Revisiting the Central Metabolism of the Bloodstream Forms of Trypanosoma brucei: Production of Acetate in the Mitochondrion Is Essential for Parasite Viability

Background

The bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei.

Methodology/Principal Findings

A comparative NMR analysis showed that the bloodstream long-slender and procyclic trypanosomes excreted equivalent amounts of acetate and succinate from glucose metabolism. Key enzymes of acetate production from glucose-derived pyruvate and threonine are expressed in the mitochondrion of the long-slender forms, which produces 1.4-times more acetate from glucose than from threonine in the presence of an equal amount of both carbon sources. By using a combination of reverse genetics and NMR analyses, we showed that mitochondrial production of acetate is essential for the long-slender forms, since blocking of acetate biosynthesis from both carbon sources induces cell death. This was confirmed in the absence of threonine by the lethal phenotype of RNAi-mediated depletion of the pyruvate dehydrogenase, which is involved in glucose-derived acetate production. In addition, we showed that de novo fatty acid biosynthesis from acetate is essential for this parasite, as demonstrated by a lethal phenotype and metabolic analyses of RNAi-mediated depletion of acetyl-CoA synthetase, catalyzing the first cytosolic step of this pathway.

Conclusions/Significance

Acetate produced in the mitochondrion from glucose and threonine is synthetically essential for the long-slender mammalian forms of T. brucei to feed the essential fatty acid biosynthesis through the “acetate shuttle” that was recently described in the procyclic insect form of the parasite. Consequently, key enzymatic steps of this pathway, particularly acetyl-CoA synthetase, constitute new attractive drug targets against trypanosomiasis.     

Whole Genome Sequencing of Field Isolates Reveals a Common Duplication of the Duffy Binding Protein Gene in Malagasy Plasmodium vivax Strains

Background

Plasmodium vivax is the most prevalent human malaria parasite, causing serious public health problems in malaria-endemic countries. Until recently the Duffy-negative blood group phenotype was considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human erythrocytes.

Methods/Principal Findings

Through recent whole genome sequencing we obtained ≥70× coverage of the P. vivax genome from five field-isolates, resulting in ≥93% of the Sal I reference sequenced at coverage greater than 20×. Combined with sequences from one additional Malagasy field isolate and from five monkey-adapted strains, we describe here identification of DNA sequence rearrangements in the P. vivax genome, including discovery of a duplication of the P. vivax Duffy binding protein (PvDBP) gene. A survey of Malagasy patients infected with P. vivax showed that the PvDBP duplication was present in numerous locations in Madagascar and found in over 50% of infected patients evaluated. Extended geographic surveys showed that the PvDBP duplication was detected frequently in vivax patients living in East Africa and in some residents of non-African P. vivax-endemic countries. Additionally, the PvDBP duplication was observed in travelers seeking treatment of vivax malaria upon returning home. PvDBP duplication prevalence was highest in west-central Madagascar sites where the highest frequencies of P. vivax-infected, Duffy-negative people were reported.

Conclusions/Significance

The highly conserved nature of the sequence involved in the PvDBP duplication suggests that it has occurred in a recent evolutionary time frame. These data suggest that PvDBP, a merozoite surface protein involved in red cell adhesion is rapidly evolving, possibly in response to constraints imposed by erythrocyte Duffy negativity in some human populations.     

Evidence of Habitat Structuring Aedes albopictus Populations in Réunion Island

Arbovirus vector dynamics and spread are influenced by climatic, environmental and geographic factors. Major Chikungunya and Dengue fever outbreaks occurring the last 10 years have coincided with the expansion of the mosquito vector Aedes albopictus to nearly all the continents. We characterized the ecological (larval development sites, population dynamics, insemination and daily survival rates) and genetic (diversity, gene flow, population structure) features of two Aedes albopictus populations from distinct environments (rural and urban) on Réunion Island, in the South-West Indian Ocean. Microsatellite analysis suggests population sub-structuring Ae. albopictus populations. Two genetic clusters were identified that were significantly linked to natural versus urban habitats with a mixed population in both areas. Ae. albopictus individuals prefer urban areas for mating and immature development, where hosts and containers that serve as larval development sites are readily available and support high population densities, whereas natural environments appear to serve as reservoirs for the mosquito.     

Sensitivity and Specificity of a New Vertical Flow Rapid Diagnostic Test for the Serodiagnosis of Human Leptospirosis

Background: Leptospirosis is a growing public health concern in many tropical and subtropical countries. However, its diagnosis is difficult because of non-specific symptoms and concurrent other endemic febrile diseases. In many regions, the laboratory diagnosis is not available due to a lack of preparedness and simple diagnostic assay or difficult access to reference laboratories. Yet, an early antibiotic treatment is decisive to the outcome. The need for Rapid Diagnostic Tests (RDTs) for bedside diagnosis of leptospirosis has been recognized. We developed a vertical flow immunochromatography strip RDT detecting anti-Leptospira human IgM and evaluated it in patients from New Caledonia, France, and French West Indies. Methodology/Principal Findings: Whole killed Leptospira fainei cells were used as antigen for the test line and purified human IgM as the control line. The mobile phase was made of gold particles conjugated with goat anti-human IgM. Standards for Reporting of Diagnostic Accuracy criteria were used to assess the performance of this RDT. The Microscopic Agglutination Test (MAT) was used as the gold standard with a cut-off titer of ≥400. The sensitivity was 89.8% and the specificity 93.7%. Positive and negative Likelihood Ratios of 14.18 and 0.108 respectively, and a Diagnostic Odds Ratio of 130.737 confirmed its usefulness. This RDT had satisfactory reproducibility, repeatability, thermal tolerance and shelf-life. The comparison with MAT evidenced the earliness of the RDT to detect seroconversion. When compared with other RDT, the Vertical Flow RDT developed displayed good diagnostic performances.

Conclusions/Significance

This RDT might be used as a point of care diagnostic tool in limited resources countries. An evaluation in field conditions and in other epidemiological contexts should be considered to assess its validity over a wider range of serogroups or when facing different endemic pathogens. It might prove useful in endemic contexts or outbreak situations.     

Overview of the Oldest Existing Set of Substrate-optimized Anaerobic Processes: Digestive Tracts

Over millions of years, living organisms have explored and optimized the digestion of a wide variety of substrates. Engineers who develop anaerobic digestion processes for waste treatment and energy production can learn much from this accumulated ‘experience’. The aim of this work is a survey based on the comparison of 190 digestive tracts (vertebrate and insect) considered as ‘reactors’ and their anaerobic processes. Within a digestive tract, each organ is modeled as a type of reactor (continuous stirred-tank, such reactors in series, plug-flow or batch) associated with chemical aspects such as pH or enzymes. Based on this analysis, each complete digestion process has been rebuilt and classified in accordance with basic structures which take into account the relative size of the different reactors. The results show that all animal digestive structures can be grouped within four basic types. Size and/or position in the structure of the different reactors (pre/post treatment and anaerobic microbial digestion) are closely correlated to the degradability of the feed (substrate). Major common features are: (i) grinding, (ii) an extreme pH compartment, and (iii) correlation between the size of the microbial compartment and the degradability of the feed. Thus, shared answers found by animals during their evolution can be a source of inspiration for engineers in designing optimal anaerobic processes.     

Non-contiguous finished genome sequence and description of Bartonella senegalensis sp. nov.

Bartonella senegalensis sp. nov. strain OS02^T is the type strain of B. senegalensis sp. nov., a new species within the genus Bartonella. This strain, whose genome is described here, was isolated in Senegal from the soft tick Ornithodoros sonrai, the vector of relapsing fever. B. senegalensis is an aerobic, rod-shaped, Gram-negative bacterium. Here we describe the features of this organism, together with the complete genome sequence and its annotation. The 1,966,996 bp-long genome contains 1,710 protein-coding and 46 RNA genes, including 6 rRNA genes.     

Non-contiguous genome sequence of Mycobacterium simiae strain DSM 44165T

Mycobacterium simiae is a non-tuberculosis mycobacterium causing pulmonary infections in both immunocompetent and imunocompromized patients. We announce the draft genome sequence of M. simiae DSM 44165^T. The 5,782,968-bp long genome with 65.15% GC content (one chromosome, no plasmid) contains 5,727 open reading frames (33% with unknown function and 11 ORFs sizing more than 5000 -bp), three rRNA operons, 52 tRNA, one 66-bp tmRNA matching with tmRNA tags from Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium marinum, and Mycobacterium africanum and 389 DNA repetitive sequences. Comparing ORFs and size distribution between M. simiae and five other Mycobacterium species M. simiae clustered with M. abscessus and M. smegmatis. A 40-kb prophage was predicted in addition to two prophage-like elements, 7-kb and 18-kb in size, but no mycobacteriophage was seen after the observation of 10⁶ M. simiae cells. Fifteen putative CRISPRs were found. Three genes were predicted to encode resistance to aminoglycosides, betalactams and macrolide-lincosamide-streptogramin B. A total of 163 CAZYmes were annotated. M. simiae contains ESX-1 to ESX-5 genes encoding for a type-VII secretion system. Availability of the genome sequence may help depict the unique properties of this environmental, opportunistic pathogen.     

Non-contiguous finished genome sequence and description of Kallipyga massiliensis gen. nov., sp. nov., a new member of the family Clostridiales Incertae Sedis XI

Kallipyga massiliensis strain ph2^T is the type strain of Kallipyga massiliensis gen. nov., sp. nov., the type species of the new genus Kallipyga within the family Clostridiales Incertae Sedis XI. This strain, whose genome is described here, was isolated from the fecal flora of a 26-year-old woman suffering from morbid obesity. K. massiliensis is an obligate anaerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,770,679 bp long genome (1 chromosome but no plasmid) contains 1,575 protein-coding and 50 RNA genes, including 4 rRNA genes.     

Volumetric properties underlying ligand binding in a monomeric hemoglobin: A high-pressure NMR study

The 2/2 hemoglobin of the cyanobacterium Synechococcus sp. PCC 7002, GlbN, coordinates the heme iron with two histidines and exists either with a b heme or with a covalently attached heme. The binding of exogenous ligands displaces the distal histidine and induces a conformational rearrangement involving the reorganization of internal void volumes. The formation of passageways within the resulting conformation is thought to facilitate ligand exchange and play a functional role. Here we monitored the perturbation induced by pressure on the ferric bis-histidine and cyanide-bound states of GlbN using ¹H–¹⁵N HSQC NMR spectroscopy. We inspected the outcome with a statistical analysis of 170 homologous 2/2 hemoglobin sequences. We found that the compression landscape of GlbN, as represented by the variation of an average chemical shift parameter, was highly sensitive to ligand swapping and heme covalent attachment. Stabilization of rare conformers was observed at high pressures and consistent with cavity redistribution upon ligand binding. In all states, the EF loop was found to be exceptionally labile to pressure, suggesting a functional role as a semi-flexible hinge between the adjacent helices. Finally, coevolved clusters presented a common pattern of compensating pressure responses. The high-pressure dissection combined with protein sequence analysis established locations with volumetric signatures relevant to residual communication of 2/2 hemoglobins. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Palifermin for the protection and regeneration of epithelial tissues following injury: new findings in basic research and pre‐clinical models

Keratinocyte growth factor (KGF) is a paracrine‐acting epithelial mitogen produced by cells of mesenchymal origin, that plays an important role in protecting and repairing epithelial tissues. Pre‐clinical data initially demonstrated that a recombinant truncated KGF (palifermin) could reduce gastrointestinal injury and mortality resulting from a variety of toxic exposures. Furthermore, the use of palifermin in patients with hematological malignancies reduced the incidence and duration of severe oral mucositis experienced after intensive chemoradiotherapy. Based upon these findings, as well as the observation that KGF receptors are expressed in many, if not all, epithelial tissues, pre‐clinical studies have been conducted to determine the efficacy of palifermin in protecting different epithelial tissues from toxic injury in an attempt to model various clinical situations in which it might prove to be of benefit in limiting tissue damage. In this article, we review these studies to provide the pre‐clinical background for clinical trials that are described in the accompanying article and the rationale for additional clinical applications of palifermin.