Transcriptional analysis of metabolic and virulence genes associated with biofilm formation in Piscirickettsia salmonis strains.

Piscirickettsia salmonis is a facultative intracellular bacterium that generates piscirickettsiosis affecting salmonids in Chile. The bacterium has the adaptability to survive in the marine environment under multiple stressful conditions. In this sense, this work focused on the analysis of a gene battery associated with biofilm formation under different culture conditions and on the adaptability of this biofilm to different media. The results indicated that the strains LF-89, IBM-034 and IBM-040 were strong biofilm producers, evidencing adaptability to the media by increasing the amount of biofilm through successive growths. Transcript levels of six genes described in various bacteria and P. salmonis, considered to have metabolic functions, and playing a relevant role in biofilm formation, were analyzed to evaluate bacterial functionality in the biofilm. The genes mazE-mazF, implicated in biofilm and stress, were markedly overexpressed in the biofilm condition in the three strains. For its part, gene gltA, an indicator of metabolic activity and related to virulence inhibition in Salmonella typhimurium, also seems to restrain the pathogenesis process in P. salmonis by inhibiting the expression of the virulence-associated genes liso and tcf. Finally, the expression of the glnA gene suggests the use of glutamine as an essential element for the growth of the biofilm.

Identification of genomic islands in Chilean Piscirickettsia salmonis strains and analysis of gene expression involved in virulence.

Piscirickettsia salmonis, an agent of Piscirickettsiosis, is the cause of major losses in the Chilean salmon industry. We identified, characterized and bioinformatically analysed genomic islands in field strains of P. Salmonis, using the bioinformatic software PIPS, that uses the characteristics of the islands of pathogenicity to identify them. We analysed nine partially sequenced genomes in different new field strains, and compared them with the LF-89 (Type strain) genome, selecting a genomic island present in all of them. We then evaluated the relative expression of three genes present in that island. From the obtained results, we conclude that the expression of the tcf gene is directly proportional to the cytopathogenicity in vitro of the bacteria; the product of the dnsa gene could contribute to its pathogenicity, but would be potentiated by one or more factors. The product of the gene liso is necessary for the virulence process and could have functions in early stages of infection. Regarding the strains, the IBM-040 strain showed a significant increase in the expression of all the genes in the study. Contrarily, LF-89 only presented a significant increase in expression of the gene liso, which correlates with the cytopathogenicity in vitro observed in the SHK-1 cells.

Search and analysis of genes involved in antibiotic resistance in Chilean strains of Piscirickettsia salmonis.

Piscirickettsia salmonis is the pathogen causing Piscirickettsiosis. For treatment, the industry mainly uses oxytetracycline and florfenicol, so it is essential to understand the degree of susceptibility of this pathogen to these drugs. But this is still unknown for a large number of P. salmonis strains, as are the molecular mechanisms responsible for greater or lesser susceptibility. However, genes that confer resistance to these antimicrobials have been reported and characterized for this and other bacterial species, among which are membrane proteins that take out the drug. Our results identified differences in the degree of susceptibility to both antibiotics among different Chilean isolated of these bacteria. We analysed 10 available genomes in our laboratory and identified ~140 genes likely to be involved in antibiotic resistance. We analysed six specific genes, which suggests that some of them would eventually be relevant in conferring resistance to both antibiotics, as they encode for specific transporter proteins, which increase the number of transcripts when grown in media with these antibiotics. Our results were corroborated with EtBr permeability analysis, which revealed that the LF-89 strain accumulates this compound and has a reduced capacity to expulse it compared with the field strains.

Immunostimulatory effects of prolactin on TLR1 and TLR5M in SHK-1 cells infected with Piscirickettsia salmonis.

The innate immune system is the first line of defense against infection by pathogens. It consists of various elements, including Toll-like receptors (TLRs), which recognize molecular patterns associated with pathogens and trigger the immune response, through activation of important transcription factors such as NF-κB, which are usually found sequestered in the cytoplasm by IκBα until it receives the release signal. Piscirickettsia salmonis causes piscirickettsiosis or salmonid rickettsial septicemia, a disease of great importance in Chile, representing 79.4% of the secondary mortality in important species such as Salmo salar, which is reflected in the Chilean economy. Prolactin (PRL) is a peptide hormone which has immunomodulating functions in mammals and some fish. Olavarría et al. (2010, J Immunol 185:3873-3883) determined its ability to increase the respiratory burst, its relationship with the JAK/STAT pathway, and the expression of interleukin IL-1ß in Sparus aurata. Therefore, the present study was intended to establish a possible correlation and modulation between the signal transduction pathway of PRL (JAK/STAT), the pathways of NF-κB, and TLRs, in an infection caused by P. salmonis in salmon head kidney (SHK­1) cells of S. salar. Stimulus with native PRL from S. salar was performed, and gene expression was analyzed for IL-1ß, IκBα, TLR1, and TLR5M (membrane-bound form). In addition, the effect of PRL in the nuclear translocation of the transcription factor NF-κB and the possible involvement of JAK2 were analyzed by using a pharmacological inhibitor of this kinase. The results show a positive modulation of PRL in all analyzed genes and a significant increase in the translocation of NF-κB, recording a maximum at 2 h post-treatment, supporting the stimulatory hypothesis of PRL.

Molecular cloning of Salmo salar Toll-like receptors (TLR1, TLR22, TLR5M and TLR5S) and expression analysis in SHK-1 cells during Piscirickettsia salmonis infection.

In fish, the innate immune system is the primary response against infection. Toll-like receptors (TLRs) recognize pathogens through pathogen-associated molecular patterns (PAMPs), and some target molecules of TLRs are homologous between fish and mammals. Piscirickettsia salmonis is one of the main pathogens affecting the salmon industry in Chile. Better knowledge of mechanisms underlying its invasive capacity and recognition of target cells is crucial for vaccine development. Therefore, Salmo salar L. TLR1, TLR22, membrane TLR5M and soluble TLR5S sequences were cloned, and expression kinetics were analysed by RT-qPCR in salmon head kidney cells (SHK-1) infected with three different P. salmonis preparations: alive, formaldehyde treated, extract. Clearly, all analysed TLRs were expressed and transcription level changes were revealed at 2 hpi, 12 or 16 hpi and 24 hpi depending on P. salmonis infection scheme. Increased IL1-beta expression confirmed TLR pathway response. Furthermore, significant expression modulations of several members of the TLR pathway in this in vitro model suggest that P. salmonis extract rather than formaldehyde-inactivated bacteria might strengthen the salmon immune system.

Immunostimulatory effect of salmon prolactin on expression of Toll-like receptors in Oncorhynchus mykiss infected with Piscirickettsia salmonis.

In aquaculture, antibiotics are the traditional treatment used against bacterial infections. However, their use has increasingly come into question given their effects on fish and, possibly, on human health. Consequently, there is interest in developing alternative treatments aimed at stimulating the innate immune response of fish, which is the first line of defense against pathogens. In relation to this, the Toll-like receptors (TLR) aid in the selective identification of pathogens. The present study evaluated immunostimulatory activity of prolactin (PRL) hormone on expression levels of TLR1, 9, and 22, MyD88, and IL-1ß during in vitro infection with the fish pathogen Piscirickettsia salmonis, in primary cultures of Oncorhynchus mykiss head kidney cells. Results indicated that PRL increased expression of TLRs and MyD88 during the first hours of bacterial infection, while a constant increase in expression was found for IL-1ß. These findings suggest that PRL indirectly modulates expression of TLRs by activating expression of suppressors of cytokine signaling, thereby regulating immune response over long periods of time during bacterial infection.

Identification of the clpB and bipA genes and an evaluation of their expression as related to intracellular survival for the bacterial pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is the pathogen responsible for salmonid rickettsial septicemia (SRS), a disease that affects a wide variety of marine cultivated fish species and causes economic losses for the aquaculture industry worldwide. Many in vitro studies have reported on the capacity of this microorganism to replicate in the interior of cytoplasmic vesicles from varied fish cell lines. However, the mechanisms used by this bacteria to survive, replicate, and propagate in cell lines, especially in macrophages and monocytes, are unknown. A number of studies have described the diverse proteins in pathogens such as Legionella pneumophila, Coxiella burnetii, and Francisella tularensis which allow these to evade the cellular immune response and replicate in the interior of macrophages in different hosts. Some of these proteins are the virulence factor BipA/TypA and the heat shock protein ClpB, both of which have been widely characterized. The results of the current study present the complete coding sequence of the genes clpB and bipA from the P. salmonis genome. Moreover, the experimental results suggest that during the infectious process of the SHK-1 cellular line in P. salmonis, the pathogen significantly increases the expression of proteins ClpB and BipA. This would permit the pathogen to adapt to the hostile conditions produced by the macrophage and thus evade mechanisms of cellular degradation while facilitating replication in the interior of this salmon cell line.

Glandular kallikrein in the innate immune system of Atlantic salmon (Salmo salar).

Glandular Kallikrein is a serine-protease with trypsin-like activity and is able to generate bioactive peptides from inactive precursors. We have evaluated the presence of this protease in the different organs of the Atlantic salmon (Salmo salar). The results clearly indicate that GK and PRL are generated in the same pituitary cells based on a co-localization by confocal microscopy. Based on probed cross-reactivity between C. striata and C. carpio glandular anti-GK antibodies, we used a homologous antibody to detect the presence of GK in several salmon tissues. We have evaluated the GK expression in healthy and defied fish. P. salmonis and V. ordalii. The GK immunoreaction in organs such as leukocytes, gills and skin is considerably increased in defied fish compared to healthy fish. This increase was present in the cells of the excretory kidney and in the intercellular tissue, where the development of hematopoietic and lymphocytic lines in fish take place. One of the most interesting organs to study was the skin, bearing in mind that this is a primary barrier to all pathogens. The skin of the defied fish exhibited an increase in immunoreactivity for glandular kallikrein similar to the protease found in mucus. An immunoreactive tissue kallikrein-like protein was identified and partially separated by perfusion chromatography. Enzymatic activity of salmon muscle prokallikrein was determined before and after trypsin activation. Kallikrein activity was characterized with respect to their ability to cleave the chromogenic leaving group, p-nitroanilide, from the peptidyl kallikrein and trypsin substrate. These findings constitute a important contribution to reveal the role of kallikrein in the innate immune system of fish.

Glandular kallikreins in the teleost Cyprinus carpio: tissue distribution, possible involvement in prolactin processing, and effect of 17 beta-estradiol in vivo.

We examined glandular kallikrein (GK), a putative prolactin processing protease, in the teleost Cyprinus carpio. When employing an anti-Centropristis striata GK antibody proteins of 39 kDa in muscle, 52 kDa in gill, 52 kDa in kidney, and two proteins of 46 and 72 kDa in pituitary gland were detected. Immunoreactive kallikreins were recognized in intermuscle cell tissue, epithelial gill cells, apical region of tubular cells, and prolactin producing lactotrophs in pituitary gland, suggesting a osmoregulatory role for this enzyme. We found three prolactin (PRL) variants using anti-tilapia PRL antibodies, in pituitary gland 23 and 16 kDa, and in plasma 23 and 22 kDa forms. Clearly co-localization of GK and PRL in lactotrophs could be demonstrated. In winter-acclimatized male carp, where the pituitary PRL level is low, 17beta-estradiol treatment increased PRL but not GK immunoreactivity. In contrast to GK and PRL co-regulation by estrogen in mammalian pituitary gland, no similar effect on immunoreactive PRL and GK was observed in the ichtyc pituitary. No changes in GK immunostaining occurred in gill or muscle tissue in response to estrogen treatment. These results, taken with the observation of significantly increased GK immunoreactivity in the apical region of kidney tubular cells in estrogen treated male carp, indicate that the regulation of GK expression in pituitary and kidney could be different in fish with respect to mammals.

Differential effects of quinaprilat and enalaprilat on endothelial function of conduit arteries in patients with chronic heart failure.

BACKGROUND: Chronic heart failure (CHF) is associated with endothelial dysfunction, including impaired flow-dependent (endothelium-mediated) dilation (FDD). We have previously shown that ACE inhibition improves endothelium-mediated vasodilation in healthy volunteers. The present study was designed to determine whether ACE inhibition improves the impaired FDD in patients with CHF. Because their affinity to tissue ACE may influence the ability of ACE inhibitors to affect endothelial function, we compared the effects of quinaprilat (high affinity to tissue ACE) and enalaprilat (low affinity to tissue ACE) on FDD in patients with CHF. METHODS AND RESULTS: High-resolution ultrasound and Doppler were used to measure radial artery diameter and blood flow in patients with CHF. The effects of intra-arterial infusion of quinaprilat 1.6 microg/min (n=15) and enalaprilat 5 microg/min (n=15) were determined at rest and during reactive hyperemia (causing endothelium-mediated dilation) before and after N-monomethyl-L-arginine (L-NMMA) to inhibit endothelial synthesis of nitric oxide. Quinaprilat improved FDD by >40% (10.2+/-0.6% versus 6.9+/-0.6%; P<0.01), whereas enalaprilat had no effect. In particular, the part of FDD mediated by nitric oxide (ie, inhibited by L-NMMA) was increased by >100% with quinaprilat (5.6+/-0.5% versus 2.5+/-0.5%; P<0.01). Enalaprilat had no effect on FDD even when it was infused twice in the same dose (5 microg/min) and up to 30 microg/min. The effect of sodium nitroprusside on radial artery diameter and blood flow was similar in patients treated with quinaprilat, enalaprilat, and placebo. CONCLUSIONS: Quinaprilat improves FDD in patients with CHF as the result of increased availability of nitric oxide, whereas enalaprilat does not. This observation suggests that intrinsic differences exist between quinaprilat and enalaprilat that determine the ability to improve endothelium-mediated vasodilation, ie, their different affinity to tissue ACE.