Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities.

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell-cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions.

Acquisition of haemoglobin-bound iron by strains of the Actinobacillus minor/"porcitonsillarum" complex.

Members of the Actinobacillus minor/"porcitonsillarum" complex are common inhabitants of the swine respiratory tract. Although avirulent or of low virulence for pigs, these organisms, like pathogens, do grow in vivo and must, therefore, be able to acquire iron within the host. Here, we investigated the abilities of six members of the A. minor/"porcitonsillarum" complex to acquire iron from transferrin and various haemoglobins. Using growth assays, all six strains were shown to acquire iron from porcine, bovine and human haemoglobins but not from porcine transferrin. Analyses of whole genome sequences revealed that A. minor strains NM305(T) and 202, unlike the swine-pathogenic actinobacilli, A. pleuropneumoniae and A. suis, lack not only the transferrin-binding protein genes, tbpA and tbpB, but also the haemoglobin-binding protein gene, hgbA. Strains NM305(T) and 202, however, were found to possess other putative haemin/haemoglobin-binding protein genes that were predicted to encode mature proteins of ∼ 72 and ∼ 75 kDa, respectively. An affinity procedure based on haemin-agarose allowed the isolation of ∼ 65 and ∼ 67 kDa iron-repressible outer membrane polypeptides from membranes derived from strains NM305(T) and 202, respectively, and mass spectrometry revealed that these polypeptides were the products of the putative haemin/haemoglobin-binding protein genes. PCR approaches allowed the amplification and sequencing of homologues of both haemin/haemoglobin-binding protein genes from each of the other four strains, strains 33PN and 7ATS of the A. minor/"porcitonsillarum" complex and "A. porcitonsillarum" strains 9953L55 and 0347, suggesting that such proteins are involved in the utilization of haemoglobin-bound iron, presumably as surface receptors, by all six strains investigated.

Production of haemolysins by strains of the Actinobacillus minor/"porcitonsillarum" complex.

Actinobacillus minor and "Actinobacillus porcitonsillarum" are distinguished by their haemolytic activities, the latter organism being haemolytic and the former, non-haemolytic. Analysis of a whole genome shotgun sequence, however, revealed that A. minor strain 202, like "A. porcitonsillarum", possesses a haemolysin-encoding apxII operon. The purpose of this study was therefore to investigate haemolysin production by this organism and also by three additional members of the A. minor/"porcitonsillarum" complex, strains 33PN and 7ATS and A. minor strain NM305(T). Primers based on sequences within the apxII genes of strain 202 allowed the amplification of appropriately sized fragments from DNA from strain 33PN suggesting that this organism also possesses an apxII operon. Analysis of a whole genome shotgun sequence failed to reveal any trace of an apxII operon in strain NM305(T) and attempts to amplify apxII genes from DNA from strain 7ATS also failed. Strains 202 and 33PN, and surprisingly, the type strain of A. minor and strain 7ATS, were all found to be haemolysin-positive as growth media from cultures of these organisms could promote the lysis of erythrocytes in suspension. The erythrocyte specificities of the haemolysins produced by strains 202 and 33PN indicated that the haemolytic activities exhibited by these organisms were due to ApxII. In keeping with the apparent lack of apxII genes in strains NM305(T) and 7ATS, the haemolysins produced by these organisms were not erythrocyte-specific and with both organisms, haemolytic activity appeared to be due to a combination of heat-stable and heat-labile components. The identities of these components, however, remain unknown.

Surface antigens of Xenorhabdus nematophila (F. Enterobacteriaceae) and Bacillus subtilis (F. Bacillaceae) react with antibacterial factors of Malacosoma disstria (C. Insecta: O. Lepidoptera) hemolymph.

Previous research established different interactions of the insect pathogen, Xenorhabdus nematophila and nonpathogen, Bacillus subtilis, with antimicrobial hemocytes and humoral factors of larval Malacosoma disstria [Giannoulis, P., Brooks, C.L., Dunphy, G.B., Mandato, C.A., Niven, D.F., Zakarian, R.J., 2007. Interaction of the bacteria Xenorhabdus nematophila (Enterobacteriaceae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasicocampidae). J. Invertebr. Pathol. 94, 20-30]. The antimicrobial systems were inhibited by X. nematophila and stimulated by B. subtilis. The bacterial surface antigens participating in these reactions were unknown. Thus, herein the effects of lipopolysaccharide (endotoxin) from X. nematophila and lipoteichoic acid from B. subtilis on the larval M. disstria immune factors, the hemocytes and phenoloxidase, were determined. Endotoxin elevated the level of damaged hemocytes limiting the removal of X. nematophila from the hemolymph and enhancing the rapid release of bacteria trapped by nodulation. Similar effects were observed with the lipid A moiety of the endotoxin. The effects of lipopolysaccharide and lipid A on the hemocyte activities were abrogated by polymyxin B (an antibiotic that binds to lipid A) confirming lipopolysaccharide as the hemocytotoxin by virtue of the lipid A moiety. Lipoteichoic acid elicited nodulation and enhanced phenoloxidase activation and/or activity. Although lipoidal endotoxin and lipid A inhibited phenoloxidase activation they enhanced the activity of the enzyme. Apolipophorin-III precluded the effects of lipopolysaccharide, lipid A, and lipoteichoic acid on the hemocytes and prophenoloxidase until the antigens exceeded a critical threshold.

Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasiocampidae).

Malacosoma disstria larvae are a pest of deciduous trees. Little is known on the interaction of bacteria with the immediate hemocytic antimicrobial responses of these insects. Incubating dead Xenorhabdus nematophila and Bacillus subtilis with a mixture of serum-free granular cells and plasmatocytes in vitro revealed differential bacterial-hemocyte adhesion and differential discharge of lysozyme and phenoloxidase but not total protein. Although active phenoloxidase adhered equally to both bacterial species, X. nematophila limited enzyme activation whereas B. subtilis enhanced activation. Serum with active phenoloxidase (as opposed to tropolone-inhibited phenoloxidase) and purified insect lysozyme increased bacterial-hemocyte adhesion of both bacterial species. An apolipophorin-III-like protein when incubated with hemocytes, limited their responses to glass slides and bacterial adhesion. However, initial binding of the protein to both bacteria increased granular cell levels with bacteria while lowering the plasmatocyte levels with adhering procaryotes. The protein also increased lysozyme and phenoloxidase activities. Although B. subtilis in vivo elicited a nodulation-based decline in total hemocyte counts and did not affect hemocyte viability, dead X. nematophila elevated hemocyte counts and damaged the hemocytes as lipopolysaccharide levels increased and X. nematophila emerged into the hemolymph. Apolipophorin-III-like protein once bound to the bacteria slowed their removal from the hemolymph.

Acquisition of haemoglobin-bound iron by Histophilus somni.

Histophilus somni is an important pathogen of cattle and sheep. H. somni requires iron and can use ruminant transferrins as iron sources for growth. Here, we investigated the abilities of bovine (strains 649 and 2,336) and ovine (strains 9L and 3384Y) isolates of H. somni to acquire iron from haemoglobins. Using growth assays, the bovine isolates were shown to acquire iron from bovine haemoglobin, but not from ovine, porcine or human haemoglobins; the ovine isolates, however, failed to use any of these haemoglobins as iron sources for growth. In solid phase binding assays, the bovine isolates, grown under iron-restricted conditions in the presence of bovine haemoglobin, bound not only bovine but also ovine and human haemoglobins. Competition binding assays indicated that all three haemoglobins were bound by the same receptor(s) and SDS-PAGE of membrane fractions revealed that expression of haemoglobin-binding activity was associated with the production of an approximately 120-kDa outer membrane protein. PCR approaches allowed the amplification and sequencing of hgbA, and also hugX and hugZ homologues from strains 649, 9L and 3384Y. While hgbA of strain 649 was predicted to encode an HgbA precursor that is processed to yield a mature, 123.9-kDa haemoglobin-binding protein, the hgbA genes of strains 9L and 3384Y were predicted to give rise to truncated products. RT-PCR experiments revealed that in strain 649, hugX, hugZ and hgbA are co-transcribed and iron-regulated and additional sequencing suggested that in strain 2336, expression of HgbA is subject to phase variation involving a poly C tract within hgbA.

Iron acquisition by Actinobacillus suis: identification and characterization of a single-component haemoglobin receptor and encoding gene.

Actinobacillus suis is an important swine pathogen. As with other pathogens, the ability of A. suis to acquire iron within the host is crucial for virulence. Here, we investigated the ability of seven strains of A. suis to acquire iron from haemoglobins. In growth assays, all strains could use porcine, bovine and human haemoglobins as iron sources for growth. Using solid phase binding assays, membranes derived from all strains, grown under iron-restricted conditions, were shown to bind all three haemoglobins. Competition binding assays indicated that these haemoglobins were bound by the same receptor and an affinity procedure allowed the isolation and identification of an iron-repressible, haemoglobin-binding polypeptide (approximately 105 kDa) from all strains. Nucleotide sequence analyses revealed that A. suis possesses a gene (hgbA) that encodes a homologue of the Actinobacillus pleuropneumoniae haemoglobin-binding protein, HgbA. hgbA, encoding a mature protein of 105 kDa, was shown to be preceded by a hugZ homologue; putative promoter sequences and a putative Fur box were located upstream of hugZ and RT-PCR revealed that hugZ and hgbA are co-transcribed and iron-repressible. It is concluded that the acquisition of haemoglobin-bound iron by A. suis involves a single-component receptor that is up-regulated in response to iron restriction.

Haemophilus somnus possesses two systems for acquisition of transferrin-bound iron.

Haemophilus somnus strain 649 was found to acquire iron from ovine, bovine, and goat transferrins (Tfs). Expression of Tf receptors, as evaluated by solid-phase binding assays, required the organisms to be grown under iron-restricted conditions in the presence of Tf. Competition binding assays revealed the presence of two distinct Tf-binding receptor systems, one specific for bovine Tf and the other capable of binding all three ruminant Tfs. Affinity isolation procedures using total membranes yielded three putative bovine Tf-binding polypeptides and one putative ovine and goat Tf-binding polypeptide. PCR amplification followed by DNA sequence analyses revealed that H. somnus strain 649 possesses genes that encode a bipartite TbpA-TbpB receptor along with a homolog of the Histophilus ovis single-component TbpA receptor. Expression of TbpB and the single-component TbpA would appear to be subject to a form of phase variation involving homopolymeric nucleotide tracts within the structural genes.

Levels of nicotinamide adenine dinucleotide in extracellular body fluids of pigs may be growth-limiting for Actinobacillus pleuropneumoniae and Haemophilus parasuis.

During infection, nutrient deprivation can alter bacterial phenotype. This, in turn, may have implications for pathogenesis and prophylaxis. Actinobacillus pleuropneumoniae (biotype 1) and Haemophilus parasuis, respiratory tract pathogens of swine, are both V-factor-dependent. The concentrations of V factor in the extracellular fluids of pigs are unknown and may limit the growth of these bacteria in vivo. The aim of this study was to determine the concentrations of nicotinamide adenine dinucleotide (NAD) in select porcine body fluids and to compare the availability of NAD in vivo with the affinities of the organisms for this compound. Levels in plasma, tissue fluids (peritoneal, pleural, synovial, and cerebrospinal), and laryngeal, tracheal, and lung washings were determined with an enzymatic cycling assay. We concluded that, although the NAD supply in the respiratory tract is probably not growth-limiting, it may become limiting if the organisms are disseminated.

Iron acquisition by Actinobacillus suis: identification and characterization of transferrin receptor proteins and encoding genes.

Actinobacillus suis is an important pathogen of swine, especially in high-health-status herds. A published report mentioning the binding of porcine transferrin (Tf) by at least one strain of A. suis suggested that A. suis, like other members of the Pasteurellaceae, can acquire Tf-bound iron by means of a siderophore-independent, receptor-mediated mechanism. The objective of the present study was to characterize the components involved in this process, if present. Growth assays, with seven strains, confirmed that A. suis can use porcine (but not human or bovine) Tf as an iron source for growth. In solid phase binding assays, total membranes derived from all strains exhibited strong binding of porcine Tf, but only if the membranes were from organisms grown under iron-restricted conditions. An affinity-isolation procedure allowed the isolation of putative Tf-binding polypeptides ( approximately 100 and approximately 63 kDa) from comparable membranes from all strains. PCR approaches allowed the amplification, cloning and sequencing of A. suis tonB, exbB, exbD, tbpB and tbpA homologues. Reverse transcription (RT)-PCR, using RNA from organisms grown under iron-replete and iron-restricted conditions, revealed that tonB, exbB, exbD, tbpB and tbpA are transcribed as a single unit with expression being up-regulated in response to iron restriction. The calculated molecular masses of the predicted, mature TbpA (104.3 kDa) and TbpB (63.4 kDa) proteins suggest strongly that the affinity-isolated, approximately 100 and approximately 63 kDa Tf-binding polypeptides represent TbpA and TbpB, respectively. It is concluded that the acquisition of Tf-bound iron by A. suis involves mechanisms analogous to those found in other members of the Pasteurellaceae.

Transferrin-dependent expression of TbpA by Histophilus ovis involves a poly G tract within tbpA.

A poly G tract in tbpA of Histophilus ovis strain 3384Y was suspected of being responsible for the transferrin (Tf)-dependent expression of TbpA. The region encompassing the poly G tract was amplified using DNA from H. ovis strains 9L and 3384Y grown under iron-replete conditions and under iron-restricted conditions in the presence of bovine Tf. Sequence analysis of the amplification products revealed that regardless of the growth conditions, the poly G tract in strain 9L contained eight Gs, a situation that maintains the correct reading frame of the gene. Similarly, the poly G tract in strain 3384Y contained eight Gs when the organisms were grown under iron-restricted conditions in the presence of bovine Tf but when grown under iron-replete conditions, the poly G tract contained nine Gs resulting in a frame shift and the introduction of a premature stop codon. It is concluded that the Tf-dependent expression of TbpA in H. ovis strain 3384Y is due to a form of phase variation.

Identification of fur and fldA homologs and a Pasteurella multocida tbpA homolog in Histophilus ovis and effects of iron availability on their transcription.

tbpA, fur, and fldA homologs from two strains (9L and 3384Y) of the sheep pathogen Histophilus ovis were sequenced. The predicted TbpA proteins of these strains are homologs of the Pasteurella multocida TbpA protein and collectively represent the second example of a new subfamily of TonB-dependent receptors. tbpA transcripts were readily detected by reverse transcription (RT)-PCR with RNA isolated from strain 9L grown under iron-restricted conditions in the presence or absence of bovine transferrin (Tf). However, with strain 3384Y and depending on the primer pair, tbpA transcripts were detected by RT-PCR predominantly when the RNA was from cells grown under iron-restricted conditions in the presence of bovine Tf. In both strains, the fldA homolog was found to be immediately upstream of fur and, based on RT-PCR, these genes are transcribed as a single unit; the availability of iron and the presence or absence of bovine Tf in the growth medium had no apparent effect on the relative amounts of the fldA-fur transcripts.

Iron contributes to the antibacterial functions of the haemolymph of Galleria mellonella.

Studies with Galleria mellonella larvae and the iron chelating agent EDDA showed that iron was essential for the removal of dead Xenorhabdas nematophila and Bacillus subtilis from the haemolymph. The delay in removal of the bacteria from the iron-restricted haemolymph was attributed to reduced adhesiveness of the haemocytes and prophenoloxidase activity. Iron augmentation returned these activities to control levels. Whereas dead B. subtilis had no effect on the concentration of ferrozine-detectable iron (henceforth iron) in the haemolymph, dead X. nematophila was associated with substantially lower levels of iron as the number of damaged haemocytes increased. Haemocyte lysate lowered the concentrations of iron in both FeCl(3) solutions and deproteinized larval serum independent of serum lipids. Haemocyte lysate added to tryptic soybroth lowered the level of iron and limited the growth of X. nematophila. X. nematophila limited iron availability in the plasma by releasing lipopolysaccharides; such a mechanism may be a means of impairing the antimicrobial defences of the insects.