unstable genetic element, probably an intermediate in the pathway of lateral transfer of such pathogenicity islands among enteric gram-negative bacteria.     

The helminth community of the wood mouse, Apodemus sylvaticus, in the Sierra Espuna, Murcia, Spain

The helminth community of the wood mouse, Apodemus sylvaticus, in the Sierra Espuna was characterized after a complete analysis of its helminth community component and infracommunity structure relative to host age, sex and year of capture. The helminth community comprised 13 species: one trematode, four cestodes and eight nematodes. The cestode Pseudocatenotaenia matovi and the nematode Syphacia frederici were the most prevalent and abundant helminth species, respectively. Sixty four percent of mice analysed presented helminths with a direct cycle and 42% presented helminths with an indirect cycle. The helminth community presents a low diversity with infracommunities usually made up of only one or two helminth species. Host age and year of capture seem to play a major role in determining species richness and helminth diversity, but not in determining the abundance of helminths. Host sex does not seem to affect the infection rate nor the diversity. Further studies on more samples of wood mice and other small mammal species in this regional park are needed to explore any possible interactions between helminth communities in the host populations.