ABSTRACT
High glucose concentration in the airway surface liquid (ASL) is an important feature of diabetes that predisposes to respiratory infections. We investigated the role of alveolar epithelial SGLT1 activity on ASL glucose concentration and bacterial proliferation. Non-diabetic and diabetic rats were intranasally treated with saline, isoproterenol (to increase SGLT1 activity) or phlorizin (to decrease SGLT1 activity); 2 hours later, glucose concentration and bacterial proliferation (methicillin-resistant Sthaphylococcus aureus, MRSA and Pseudomonas aeruginosa, P. aeruginosa) were analyzed in bronchoalveolar lavage (BAL); and alveolar SGLT1 was analyzed by immunohistochemistry. BAL glucose concentration and bacterial proliferation increased in diabetic animals: isoproterenol stimulated SGLT1 migration to luminal membrane, and reduced (50%) the BAL glucose concentration; whereas phlorizin increased the BAL glucose concentration (100%). These regulations were accompanied by parallel changes of in vitro MRSA and P. aeruginosa proliferation in BAL (r = 0.9651 and r = 0.9613, respectively, Pearson correlation). The same regulations were observed in in vivo P. aeruginosa proliferation. In summary, the results indicate a relationship among SGLT1 activity, ASL glucose concentration and pulmonary bacterial proliferation. Besides, the study highlights that, in situations of pulmonary infection risk, such as in diabetic subjects, increased SGLT1 activity may prevent bacterial proliferation whereas decreased SGLT1 activity can exacerbate it.
Subject(s)
Diabetes Mellitus, Experimental/metabolism , Glucose/metabolism , Lung/metabolism , Pneumonia, Bacterial/metabolism , Pseudomonas Infections/metabolism , Sodium-Glucose Transporter 1/genetics , Staphylococcal Infections/metabolism , Alloxan , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/microbiology , Animals , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/microbiology , Bronchodilator Agents/pharmacology , Colony Count, Microbial , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/genetics , Gene Expression Regulation , Isoproterenol/pharmacology , Lung/drug effects , Lung/microbiology , Lung/pathology , Male , Methicillin-Resistant Staphylococcus aureus/drug effects , Methicillin-Resistant Staphylococcus aureus/growth & development , Phlorhizin/pharmacology , Pneumonia, Bacterial/complications , Pneumonia, Bacterial/genetics , Pneumonia, Bacterial/pathology , Pseudomonas Infections/complications , Pseudomonas Infections/genetics , Pseudomonas Infections/pathology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/growth & development , Rats , Rats, Wistar , Sodium-Glucose Transporter 1/agonists , Sodium-Glucose Transporter 1/antagonists & inhibitors , Sodium-Glucose Transporter 1/metabolism , Staphylococcal Infections/complications , Staphylococcal Infections/genetics , Staphylococcal Infections/pathologyABSTRACT
The histone H4 from Trypanosomatids diverged from other eukaryotes in the N-terminus, a region that undergoes post-translation modifications involved in the control of gene expression, DNA replication, and chromatin assembly. Nonetheless, the N-terminus of Trypanosoma cruzi histone H4 is mainly acetylated at lysine 4. The lysines 10 and 14 are also acetylated, although at less extent, increasing during the S-phase or after DNA damage, which suggests a regulatory function. Here, we investigated the roles of these acetylations by expressing non-acetylated forms of histone H4 in T. cruzi. We found that histone H4 containing arginines at positions 10 or 14, to prevent acetylation were transported to the nucleus and inserted into the chromatin. However, their presence, even at low levels, interfered with DNA replication and transcription, causing a significant growth arrest of the cells. The absence of acetylation also increased the amount of soluble endogenous histones H3 and H4 and affected the interaction with Asf1, a histone chaperone. Therefore, acetylation of lysines 10 and 14 of the histone H4 in trypanosomes could be required for chromatin assembly and/or remodeling required for transcription and replication.
Subject(s)
DNA Replication , Histone Chaperones/metabolism , Histones/metabolism , Protozoan Proteins/metabolism , Transcription, Genetic , Trypanosoma cruzi/genetics , Acetylation , Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Chromatin Assembly and Disassembly , Gene Expression Regulation , Histone Acetyltransferases/metabolism , Lysine/chemistry , Protein Processing, Post-TranslationalABSTRACT
The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans-splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. In the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing.
