Characterization of the Temporal Pattern of Blood Protein Digestion in Rhodnius prolixus: First Description of Early and Late Gut Cathepsins

Rhodnius prolixus is one important vector for the parasite Trypanosoma cruzi in Latin America, where Chagas disease is a significant health issue. Although R. prolixus is a model for investigations of vector–parasite interaction and transmission, not much has been done recently to further comprehend its protein digestion. In this work, gut proteolysis was characterized using new fluorogenic substrates, including optimum pH, inhibition profiles, and tissue and temporal expression patterns. Each protease possessed a particular tissue prevalence and activity cycle after feeding. Cathepsin L had a higher activity in the posterior midgut lumen, being characterized by a plateau of high activities during several days in the intermediate phase of digestion. Cathepsin D showed high activity levels in the tissue homogenates and in the luminal content of the posterior midgut, with a single peak 5 days after blood feeding. Aminopeptidases are highly associated with the midgut wall, where the highest activity is located. Assays with proteinaceous substrates as casein, hemoglobin, and serum albumin revealed different activity profiles, with some evidence of biphasic temporal proteolytic patterns. Cathepsin D genes are preferentially expressed in the anterior midgut, while cathepsin L genes are mainly located in the posterior portion of the midgut, with specific sets of genes being differently expressed in the initial, intermediate, or late phases of blood digestion.Significance StatementThis is the first description in a non-dipteran hematophagous species of a sequential protease secretion system based on midgut cathepsins instead of the most common insect digestive serine proteases (trypsins and chymotrypsins). The midgut of R. prolixus (Hemiptera) shows a different temporal expression of proteases in the initial, intermediate, and late stages of blood digestion. In this respect, a different timing in protease secretion may be an example of adaptative convergence in blood-sucking vectors from different orders. Expanding the knowledge about gut physiology in triatomine vectors may contribute to the development of new control strategies, aiming the blocking of parasite transmission.

Mechano-Chemo Signaling Interactions Modulate Matrix Production by Cardiac Fibroblasts

Extracellular matrix remodeling after myocardial infarction occurs in a dynamic environment in which local mechanical stresses and biochemical signaling species stimulate the accumulation of collagen-rich scar tissue. It is well-known that cardiac fibroblasts regulate post-infarction matrix turnover by secreting matrix proteins, proteases, and protease inhibitors in response to both biochemical stimuli and mechanical stretch, but how these stimuli act together to dictate cellular responses is still unclear. We developed a screen of cardiac fibroblast-secreted proteins in response to combinations of biochemical agonists and cyclic uniaxial stretch in order to elucidate the relationships between stretch, biochemical signaling, and cardiac matrix turnover. We found that stretch significantly synergized with biochemical agonists to inhibit the secretion of matrix metalloproteinases, with stretch either amplifying protease suppression by individual agonists or antagonizing agonist-driven upregulation of protease expression. Stretch also modulated fibroblast sensitivity towards biochemical agonists by either sensitizing cells towards agonists that suppress protease secretion or de-sensitizing cells towards agonists that upregulate protease secretion. These findings suggest that the mechanical environment can significantly alter fibrosis-related signaling in cardiac fibroblasts, suggesting caution when extrapolating in vitro data to predict effects of fibrosis-related cytokines in situations like myocardial infarction where mechanical stretch occurs.

Hypermutation as an Evolutionary Mechanism for Achromobacter xylosoxidans in Cystic Fibrosis Lung Infection

Achromobacter xylosoxidans can cause chronic infections in the lungs of patients with cystic fibrosis (CF) by adapting to the specific environment. The study of longitudinal isolates allows to investigate its within-host evolution to unravel the adaptive mechanisms contributing to successful colonization. In this study, four clinical isolates longitudinally collected from two chronically infected patients underwent whole genome sequencing, de novo assembly and sequence analysis. Phenotypic assays were also performed. The isolates coming from one of the patients (patient A) presented a greater number of genetic variants, diverse integrative and conjugative elements, and different protease secretion. In the first of these isolates (strain A1), we also found a large deletion in the mutS gene, involved in DNA mismatch repair (MMR). In contrast, isolates from patient B showed a lower number of variants, only one integrative and mobilizable element, no phenotypic changes, and no mutations in the MMR system. These results suggest that in the two patients the establishment of a chronic infection was mediated by different adaptive mechanisms. While the strains isolated from patient B showed a longitudinal microevolution, strain A1 can be clearly classified as a hypermutator, confirming the occurrence and importance of this adaptive mechanism in A. xylosoxidans infection.

Endocytosis and Digestion in Carnivorous Pitcher Plants of the Family Sarraceniaceae

Highly evolved carnivorous plants secrete digestive enzymes for degradation of trapped animals and absorb whole macromolecules from their prey by means of endocytosis. (1) Background: In the pitcher-plant family Sarraceniaceae, the production of enzymes is dubious and no evidence for endocytosis is known so far. (2) Methods: Heliamphora nutans, Darlingtonia californica, and nine taxa of Sarracenia are tested for cuticular pores, and for protease and endocytosis of the fluorescent protein analogue FITC-BSA, after 10–48 h of stimulation. (3) Results: Cuticular pores as a prerequisite for enzyme secretion and nutrient uptake are present in all tested species. Permeable cells form clusters in the inner epidermis of the pitchers, but are only little differentiated from impermeable epidermis cells. Proteases are found in S. psittacina, S. leucophylla, S. minor, S. oreophila, S. alabamensis, H. nutans, D. californica lacking only in S. flava and in S. purpurea ssp. purpurea, S. purpurea ssp. venosa, S. rosea, where enzyme production is possibly replaced by degradation via the extraordinary diverse inquiline fauna. S. leucophylla, S. minor, S. oreophila exhibit both protease production and endocytosis; S. psittacina, S. alabamensis, H. nutans, D. californica produce proteases only; no single species shows endocytosis without protease production. (4) Conclusions: Protease secretion seems to be a prerequisite for endocytotic nutrient uptake. Transport of FITC-BSA absorbed by endocytosis towards the vascular tissue of the trap leaves suggests that endocytosis of nutrients is more than a side effect of enzyme secretion.