Modulation of Vacuolar pH Is Required for Replication of Edwardsiella ictaluri in Channel Catfish Macrophages

Previous in vitro work demonstrated that Edwardsiella ictaluri produces an acid-activated urease that can modulate environmental pH through the production of ammonia from urea. Additional work revealed that expression of the E. ictaluri type III secretion system (T3SS) is upregulated by acidic pH. Both the urease and the T3SS were previously shown to be essential to intracellular replication. In this work, fluorescence microscopy with LysoTracker Red DND-99 (LTR) indicated that E. ictaluri-containing vacuoles (ECV) became acidified following ingestion by head kidney-derived macrophages (HKDM). In vivo ratiometric imaging demonstrated a lowered ECV pH, which fell to as low as pH 4 but subsequently increased to pH 6 or greater. Inhibition of vacuolar H⁺-ATPases by use of the specific inhibitor bafilomycin A₁ abrogated both ECV acidification and intracellular replication in HKDM. Failure of an E. ictaluri urease knockout mutant to increase the ECV pH in the in vivo ratiometric assay suggests that ammonia produced by the urease reaction mediates the pH increase. Additionally, when the specific arginase inhibitor l-norvaline was used to treat E. ictaluri-infected HKDM, the ECV failed to neutralize and E. ictaluri was unable to replicate. This indicates that the HKDM-encoded arginase enzyme produces the urea used by the E. ictaluri urease enzyme. Failure of the ECV to acidify would prevent both upregulation of the T3SS and activation of the urease enzyme, either of which would prevent E. ictaluri from replicating in HKDM. Failure of the ECV to neutralize would result in a vacuolar pH too low to support E. ictaluri replication.     

Activation of TLR3 and TLR5 in channel catfish exposed to virulent Edwardsiella ictaluri

Two toll-like receptors (TLR3 and TLR5) were identified from a catfish cDNA fry library based on sequence similarity to other vertebrate TLR genes. Expression (using real-time PCR) of TLR3 and TLR5 was measured for two strains of channel catfish in previously non-exposed fish 2, 5, 8, and 21 days after experimental Edwardsiella ictaluri challenge to determine if TLRs are associated with host response to E. ictaluri infection. Expression of TLR5 was higher than TLR3 (P<0.0001). TLR3 expression in kidney was elevated in Norris strain (P=0.480) and differed over time in spleen (P=0.0134). Fold induction of TLR5 compared to non-exposed fish increased on days 5 (Norris; 154.72+/-62.12 fold induction) and 8 (USDA103; 164.65+/-50.56) post-exposure in liver and was slightly increased on day 5 (Norris; 10.17+/-24.73, USDA103; 42.56+/-24.73) in kidney. Upregulation of TLR3 suggests a more widespread function in primitive fish. TLR5 was highly expressed in liver tissue, which may be due to macrophage aggregation during ESC infection. This suggests that toll-like receptors are an important component of the innate immune system of catfish.     

In vitro responses of channel catfish, Ictalurus punctatus, neutrophils to Edwardsiella ictaluri

The in vitro responses of channel catfish peripheral blood neutrophils to Edwardsiella ictaluri were examined through the use of phagocytic, bactericidal, and chemiluminescent assays. Evidence from both light and electron microscopy indicate that catfish neutrophils appeared to phagocytose E. ictaluri. Although extracellular killing of E. ictaluri was observed, bactericidal assays did not demonstrate intracellular killing of E. ictaluri by neutrophils. Catfish neutrophils mount a chemiluminescent response to E. ictaluri which is enhanced by the presence of specific antibody. Incubation of catfish neutrophils with LPS from E. ictaluri appeared to suppress the chemiluminescent response to opsonized zymosan.     

Attenuation, persistence, and vaccine potential of an Edwardsiella ictaluri purA mutant.

In this study, an adenine-auxotrophic strain of Edwardsiella ictaluri was constructed and its virulence, tissue persistence, and vaccine efficacy were evaluated. A clone containing the purA gene was isolated from an E. ictaluri genomic library, sequenced, and shown to have an overall sequence identity of 79.3% at the nucleotide level and 85.7% at the amino acid level with the Escherichia coli purA gene. The cloned E. ictaluri purA gene was mutated by deleting a 598-bp segment of the gene and inserting the kanamycin resistance gene from Tn903 into the gap. The delta purA::Km(r) gene was subcloned into the suicide plasmid pGP704, and the resulting plasmid was used to deliver the modified gene into a virulent strain of E. ictaluri by conjugation. Homologous recombination replaced the chromosomal purA gene with the mutated gene to create an adenine-auxotrophic strain (LSU-E2). Compared to wild-type E. ictaluri, LSU-E2 was highly attenuated by the injection, immersion, and oral routes of exposure. By the injection route, LSU-E2 had a 50% lethal dose (LD50) that was greater than 5 logs10 higher than the LD50 for wild-type E. ictaluri. In a tissue persistence study, LSU-E2 was able to invade channel catfish by the immersion route and persist in internal organs for at least 48 h. Channel catfish that were vaccinated with a single immersion dose of LSU-E2 had mortality significantly lower (P < 0.01) following a wild-type E. ictaluri challenge than that of nonvaccinated fish.     

Sequence analysis and expression of a CXC chemokine in resistant and susceptible catfish after infection of Edwardsiella ictaluri

Chemokines represent a superfamily of chemotactic cytokines involved in recruitment, activation and adhesion of a variety of leukocyte types to inflammatory foci. We cloned and sequenced the cDNA of a CXC chemokine that is most similar to CXCL10 from channel catfish and blue catfish. Sequence analysis of PCR amplicons from a single F1 hybrid catfish indicated that channel catfish and blue catfish may have a multigene family for the CXC chemokine. The catfish CXC chemokine was expressed in a wide range of tissues including head kidney, spleen, liver, gill, skin, stomach, and intestine, but not in the muscle. Fish challenged with intracellular bacterium Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), showed dramatically elevated levels of the CXC chemokine expression, as quantified with real time RT-PCR. Differential expression profiles were observed between resistant and susceptible channel catfish strains and blue catfish. Blue catfish were characterized by only modest induction in comparison to the drastic elevation of the CXC chemokine in channel catfish.     

Lactoferrin Protects Gut Mucosal Integrity During Endotoxemia Induced by Lipopolysaccharide in Mice

The hypothesis that lactoferrin protects mice against lethal effects of bacterial lipopolysaccharide (LPS) is the subject of experimental investigations described in this article. Lipopolysaccharide is a powerful toxin produced by Gram negative bacteria that when injected into humans or experimental animals reproduce many of the pathophysiologic and immune responses caused by live bacteria. Lactoferrin administered intraperitoneally 1 hr prior to injection of LPS significantly enhanced the survival of mice, reducing LPS-induced mortality from 83.3% to 16.7%. Changes in locomotor and other behavioral activities resulting from LPS injection were not present in mice treated with lactoferrin. Also, histological examination of intestine revealed remarkable resistance to injury produced by LPS if mice were pretreated with lactoferrin. Severe villus atrophy, edema and epithelial vacuolation were observed in LPS-treated animals but not in lactoferrin-treated counterparts. Electrophysiological parameters were used to assess secretory and absorptive functions in the small intestine. In mice treated with LPS, transmural electrical resistance was reduced and absorption of glucose was increased. Lactoferrin treatment had no significant influence on basal electrophysiological correlates of net ion secretion or glucose absorption nor on changes induced by LPS. Collectively, these results suggest that lactoferrin attenuates the lethal effect of LPS and modulates behavioral and histopathological sequela of endotoxemia.     

Fur-regulated iron uptake system of Edwardsiella ictaluri and its influence on pathogenesis and immunogenicity in the catfish host

The ability of bacterial pathogens to take up iron from the host during infection is necessary for their multiplication within the host. However, host high-affinity iron binding proteins limit levels of free iron in fluids and tissues. To overcome this deficiency of iron during infection, bacterial pathogens have developed iron uptake systems that are upregulated in the absence of iron, typically tightly controlled by the ferric uptake regulator (Fur) protein. The iron uptake system of Edwardsiella ictaluri, a host-restricted pathogen of channel catfish (Ictalurus punctatus) and the main pathogen of this fish in aquaculture, is unknown. Here we describe the E. ictaluri Fur protein, the iron uptake machinery controlled by Fur, and the effects of fur gene deletion on virulence and immunogenicity in the fish host. Analysis of the E. ictaluri Fur protein shows that it lacks the N-terminal region found in the majority of pathogen-encoded Fur proteins. However, it is fully functional in regulated genes encoding iron uptake proteins. E. ictaluri grown under iron-limited conditions upregulates an outer membrane protein (HemR) that shows heme-hemoglobin transport activity and is tightly regulated by Fur. In vivo studies showed that an E. ictaluri Δfur mutant is attenuated and immune protective in zebrafish (Danio rerio) and catfish (Ictalurus punctatus), triggering systemic immunity. We conclude that an E. ictaluri Δfur mutant could be an effective component of an immersion-oral vaccine for the catfish industry.     

OmpN,outer membrane proteins of Edwardsiella ictaluri are potential vaccine candidates for channel catfish (Ictalurus punctatus)

Outer membrane proteins (OMPs) are a class of proteins that reside in the outer membrane of Gram-negative bacteria. OMPs act as epitopes and are potential vaccine candidates. Outer membrane protein N (OmpN) is a component of the outer membrane of Edwardsiella ictaluri (E. ictaluri). In a previous study, the OmpN1-, OmpN2-, OmpN3-encoding genes of E. ictaluri were cloned, and here they were expressed in Escherichia coli. Western blotting showed that these three proteins had molecular weights of ∼60 kDa. Channel catfish were immunized with recombinant OmpNs (rOmpNs) and then challenged with E. ictaluri. The results showed that rOmpN1, rOmpN2, and rOmpN3, as well as a mixture of all three proteins (in a ratio of 1:1:1) generated moderate immune protection (relative percentage of survival = 62.5, 62.5, 67.5, and 75%, respectively). In an agglutination antibody titer assay, fish antisera showed an antibody titer of 1:128. Furthermore, each of the proteins stimulated high levels of lysozyme activity. In addition, a real-time polymerase chain reaction analysis revealed significant up-regulation of immune-related genes encoding major histocompatibility complex class I (MHC I), MHC II, CD4L, tumor necrosis factor-α, and interferon-γ after 24 and 48 h of challenge, compared with the levels stimulated by phosphate-buffered saline. Taken together, we conclude that rOmpNs may elicit immune responses and generate protection against E. ictaluri in channel catfish. Thus, rOmpNs could be promising vaccine candidates against E. ictaluri.     

Immunomodulatory Effects of Bacterial Lipopolysaccharide

Abstract

The most easily discernible and characteristic result of interaction of LPS with the immune system are the activation of B cells to divide and/or synthesize and secrete immunoglobulin. LPS as an antigen or carrier for chemically defined haptens induces a T independent antibody response, while antibody responses to unrelated T dependent antigens are modulated by LPS. Such regulation can be explained both by increased susceptibility of B cells to regulation after interaction with LPS, and increased activity of regulatory cells after their interaction with LPS. Studies of these mechanisms have exploited the availability of several strains of mice which are genetically poorly responsive to LPS and thus have helped to define the genetic basis of LPS responses. In addition, these studies have demonstrated the existence of other bacterial cell wall components which have some of the immunological properties of LPS but are not susceptible to the same genetic control. Future studies on the molecular nature of the interaction of LPS with target cells and the biochemical basis of cell triggering should yield interesting information on the cell biology of cell activation.     

Immunomodulatory Effects of Bacterial Lipopolysaccharide

The most easily discernible and characteristic result of interaction of LPS with the immune system are the activation of B cells to divide and/or synthesize and secrete immunoglobulin. LPS as an antigen or carrier for chemically defined haptens induces a T independent antibody response, while antibody responses to unrelated T dependent antigens are modulated by LPS. Such regulation can be explained both by increased susceptibility of B cells to regulation after interaction with LPS, and increased activity of regulatory cells after their interaction with LPS. Studies of these mechanisms have exploited the availability of several strains of mice which are genetically poorly responsive to LPS and thus have helped to define the genetic basis of LPS responses. In addition, these studies have demonstrated the existence of other bacterial cell wall components which have some of the immunological properties of LPS but are not susceptible to the same genetic control. Future studies on the molecular nature of the interaction of LPS with target cells and the biochemical basis of cell triggering should yield interesting information on the cell biology of cell activation.     

The two channel catfish intelectin genes exhibit highly differential patterns of tissue expression and regulation after infection with Edwardsiella ictaluri

Intelectins (IntL) are Ca(2+)-dependent secretory glycoproteins that play a role in the innate immune response. The mammalian IntL is also known as lactoferrin receptor (LfR) that is involved in iron metabolism. The objective of this study was to characterize the intelectin genes in both channel catfish and blue catfish, to determine their genomic organization and copy numbers, to determine their patterns of tissue expression, and to establish if they are involved in defense responses of catfish after bacterial infection. Two types of IntL genes have been identified from catfish, and IntL2 was completely sequenced. The genomic structure and organization of IntL2 were similar to those of the mammalian species and of zebrafish and grass carp, but orthologies cannot be established with mammalian IntL genes. The IntL genes are highly conserved through evolution. Sequence analysis also indicated the presence of the fibrinogen-related domain in the catfish IntL genes, suggesting their structural conservations. Phylogenetic analysis suggested the presence of at least two prototypes of IntL genes in teleosts, but only one in mammals. The catfish IntL genes exhibited drastically different patterns of expression as compared to those of the mammalian species, or even with the grass carp gene. The catfish IntL1 gene is widely expressed in various tissues, whereas the channel catfish IntL2 gene was mainly expressed in the liver. While the catfish IntL1 is constitutively expressed, the catfish IntL2 was drastically induced by intraperitoneal injection of Edwardsiella ictaluri and/or iron dextran. Such induction was most dramatic when the fish were treated with both the bacteria and iron dextran. While IntL1 was expressed in all leukocyte cell lines, no expression of IntL2 was detected in any of the leukocyte cell lines, suggesting that the up-regulated channel catfish IntL2 expression after bacterial infection may be a consequence of the initial immune response, and/or a downstream immune response rather than a part of the primary immune responses.     

Effect of Ichthyophthirius multifiliis parasitism on the survival, hematology and bacterial load in channel catfish previously exposed to Edwardsiella ictaluri

The effect of Ichthyophthirius multifiliis (Ich) parasitism on survival, hematology and bacterial load in channel catfish, Ictalurus punctatus, previously exposed to Edwardsiella ictaluri was studied. Fish were exposed to E. ictaluri 1?day prior to Ich in the following treatments: (1) infected by E. ictaluri and Ich at 2,500 theronts/fish; (2) infected by E. ictaluri only; (3) infected by Ich at 2,500 theronts/fish only; and (4) non infected control. Mortality was significantly higher in fish previously exposed to E. ictaluri and then infected by Ich (71.1?%). Mortalities were 26.7?%, 28.9?% and 0?% for fish infected by E. ictaluri only, by Ich only and non-infected control, respectively. Quantitative polymerase chain reaction demonstrated the presence of E. ictaluri in the brain, gill, kidney and liver of fish infected with E. ictaluri regardless of Ich parasitism. At day 8, E. ictaluri parasitized fish had significantly more bacteria present in the brain, gill and liver, with no bacteria detected in these organs in the E. ictaluri-only treatment, suggesting that the bacteria persisted longer in parasitized fish. Decreased red blood cells count and hematocrit in fish at days 8 and 19 after co-infection suggests chronic anemia. Lymphocyte numbers significantly decreased in all infected treatments versus the non-infected controls at days 2, 8 and 19. Lymphopenia suggests that lymphocytes were actively involved in the immune response. Bacterial clearance was probably influenced by the stress of parasitism and/or the mucosal response induced by ectoparasitic Ich that resulted in the higher mortality seen in the co-infected treatment.     

Strain-Dependent Activation of Monocytes and Inflammatory Macrophages by Lipopolysaccharide of Porphyromonas gingivalis

Porphyromonas gingivalis is one of the pathogens associated with periodontal diseases, and its lipopolysaccharide (LPS) has been suggested as a possible virulence factor, acting by stimulation of host cells to secrete proinflammatory mediators. However, recent studies have shown that P. gingivalis LPS inhibited some components of the inflammatory response. The present study was designed to test the hypothesis that there are strain-dependent variations in the ability of P. gingivalis LPS to elicit the host inflammatory response. By using LPS preparations from two strains of P. gingivalis, W50 and A7346, the responses of mouse macrophages and human monocytes were evaluated by measuring the secretion of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α). Both direct and indirect (priming) effects were investigated. LPS from Salmonella typhosa was used as a reference LPS. P. gingivalis A7436 LPS induced lower secreted levels of NO from the tested cells than S. typhosa LPS but induced similar levels of TNF-α. In contrast, LPS from P. gingivalis W50 did not induce NO or TNF-α secretion. Preincubation of macrophages with LPS from S. typhosa or P. gingivalis A7436 prior to stimulation with S. typhosa LPS upregulated NO secretion and downregulated TNF-α secretion, while preincubation with P. gingivalis W50 LPS enhanced both TNF-α and NO secretory responses. These results demonstrate that LPSs derived from different strains of P. gingivalis vary in their biological activities in vitro. The findings may have an impact on our understanding of the range of P. gingivalis virulence in vivo.     

In Situ Expression of Interleukin-10 in Noninflamed Human Gut and in Inflammatory Bowel Disease

A dysregulated secretion of contra-inflammatory cytokines such as interleukin-10 (IL-10) could play a role in the pathogenesis of inflammatory bowel disease (IBD). We have investigated the expression of IL-10 in gut tissues from patients with Crohn’s disease (CD), ulcerative colitis (UC) and controls by mRNA in situ hybridization and immunohistochemistry. Intestinal epithelial cells were found to express IL-10 mRNA and IL-10 protein in all of the tissues investigated without any major differences in the expression patterns. However, compared with noninflamed gut, significantly increased numbers of mononuclear cells (MNCs) producing IL-10 were present in inflamed gut, both in CD and UC. This cytokine was expressed most prominently by inflammatory infiltrates enriched in macrophages, although T cells seem to contribute to its production as well. Elevated IL-10 expression in IBD was mainly detected in the submucosa, whereas IL-10 production by lamina propria cells remained comparably low. In contrast, the expression of IL-1β mRNA was preferentially increased in the lamina propria. Our data argue against a general deficiency in IL-10 production in IBD. The results suggest rather that the local production of IL-10 by mucosal MNCs in IBD is insufficient to down-regulate pro-inflammatory cytokines such as IL-1β in the lamina propria compartment.