Bacterial infection and tissue-specific Hsp72, -73 and -90 expression in western painted turtles

https://doi.org/10.1016/j.cca.2004.06.007Get rights and content

Abstract

Heat shock proteins (Hsps) are molecular chaperones that assist intracellular folding, assembly and translocation of proteins in prokaryotic and eukaryotic cells. A variety of stresses including hyperthermia, radiation, heavy metals, ischemia, anoxia and reoxygenation have been shown to increase the expression of Hsps. Likewise, bacterial infection represents a stress for the host cell. In this study, expression of the constitutive (Hsp73) and inducible (Hsp72) isoforms of Hsp70 and Hsp90 was monitored in brain, heart, liver and skeletal muscle from the western painted turtle Chrysemys picta bellii diagnosed with Septicemic Cutaneous Ulcerative Dermatitis (SCUD). This disease is caused by a gram-negative bacterium probably belonging to the Citrobacter spp. The expression of Hsp73 increased 1.8-fold in brain and liver, 2.2-fold in heart but did not change in skeletal muscle; Hsp72 expression increased 5.5-fold in brain and 3-fold in liver but did not change in heart or skeletal muscle; Hsp90 expression increased 9-fold in brain, 2.7-fold in heart and 2.4-fold in skeletal muscle but did not change in liver. These results suggest a tissue-specific Hsp response during bacterial infection and a role for Hsps in immunopathological events in reptiles.

Introduction

Originally characterized in Drosophila melanogaster, heat shock proteins (Hsps) have been described in many species ranging from bacteria to mammals (Lindquist and Craig, 1988). Hsps are constitutively present in the cell but their expression increases in response to a variety of stresses including hyperthermia, radiation, heavy metals, ischemia, anoxia and reoxygenation (Lindquist and Craig, 1988). Pathological events such as inflammation represent a stress not only for the bacteria facing the immune response but also for the host cell. However, little is known about the effects of bacterial infection on the expression of Hsps.

Under physiological conditions, Hsps act as molecular chaperones, preventing premature folding of proteins and aiding in translocation to organelles. Under conditions of stress, they are believed to bind to damaged or misfolded polypeptides, either facilitating their repair or targeting irreparably damaged proteins for degradation by the ubiquitin/proteasome-dependent pathway (Lindquist and Craig, 1988). The 70-kDa family of Hsps includes several different isoforms and is the best characterized Hsp group. These Hsps are highly conserved from prokaryotes to mammals. The constitutive (Hsp73) and inducible (Hsp72) forms of Hsp70 are both present in the unstressed cell. Under conditions of stress, the expression of Hsp73 has been shown to be moderately up-regulated whereas Hsp72 is highly induced (Manzerra et al., 1997, Lindquist and Craig, 1988). In contrast to the widely described 70-kDa family of stress proteins, the 90-kDa Hsp has been less well characterized. Hsp90 is expressed in the cell as two different isoforms, Hsp90α and Hsp90β, encoded by separate genes. Under physiological conditions, Hsp90 is involved in cell growth and differentiation, intracellular transport and regulation of nuclear hormone receptor activity as well as modulation of protein kinases (Mayer and Bukau, 1999). At moderately high temperatures, Hsp90 is protective during growth (Borkovich et al., 1989). In addition, Hsp90 expression in rat kidney has been shown to increase in response to oxidative stress (Fukuda et al., 1996).

The induced expression of Hsps as a result of bacterial infection has been reported several times in mammals but only once in a non-mammalian species. Incubation with Escherichia coli or endotoxins induces Hsp72 expression in human enterocytes (Deitch et al., 1995). Phagocytosis of Staphylococcus aureus or opsonized red blood cells induces the synthesis of Hsp70 and Hsp90 in human monocytes-macrophages (Kantengwa and Polla, 1993, Clerget and Polla, 1990). In addition, Mistry et al. (1992) reported an increase in the expression of Hsp72 in murine and monkey Schwann cells infected with Mycobacterium leprae. Coho salmon given an intraperitoneal injection of the bacterium Renibacterium salmoninarum demonstrated significantly increased Hsp72 levels in liver and kidney compared to controls (Forsyth et al., 1997). More recently, Hsp72 up-regulation has been shown to occur in response to rheumatoid arthritis (Schett et al., 1998) and hypersensitivity pneumonitis (Racine et al., 1999).

We have previously demonstrated in tissues from the western painted turtle (Chrysemys picta bellii) that expression of Hsp72 increases in response to thermal stress and long-term anoxia (Scott et al., 2003). Since the freshwater turtle is able to tolerate anoxia for months, we suggested that the up-regulation of stress proteins, even when energy saving is critical to survival, could play a role in promoting anoxia tolerance. However, in an attempt to further investigate the molecular mechanisms underlying anoxic survival, we diagnosed the experimental turtles with Septisemic Cutaneous Ulcerative Dermatitis (SCUD).

SCUD is also known as Shell Rot or Blood Poisoning. The etiologic agent of SCUD is the gram-negative bacterium Citrobacter freundii, but other bacteria such as Aeromonas hydrophila and Beneckea chitinovora have been associated with ulcerative lesions in freshwater turtles (Boyer, 1996). The disease causes cutaneous ulceration, anorexia, lethargy, hepatic necrosis and finally death (Barten, 1996). Poor nutrition and maintenance in contaminated water are the suspected origin of the disease. In addition, bacteria of the genus Serratia are able to secrete proteolytic enzymes and may contribute to the origin of the disease allowing the entry of the etiologic agent through the skin (Boyer, 1996).

The primary aim of this study is to investigate the expression of Hsp72, Hsp73 and Hsp90 in response to heat shock and bacterial infection in brain, heart, liver and skeletal muscle from the western painted turtle. This is only the second report where the expression of stress proteins resulting from bacterial infection has been monitored in a non-mammalian species.

Section snippets

Animals

Male and female turtles (C. picta bellii, Schneider 1783), weighing between 250 and 750 g, were purchased from Lemberger (Oshkosh, WI, USA). Animals were housed in an indoor pond (2×4×1.5 m) equipped with basking platform, heating lamp and a flow-through dechlorinated fresh water system. A supplementary tank was also available to house the animals during periodical cleaning of the pond. In both pools, the water temperature was maintained at approximately 17 °C. Turtles were given continuous

Hsp73, Hsp72 and Hsp90 expression following heat shock

Although termed stress proteins, heat shock proteins derive their original name from their ability to up-regulate in response to high temperatures. Therefore, as a positive control, we tested the effects of a 40 °C heat shock on the expression of Hsp73, Hsp72 and Hsp90 in turtle brain, heart, liver and skeletal muscle.

Following heat shock, Western blot analysis and quantification by densitometry revealed no significant change in the expression of Hsp73 in brain (38.6±1.9 pg μg tissue−1), liver

Discussion

A great deal of literature has focused on the effects of various stressors on Hsp expression. Hyperthermia, radiation, heavy metals, ischemia, anoxia and reoxygenation are by far the most studied inducers of the stress response (Lindquist and Craig, 1988). Pathogens such as viruses and bacteria also represent a stress for the host cell. Although a number of studies have reported an increase in the level of Hsps as a result of viral infection (Bolt, 2001, Melcher et al., 1999, Cho et al., 1997,

References (51)

  • M.A. Yenari et al.

    The neuroprotective potential of heat shock protein 70 (HSP70)

    Mol. Med. Today

    (1999)
  • S. Airaksinen et al.

    Effects of heat shock and hypoxia on protein synthesis in rainbow trout (Oncorhynchus mykiss) cells

    J. Exp. Biol.

    (1998)
  • J.C. Bardwell et al.

    Eukaryotic Mr 83,000 heat shock protein has a homologue in Escherichia coli

    Proc. Natl. Acad. Sci. U. S. A.

    (1987)
  • S.L. Barten

    Shell damage

  • D.A. Bechtold et al.

    Localization of the heat-shock protein Hsp70 to the synapse following hyperthermic stress in the brain

    J. Neurochem.

    (2000)
  • G. Bolt

    The measles virus (MV) glycoproteins interact with cellular chaperones in the endoplasmic reticulum and MV infection upregulates chaperone expression

    Arch. Virol.

    (2001)
  • K.A. Borkovich et al.

    hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures

    Mol. Cell. Biol.

    (1989)
  • T.H. Boyer

    Turtles, tortoises and terrapins

  • M. Clerget et al.

    Erythrophagocytosis induces heat shock protein synthesis by human monocytes-macrophages

    Proc. Natl. Acad. Sci. U. S. A.

    (1990)
  • A. De Maio et al.

    Induction of translational thermotolerance in liver of thermally stressed rats

    Eur. J. Biochem.

    (1993)
  • A. De Maio et al.

    Heat shock gene expression and development of translational thermotolerance in human hepatoblastoma cells

    Circ. Shock

    (1993)
  • E.A. Deitch et al.

    Induction of heat shock gene expression in colonic epithelial cells after incubation with Escherichia coli or endotoxin

    Crit. Care Med.

    (1995)
  • Y.R. Donati et al.

    Phagocytosis and heat shock response in human monocytes-macrophages

    Pathobiology

    (1991)
  • S.W. Flanagan et al.

    Tissue-specific HSP70 response in animals undergoing heat stress

    Am. J. Physiol.

    (1995)
  • R.B. Forsyth et al.

    Stress protein expression in coho salmon with bacterial kidney disease

    J. Aquat. Anim. Health

    (1997)
  • Cited by (59)

    • Molecular characterization of GRP94 and HSP90α from Trachinotus ovatus, Linnaeus 1758 and their expression responses to various levels of stocking density stress and Cryptocaryon irritans infection

      2020, Aquaculture
      Citation Excerpt :

      Jiang et al. (2019) found that the expression level of HSP90α in the skin of Siganus oramin was significantly upregulated at 12 h after C. irritans infection, suggesting that C. irritans infection triggered an immune response in skin cells. Therefore, upregulation of HSPs may be a self-protection mechanism (Ramaglia et al., 2004). In this study, the expression of the ToGRP94 gene under C. irritans infection was analysed, resulting in a significant increase in liver, spleen, and kidney tissues at 24 h post-infection.

    View all citing articles on Scopus
    View full text