Limits...
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.

Show MeSH

Related in: MedlinePlus

Attachment and invasion of  H. vermiformis by L. pneumophila induces  tyrosine dephosphorylation of different  host proteins. H. vermiformis cell extracts  were prepared from uninfected cells (A)  or from cells infected with L. pneumophila for 1, 3, 5, 15, and 30 min (B, lanes  1–5), subjected to SDS-PAGE, and  probed with antiphosphotyrosine antibody using the enhanced chemiluminescence kit as described in Materials and  Methods. Lane 6 is a cell extract from  uninfected cells incubated at 37°C for 30  min. Lane 7 represents samples prepared  from H. vermiformis infected for 30 min  following which the extracellular bacteria were washed away and cells were incubated at 37°C for 15 min.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199037&req=5

Figure 1: Attachment and invasion of H. vermiformis by L. pneumophila induces tyrosine dephosphorylation of different host proteins. H. vermiformis cell extracts were prepared from uninfected cells (A) or from cells infected with L. pneumophila for 1, 3, 5, 15, and 30 min (B, lanes 1–5), subjected to SDS-PAGE, and probed with antiphosphotyrosine antibody using the enhanced chemiluminescence kit as described in Materials and Methods. Lane 6 is a cell extract from uninfected cells incubated at 37°C for 30 min. Lane 7 represents samples prepared from H. vermiformis infected for 30 min following which the extracellular bacteria were washed away and cells were incubated at 37°C for 15 min.

Mentions: To understand the host cell processes which are activated following adherence of L. pneumophila to protozoa, we investigated the involvement of host tyrosine phosphorylation. H. vermiformis were coincubated with L. pneumophila for various time intervals, and amebal proteins were visualized by immunoblots probed with a recombinant antiphosphotyrosine antibody. Several tyrosine phosphorylated proteins of apparent molecular mass (in kilodaltons) including 200, 170, 150, 130, 90, 80, 60–65, 45–50, 40, 30–35, and 28 were detected in uninfected H. vermiformis (Fig. 1 A and B, lane 6). Following coincubation, dephosphorylation of all of the most strongly tyrosine phosphorylated proteins, including those with molecular masses of 170, 130, and 60– 65 kD, was evident as early as 5 min, and was complete by 15 min for the 170-kD protein (Fig. 1 B). The antiphosphotyrosine antibody did not bind any L. pneumophila proteins (data not shown). Although the level of dephosphorylation at each time interval varied slightly in multiple experiments, and occasionally was very prominent at 5 min (data not shown), this pattern of time-dependent tyrosine dephosphorylation was consistently observed. Identical protein tyrosine phosphorylation patterns were obtained using another antiphosphotyrosine antibody, clone 4G10 (data not shown), thus confirming the specificity of the RC-20 antibody.


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)

Attachment and invasion of  H. vermiformis by L. pneumophila induces  tyrosine dephosphorylation of different  host proteins. H. vermiformis cell extracts  were prepared from uninfected cells (A)  or from cells infected with L. pneumophila for 1, 3, 5, 15, and 30 min (B, lanes  1–5), subjected to SDS-PAGE, and  probed with antiphosphotyrosine antibody using the enhanced chemiluminescence kit as described in Materials and  Methods. Lane 6 is a cell extract from  uninfected cells incubated at 37°C for 30  min. Lane 7 represents samples prepared  from H. vermiformis infected for 30 min  following which the extracellular bacteria were washed away and cells were incubated at 37°C for 15 min.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Attachment and invasion of H. vermiformis by L. pneumophila induces tyrosine dephosphorylation of different host proteins. H. vermiformis cell extracts were prepared from uninfected cells (A) or from cells infected with L. pneumophila for 1, 3, 5, 15, and 30 min (B, lanes 1–5), subjected to SDS-PAGE, and probed with antiphosphotyrosine antibody using the enhanced chemiluminescence kit as described in Materials and Methods. Lane 6 is a cell extract from uninfected cells incubated at 37°C for 30 min. Lane 7 represents samples prepared from H. vermiformis infected for 30 min following which the extracellular bacteria were washed away and cells were incubated at 37°C for 15 min.
Mentions: To understand the host cell processes which are activated following adherence of L. pneumophila to protozoa, we investigated the involvement of host tyrosine phosphorylation. H. vermiformis were coincubated with L. pneumophila for various time intervals, and amebal proteins were visualized by immunoblots probed with a recombinant antiphosphotyrosine antibody. Several tyrosine phosphorylated proteins of apparent molecular mass (in kilodaltons) including 200, 170, 150, 130, 90, 80, 60–65, 45–50, 40, 30–35, and 28 were detected in uninfected H. vermiformis (Fig. 1 A and B, lane 6). Following coincubation, dephosphorylation of all of the most strongly tyrosine phosphorylated proteins, including those with molecular masses of 170, 130, and 60– 65 kD, was evident as early as 5 min, and was complete by 15 min for the 170-kD protein (Fig. 1 B). The antiphosphotyrosine antibody did not bind any L. pneumophila proteins (data not shown). Although the level of dephosphorylation at each time interval varied slightly in multiple experiments, and occasionally was very prominent at 5 min (data not shown), this pattern of time-dependent tyrosine dephosphorylation was consistently observed. Identical protein tyrosine phosphorylation patterns were obtained using another antiphosphotyrosine antibody, clone 4G10 (data not shown), thus confirming the specificity of the RC-20 antibody.

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