Regulation of antibacterial protein synthesis following infection and during metamorphosis of Manduca sexta.

Larvae of the tobacco hornworm, Manduca sexta, respond to intrahemocoelic injection of bacteria or bacterial cell wall peptidoglycan with induced synthesis of a suite of antibacterial proteins. Previous studies have demonstrated peptidoglycan regulation of the synthesis of these antibacterial proteins. In addition to eliciting enhanced synthesis of antibacterial proteins, peptidoglycan fragments also elicit a "malaise syndrome" characterized by decreased feeding and growth, delayed metamorphosis, and altered excretion. We speculate that these symptoms may be components of a mechanism to flush out and sterilize the midgut lumen, one of the primary sources of bacterial infection in insects. Studies of naive larvae have demonstrated the accumulation of lysozyme in the differentiating pupal midgut epithelium and release of lysozyme into the pupal midgut lumen after the larval midgut epithelium has been sloughed off. These observations have been extended by the identification of potent bactericidal activity against E. coli and immunoreactive hemolin, together with lysozyme, in the lumen of the newly differentiated pupal midgut.

Proline transport into brush border membrane vesicles from the midgut of Manduca sexta larvae.

The unidirectional transport of proline by midgut epithelium cells of the tobacco hornworm, Manduca sexta, was investigated in brush border membrane vesicles. Both K.(+)-stimulated and K(+)-insensitive transport pathways were identified. Analyses of K(+)-dependent proline transport revealed 1:1 ratio of K+ to proline, a Km of 13 mM for K+ and a decrease in both Km (from 18 mM to 3 mM) and Vmax (from 37 nmol/mg protein/min to 10 nmol/mg protein/min) for proline in the presence of a K+ gradient. The profiles of cis-inhibition by other amino acids demonstrated that proline is transported into midgut cells by a transport system that is shared by other neutral amino acids.

Structure and induction of a lysozyme gene from the tobacco hornworm, Manduca sexta.

Lysozyme is hypothesized to play a central role in initiating and maintaining the antibacterial defense response of Manduca sexta. We isolated a cDNA clone encoding a M. sexta lysozyme. Results of Northern blot analyses using this cDNA as a probe indicated that the abundance of lysozyme transcripts increased in seven tissues following treatment with peptidoglycan, with the highest level of accumulation occurring in the fat body. An analysis of the kinetics of accumulation of the transcripts in the fat body demonstrated low levels of transcripts in the naive larvae which increased rapidly after treatment and remained elevated over several days. A genomic fragment containing a lysozyme gene was also isolated and the nucleotide sequence and transcription start site of the gene was determined.

Differential inhibition by Bacillus thuringiensis delta endotoxin of leucine and aspartic acid uptake into BBMV from midgut of Manduca sexta.

Pre-incubation of brush border membrane vesicles (BBMV) isolated from the midgut of Manduca sexta with activated Bacillus thuringiensis delta endotoxin for a short period resulted in differential inhibition of K(+)-dependent transport of leucine relative to the effect on K(+)-dependent transport of aspartic acid. The difference in I1/2 (5 fold greater for aspartic acid than for leucine) is interpreted as the result of enhanced binding of the B. thuringiensis delta endotoxin to the leucine transport system.

Lysozyme in the midgut of Manduca sexta during metamorphosis.

Low levels of lysozyme were found in the midgut epithelium of the tobacco hornworm, Manduca sexta, during the early part of the fifth larval stadium. This was observed in control insects as well as in bacterially challenged insects. No lysozyme was detected in the gut contents of either group of insects which were actively eating or in the early stages of metamorphosis. However, high levels of lysozyme activity were detected in homogenates of midgut tissue collected from insects later in the stadium. Immunocytochemical studies demonstrated that lysozyme accumulates in large apical vacuoles in regenerative cells of the midgut during the larval-pupal molt. These cells, initially scattered basally throughout the larval midgut epithelium, multiply and form a continuous cell layer underneath the larval midgut cells. At the larval/pupal ecdysis the larval midgut epithelium is sloughed off and the regenerative cells, now forming the single cell layer of the midgut, release the contents of their vacuoles into the midgut lumen. This release results in high lysozyme activity in the lumen of the pupal midgut and is thought to confer protection from bacterial infection. This is the first indication that the lysozyme gene may be developmentally regulated in a specific tissue in the absence of a bacterial infection.

Specificity of Bacillus thuringiensis for lepidopteran larvae: factors involved in vivo and in the structure of a purified protoxin.

The relative LD50 values in two test Lepidoptera of Bacillus thuringiensis subspecies kurstaki HD1, which contains three crylA protoxin genes, was the same as a plasmid-cured derivative or a Bacillus cereus transcipient containing only one of the three genes. Differential rates of transcription of these genes in the original strain could account, at least partly, for this result. Strains containing only the single protoxin gene (crylA(b] produced inclusions when grown at 25 degrees C but not 32 degrees C, despite transcription of this gene at both temperatures. The instability of the crylA(b) protoxin was not found in the parental B. thuringiensis subsp. kurstaki HD1 strain grown at either temperature, however, so kurstaki HD1 strains with multiple protoxin genes must produce some stabilizing factor, perhaps another protoxin. The cryl protoxins contain a highly conserved carboxyl half which is proteolytically removed upon conversion to toxin. All of the protoxin cysteines are present in protease-sensitive regions and they are oxidized in inclusions. Most of the disulphides appear to be essential for specificity since their reduction in the crylA(b) protoxin resulted in loss of selectivity for one of the test insects. This lack of specificity was also found for this protoxin produced by an Escherichia coli clone, probably because of the reducing conditions in these cells. Specificity was restored by reoxidation of the pure protoxin, by removal of the carboxyl half of oxidized protoxin with trypsin, or by subcloning of the toxin portion. The oxidized form of protoxins must be important for specificity, for the formation of crystalline inclusions, and probably for interactions required for the stabilization of some protoxins.

Effect of parasitism by Cotesia congregata on the susceptibility of Manduca sexta larvae to bacterial infection.

Eggs and larvae of the braconid Cotesia congregata are not encapsulated within the hemocoel of their habitual host, Manduca sexta. Experiments were performed to evaluate the status of antibacterial defensive responses in M. sexta larvae parasitized by this gregarious endoparasitoid. Previous investigations have shown that immunologically naive, nonparasitized M. sexta larvae are resistant to infection by Pseudomonas aeruginosa. Studies using naive, parasitized larvae demonstrated that inoculation with 10 colony forming units of P. aeruginosa 9027, one hour after oviposition, resulted in greater than 90% host mortality. Examination of the fate of P. aeruginosa and Escherichia coli injected into parasitized larvae suggested that the cellular antibacterial defenses of the host, nodule formation, and phagocytosis, were impaired.

Plasmatocyte depletion in larvae of Manduca sexta following injection of bacteria.

Injection of bacteria and bacterial cell walls into larvae of the tobacco hornworm elicits a rapid, specific depletion of plasmatocytes from circulation. Plasmatocyte depletion in response to injection of bacteria is dose dependent with a threshold for response between 10(3)-10(4) bacteria per insect and a maximal depletion at greater than 10(7) bacteria per insect. Injection of saline, latex beads that are phagocytized, or fragments of peptidoglycan that elicit antibacterial protein synthesis do not affect plasmatocyte abundance.

A family of bacteria-regulated, cecropin D-like peptides from Manduca sexta.

Manduca sexta larvae respond to bacterial challenge by synthesizing a set of antibacterial hemolymph proteins. We have purified and sequenced three members of a family of cecropin D-like bactericidal peptides and isolated a cDNA clone complementary to a closely related bactericidin. Results obtained by Northern hybridization and RNase protection analysis showed that the increased synthesis of bactericidins is due to induction of their mRNA levels and that the synthesis of these peptides is not strictly tissue-specific, in contrast to previous beliefs. Although fat body was a richer source, the relative amounts of bactericidin mRNA were significant in seven other tissues examined.

Hemostasis in larvae of Manduca sexta: formation of a fibrous coagulum by hemolymph proteins.

Little is known of the participation of insect hemolymph proteins in wound healing and clot formation. We describe the assembly of purified hemolymph protein from the tobacco hornworm into an extended fibrous coagulum in the absence of hemocytes. This coagulum resembles the clot formed from bovine fibrinogen and thrombin. Structural components of the coagulum are present in hemolymph, however, spontaneous assembly occurs only in hemolymph collected through a wound. The fibrous coagulum assembles from purified structural protein(s) following addition of a non-protein factor from hemolymph, which is also present in Grace's insect cell culture medium.

Soluble peptidoglycan fragments stimulate antibacterial protein synthesis by fat body from larvae of Manduca sexta.

Both hemocytes and fat body from larvae of Manduca sexta, which have been injected with inducers of antibacterial protein synthesis, contain immunoreactive lysozyme. However, fat body is a richer source and has been demonstrated to synthesize and release lysozyme and cecropin-like peptides (bactericidins) in vitro. Fat body secretion of lysozyme and bactericidins is stimulated by addition of soluble peptidoglycan fragments to culture medium. The rate of lysozyme secretion by fat body varies as a function of peptidoglycan inducer concentration. These data are consistent with the hypothesis that, in vivo, bacteria must be phagocytized and partially degraded (processed) by hemocytes to generate a signal (peptidoglycan) that subsequently induces antibacterial protein synthesis by fat body.

Binding specificity of the juvenile hormone carrier protein from the hemolymph of the tobacco hornworm Manduca sexta Johannson (Lepidoptera: Sphingidae).

A series of analogues of insect juvenile hormone (four geometric isomers of methyl epoxyfarnesenate, several para-substituted epoxygeranyl phenyl ethers, and epoxyfarnesol and its acetate and haloacetate derivatives) was prepared to investigate the binding specificity of the hemolymph juvenile hormone binding protein from the tobacco hornworm Manduct sexta. The relative binding affinities were determined by a competition assay against radiolabeled methyl (E,E)-3,11-dimethyl-7-ethyl-cis-10,11-epoxytrideca-2,6-dienoate (JH I). The ratio of dissociation constants was estimated by plotting competitor data according to a linear transformation of the dissociation equations describing competition of two ligands for a binding protein. The importance of the geometry of the sesquiterpene hydrocarbon chain is indicated by the fact that the binding affinity is decreased as Z (cis) double bonds are substituted for E (trans) double bonds in the methyl epoxyfarnesenate series; the unepoxidized analogues do not bind. A carboxylic ester function is important although its orientation can be reversed, as indicated by the good binding of epoxyfarnesyl acetate. In the monoterpene series, methyl epoxygeranoate shows no affinity for the binding protein, but substitution of a phenyl or p-carbomethoxyphenyl ether for the ester function imparts a low, but significant affinity. These data taken together with earlier results indicate that the binding site for juvenile hormone in the hemolymph binding protein is characterized by a sterically defined hydrophobic region with polar sites that recognize the epoxide and the ester functions.

Purification and characterization of the carrier protein for juvenile hormone from the hemolymph of the tobacco hornworm Manduca sexta Johannson (Lepidoptera: Sphingidae).

The larval hemolymph of the tobacco hornworm, Manduca sexta, contains a carrier protein that binds specifically and with high affinity the juvenile hormone, an important regulator of insect development. This protein serves to transport the hormone and to protect it from the action of degradative enzymes during early larval stages. Using hemolymph from the last larval stage, we have isolated a pure carrier protein using acetone precipitation, gel filtration, ion exchange chromatography, and preparative isoelectric focusing. Gel filtration, polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and equilibrium ultracentrifugation established that the carrier protein is a single chain polypeptide of approximately 28,000 daltons. The amino acid composition is unexceptional, and no evidence for hexosamine has been obtained. An ion exchange filter disc assay method was used to determine the formation of the complex between the carrier protein and isotopically labeled juvenile hormone. With this technique it was shown that each carrier protein binds one hormone molecule with a dissociation constant of 4.4 +/- 0.2 X 10(-7) M at 0 degrees.