Changes in ribosome function induced by protein kinase associated with ribosomes of Streptomyces collinus producing kirromycin.

Protein kinase associated with ribosomes of streptomycetes phosphorylates 11 ribosomal proteins. Phosphorylation activity of protein kinase reaches its maximum at the end of exponential phase of growth. When (32)P-labeled cells from the end of exponential phase of growth were transferred to a fresh medium, after 2 h of cultivation ribosomal proteins lost more than 90% of (32)P and rate of polypeptide synthesis increases twice. Protein kinase cross-reacting with antibody raised against protein kinase C was partially purified from 1 M NH(4)Cl wash of ribosomes and used to phosphorylation of ribosomes. Phosphorylation of 50S subunits (L2, L3, L7, L16, L21, L23, and L27) had no effect on the integrity of subunits but affects association with 30 to 70S monosomes. In vitro system derived from ribosomal subunits was used to examine the activity of phosphorylated 50S at poly(U) translation. Replacement unphosphorylated 50S with 50S possessed of phosphorylated r-proteins leads to the reduction of polypeptide synthesis of about 52%. The binding of N-Ac[(14)C]Phe-tRNA to A-site of phosphorylated ribosomes is not affected but the rate of peptidyl transferase is more than twice lower than that in unphosphorylated ribosomes. These results provide evidence that phosphorylation of ribosomal proteins is involved in mechanisms regulating the translational system of Streptomyces collinus.

Iron enhancement of experimental infection of mice by Tritrichomonas foetus.

The ability of a microbial invader to acquire iron from its vertebrate host has been recognized as an important virulence mechanism in some pathogenic bacteria. We examined the involvement of similar mechanisms in an experimental infection of mice by a protozoan pathogen of cattle, Tritrichomonas foetus. In a series of experiments, outbred ICR mice were inoculated intraperitoneally with two strains of T. foetus, the moderately virulent KV-1 (approximately 5% mortality rate) and the highly virulent LUB-1MIP (approximately 80% mortality rate). Treatment of mice with ferric ammonium citrate (FeAC) (100 mg/kg per day intraperitoneally) increased the mortality rate caused by the KV-1 infection up to the level determined for the highly virulent strain. The treatment effect was dose dependent and required early administration of FeAC after inoculation of parasites and its continued supply for at least 3 subsequent days. Daily sampling of peritoneal exudate showed that the infection-enhancing effect of iron overload was associated with a stimulation of parasite multiplication, which in the case of KV-1 infection was strongly suppressed in untreated mice. Consistent with these findings, the strain of lower virulence (KV-1) showed considerably lower efficiency accumulating radiolabeled iron from transferrin and a low-molecular source [Fe(III)nitrilotriacetic acid] in vitro. The results indicate an involvement of iron uptake mechanisms by the parasite as a virulence factor in T. foetus infection.

The host-protein-independent iron uptake by Tritrichomonas foetus.

Iron uptake from a low-molecular-weight chelate Fe(III)-nitriloacetate (Fe-NTA) by anaerobic protozoan parasite Tritrichomonas foetus was investigated and compared with that from iron-saturated lactoferrin and transferrin. The results showed that the iron uptake from Fe-NTA was saturable (Km = 2.7 microM, Vmax = 21.7 fmol. microg-1.min-1) and time, and temperature dependent, thus suggesting involvement of a membrane transport carrier. The accumulation of iron from 59Fe-NTA was inhibited by NaF and iron chelators. Amilorid and inhibitors of endosome acidification did not influence the process. Ascorbate stimulated the uptake while a membrane impermeable chelator of bivalent iron (bathophenanthroline disulfonic acid) was inhibitory, suggesting that prior to transport iron is reduced extracellularly. In accord with this assumption, the reduction of ferric to ferrous iron in the presence of intact T. foetus cells was demonstrated. Dynamics and properties of uptake of iron released from transferrin were similar to those from Fe-NTA, indicating involvement of common mechanisms. Iron uptake from lactoferrin displayed profoundly different characteristics consistent with receptor-mediated endocytosis. Metronidazole-resistant derivative of the investigated T. foetus strain showed marked deficiency in iron acquisition from Fe-NTA and transferrin while its iron uptake from lactoferrin was higher than that of the parent strain. The results presented show that T. foetus possesses at least two independent mechanisms that mediate acquisition of iron.

Tritrichomonas foetus: iron acquisition from lactoferrin and transferrin.

Acquisition of iron from lactoferrin and transferrin by a parasitic protozoon Tritrichomonas foetus has been studied in vitro. Specific, time-dependent, and saturable binding of iodinated ligands to the outer membrane of T. foetus at 4 degrees C was demonstrated for 125I-labeled lactoferrin only. About 1.7 x 10(5) binding sites of a single class with Kd approximately equal to 3.6 microM was estimated by means of Scatchard analysis. Internalization of the bound lactoferrin was observed at 37 degrees C. The cell-associated radioactivity after 30 min incubation of the parasite with 125I-lactoferrin at 37 degrees C was about 3.5-fold higher than the amount bound at 4 degrees C. The majority of internalized 125I-lactoferrin was released within 15 min of cell reincubation at 37 degrees C in the presence of a 100-fold excess of nonlabeled lactoferrin. Released lactoferrin displayed unchanged mobility on autoradiography. In contrast to lactoferrin, binding of 125I-transferrin was nonspecific and did not display saturable kinetics. The growth of T. foetus in iron-restricted media was stimulated by both lactoferrin and transferrin. The ability of the cells to remove and accumulate iron from both proteins was therefore examined using 59Fe-saturated lactoferrin and transferrin. It was found that trichomonads acquired a comparable amount of iron from both lactoferrin and transferrin during 60 min incubation at 37 degrees C (495 and 577 pmole Fe/mg of protein, respectively). The pH of the assay medium (PBS) decreased from pH 7.4 to 5.6 after incubation with trichomonads. At this pH, marked release of iron from transferrin (up to 47%) but not from lactoferrin (4%) was determined in cell-free media. These results indicate that T. foetus is able to utilize both lactoferrin and transferrin to cover its iron requirements. However, mechanisms of iron acquisition from these host proteins appear to be different. Specific binding and internalization of lactoferrin suggests the possible involvement of receptor-mediated endocytosis in the acquisition of lactoferrin-bound iron, while retrieval of iron from transferrin may depend on the extracellular release of iron from this ligand.