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Caspian seal die-off is caused by canine distemper virus
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News & Comment
TRENDS in Microbiology Vol.9 No.3 March 2001
Journal Club
Necrotizing bacteria and protein targeting A type III-like secretion mechanism for injection of effector molecules into the cytoplasm of host cells has not yet been described for Gram-positive bacteria. These bacteria are known to secrete multiple proteins into their environments, such as those secreted by Streptococcus pyogenes, the agent responsible for strep throat and necrotizing fasciitis of soft tissues. Previous work using keratinocytes demonstrated that mutants defective in production of the hemolysin streptolysin O (SLO) lost the ability to manipulate host signaling pathways. SLO also has characteristics that would suggest it has a role in translocation. SLO is a member of a highly conserved family of pore-forming cytolysins known as cholesterol-dependent cytolysins (CDCs). Analysis of their pore size suggests that CDCs could translocate effector molecules from Gram-positive bacteria. Madden and colleagues1 recently presented evidence that a molecule of streptococcal origin with eukaryotic signal transduction properties is translocated into the keratinocyte cytosol in an SLO-dependent manner. Crossreactivity with a commercially available anti-streptolysin O antiserum revealed the presence of a 52-kDa streptococcal protein in infected keratinocytes, which was not present in SLO-deficient mutants or uninfected host cells. Using cytoplasmic fractions prepared
from host cells under conditions that lyse keratinocytes but not bacteria, the 52-kDa protein was found to be translocated in an SLO-dependent manner. Other host cell types were analyzed and similar results were obtained. A streptococcal NADglycohydrolase (SPN)-deficient mutant did not translocate the 52-kDa protein into host cells however, indicating that this particular protein was derived from the bacteria and was not of host origin. Co-infection with bacteria that were deficient in either SLO or SPN did not rescue protein transport. These observations suggest that SPN enters the cytosol through the SLO pore. The SPN protein is biochemically active in the host cell. The presence of the microfilament inhibitor cytochalasin D did not alter enzymatic activity in wild-type cells, indicating that the presence of SPN activity was not the result of endocytosis or bacterial invasion. Furthermore, SPN activity was dependent on SLO and was not attributed to an endogenous NAD-glycohydrolase. To demonstrate that SPN and SLO work in a synergistic manner, a cytotoxic response assay was used where keratinocytes were infected with either the wild-type strain, the SLO-mutant or the SPNmutant. At various times, samples were removed and stained with the fluorescent probes calcein AM (activated by cellular esterases) and ethidium homodimer-1 (EtH-1) (which stains the nucleus). Loss of staining
with calcein AM and positive staining with EtH-1 would be indicative of membrane damage. By 3 h, ~35% of the wild-type cells have lost their ability to exclude EtH-1. At all time points, infection with SLO-deficient mutants resembled SPN-deficient mutants in terms of the number of cells damaged and number of cells that remained adherent. Both mutants had reduced levels of permeability compared with the wild-type cell. These data are consistent with a model where SLO is required for the translocation of SPN into the cytosol of the host cell. With these data and previous studies of protein targeting in Gram-positive bacteria, a working model of protein translocation suggests that effector molecules are exported across the bacterial cellular membrane and cell wall via the generalized secretion pathway and translocated into the target cell using a CDC. Other than in Listeria, little is known about the function of CDCs in pathogenesis. These interesting and novel findings set the stage for analysis of function of virulence factors secreted from Gram-positive pathogens. 1 Madden, J.C. et al. (2001) Cytolysin-mediated translocation (CMT): a functional equivalent of type III secretion in Gram-positive bacteria. Cell 104, 143–152
Kirkwood M. Land [email protected]
Caspian seal die-off is caused by canine distemper virus The primary cause of the deaths of 10 000 seals in the Caspian Sea during the spring of 2000 is now reported by an international team of scientists to be canine distemper virus1. Caspian seals (Phoca caspica) are listed as vulnerable species (endangered) and live in the Caspian Sea bordered by Russia, Kazakhstan, Turkmenistan, Iran and Azerbaijan. The highest death rates were reported during April and May as the disease spread south from the mouth of the Ural river to the Turkmenistan coast. Animals exhibited symptoms of debilitation, muscle spasms, ocular and nasal exudation and sneezing. Necroscopies showed no consistent gross lesions but microscopic examination of various tissues showed
broncho-interstitial pneumonia, encephalitis, pancreatitis and lymphocytic depletion in lymphoid tissues, among other features. The picture was characteristic of a morbillivirus infection. Morbillivirus antigen was detected in multiple tissues using a monoclonal antibody against the nucleoprotein of phocine distemper virus. The findings were characteristic of distemper in terrestrial and aquatic animals. Tissues were also examined for morbillivirus nucleic acid by RT-PCR yielding the expected products. Fragments were sequenced and subjected to phylogenetic analyses. Serum specimens were also examined for canine distemper virus-specific IgM and IgG antibodies by direct and indirect ELISA tests. The data indicated that the seals from widely
separated regions of the Caspian Sea were infected by the same virus. The origin of the virus is unknown but there are suspicions of terrestrial carnivores being involved. Such suspicions were aroused by earlier reports of die-offs in pinniped populations attributed to some association with domestic dogs. The contributory role of high levels of pollutants in the Caspian Sea is also being investigated. 1 Kennedy, S. et al. (2000) Mass die-off of Caspian seals caused by canine distemper virus. Emerg. Infect. Dis. 6, 637–639
J. Dennis Pollack [email protected]
http://tim.trends.com 0966-842X/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
News & Comment
TRENDS in Microbiology Vol.9 No.3 March 2001
Journal Club
Necrotizing bacteria and protein targeting A type III-like secretion mechanism for injection of effector molecules into the cytoplasm of host cells has not yet been described for Gram-positive bacteria. These bacteria are known to secrete multiple proteins into their environments, such as those secreted by Streptococcus pyogenes, the agent responsible for strep throat and necrotizing fasciitis of soft tissues. Previous work using keratinocytes demonstrated that mutants defective in production of the hemolysin streptolysin O (SLO) lost the ability to manipulate host signaling pathways. SLO also has characteristics that would suggest it has a role in translocation. SLO is a member of a highly conserved family of pore-forming cytolysins known as cholesterol-dependent cytolysins (CDCs). Analysis of their pore size suggests that CDCs could translocate effector molecules from Gram-positive bacteria. Madden and colleagues1 recently presented evidence that a molecule of streptococcal origin with eukaryotic signal transduction properties is translocated into the keratinocyte cytosol in an SLO-dependent manner. Crossreactivity with a commercially available anti-streptolysin O antiserum revealed the presence of a 52-kDa streptococcal protein in infected keratinocytes, which was not present in SLO-deficient mutants or uninfected host cells. Using cytoplasmic fractions prepared
from host cells under conditions that lyse keratinocytes but not bacteria, the 52-kDa protein was found to be translocated in an SLO-dependent manner. Other host cell types were analyzed and similar results were obtained. A streptococcal NADglycohydrolase (SPN)-deficient mutant did not translocate the 52-kDa protein into host cells however, indicating that this particular protein was derived from the bacteria and was not of host origin. Co-infection with bacteria that were deficient in either SLO or SPN did not rescue protein transport. These observations suggest that SPN enters the cytosol through the SLO pore. The SPN protein is biochemically active in the host cell. The presence of the microfilament inhibitor cytochalasin D did not alter enzymatic activity in wild-type cells, indicating that the presence of SPN activity was not the result of endocytosis or bacterial invasion. Furthermore, SPN activity was dependent on SLO and was not attributed to an endogenous NAD-glycohydrolase. To demonstrate that SPN and SLO work in a synergistic manner, a cytotoxic response assay was used where keratinocytes were infected with either the wild-type strain, the SLO-mutant or the SPNmutant. At various times, samples were removed and stained with the fluorescent probes calcein AM (activated by cellular esterases) and ethidium homodimer-1 (EtH-1) (which stains the nucleus). Loss of staining
with calcein AM and positive staining with EtH-1 would be indicative of membrane damage. By 3 h, ~35% of the wild-type cells have lost their ability to exclude EtH-1. At all time points, infection with SLO-deficient mutants resembled SPN-deficient mutants in terms of the number of cells damaged and number of cells that remained adherent. Both mutants had reduced levels of permeability compared with the wild-type cell. These data are consistent with a model where SLO is required for the translocation of SPN into the cytosol of the host cell. With these data and previous studies of protein targeting in Gram-positive bacteria, a working model of protein translocation suggests that effector molecules are exported across the bacterial cellular membrane and cell wall via the generalized secretion pathway and translocated into the target cell using a CDC. Other than in Listeria, little is known about the function of CDCs in pathogenesis. These interesting and novel findings set the stage for analysis of function of virulence factors secreted from Gram-positive pathogens. 1 Madden, J.C. et al. (2001) Cytolysin-mediated translocation (CMT): a functional equivalent of type III secretion in Gram-positive bacteria. Cell 104, 143–152
Kirkwood M. Land [email protected]
Caspian seal die-off is caused by canine distemper virus The primary cause of the deaths of 10 000 seals in the Caspian Sea during the spring of 2000 is now reported by an international team of scientists to be canine distemper virus1. Caspian seals (Phoca caspica) are listed as vulnerable species (endangered) and live in the Caspian Sea bordered by Russia, Kazakhstan, Turkmenistan, Iran and Azerbaijan. The highest death rates were reported during April and May as the disease spread south from the mouth of the Ural river to the Turkmenistan coast. Animals exhibited symptoms of debilitation, muscle spasms, ocular and nasal exudation and sneezing. Necroscopies showed no consistent gross lesions but microscopic examination of various tissues showed
broncho-interstitial pneumonia, encephalitis, pancreatitis and lymphocytic depletion in lymphoid tissues, among other features. The picture was characteristic of a morbillivirus infection. Morbillivirus antigen was detected in multiple tissues using a monoclonal antibody against the nucleoprotein of phocine distemper virus. The findings were characteristic of distemper in terrestrial and aquatic animals. Tissues were also examined for morbillivirus nucleic acid by RT-PCR yielding the expected products. Fragments were sequenced and subjected to phylogenetic analyses. Serum specimens were also examined for canine distemper virus-specific IgM and IgG antibodies by direct and indirect ELISA tests. The data indicated that the seals from widely
separated regions of the Caspian Sea were infected by the same virus. The origin of the virus is unknown but there are suspicions of terrestrial carnivores being involved. Such suspicions were aroused by earlier reports of die-offs in pinniped populations attributed to some association with domestic dogs. The contributory role of high levels of pollutants in the Caspian Sea is also being investigated. 1 Kennedy, S. et al. (2000) Mass die-off of Caspian seals caused by canine distemper virus. Emerg. Infect. Dis. 6, 637–639
J. Dennis Pollack [email protected]
http://tim.trends.com 0966-842X/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.