Combinations of host biomarkers predict mortality among Ugandan children with severe malaria: a retrospective case-control study

Background

Severe malaria is a leading cause of childhood mortality in Africa. However, at presentation, it is difficult to predict which children with severe malaria are at greatest risk of death. Dysregulated host inflammatory responses and endothelial activation play central roles in severe malaria pathogenesis. We hypothesized that biomarkers of these processes would accurately predict outcome among children with severe malaria.

Methodology/Findings

Plasma was obtained from children with uncomplicated malaria (n = 53), cerebral malaria (n = 44) and severe malarial anemia (n = 59) at time of presentation to hospital in Kampala, Uganda. Levels of angiopoietin-2, von Willebrand Factor (vWF), vWF propeptide, soluble P-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), soluble endoglin, soluble FMS-like tyrosine kinase-1 (Flt-1), soluble Tie-2, C-reactive protein, procalcitonin, 10 kDa interferon gamma-induced protein (IP-10), and soluble triggering receptor expressed on myeloid cells-1 (TREM-1) were determined by ELISA. Receiver operating characteristic (ROC) curve analysis was used to assess predictive accuracy of individual biomarkers. Six biomarkers (angiopoietin-2, soluble ICAM-1, soluble Flt-1, procalcitonin, IP-10, soluble TREM-1) discriminated well between children who survived severe malaria infection and those who subsequently died (area under ROC curve>0.7). Combinational approaches were applied in an attempt to improve accuracy. A biomarker score was developed based on dichotomization and summation of the six biomarkers, resulting in 95.7% (95% CI: 78.1–99.9) sensitivity and 88.8% (79.7–94.7) specificity for predicting death. Similar predictive accuracy was achieved with models comprised of 3 biomarkers. Classification tree analysis generated a 3-marker model with 100% sensitivity and 92.5% specificity (cross-validated misclassification rate: 15.4%, standard error 4.9%).

Conclusions

We identified novel host biomarkers of pediatric severe and fatal malaria (soluble TREM-1 and soluble Flt-1) and generated simple biomarker combinations that accurately predicted death in an African pediatric population. While requiring validation in further studies, these results suggest the utility of combinatorial biomarker strategies as prognostic tests for severe malaria.     

Seasonal and sex differences in the fission–fusion dynamics of spider monkeys (<Emphasis Type="Italic">Ateles geoffroyi yucatanensis</Emphasis>) in Belize

Spider monkeys (Ateles sp.) are characterized by high fission–fusion dynamics, meaning their social grouping pattern is fluid and consists of subgroups that vary in size, composition, and spatial cohesion over time. In this study, we quantify the fission–fusion dynamics of a group of spider monkeys at Runaway Creek Nature Reserve in Belize by measuring subgroup size, spatial cohesion, and stability using data spanning 5 years. We then test whether variation in these three subgroup measures differ according to season, subgroup sex composition, and the reproductive status of female subgroup members. Our results show that subgroups were larger in size and less stable in membership during the wet season compared to the dry season. All-female subgroups were less spatially cohesive but more stable in membership than all-male subgroups. Finally, we report that subgroups with one or more non-lactating females (i.e., without nursing young) were smaller on average than subgroups containing lactating females with nursing young. These data contribute to a growing body of research documenting the ecological and social dimensions along which grouping patterns might vary.     

Characterization of Francisella tularensis outer membrane proteins

Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs.     

Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA,EsxB, and EspA

Mycobacterium tuberculosis EsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (∼100 amino acids) and conserved WXG amino acid motif. M. tuberculosis contains 11 tandem pairs of WXG genes; each gene pair is thought to be coexpressed to form a heterodimer. The precise role of these proteins in the biology of M. tuberculosis is unknown, but several of the heterodimers are secreted, which is important for virulence. However, WXG proteins are not simply virulence factors, since nonpathogenic mycobacteria also express and secrete these proteins. Here we show that three WXG heterodimers have structures and properties similar to those of the M. tuberculosis EsxBA (MtbEsxBA) heterodimer, regardless of their host species and apparent biological function. Biophysical studies indicate that the WXG proteins from M. tuberculosis (EsxG and EsxH), Mycobacterium smegmatis (EsxA and EsxB), and Corynebacterium diphtheriae (EsxA and EsxB) are heterodimers and fold into a predominately α-helical structure. An in vivo protein-protein interaction assay was modified to identify proteins that interact specifically with the native WXG100 heterodimer. MtbEsxA and MtbEsxB were fused into a single polypeptide, MtbEsxBA, to create a biomimetic bait for the native heterodimer. The MtbEsxBA bait showed specific association with several esx-1-encoded proteins and EspA, a virulence protein secreted by ESX-1. The MtbEsxBA fusion peptide was also utilized to identify residues in both EsxA and EsxB that are important for establishing protein interactions with Rv3871 and EspA. Together, the results are consistent with a model in which WXG proteins perform similar biological roles in virulent and nonvirulent species.The WXG100 (WXG; pfam06013) proteins are a class of effector molecules found in gram-positive bacteria (26). WXG proteins are characterized by their small size (∼ 100 amino acids [aa]) and the presence of a WXG motif, or its structural equivalent, near the midpoint of their primary sequence (26). Bioinformatic analyses have shown that one WXG gene is frequently positioned near, or directly adjacent to, a second, related, WXG gene (14). The gene pairs characterized thus far encode proteins that associate to form 1:1 complexes (20, 31). The WXG proteins were once thought to be restricted to the mycobacteria, but homologues have now been detected in species of Bacillus, Listeria, Streptomyces, and Corynebacterium, among others, and the Pfam server lists >89 distinct WXG-encoding species and strains (10).The identification of WXG proteins encoded by the pathogens Mycobacterium tuberculosis (15, 17, 19, 36), Mycobacterium marinum (13), and Staphylococcus aureus (5) has created significant interest in the proteins'' biological activity. Nevertheless, these proteins are not a priori virulence factors (39), since organisms expressing WXG proteins are not necessarily capable of causing disease. In addition to pathogenesis, the WXG proteins are associated with processes as disparate as zinc homeostasis (24) and conjugal gene transfer (9, 11). A model for the mechanism(s) of action of these proteins that includes an explanation for their apparent functional versatility is at present lacking. One reason for this ambiguity may be the near-absence of studies comparing virulence-associated and non-virulence-associated WXG proteins, which is a goal of this study.The M. tuberculosis secreted virulence factors EsxA (also called ESAT-6, or Rv3875) and EsxB (CFP-10; Rv3874) are the founding members of the WXG family, and M. tuberculosis derivatives defective in EsxA and EsxB are attenuated (17, 19, 36). The results of biochemical and structural studies indicate that EsxA and EsxB form a tightly associated heterodimer, EsxAB (25, 30, 31). The M. tuberculosis genome contains 23 WXG genes, named esxA to esxW, and the majority of these are expressed as tandem pairs (26). Of the pairs, five, including esxA and esxB, are contained within larger, highly conserved genetic loci, called esx-1 to esx-5 (Fig. ​(Fig.1).1). These loci have been the focus of much research, since mutants of esx-1 are attenuated, and esx-3 and esx-5 are necessary for in vitro growth of M. tuberculosis and M. marinum (1, 2, 32-34). The esx loci are proposed to encode secretory apparatuses dedicated to the secretion of their cognate WXG proteins (1).Open in a separate windowFIG. 1.Genetic map of the esx-1 loci of M. tuberculosis and M. smegmatis. The M. tuberculosis esx-1 genes discussed in the text are indicated by white arrows, as are their M. smegmatis homologues. The M. tuberculosis map also shows the Rv3884 and Rv3885 genes, which are part of the adjacent esx-2 locus. pRD1-2F9 is the cosmid that was used to create an esx-1-specific prey library. pRD1-2F9 includes the Rv3860 to Rv3885 genes, thus encompassing the entire esx-1 locus and part of esx-2. The four genes below the M. smegmatis map include defective insertion sequences (ISs) inserted into MSMEG_0075.Although the majority of genes required for the secretion of the EsxAB heterodimer are encoded from within esx-1, additional non-esx-1 genes are necessary for secretion. In particular, one M. tuberculosis locus, esp, encodes three proteins essential for EsxAB secretion (12, 23). The first gene of the operon encodes a protein, EspA, that is cosecreted with EsxAB via the ESX-1 apparatus (12). Although no direct physical evidence has been presented, the inference from the interdependent cosecretion of the three proteins is that they likely form a complex, which is secreted by the ESX-1 apparatus. In this paper we provide the first genetic evidence that these three proteins interact.The lack of a genetic assay for the study of ESX-1 activity in M. tuberculosis has hindered the identification of all of the protein components of the apparatus and all of the substrates that it secretes. However, the fast-growing, nonpathogenic organism Mycobacterium smegmatis has a conserved esx-1 locus that is essential for DNA transfer, and we have exploited this requirement for genetic studies (9). These analyses have shown that the M. smegmatis ESX-1 apparatus is functionally related to that of M. tuberculosis (11) and that M. smegmatis encodes non-esx-1 genes necessary for the secretion of the EsxAB heterodimer, including orthologues of EspA (9).Here we have examined whether the secondary and quaternary structures of M. tuberculosis EsxA and EsxB are prototypical for other, functionally distinct and evolutionarily distant members of the WXG family (Fig. ​(Fig.2A).2A). Comparisons were made to homologues encoded by M. smegmatis (esxA and esxB), Corynebacterium diphtheriae (esxA and esxB), and an additional non-virulence-related pair from M. tuberculosis (esxG and esxH, encoded from the esx-3 locus). Structural characterization of these proteins establishes that their secondary and quaternary structures are conserved, with each pair folding into a predominately α-helical structure and associating to form a heterodimer. We next devised and tested the utility of a novel strategy to identify proteins that interact specifically with these WXG heterodimers. This involved fusing EsxB and EsxA to create a biomimetic heterodimer for use in mycobacterial two-hybrid experiments. We reasoned that the use of this unique bait would allow the detection of proteins that interact with both components of the native heterodimer and that these proteins would normally go undetected in the conventional, single-protein two-hybrid screens. Indeed, using this approach, we identified novel protein partners of M. tuberculosis EsxBA (MtbEsxBA). We show for the first time that EspA proteins from M. tuberculosis and M. smegmatis interact with the EsxBA heterodimer (from both species) but not with EsxA or EsxB alone. We also provide evidence for promiscuity between the different M. tuberculosis ESX apparatuses by showing that EsxBA, encoded by esx-1, can interact with Esx proteins encoded by esx-2. Taken together, our studies suggest that the WXG proteins possess similar structures and properties, regardless of the host species and the apparent biological function.Open in a separate windowFIG. 2.Sequence alignment of WXG proteins characterized in this study and the strategy used to facilitate their expression. (A) Amino acid sequence alignment of four pairs of WXG proteins. Conserved sequences are in boldface, and the signature WXG motif is indicated with asterisks. Three residues in Rv3874 (EsxB) and a single residue in Rv3875 (EsxA) are underlined; they are the sites of amino acid substitutions discussed in the text that abrogate Rv3871 interactions. (B) (Bottom) Scheme for coexpression of tandemly arranged WXG genes. (Top) The ribbon cartoon (30) shows how the two monomers are freed from the expressed fusion protein by thrombin cleavage (scissors) at the peptide tether (balls and sticks).     

The bacterial parasite Pasteuria ramosa is not killed if it fails to infect: implications for coevolution

Strong selection on parasites, as well as on hosts, is crucial for fueling coevolutionary dynamics. Selection will be especially strong if parasites that encounter resistant hosts are destroyed and diluted from the local environment. We tested whether spores of the bacterial parasite Pasteuria ramosa were passed through the gut (the route of infection) of their host, Daphnia magna, and whether passaged spores remained viable for a “second chance” at infecting a new host. In particular, we tested if this viability (estimated via infectivity) depended on host genotype, whether or not the genotype was susceptible, and on initial parasite dose. Our results show that Pasteuria spores generally remain viable after passage through both susceptible and resistant Daphnia. Furthermore, these spores remained infectious even after being frozen for several weeks. If parasites can get a second chance at infecting hosts in the wild, selection for infection success in the first instance will be reduced. This could also weaken reciprocal selection on hosts and slow the coevolutionary process.     

Population-based biochemistry, immunologic and hematological reference values for adolescents and young adults in a rural population in Western Kenya

Background

There is need for locally-derived age-specific clinical laboratory reference ranges of healthy Africans in sub-Saharan Africa. Reference values from North American and European populations are being used for African subjects despite previous studies showing significant differences. Our aim was to establish clinical laboratory reference values for African adolescents and young adults that can be used in clinical trials and for patient management.

Methods and Findings

A panel of 298, HIV-seronegative individuals aged 13–34 years was randomly selected from participants in two population-based cross-sectional surveys assessing HIV prevalence and other sexually transmitted infections in western Kenya. The adolescent (<18 years)-to-adults (≥18 years) ratio and the male-to-female ratio was 1∶1. Median and 95% reference ranges were calculated for immunohematological and biochemistry values. Compared with U.S-derived reference ranges, we detected lower hemoglobin (HB), hematocrit (HCT), red blood cells (RBC), mean corpuscular volume (MCV), neutrophil, glucose, and blood urea nitrogen values but elevated eosinophil and total bilirubin values. Significant gender variation was observed in hematological parameters in addition to T-bilirubin and creatinine indices in all age groups, AST in the younger and neutrophil, platelet and CD4 indices among the older age group. Age variation was also observed, mainly in hematological parameters among males. Applying U.S. NIH Division of AIDS (DAIDS) toxicity grading to our results, 40% of otherwise healthy study participants were classified as having an abnormal laboratory parameter (grade 1–4) which would exclude them from participating in clinical trials.

Conclusion

Hematological and biochemistry reference values from African population differ from those derived from a North American population, showing the need to develop region-specific reference values. Our data also show variations in hematological indices between adolescent and adult males which should be considered when developing reference ranges. This study provides the first locally-derived clinical laboratory reference ranges for adolescents and young adults in western Kenya.     

Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

TransMEMbrane 16A (TMEM16A) is a Ca²⁺-activated Cl⁻ channel that plays critical roles in regulating diverse physiologic processes, including vascular tone, sensory signal transduction, and mucosal secretion. In addition to Ca²⁺, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). However, the structural determinants mediating this interaction are not clear. Here, we interrogated the parts of the PI(4,5)P₂ head group that mediate its interaction with TMEM16A by using patch- and two-electrode voltage-clamp recordings on oocytes from the African clawed frog Xenopus laevis, which endogenously express TMEM16A channels. During continuous application of Ca²⁺ to excised inside–out patches, we found that TMEM16A-conducted currents decayed shortly after patch excision. Following this rundown, we show that the application of a synthetic PI(4,5)P₂ analog produced current recovery. Furthermore, inducible dephosphorylation of PI(4,5)P₂ reduces TMEM16A-conducted currents. Application of PIP₂ analogs with different phosphate orientations yielded distinct amounts of current recovery, and only lipids that include a phosphate at the 4′ position effectively recovered TMEM16A currents. Taken together, these findings improve our understanding of how PI(4,5)P₂ binds to and potentiates TMEM16A channels.     

Thrombospondin-1 expression and modulation of Wnt and hippo signaling pathways during the early phase of Trypanosoma cruzi infection of heart endothelial cells

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/β-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/β-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/β-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3β at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3β was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of β-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of β-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating β-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a β-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the β-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.