ABSTRACT
Two isoforms of MTF-1, MTF-1L (long form) and MTF-1S (short form), were cloned in tilapia (Ti) and characterized in a tilapia liver cell line, Hepa-T1. The cloned tiMTF-1L has the characteristics of all of the tiMTF-1S identified so far with the zinc finger domain having six fingers, the acidic-rich, proline-rich, and serine/threonine-rich domains; however, the short form encodes for the zinc finger domain with five zinc fingers only and no other domains. The transient transfection of tiMTF-1L into human HepG2 cells showed both constitutive and zinc-induced metal-responsive-element (MRE)-driven reporter gene expression. However, the transfection of tiMTF-1S (which lacks all three transactivation domains) into a human cell line showed reduced transcriptional activities compared with an endogenous control in both basal- and Zn(2+)-induced conditions. The tiMTF-1 isoforms were tagged with GFP and transfected into Hepa-T1 cells (tilapia hepatocytes). The nuclear translocation of tiMTF-1L was observed when the cells were exposed to a sufficient concentration of metals for 6h. However, tiMTF-1S, was localized in the nucleus with or without metal treatment. Electrophoretic mobility shift assay (EMSA) confirmed that both of the isoforms were able to bind to the MRE specifically in vitro. Tissue distribution studies showed that tiMTF-1L was more abundant than tiMTF-1S in all of the tissues tested.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Response Elements/genetics , Tilapia/genetics , Tilapia/metabolism , Transcription Factors/metabolism , Zinc/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Line, Tumor , DNA-Binding Proteins/genetics , Electrophoretic Mobility Shift Assay , Gene Expression Regulation/drug effects , Genes, Reporter/genetics , Hep G2 Cells , Humans , Metallothionein/genetics , Metallothionein/metabolism , Molecular Sequence Data , Protein Isoforms/genetics , Protein Isoforms/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transcription Factors/genetics , Transcriptional Activation/drug effects , Transcriptional Activation/genetics , Zinc/metabolism , Zinc Fingers/genetics , Zinc Fingers/physiology , Transcription Factor MTF-1ABSTRACT
Pathogenicity and symbiosis are central to bacteria-host interactions. Although several human pathogens have been subjected to functional genomic analysis, we still understand little about bacteria-invertebrate interactions despite their ecological prevalence. Advances in our knowledge of this area are often hindered by the difficulty of isolating and working with invertebrate pathogenic bacteria and their hosts. Here we review studies on pathogenicity and symbiosis in an insect pathogenic bacterium Photorhabdus and its entomopathogenic nematode vector and model insect hosts. Whilst switching between these hosts, Photorhabdus changes from a state of symbiosis with its nematode vector to one of pathogenicity towards its new insect host and both the bacteria and the nematode then cooperatively exploit the dying insect. We examine candidate genes involved in symbiosis and pathogenicity, their secretion and expression patterns in culture and in the host, and begin to dissect the extent of their genetic coregulation. We describe the presence of several large genomic islands, putatively involved in pathogenicity or symbiosis, within the otherwise Yersinia-like backbone of the Photorhabdus genome. Finally, we examine the emerging comparative genomics of the Photorhabdus group and begin to describe the interrelationship between anti-invertebrate virulence factors and those used against vertebrates.
Subject(s)
Genome, Bacterial , Photorhabdus , Symbiosis/genetics , Animals , Bacteriocins/genetics , Bacteriocins/metabolism , Bacteriophages/genetics , Bacteriophages/physiology , Bacteriophages/ultrastructure , Insecta/metabolism , Insecta/microbiology , Insecta/ultrastructure , Life Cycle Stages , Models, Genetic , Nematoda/genetics , Nematoda/microbiology , Nematoda/physiology , Photorhabdus/genetics , Photorhabdus/isolation & purification , Photorhabdus/pathogenicity , Photorhabdus/physiology , Virulence Factors/biosynthesis , Virulence Factors/geneticsABSTRACT
Invertebrates, including insects, are being developed as model systems for the study of bacterial virulence. However, we understand little of the interaction between bacteria and specific invertebrate tissues or the immune system. To establish an infection model for Photorhabdus, which is released directly into the insect blood system by its nematode symbiont, we document the number and location of recoverable bacteria found during infection of Manduca sexta. After injection into the insect larva, P. luminescens multiplies in both the midgut and haemolymph, only later colonizing the fat body and the remaining tissues of the cadaver. Bacteria persist by suppressing haemocyte-mediated phagocytosis and culture supernatants grown in vitro, as well as plasma from infected insects, suppress phagocytosis of P. luminescens. Using GFP-labelled bacteria, we show that colonization of the gut begins at the anterior of the midgut and proceeds posteriorly. Within the midgut, P. luminescens occupies a specific niche between the extracellular matrix and basal membrane (lamina) of the folded midgut epithelium. Here, the bacteria express the gut-active Toxin complex A (Tca) and an RTX-like metalloprotease PrtA. This close association of the bacteria with the gut, and the production of toxins and protease, triggers a massive programmed cell death of the midgut epithelium.
