Proteomic analysis reveals Renibacterium salmoninarum grown under iron-limited conditions induces iron uptake mechanisms and overproduction of the 57-kDa protein.

Renibacterium salmoninarum, a slow-growing facultative intracellular pathogen, is the causative agent of bacterial kidney disease, a chronic, progressive and granulomatous infection that threatens farmed and wild salmonids worldwide. Pathogenic R. salmoninarum colonizes tissues and invades the host through cell surface-associated and secreted proteins. While correlations between iron acquisition genes and virulence have been demonstrated in vitro, these mechanisms have not undergone proteomic characterization. The present study applied a proteomic approach to elucidate the differences between the virulent Chilean R. salmoninarum H-2 strain and the type strain ATCC 33209T . Analyses were conducted under normal (control) and iron-limited conditions (DIP) emulating the host environment. Interestingly, strain H-2 apparently responded better to the iron-limited condition-for example, only this strain presented a significantly enriched iron ion homeostasis pathway. Furthermore, key virulence factors related to an iron-limited environment were more abundant in strain H-2. Importantly, the lack of iron favoured the expression of the 57-kDa protein in strain H-2, the principal virulence factor for R. salmoninarum. Our findings can be employed in the design and development of treatments targeted to iron uptake mechanisms (e.g. siderophore synthesis or haem uptake), which represents a promising therapeutic approach for treating this persistent fastidious bacterium.

Renibacterium salmoninarum iron-acquisition mechanisms and ASK cell line infection: Virulence and immune response.

Renibacterium salmoninarum is the aetiological agent of bacterial kidney disease (BKD) in salmonid farms. This pathogen possesses at least three iron-acquisition mechanisms, but the link between these mechanisms and virulence is unclear. Therefore, this study used RT-qPCR to assess the effects of normal and iron-limited conditions on iron-uptake genes controlled by IdeR and related to iron acquisition in Chilean R. salmoninarum strain H-2 and the type strain DSM20767T . Further evaluated was the in vitro immune-related response of the Atlantic Salmon Kidney (ASK) cell line, derived from the primary organ affected by BKD. R. salmoninarum grown under iron-limited conditions overexpressed genes involved in haemin uptake and siderophore transport, with overexpression significantly higher in H-2 than DSM20767T . These overexpressed genes resulted in higher cytotoxicity and an increased immune response (i.e., TNF-α, IL-1ß, TLR1 and INF-γ) in the ASK cell line. This response was significantly higher against bacteria grown under iron-limited conditions, especially H-2. These observations indicate that iron-acquisition mechanisms are possibly highly related to the virulence and pathogenic capacity of R. salmoninarum. In conclusion, treatments that block iron-uptake mechanisms or siderophore synthesis are attractive therapeutic approaches for treating R. salmoninarum, which causes significant aquaculture losses.

Comparative Genomic Analysis of Two Chilean Renibacterium salmoninarum Isolates and the Type Strain ATCC 33209T.

Renibacterium salmoninarum, a slow-growing facultative intracellular pathogen belonging to the high C + G content Actinobacteria phylum, is the causative agent of bacterial kidney disease, a progressive granulomatous infection affecting salmonids worldwide. This Gram-positive bacterium has existed in the Chilean salmonid industry for >30 years, but little or no information is available regarding the virulence mechanisms and genomic characteristics of Chilean isolates. In this study, the genomes of two Chilean isolates (H-2 and DJ2R) were sequenced, and a search was conducted for genes and proteins involved in virulence and pathogenicity, and we compare with the type strain ATCC 33209 T genome. The genome sizes of H-2 and DJ2R are 3,155,332 bp and 3,155,228 bp, respectively. They genomes presented six ribosomal RNA, 46 transcription RNA, and 25 noncodingRNA, and both had the same 56.27% G + C content described for the type strain ATCC 33209 T. A total of 3,522 and 3,527 coding sequences were found for H-2 and DJ2R, respectively. Meanwhile, the ATCC 33209 T type strain had 3,519 coding sequences. The in silico genome analysis revealed a genes related to tricarboxylic acid cycle, glycolysis, iron transport and others metabolic pathway. Also, the data indicated that R salmoninarum may have a variety of possible virulence-factor and antibiotic-resistance strategies. Interestingly, many of genes had high identities with Mycobacterium species, a known pathogenic Actinobacteria bacterium. In summary, this study provides the first insights into and initial steps towards understanding the molecular basis of antibiotic resistance, virulence mechanisms and host/environment adaptation in two Chilean R. salmoninarum isolates that contain proteins of which were similar to those of Mycobacterium. Furthermore, important information is presented that could facilitate the development of preventive and treatment measures against R. salmoninarum in Chile and worldwide.

HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress.

BACKGROUND: Heat stress proteins are implicated in stabilizing and refolding denatured proteins in vertebrates and invertebrates. Members of the Hsp70 gene family comprise the cognate heat shock protein (Hsc70) and inducible heat shock protein (Hsp70). However, the cDNA sequence and the expression of Hsp70 in the Antarctic sea urchin are unknown. METHODS: We amplified and cloned a transcript sequence of 1991 bp from the Antarctic sea urchin Sterechinus neumayeri, experimentally exposed to heat stress (5  and 10 °C for 1, 24 and 48 h). RACE-PCR and qPCR were employed to determine Hsp70 gene expression, while western blot and ELISA methods were used to determine protein expression. RESULTS: The sequence obtained from S. neumayeri showed high identity with Hsp70 members. Several Hsp70 family features were identified in the deduced amino acid sequence and they indicate that the isolated Hsp70 is related to the cognate heat shock protein type. The corresponding 70 kDa protein, called Sn-Hsp70, was immune detected in the coelomocytes and the digestive tract of S. neumayeri using a monospecific polyclonal antibody. We showed that S. neumayeri do not respond to acute heat stress by up-regulation of Sn-Hsp70 at transcript and protein level. Furthermore, the Sn-Hsp70 protein expression was not induced in the digestive tract. CONCLUSIONS: Our results provide the first molecular evidence that Sn-Hsp70 is expressed constitutively and is non-induced by heat stress in S. neumayeri.

HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress

Abstract Background: Heat stress proteins are implicated in stabilizing and refolding denatured proteins in vertebrates and invertebrates. Members of the Hsp70 gene family comprise the cognate heat shock protein (Hsc70) and inducible heat shock protein (Hsp70). However, the cDNA sequence and the expression of Hsp70 in the Antarctic sea urchin are unknown. Methods: We amplified and cloned a transcript sequence of 1991 bp from the Antarctic sea urchin Sterechinus neumayeri, experimentally exposed to heat stress (5 and 10 °C for 1, 24 and 48 h). RACE-PCR and qPCR were employed to determine Hsp70 gene expression, while western blot and ELISA methods were used to determine protein expression. Results: The sequence obtained from S. neumayeri showed high identity with Hsp70 members. Several Hsp70 family features were identified in the deduced amino acid sequence and they indicate that the isolated Hsp70 is related to the cognate heat shock protein type. The corresponding 70 kDa protein, called Sn-Hsp70, was immune detected in the coelomocytes and the digestive tract of S. neumayeri using a monospecific polyclonal antibody. We showed that S. neumayeri do not respond to acute heat stress by up-regulation of Sn-Hsp70 at transcript and protein level. Furthermore, the Sn-Hsp70 protein expression was not induced in the digestive tract. Conclusions: Our results provide the first molecular evidence that Sn-Hsp70 is expressed constitutively and is noninduced by heat stress in S. neumayeri.

Immune response of the Antarctic sea urchin Sterechinus neumayeri: cellular, molecular and physiological approach / Respuesta inmune del erizo de mar Antártico Sterechinus neumayeri: un enfoque celular, molecular y fisiológico

Abstract In the Antarctic marine environment, the water temperature is usually between 2 and - 1.9 °C. Consequently, some Antarctic species have lost the capacity to adapt to sudden changes in temperature. The study of the immune response in Antarctic sea urchin (Sterechinus neumayeri) could help us understand the future impacts of global warming on endemic animals in the Antarctic Peninsula. In this study, the Antarctic sea urchins were challenged with lipopolysaccharides and Vibrio alginolitycus. The cellular response was evaluated by the number of coelomocytes and phagocytosis. A significant increase was observed in red sphere cells and total coelomocytes in animals exposed to LPS. A significant rise in phagocytosis in animals stimulated by LPS was also evidenced. Moreover, the gene expression of three immune related genes was measured by qPCR, showing a rapid increase in their expression levels. By contrast, these immune genes showed a depression in their expression by a thermal effect at 5 and 10 °C. In addition, during bacterial injection, the oxygen consumption was higher in challenged animals. Our results showed that the immune response in the Antarctic sea urchin may be affected by acute thermal stress and that this immune response has a metabolic cost. Rev. Biol. Trop. 63 (Suppl. 2): 309-320. Epub 2015 June 01.

Resumen En el medio ambiente de la Antártica la temperatura del agua es de entre 2 y - 1.9 °C. Por consecuencia ciertas especies han perdido la capacidad de adaptarse a los cambios repentinos de la temperatura del agua. El estudio de la respuesta inmune del erizo antártico (Sterechinus neumayeri) podría ayudar a comprender los futuros impactos en los animales endémicos del cambio climático en la Península Antártica. En este estudio nosotros hemos evaluado la respuesta inmunitaria de S. neumayeri respecto de estimulaciones con bacterias (Lipopolisacáridos y Vibrio alginolitycus) asi como durante el estrés térmico a 5 y 10 °C. La respuesta del erizo fue evaluada en relación al número de celomocitos circulantes, capacidad fagocítica de estos y por el análisis de la expresión de tres genes inmunitarios. Después de la estimulación con LPS un aumento significativo de células esferoidales rojas, de amebocitos fagocíticos y de celomocitos totales fue observado después de las primeras horas de estimulación, de la misma manera que la capacidad fagocítica. Por otra parte los tres genes inmunes medidos mostraron un aumento significativo de su expresión por qPCR después de la estimulación con LPS. El estrés térmico de 5 °C produjo un aumento de la expresión de estos tres genes inmunitarios, por el contrario a una temperatura más alta (10 °C) se produce la reducción de dos de entre ellos. Adicionalmente un aumento del consumo de oxígeno fue observado durante la estimulación bacteriana. Nuestros resultados muestran que la respuesta inmunitaria en el erizo antártico puede ser afectada por el estrés térmico agudo y que la respuesta inmune en invertebrados antárticos tendría un costo metabólico.