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Identification of a Gal/GalNAc lectin in the protozoan Hartmannella vermiformis as a potential receptor for attachment and invasion by the Legionnaires' disease bacterium.

Venkataraman C, Haack BJ, Bondada S, Abu Kwaik Y - J. Exp. Med. (1997)

Bottom Line: Interestingly, inhibition of invasion by Gal and GalNAc was associated with inhibition of bacterial-induced tyrosine dephosphorylation of H. vermiformis proteins.High stringency DNA hybridization confirmed the presence of the 170-kD lectin gene in H. vermiformis.This is the first demonstration of a potential receptor used by L. pneumophila to invade protozoa.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, Kentucky 40536-0084, USA.

ABSTRACT
The Legionnaire's disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen which invades and replicates within two evolutionarily distant hosts, free-living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaire's disease. Although attachment and invasion of human macrophages by L. pneumophila is mediated in part by the complement receptors CR1 and CR3, the protozoan receptor involved in bacterial attachment and invasion has not been identified. To define the molecular events involved in invasion of protozoa by L. pneumophila, we examined the role of protein tyrosine phosphorylation of the protozoan host Hartmannella vermiformis upon attachment and invasion by L. pneumophila. Bacterial attachment and invasion were associated with a time-dependent tyrosine dephosphorylation of multiple host cell proteins. This host cell response was highly specific for live L. pneumophila, required contact with viable bacteria, and was completely reversible following washing off the bacteria from the host cell surface. Tyrosine dephosphorylation of host proteins was blocked by a tyrosine phosphatase inhibitor but not by tyrosine kinase inhibitors. One of the tyrosine dephosphorylated proteins was identified as the 170-kD galactose/N-acetylgalactosamine-inhibitable lectin (Gal/GalNAc) using immunoprecipitation and immunoblotting by antibodies generated against the Gal/GalNAc lectin of the protozoan Entamoeba histolytica. This Gal/GalNAc-inhibitable lectin has been shown previously to mediate adherence of E. histolytica to mammalian epithelial cells. Uptake of L. pneumophila by H. vermiformis was specifically inhibited by two monovalent sugars, Gal and GalNAc, and by mABs generated against the 170-kD lectin of E. histolytica. Interestingly, inhibition of invasion by Gal and GalNAc was associated with inhibition of bacterial-induced tyrosine dephosphorylation of H. vermiformis proteins. High stringency DNA hybridization confirmed the presence of the 170-kD lectin gene in H. vermiformis. We conclude that attachment of L. pneumophila to the H. vermiformis 170-kD lectin is required for invasion and is associated with tyrosine dephosphorylation of the Gal lectin and other host proteins. This is the first demonstration of a potential receptor used by L. pneumophila to invade protozoa.

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Identification of the 170-kD protein of H. vermiformis which  is dephosphorylated upon contact and invasion by L. pneumophila. (A) Extracts of 107 cell equivalents were subjected to SDS-PAGE and probed  with antilectin mAb (1G7) followed by incubation with HRP-conjugated  anti–mouse secondary antibody. (B) Lysates of 108 cell equivalents were  prepared from uninfected H. vermiformis (lane 1) or infected with L. pneumophila for 5 min (lane 2). Samples were immunoprecipitated with rabbit  antiserum of the 170-kD lectin of E. histolytica. Following SDS-PAGE,  proteins were probed with antiphosphotyrosine (anti-ptyr) antibody and  visualized with an enhanced chemiluminescence kit as described in Materials and Methods. H is heavy chain of immunoprecipitating antibody.
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Figure 3: Identification of the 170-kD protein of H. vermiformis which is dephosphorylated upon contact and invasion by L. pneumophila. (A) Extracts of 107 cell equivalents were subjected to SDS-PAGE and probed with antilectin mAb (1G7) followed by incubation with HRP-conjugated anti–mouse secondary antibody. (B) Lysates of 108 cell equivalents were prepared from uninfected H. vermiformis (lane 1) or infected with L. pneumophila for 5 min (lane 2). Samples were immunoprecipitated with rabbit antiserum of the 170-kD lectin of E. histolytica. Following SDS-PAGE, proteins were probed with antiphosphotyrosine (anti-ptyr) antibody and visualized with an enhanced chemiluminescence kit as described in Materials and Methods. H is heavy chain of immunoprecipitating antibody.

Mentions: One of the prominent amebal proteins which was dephosphorylated upon contact with L. pneumophila was ∼170 kD in molecular mass (Fig. 1 A, arrow). Search of the literature on other amebal species indicated that a 170-kD Gal/GalNAc–specific lectin is involved in adherence of E. histolytica to host epithelial cells (32, 33). This adherence can be blocked by antilectin antibodies (32–34). To test whether the 170-kD dephosphorylated protein in H. vermiformis is similar to the 170-kD lectin of E. histolytica, we performed immunoprecipitation studies using a rabbit antiserum generated against the E. histolytica Gal/GalNAc lectin (30). Following coincubation with L. pneumophila, amebal proteins were immunoprecipitated with anti–170-kD antibody and then immunoblotted with antiphosphotyrosine antibody (see Materials and Methods). The results in Fig. 3 B showed that the antiserum immunoprecipitated a 170-kD protein which was tyrosine dephosphorylated after 5 min of infection. The identity of the 170-kD lectin was further confirmed by Western blotting of total amebal lysates using a mouse mAb which recognizes the Gal/GalNAc lectin of E. histolytica (28). The mAb bound to a 170-kD protein of H. vermiformis (Fig. 3 A). These findings suggested that the 170-kD Gal lectin of H. vermiformis and E. histolytica shared similar conserved epitopes. The tyrosine dephosphorylation of the 170-kD lectin suggested that this lectin may be posttranslationally modified by signaling processes following attachment of L. pneumophila.


Identification of a Gal/GalNAc lectin in the protozoan Hartmannella vermiformis as a potential receptor for attachment and invasion by the Legionnaires' disease bacterium.

Venkataraman C, Haack BJ, Bondada S, Abu Kwaik Y - J. Exp. Med. (1997)

Identification of the 170-kD protein of H. vermiformis which  is dephosphorylated upon contact and invasion by L. pneumophila. (A) Extracts of 107 cell equivalents were subjected to SDS-PAGE and probed  with antilectin mAb (1G7) followed by incubation with HRP-conjugated  anti–mouse secondary antibody. (B) Lysates of 108 cell equivalents were  prepared from uninfected H. vermiformis (lane 1) or infected with L. pneumophila for 5 min (lane 2). Samples were immunoprecipitated with rabbit  antiserum of the 170-kD lectin of E. histolytica. Following SDS-PAGE,  proteins were probed with antiphosphotyrosine (anti-ptyr) antibody and  visualized with an enhanced chemiluminescence kit as described in Materials and Methods. H is heavy chain of immunoprecipitating antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199037&req=5

Figure 3: Identification of the 170-kD protein of H. vermiformis which is dephosphorylated upon contact and invasion by L. pneumophila. (A) Extracts of 107 cell equivalents were subjected to SDS-PAGE and probed with antilectin mAb (1G7) followed by incubation with HRP-conjugated anti–mouse secondary antibody. (B) Lysates of 108 cell equivalents were prepared from uninfected H. vermiformis (lane 1) or infected with L. pneumophila for 5 min (lane 2). Samples were immunoprecipitated with rabbit antiserum of the 170-kD lectin of E. histolytica. Following SDS-PAGE, proteins were probed with antiphosphotyrosine (anti-ptyr) antibody and visualized with an enhanced chemiluminescence kit as described in Materials and Methods. H is heavy chain of immunoprecipitating antibody.
Mentions: One of the prominent amebal proteins which was dephosphorylated upon contact with L. pneumophila was ∼170 kD in molecular mass (Fig. 1 A, arrow). Search of the literature on other amebal species indicated that a 170-kD Gal/GalNAc–specific lectin is involved in adherence of E. histolytica to host epithelial cells (32, 33). This adherence can be blocked by antilectin antibodies (32–34). To test whether the 170-kD dephosphorylated protein in H. vermiformis is similar to the 170-kD lectin of E. histolytica, we performed immunoprecipitation studies using a rabbit antiserum generated against the E. histolytica Gal/GalNAc lectin (30). Following coincubation with L. pneumophila, amebal proteins were immunoprecipitated with anti–170-kD antibody and then immunoblotted with antiphosphotyrosine antibody (see Materials and Methods). The results in Fig. 3 B showed that the antiserum immunoprecipitated a 170-kD protein which was tyrosine dephosphorylated after 5 min of infection. The identity of the 170-kD lectin was further confirmed by Western blotting of total amebal lysates using a mouse mAb which recognizes the Gal/GalNAc lectin of E. histolytica (28). The mAb bound to a 170-kD protein of H. vermiformis (Fig. 3 A). These findings suggested that the 170-kD Gal lectin of H. vermiformis and E. histolytica shared similar conserved epitopes. The tyrosine dephosphorylation of the 170-kD lectin suggested that this lectin may be posttranslationally modified by signaling processes following attachment of L. pneumophila.

Bottom Line: Interestingly, inhibition of invasion by Gal and GalNAc was associated with inhibition of bacterial-induced tyrosine dephosphorylation of H. vermiformis proteins.High stringency DNA hybridization confirmed the presence of the 170-kD lectin gene in H. vermiformis.This is the first demonstration of a potential receptor used by L. pneumophila to invade protozoa.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, Kentucky 40536-0084, USA.

ABSTRACT
The Legionnaire's disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen which invades and replicates within two evolutionarily distant hosts, free-living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaire's disease. Although attachment and invasion of human macrophages by L. pneumophila is mediated in part by the complement receptors CR1 and CR3, the protozoan receptor involved in bacterial attachment and invasion has not been identified. To define the molecular events involved in invasion of protozoa by L. pneumophila, we examined the role of protein tyrosine phosphorylation of the protozoan host Hartmannella vermiformis upon attachment and invasion by L. pneumophila. Bacterial attachment and invasion were associated with a time-dependent tyrosine dephosphorylation of multiple host cell proteins. This host cell response was highly specific for live L. pneumophila, required contact with viable bacteria, and was completely reversible following washing off the bacteria from the host cell surface. Tyrosine dephosphorylation of host proteins was blocked by a tyrosine phosphatase inhibitor but not by tyrosine kinase inhibitors. One of the tyrosine dephosphorylated proteins was identified as the 170-kD galactose/N-acetylgalactosamine-inhibitable lectin (Gal/GalNAc) using immunoprecipitation and immunoblotting by antibodies generated against the Gal/GalNAc lectin of the protozoan Entamoeba histolytica. This Gal/GalNAc-inhibitable lectin has been shown previously to mediate adherence of E. histolytica to mammalian epithelial cells. Uptake of L. pneumophila by H. vermiformis was specifically inhibited by two monovalent sugars, Gal and GalNAc, and by mABs generated against the 170-kD lectin of E. histolytica. Interestingly, inhibition of invasion by Gal and GalNAc was associated with inhibition of bacterial-induced tyrosine dephosphorylation of H. vermiformis proteins. High stringency DNA hybridization confirmed the presence of the 170-kD lectin gene in H. vermiformis. We conclude that attachment of L. pneumophila to the H. vermiformis 170-kD lectin is required for invasion and is associated with tyrosine dephosphorylation of the Gal lectin and other host proteins. This is the first demonstration of a potential receptor used by L. pneumophila to invade protozoa.

Show MeSH
Related in: MedlinePlus