Molecular studies of the humoral immune response in Hyalophora cecropia : structure, function and transcriptional regulation of three classes of immunoresponsive genes

Sammanfattning: Insects have an efficient humoral immune system, an important part of which is constituted by the antibacterial proteins, produced upon bacterial infection or wounding. In this thesis, the molecular mechanism of this immune response was investigated in the giant silkmoth Hyalophora cecropia, where a set of bacteriainducible proteins (immune proteins) had previously been characterized. These included three classes of antibacterial factors, cecropins, attacins and a lysozyme, and a functionally unknown protein named P4.cDNA sequencing revealed that P4, now named hemolin, belongs to the immunoglobulin superfamily. It binds to the surface of bacteria and is most likely involved in the recognition of non-self. Unlike the real immunoglobulins that are specified by multiple sets of gene segments, hemolin is encoded by a single gene. The genes coding for two other classes of immune proteins, the attacins and the lysozyme, were also characterized. Two attacin genes, clustered with two attacin pseudogenes, and a single lysozyme gene are present on the genome of Cecropia.Expression studies revealed that the immune genes are normally silent or, in the case of the hemolin gene, expressed at a very low level. Upon injection of bacteria or other agents such as lipopolysaccharides and phorbol esters, they are quickly induced for transcription. The induction kinetics for all the sequenced immune genes, except one, are very similar. Sequence analyses showed that these genes contained a similar decameric sequence in their promoter regions. Interestingly, this sequence element is highly homologous to the binding motifs of the mammalian nuclear factor kB (NF-kB), which is involved in the induction of quite a number of immune related genes. Furthermore, the xB-like sequences of the Cecropia immune genes are specifically bound by a nuclear protein that can be detected only in the bacteria-induced pupae but not in the normal pupae. Subsequent investigations showed that this DNA binding protein, named Cecropia immunoresponsive factor (CIF), is strongly induced by bacteria and all the other known inducers of the immune genes. The induction kinetics of CIF is well correlated to the immune gene transcription, indicating its importance in the induction of the immune genes.Finally, CIF and NF-kB share certain similarities in their DNA binding specificity, pattern of induction and migration rate on native gels. The purified CIF contains a single polypeptide chain of 65 kDa, which is identical in size to one of the NF-kB subunits.