In vitro and in vivo interaction of macrophages from vaccinated and non-vaccinated channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri

Macrophages from catfish vaccinated with an Edwardsiella ictaluri vaccine and macrophages from non-vaccinated catfish were used in in vitro and in vivo studies with red-fluorescent E. ictaluri to assess phagocytic ability, reactive oxygen and nitric oxide production and bactericidal activity. In the in vitro experiment, macrophages were harvested from vaccinated and non-vaccinated fish and then exposed to red-fluorescent E. ictaluri. Results of this study showed that E. ictaluri can survive and replicate in macrophages from non-vaccinated catfish (relative percent killing, RPK, from 0.011 to 0.620 and from ?0.904 to 0.042 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) even in the presence of reactive oxygen and nitrogen products. Macrophages from vaccinated fish were significantly (p < 0.05) more efficient in killing E. ictaluri (RPK from 0.656 to 0.978 and from 0.011 to 0.620 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) and produced significantly (p < 0.05) higher amounts of ROS (10-fold increase) and nitrogen oxide (about 10-fold increase) than macrophages from non-vaccinated fish. In the in vivo experiment, vaccinated and non-vaccinated catfish were injected with red-fluorescent E. ictaluri to allow the interaction between macrophages and other components of the immune system. After 6 h, macrophages were harvested from the fish and seeded in glass chamber slides and bactericidal activity was measured in vitro. Results showed in vivo interaction of other components of the immune system enhanced bactericidal activity of macrophages from vaccinated fish. In another set of experiments, catfish were intraperitoneally injected with fluorescent bacteria opsonized with immune serum or non-opsonized and necropsied in the first 48 h after bacterial challenge to observe localization of E. ictaluri between vaccinated and non-vaccinated catfish. Vaccinated fish were able to control the dispersion of E. ictaluri in the body and red-fluorescent bacteria were observed only in the spleen, anterior and trunk kidney. In non-vaccinated fish E. ictaluri was able to replicate and invade all organs with the exception of the brain. We further determined that macrophages seeded with E. ictaluri could cause infection in non-vaccinated fish upon reinoculation with in vitro infected-macrophages. Overall, the results indicated that macrophages from vaccinated fish are activated and responsible for rapid clearance of infection upon re-exposure to virulent E. ictaluri.     

Susceptibility of European catfish (Silurus gianis) to Edwardsiella ictaluri

European catfish (Silurus glanis) were tested for their susceptibility to the bacterium Edwardsiella ictaluri. The LD₅₀ of E. ictaluri when injected into European catfish was 5.4 × 10⁶ compared to 7.1 times; 10⁴ for channel catfish (Ictalurus punctatus). E. ictaluri was isolated from dead and moribund European catfish and the bacterium was also detected in kidney smears by an indirect fluorescent antibody (FA) technique. The bacterium was not isolated or detected by FA from surviving fish 15 days after injection. No clinical signs of E. ictaluri infection were noted in European catfish, but these were prevalent in the channel catfish. These experiments indicate that under experimental conditions European catfish are not as susceptible to E. ictaluri as channel catfish.     

Proteome analysis of Edwardsiella ictaluri

Edwardsiella ictaluri is a facultative intracellular Gram‐negative bacterium causing enteric septicemia of catfish (ESC), the most prevalent disease affecting farm‐raised channel catfish in the United States. Despite its economic importance, studies addressing high‐throughput proteomics were not possible because of lack of comprehensive protein database. Here, we report the first high‐throughput proteomics analysis of E. ictaluri using 2‐D LC ESI MS/MS and 2‐DE MALDI TOF/TOF MS. Proteins identified in this study and predicted from the whole E. ictaluri genome were clustered into functional groups using clusters of orthologous groups (COG), and their subcellular locations were predicted. Possible functional relationships among proteins were determined using pathway analysis. The total number of unique E. ictaluri proteins identified using both 2‐D LC and 2‐DE approaches was 788, of which 15.48% (122) were identified by both methods while 78.43% (618) and 6.09% (48) were unique in 2‐D LC and 2‐DE, respectively. COG groupings and subcellular localizations were quite similar between our data set and proteins predicted from the whole genome. Twelve pathways were significantly represented in our dataset (p <0.05). Results from this study provided experimental evidence for many proteins that were predicted from the E. ictaluri genome annotation, and they should accelerate future functional and comparative studies aimed at understanding virulence mechanisms of this important pathogen.     

Edwardsiella piscicida type III protein EseJ suppresses apoptosis through down regulating type 1 fimbriae,which stimulate the cleavage of caspase‐8

The type III secretion system effector EseJ plays a regulatory role inside bacteria. It suppresses the adherence of Edwardsiella piscicida (E. piscicida) to host epithelial cells by down regulating type 1 fimbriae. In this study, we observed that more macrophages infected with ΔeseJ strain of E. piscicida detached as compared with those infected with the wild‐type (WT) strain. Terminal deoxynucleotidyl transferase dUTP nick‐end labelling (TUNEL) staining and cleaved caspase‐3 examination revealed that the detachment is due to increased apoptosis, suggesting that EseJ suppresses macrophage apoptosis. However, apoptosis inhibition by EseJ is not relative to a type III secretion system (T3SS) and is not related to EseJ's translocation. Since EseJ negatively regulates type 1 fimbriae, murine J774A.1 cells were infected with ΔeseJΔfimA or ΔeseJΔfimH strains. It was demonstrated that ΔeseJ stimulates macrophage apoptosis through type 1 fimbriae. Moreover, we found that infecting J774A.1 cells with the ΔeseJ strain increased levels of cleaved caspase‐8, caspase‐9, and caspase‐3, demonstrating that EseJ inhibits apoptosis through either an extrinsic or a combination of extrinsic and intrinsic pathways. Pre‐treatment of macrophages with caspase‐8 inhibitor prior to infection with the ΔeseJ strain decreased the levels of cleaved caspase‐8, caspase‐9, and caspase‐3, indicating that the ΔeseJ strain stimulates apoptosis, mainly through an extrinsic pathway by up regulating type 1 fimbriae. Zebrafish larvae or blue gourami fish infected with the ΔeseJ strain consistently exhibited higher apoptosis than those infected with the E. piscicida WT strain or ΔeseJΔfimA strain. Taken together, we revealed that the T3SS protein EseJ of E. piscicida inhibits host apoptosis, mainly through an extrinsic pathway by down regulating type 1 fimbriae.     

Tricarboxylic Acid Cycle and One-Carbon Metabolism Pathways Are Important in Edwardsiella ictaluri Virulence

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of channel catfish (ESC). The disease causes considerable economic losses in the commercial catfish industry in the United States. Although antibiotics are used as feed additive, vaccination is a better alternative for prevention of the disease. Here we report the development and characterization of novel live attenuated E. ictaluri mutants. To accomplish this, several tricarboxylic acid cycle (sdhC, mdh, and frdA) and one-carbon metabolism genes (gcvP and glyA) were deleted in wild type E. ictaluri strain 93-146 by allelic exchange. Following bioluminescence tagging of the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, ΔgcvP, and ΔglyA mutants, their dissemination, attenuation, and vaccine efficacy were determined in catfish fingerlings by in vivo imaging technology. Immunogenicity of each mutant was also determined in catfish fingerlings. Results indicated that all of the E. ictaluri mutants were attenuated significantly in catfish compared to the parent strain as evidenced by 2,265-fold average reduction in bioluminescence signal from all the mutants at 144 h post-infection. Catfish immunized with the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, and ΔglyA mutants had 100% relative percent survival (RPS), while E. ictaluri ΔgcvP vaccinated catfish had 31.23% RPS after re-challenge with the wild type E. ictaluri.     

Edwardsiella ictaluri Encodes an Acid-Activated Urease That Is Required for Intracellular Replication in Channel Catfish (Ictalurus punctatus) Macrophages

Genomic analysis indicated that Edwardsiella ictaluri encodes a putative urease pathogenicity island containing the products of nine open reading frames, including urea and ammonium transporters. In vitro studies with wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significantly tolerant of acid conditions (pH 3.0) but that urease activity is not required for acid tolerance. Growth studies demonstrated that E. ictaluri is unable to grow at pH 5 in the absence of urea but is able to elevate the environmental pH from pH 5 to pH 7 and grow when exogenous urea is available. Substantial production of ammonia was observed for wild-type E. ictaluri in vitro in the presence of urea at low pH, and optimal activity occurred at pH 2 to 3. No ammonia production was detected for the urease mutant. Proteomic analysis with two-dimensional gel electrophoresis indicated that urease proteins are expressed at both pH 5 and pH 7, although urease activity is detectable only at pH 5. Urease was not required for initial invasion of catfish but was required for subsequent proliferation and virulence. Urease was not required for initial uptake or survival in head kidney-derived macrophages but was required for intracellular replication. Intracellular replication of wild-type E. ictaluri was significantly enhanced when urea was present, indicating that urease plays an important role in intracellular survival and replication, possibly through neutralization of the acidic environment of the phagosome.Identification of virulence factors is vitally important to an understanding of the pathogenesis of Edwardsiella ictaluri and to the development of methods for controlling the spread of disease. Although the pathogenesis of E. ictaluri was reviewed in 1993 (28, 31), recent reports demonstrated that E. ictaluri is a facultative intracellular pathogen (3) and that a type III secretion system is required for intracellular survival and replication within channel catfish head kidney-derived macrophages (HKDM) (30). Using signature-tagged mutagenesis (STM) in an immersion challenge model for E. ictaluri, Thune et al. (30) identified 50 transconjugants carrying transposon insertions in genes required for survival and replication in the channel catfish host. Two of those mutants had insertions in genes encoding homologs of UreG and UreF, proteins that are essential for the production of an active urease enzyme in other bacteria (6, 10, 14, 26). UreG is a GTP-binding accessory protein that functions in energy-dependent assembly of the urease holoenzyme (19), while UreF is a urease accessory protein that functions in the generation or delivery of carbon dioxide to the urease metallocenter assembly site (19). Both the ureG and ureF mutant strains were further characterized in a competitive challenge with the wild-type (WT) parental strain and were confirmed to be significantly attenuated (30). The identification of two mutants with insertions in urease-associated genes suggests an important role for urease activity in E. ictaluri pathogenesis, despite the fact that E. ictaluri is urease negative in standard biochemical tests. Consequently, the objectives of this study are to characterize the E. ictaluri urease pathogenicity island (PAI), to evaluate conditions for E. ictaluri urease activity, and to establish a possible role for urease in E. ictaluri pathogenesis.     

The protective effect of EGF-activated ROS in human corneal epithelial cells by inducing mitochondrial autophagy via activation TRPM2

Oxidative stress is a major pathogenesis of some ocular surface diseases. Our previous study demonstrated that epidermal growth factor (EGF)-activated reactive oxygen species (ROS) could protect against human corneal epithelial cell (HCE) injury. In the present study, we aimed to explore the role and mechanisms of oxidative stress and mitochondrial autophagy in HCE cells subjected to scratch injury. CCK-8 assays, EdU assays, Western blot analysis, wound-healing assays, and flow cytometry were conducted to determine cell viability, proliferation, protein expression, cell apoptosis, and intracellular ROS levels, respectively. The results showed that EGF could promote damage repair and inhibit cell apoptosis in scratch injured HCE cells by upregulating ROS (**p < .01, ***p < .001). EGF also induced mitochondrial autophagy and alleviated mitochondrial damage. Interestingly, the combination of the mitochondrial autophagy inhibitor and mitochondrial division inhibitor 1 (MDIVI-1) with EGF could reduce cell proliferation, viability, and the ROS level (*p < .05, **p < .01, ***p < .001). Treatment using the ROS inhibitor N-acetyl- l -cysteine abrogated the increase in mitochondrial membrane potential after EGF treatment. (*p < .05). Taken together, these findings indicated that EGF plays an important role in HCE damage repair and could activate ROS to protect against HCE injury by inducing mitochondrial autophagy via activation of TRPM2.     

Effect of genistein and coenzyme Q10 in oxidative damage and mitochondrial membrane potential in an attenuated type II mucopolysaccharidosis cellular model

Mucopolysaccharidosis type II (MPS II) is an inborn error of the metabolism resulting from several possible mutations in the gene coding for iduronate-2-sulfatase (IDS), which leads to a great clinical heterogeneity presented by these patients. Many studies demonstrate the involvement of oxidative stress in the pathogenesis of inborn errors of metabolism, and mitochondrial dysfunction and oxidative stress can be related since most of reactive oxygen species come from mitochondria. Cellular models have been used to study different diseases and are useful in biochemical research to investigate them in a new promising way. The aim of this study is to develop a heterozygous cellular model for MPS II and analyze parameters of oxidative stress and mitochondrial dysfunction and investigate the in vitro effect of genistein and coenzyme Q10 on these parameters for a better understanding of the pathophysiology of this disease. The HP18 cells (heterozygous c.261_266del6/c.259_261del3) showed almost null results in the activity of the IDS enzyme and presented accumulation of glycosaminoglycans (GAGs), allowing the characterization of this knockout cellular model by MPS II gene editing. An increase in the production of reactive species was demonstrated (p < .05 compared with WT vehicle group) and genistein at concentrations of 25 and 50 µm decreased in vitro its production (p < .05 compared with HP18 vehicle group), but there was no effect of coenzyme Q10 in this parameter. There was a tendency for lysosomal pH change in HP18 cells in comparison to WT group and none of the antioxidants tested demonstrated any effect on this parameter. There was no increase in the activity of the antioxidant enzymes superoxide dismutase and catalase and oxidative damage to DNA in HP18 cells in comparison to WT group and neither genistein nor coenzyme q10 had any effect on these parameters. Regarding mitochondrial membrane potential, genistein induced mitochondrial depolarization in both concentrations tested (p < .05 compared with HP18 vehicle group and compared with WT vehicle group) and incubation with coenzyme Q10 demonstrated no effect on this parameter. In conclusion, it is hypothesized that our cellular model could be compared with a milder MPS II phenotype, given that the accumulation of GAGs in lysosomes is not as expressive as another cellular model for MPS II presented in the literature. Therefore, it is reasonable to expect that there is no mitochondrial depolarization and no DNA damage, since there is less lysosomal impairment, as well as less redox imbalance.     

Immune system of channel catfish: An overture on immunity to Edwardsiella ictaluri

Edwardsiella ictaluri is a gram negative rod that causes enteric septicemia of catfish (ESC). The E. ictaluri disease complex includes acute and chronic ESC. One of the most economically important diseases of channel catfish, Ictalurus punctatus, ESC is difficult to control. Although the bacterium is usually susceptible to antibiotics, resistant strains are emerging. Immunization is a more attractive approach to control. Vaccine efficacy is not well documented, in part due to a lack of basic information about immune system components and their interactions after E. ictaluri infection. Channel catfish vaccinated by immersion in E. ictaluri bacterin are only partly protected against ESC. Specific antibody responses are poor in the vaccinates. E. ictaluri infection causes both humoral and cell-mediated immune responses. A vaccine that stimulates both types of immunity may provide better ESC immunity. Diagnosis of E. ictaluri infection is accomplished by detection of specific antibody. Recently the discovery of an immunodominant E. ictaluri exoantigen is allowing the development of accurate and rapid diagnostic tests that can detect infection before clinical ESC occurs. Health management programs for food animals depend on serological tests to detect early infections and carriers; the value of these tests is now realized in the channel catfish industry. This review documents factors that influence the immune responses of channel catfish. These factors include the influence of water temperature, seasons, stressful conditions, diet, and E. ictaluri carrier status.     

Identification and characterization of bacteriophages specific to the catfish pathogen,Edwardsiella ictaluri

Aims: To identify and characterize bacteriophages specific for Edwardsiella ictaluri, the causative agent for enteric septicemia of catfish (ESC). Methods and Results: Two bacteriophages were isolated that infect Edw. ictaluri. They both produce clear plaques, have icosahedral heads with a non‐rigid tail, and are tentatively classified as Siphoviridae. Phages ΦeiDWF and ΦeiAU are dsDNA viruses with approximate genome sizes of 40 and 45 kb, respectively. The addition of 500 μmol l^?1 CaCl₂ enhanced phage titres. Both phages have a latent period of 40 min and an estimated burst size of 270. Every Edw. ictaluri strain tested was susceptible to phage infection with variable plaquing efficiencies and with no evidence of lysogeny, with no plaques detected on other bacterial species. Conclusions: Two unique bacteriophages were isolated that show host‐specificity for Edw. ictaluri, have temperature and metal cation‐dependent infectivity, and are tentatively placed within the family Siphoviridae. Significance and Impact of the Study: This is the first report of bacteriophages specific to Edw. ictaluri, an important fish pathogen affecting farm‐raised channel catfish. Initial characterization of these bacteriophages has demonstrated their potential use as biotherapeutic and diagnostic agents associated with ESC.     

Morphological variations among four riverine populations (Bangladesh) of endangered catfish Rita rita (Hamilton, 1822)

The morphological variation of the endangered catfish Rita rita was studied based on meristic, length–weight relationship (LWR), traditional (TRA) and truss (TRU) distance based morphometric analysis of 200 juveniles collected from the Old Brahmaputra, Jamuna, Meghna and Kangsa rivers. Data were subjected to Kruskal–Wallis test for meristic counts, t test for LWR, univariate ANOVA, multivariate discriminant function analysis and canonical analysis (CA) both for TRA and TRU morphometric characters to discriminate the populations. Kruskal–Wallis test denoted that only one (caudal fin rays) out of six meristic counts was significantly (p < .05) variable among the stocks. One sample t test clarified that all parameters of LWR were significantly (p < .05) differentiated in all stocks. Univariate ANOVA revealed significant (p < .001, p < .01, and p < .05) differences among the four stocks in 14 of 15 standardized TRA traits and 17 of 25 TRU distances, respectively. The first discriminant function (DF1) explained 59.4% and 68.0% of total variance, while 30.6% and 23.1% by DF2, 10.0% and 8.9% by DF3 for TRA and TRU features, respectively among the stocks. CA showed a clear separation of the stocks both for TRA and TRU measurements. The overall random assignment of individuals into their original groups was high as 78.5% for TRA and 78.0% for TRU characters, respectively, indicating that all the four stocks are highly divergent from each other. The results exposed existence of four distinct phenotypic stocks of R. rita in the tested rivers.     

Humoral immune responses of channel catfish (Ictalurus punctatus) fry and fingerlings exposed toEdwardsiella ictaluri

The ability of channel catfish to develop antibody responses to Edwardsiella ictaluri was evaluated. Fish were produced and reared under specific pathogen free (SPF) conditions. At the ages of 1, 2, 3 and 4 weeks and 2, 3, 4, 5, and 6 months post-hatch, a group of these naive fish were given a primary immersion exposure to E. ictaluri (mean doses of 6·4×10⁴ cfu ml⁻¹of tank water). Each group received a secondary immersion exposure 4 weeks after the primary exposure and were sampled 2 weeks after each exposure. Control groups were exposed to sterile culture medium. Specific antibody titres were first detected in fish exposed at 4 weeks post-hatch at an average weight of 85 mg. A secondary response was first demonstrated by fry that received a primary exposure at 4 weeks post-hatch and a secondary exposure at 8 weeks post-hatch. However, a true boosting effect was first demonstrated in fish that received their primary exposure at 2 months of age. Fish given a primary exposure before 4 weeks of age and a secondary exposure failed to produce a significant antibody response, even though they were the same age at secondary exposure as fish that produced strong antibody responses upon primary exposure. This phenomenon suggests that immunological tolerance was induced and indicates that channel catfish may not be capable of generating a humoral immune response before four weeks of age.     

Effect of phototherapy on oxidant/antioxidant status: a randomized controlled trial

In order to evaluate the effect of different types of phototherapy on oxidant/antioxidant status in hyperbilirubinemic neonates, an interventional randomized control trial was conducted on 120 neonates ≥35 weeks’ gestational age with indirect hyperbilirubinemia reaching phototherapy level. This study is registered with ClinicalTrials.gov as NCT03074292. Neonates were assigned to three groups; 40 neonates received conventional phototherapy, 40 received intensive phototherapy and 40 received blue light-emitting diodes (LED) phototherapy. Complete blood count (CBC), total serum bilirubin (TSB), total antioxidant capacity (TAC), malondialdehyde (MDA), nitric oxide (NO), copper (Cu), zinc (Zn), and iron (Fe) levels were measured before and 24?hours after phototherapy. TSB decreased postphototherapy in all three groups (p < .05 for all), with significantly lower levels following intensive and LED phototherapy compared to conventional phototherapy (p < .05 for both). TAC decreased postphototherapy in the three groups (p < .05 for all). MDA and NO increased postphototherapy (p < .05 for all), with the intensive phototherapy group having the highest levels followed by the conventional while LED phototherapy group showed the lowest levels in comparison to the other groups (p < .05). Cu, Zn and Fe increased postphototherapy in all three groups (p < .05 for all). Positive correlations were found between postphototherapy TSB with TAC, Cu and Zn (p < .05) and negative correlations with MDA, NO and Fe (p < .05) among neonates of the 3 studied groups. In conclusion, different photo therapies have an impact on oxidant/antioxidant balance and are associated with increased oxidative stress markers with the LED phototherapy being the safest.     

Depleted folate pool and dysfunctional mitochondria associated with defective mitochondrial folate proteins sensitize Chinese ovary cell mutants to tert-butylhydroperoxide-induced oxidative stress and apoptosis

The functional role of mitochondrial (mt) folate-associated proteins in mammalian cells is not clearly understood. We investigated the respiratory function and apoptosis phenotype of Chinese hamster ovary (CHO) mutant cells with defective mt serine hydroxymethyltransferase (SHMT) activities (glyA) or with defective mt folate transporter (glyB) in the absence/presence of oxidant challenge. The mechanisms underlying their aberrant phenotypes were explored. Compared with CHOK1 wild-type cells, both mutants carried dysfunctional mitochondria with reduced respiratory complex IV activity and depolarized mt membrane potential (P<.05). Elevated superoxide levels and accumulated mtDNA large deletions were observed in glyB in association with a depleted compartmental folate pool (P<.05). tert-Butylhydroperoxide (tBH) treatment at 50 μM for 72 h significantly depleted mt and cytosolic folate levels, impaired antioxidant defenses, and aggravated mt oxidative dysfunction in both mutants (P<.05), more severely in glyB. Only tBH-treated glyB cells displayed an elevated ratio of mt Bax/Bcl-2, activation of procaspases 9 and 3, and apoptosis promotion. The apoptotic phenotype of tBH-treated glyB could be partially corrected by folate supplementation (10–1000 μM), which enriched compartmental folate levels, restored antioxidant defenses, eliminated mt oxidative injuries, and normalized mt membrane function. Our data identify previously unrecognized roles of mt folate-associated proteins in the protection of mitochondria against oxidative insults. Defective mt folate transporter sensitized glyB cells to elevated oxidative stress and tBH-induced apoptosis, partly mediated by depleted compartmental folate and mt dysfunction. Defective mt SHMT sensitized glyA to respiratory dysfunction and tBH-induced oxidative injury without apoptosis promotion.     

Identification of Bacillus Strains for Biological Control of Catfish Pathogens

Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10⁷ CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05). A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.     

Effective generation of reactive oxygen species in the mycobacterial phagosome requires K+ efflux from the bacterium

Efficient killing of mycobacteria by host macrophages depends on a number of mechanisms including production of reactive oxygen species (ROS) by the phagosomal NADPH oxidase, NOX2. Survival of pathogenic mycobacteria in the phagosome relies on the ability to control maturation of the phagosome such that it is biologically and chemically altered in comparison to phagosomes containing non‐pathogenic bacteria. In this study we show that the action of NOX2 to produce ROS in the mycobacterial phagosome is paradoxically dependent on a bacterial potassium transporter. We show that a Mycobacterium bovis BCG mutant (BCGΔkef), deficient in a Kef‐type K+ transporter, exhibits an increased intracellular survival phenotype in resting and activated macrophages, yet retains the ability to inhibit phagosome acidification, and does not show increased resistance to acidic conditions or ROS. Addition of a ROS scavenger replicates this phenotype in macrophages infected with wild‐type BCG, and the production of ROS by macrophages infected with BCGΔkef is substantially decreased compared with those infected with wild‐type BCG. Our results suggest that increased intracellular survival of BCGΔkef is mediated by inducing a decreased macrophage oxidative burst, and are consistent with Kef acting to alter the ionic contents of the phagosome and promoting NOX2 production of ROS.     

Attenuation of a virulent Aeromonas hydrophila with novobiocin and pathogenic characterization of the novobiocin‐resistant strain

Aim

To determine whether novobiocin resistance strategy could be used to attenuate a virulent Aeromonas hydrophila AH11P strain and to characterize the growth and pathogenic differences between the novobiocin‐resistant strain and its virulent parent strain AH11P.

Methods and Results

A novobiocin‐resistant strain AH11NOVO was obtained from a virulent Aer. hydrophila strain AH11P through selection of resistance to novobiocin. AH11NOVO was found to be avirulent to channel catfish (Ictalurus punctatus), whereas AH11P was virulent. When AH11NOVO vaccinated channel catfish were challenged with AH11P at 14 days postvaccination, relative per cent of survival of vaccinated fish was 100%. The cell proliferation rate of AH11NOVO was found to be significantly (P < 0·05) less than that of AH11P. In vitro motility assay revealed that AH11NOVO was nonmotile, whereas AH11P was motile. AH11NOVO had significantly (P < 0·05) lower in vitro chemotactic response to catfish mucus than that of AH11P. Although the ability of AH11NOVO to attach catfish gill cells was similar to that of AH11P, the ability of AH11NOVO to invade catfish gill cells was significantly (P < 0·05) lower than that of AH11P.

Conclusions

The novobiocin‐resistant AH11NOVO is attenuated and different from its parent AH11P in pathogenicity.

Significance and Impact of the Study

The significantly lower chemotactic response and invasion ability of AH11NOVO compared with that of its virulent parent strain AH11P might shed light on the pathogenesis of Aer. hydrophila.     

Cytostatic/Cytotoxic Effects of 5‐Fluorouridine Nucleolipids on Colon,Hepatocellular, and Renal Carcinoma Cells: in vitro Identification of a Potential Cytotoxic Multi‐Anticancer Drug

The insufficient penetration through the cell membranes is one of the major drawbacks of chemotherapeutics such as 5‐fluorouracil (5‐FU; 1 ). To improve the penetration, a useful strategy is the attachment of lipophilic moieties. Thus, we have synthesized a series of nucleolipid derivatives of 5‐fluorouridine (5‐FUrd; 2a ), carrying lipophilic moieties at N(3) and/or at the 2′,3′‐O position, i.e., 3a, 3b, 4 – 7 , and tested their cytostatic/cytotoxic activities towards three carcinoma cell lines (colon (HT‐29), hepatocellular (HepG2), and renal (RENCA)) in comparison with 5‐FU ( 1 ) and 5‐FUrd ( 2a ). After 48 h of incubation, four derivatives, 3a, 3b, 5 , and 7 , showed inhibitory effects on the survival of HT‐29, HepG2, and RENCA cells. Additionally, to differentiate between anticancer and side‐effects, we tested the cytotoxicity of the derivatives in human macrophages. Interestingly, the derivatives 4, 5 , and 6 did not exhibit any effects on survival of THP‐1 macrophages. Furthermore, we investigated the apoptosis induction of compound 1 and 2a , and the above‐mentioned derivatives in HT‐29 cells. Derivative 5 showed the highest significant (p<0.05; p<0.01) increase of the apoptosis at 80 μM after 2‐h or 4‐h treatment, as well as after 6‐h incubation at 40 μM (p<0.05). Real‐time PCR revealed that 40‐μM derivative 5 showed a 1.8‐fold increase of the pro‐apoptotic caspase‐3 gene and a twofold significant increase (p<0.01 and p<0.05 vs. control and 1 , resp.) of the tumor suppressor TP53 gene, whereas the other compounds did not show any effect. We demonstrated that some 5‐FUrd derivatives such as compound 5 are more effective than 5‐FU or 5‐FUrd concerning a cytotoxic (vs. cytostatic (5‐FU, 5‐FUrd)) effect on different cancer cell lines, but without cytotoxic side‐effects on differentiated macrophages. Thus, compound 5 is suggested as a novel potent cytotoxic multi‐anti‐cancer drug.     

Focal adhesion kinase regulates tractional collagen remodeling,matrix metalloproteinase expression,and collagen structure,which in turn affects matrix-induced signaling

Focal adhesion kinase (FAK) is critical for collagen expression but its regulation of collagen remodeling is not defined. We examined the role of FAK in the degradation and reorganization of fibrillar collagen. Compared with wild-type (WT) mouse embryonic fibroblasts, FAK null (FAK^−/−) fibroblasts generated twofold (p < .0001) higher levels of ¾ collagen I fragment and expressed up to fivefold more membrane-type matrix metalloproteinase (MMP). When plated on stiff collagen substrates, compared with WT, FAK^−/− cells were smaller (threefold reduced cell surface area; p < .0001) and produced fivefold fewer cell extensions (p < .0001) that were 40% shorter (p < .001). When cultured on soft collagen gels (stiffness of ~100 Pa) for 6–48 hr, cell spreading and cell extension formation were reduced by greater than twofold (p < .05 and p < .0001, respectively) while collagen compaction and alignment were reduced by approximately 30% (p < .0001) in FAK^−/− cells. Similar results were found after treatment with PF573228, a FAK inhibitor. Reconstitution of FAK^−/− cells with FAK mutants showed that compared with WT, cell extension formation was reduced twofold (p < .0001) in the absence of the kinase domain and sixfold (p < .0001) with a Y397F mutant. Enhanced collagen degradation was exhibited by the mutants (~threefold increase; p < .0001 of ¾ collagen fragments without kinase domain or Y397F mutant; p < .01). Compared with FAK^+/+ cells, matrices produced by FAK^−/− cells generated higher levels of β₁ integrin activation (p < 0.05), extracellular-signal-regulated kinase (ERK) phosphorylation, and production of ¾ collagen I fragment by human gingival fibroblasts. Collectively these data indicate that (a) the kinase activity of FAK enhances collagen remodeling by tractional forces but inhibits collagen degradation by MMPs; (b) FAK influences the biological activity of fibroblast-secreted extracellular matrices, which in turn impacts β1 integrin and ERK signaling, and collagen degradation.