Innate immunomodulation to trypanosomatid parasite infections.

The recognition of invading pathogens by the innate immune system is essential for host protection against human parasites and the initiation of an effective adaptive immune response. Innate immune cells such as macrophages and dendritic cells (DCs) are involved in the first line of defense against protozoan parasites via sensing the invaders through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs). Activation of macrophages and dendritic cells starts with the interaction between microbial ligands (pathogen-associated molecular patterns - PAMPs) and PRRs, and these activated cells influence the overall immune response. Trypanosomatid PAMPs are sensed by TLRs; for example, TLR2 recognizes alkylacylglycerol and lipophosphoglycan in Trypanosoma cruzi and Leishmania, respectively; TLR2/TLR4 recognize glycoisnositolphospholipids and glycosylphosphatidyl inositol in Trypanosoma species; and TLR9 recognizes genomic DNA in Trypanosoma. TLR signaling includes the recruitment of different adaptor molecules that activate various transcription factors, such as NF-kB, IRF3/7, and MAP kinases, to induce the production of pro-inflammatory cytokines and type I interferons. Moreover, activated macrophages and dendritic cells produce ROS and NOS, which limit pathogen survival, and large amounts of cytokines; additionally, antigen presentation enhances the adaptive immune response. In this review, we highlight the recent findings on PAMP recognition in trypanosomatid infections and the signaling pathways activated by PRRs.

Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K(+) cycling and to active Na(+) extrusion.

BACKGROUND: Orthophosphate (Pi) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of Pi acquisition by Trypanosoma cruzi. METHODS: (32)Pi influx was measured in T. cruzi epimastigotes. The expression of Pi transporter genes and the coupling of the uptake to Na(+), H(+) and K(+) fluxes were also investigated. The transport capacities of different evolutive forms were compared. RESULTS: Epimastigotes grew significantly more slowly in 2mM than in 50mM Pi. Influx of Pi into parasites grown under low Pi conditions took place in the absence and presence of Na(+). We found that the parasites express TcPho84, a H(+):Pi-symporter, and TcPho89, a Na(+):Pi-symporter. Both Pi influx mechanisms showed Michaelis-Menten kinetics, with a one-order of magnitude higher affinity for the Na(+)-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of Pi. Valinomycin (K(+) ionophore) or SCH28028 (inhibitor of (H(+)+K(+))ATPase) significantly inhibited Pi uptake, indicating that an inwardly-directed H(+) gradient energizes uphill Pi entry and that K(+) recycling plays a key role in Pi influx. Furosemide, an inhibitor of the ouabain-insensitive Na(+)-ATPase, decreased only the Na(+)-dependent Pi uptake, indicating that this Na(+) pump generates the Na(+) gradient utilized by the symporter. Trypomastigote forms take up Pi inefficiently. CONCLUSIONS: Pi starvation stimulates membrane potential-sensitive Pi uptake through different pathways coupled to Na(+) or H(+)/K(+) fluxes. GENERAL SIGNIFICANCE: This study unravels the mechanisms of Pi acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.

Na+-dependent and Na+-independent mechanisms for inorganic phosphate uptake in Trypanosoma rangeli.

BACKGROUND: Trypanosoma rangeli is dependent on the presence of exogenous orthophosphate (Pi) for maximal growth and ecto-phosphatase activity is responsible for Pi supply under low Pi. Here we investigated the mechanisms of Pi uptake. METHODS: We investigated the kinetics of 32Pi transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:Pi cotransporter and Na+-ATPase. RESULTS: T. rangeli grown under limiting Pi transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a Pi transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of 32Pi influx by 80-95%. The H+-ATPase inhibitor, bafilomycin A1, inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of 32Pi to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high Pi had a lower Pi influx than that found in those grown in low Pi, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by Pi starvation. CONCLUSIONS: Two modes of Pi transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for Pi acquisition during development of T. rangeli. GENERAL SIGNIFICANCE: This study provides the first description of the mechanism of Pi transport across the plasma membrane of trypanosomatids.