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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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(A) Invasion of H. vermiformis by L. pneumophila in the presence of anti–170-kD Gal lectin mAbs. Amebas were preincubated with the 50  μg/ml of anti–170-kD Gal lectin mAbs, and infected with L. pneumophila for 2 h, followed by gentamicin treatment to kill extracellular bacteria. Intracellular bacteria were released by mild lysis of the amebas (0.04% Triton X-100), and plated for colony enumeration. The percentage of  invasion was  derived from the relative number of intracellular bacteria in the presence of mAbs compared to untreated cultures. Values are the means of triplicate samples, and error bars represent standard deviations. (B) Invasion of H. vermiformis by L. pneumophila in the presence of increasing concentrations of the anti– 170-kD Gal lectin mAb, H85. The number of intracellular bacteria and percentage of invasion were obtained exactly as in Fig. 3.
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Figure 5: (A) Invasion of H. vermiformis by L. pneumophila in the presence of anti–170-kD Gal lectin mAbs. Amebas were preincubated with the 50 μg/ml of anti–170-kD Gal lectin mAbs, and infected with L. pneumophila for 2 h, followed by gentamicin treatment to kill extracellular bacteria. Intracellular bacteria were released by mild lysis of the amebas (0.04% Triton X-100), and plated for colony enumeration. The percentage of  invasion was derived from the relative number of intracellular bacteria in the presence of mAbs compared to untreated cultures. Values are the means of triplicate samples, and error bars represent standard deviations. (B) Invasion of H. vermiformis by L. pneumophila in the presence of increasing concentrations of the anti– 170-kD Gal lectin mAb, H85. The number of intracellular bacteria and percentage of invasion were obtained exactly as in Fig. 3.

Mentions: To test that the Gal/GalNAc lectin was used for L. pneumophila entry, H. vermiformis were preincubated with various antilectin mAbs prior to infection. The number of intracellular bacteria was determined after gentamicin treatment to kill extracellular bacteria, followed by lysis of amebas and plating of intracellular bacteria for colony enumeration. Three mAbs, BC6, H85, and 1G7, inhibited invasion by 62, 59, and 49%, respectively (Fig. 5 A). These results are similar to the blocking effect of various antilectin mAbs on the adherence of E. histolytica to epithelial cells and colonic mucins (32–34). In contrast, normal mouse IgG had no effect on invasion of H. vermiformis by L. pneumophila. The H85 and 1G7 mAbs have been shown to be specific for the surface-exposed cysteine-rich epitope of the 170-kD lectin, and both mAbs inhibit adherence of E. histolytica to human epithelial cells (28, 33). Moreover, the E. histolytica 170-kD–specific mAb 7F4, which does not inhibit adherence of E. histolytica to epithelial cells, did not have any detectable effect on invasion of H. vermiformis by L. pneumophila (Fig. 5 A). These data indicated a high level of antigenic conservation in the surface-exposed epitopes of the 170-kD Gal lectin in both protozoa. One of the mAbs (8C12), which inhibits adherence of E. histolytica to epithelial cells, did not have any detectable effect on the ability of L. pneumophila to invade H. vermiformis. These data indicated that although the 170-kD Gal lectin was antigenically conserved, certain epitopes may be different in the two protozoa.


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)

(A) Invasion of H. vermiformis by L. pneumophila in the presence of anti–170-kD Gal lectin mAbs. Amebas were preincubated with the 50  μg/ml of anti–170-kD Gal lectin mAbs, and infected with L. pneumophila for 2 h, followed by gentamicin treatment to kill extracellular bacteria. Intracellular bacteria were released by mild lysis of the amebas (0.04% Triton X-100), and plated for colony enumeration. The percentage of  invasion was  derived from the relative number of intracellular bacteria in the presence of mAbs compared to untreated cultures. Values are the means of triplicate samples, and error bars represent standard deviations. (B) Invasion of H. vermiformis by L. pneumophila in the presence of increasing concentrations of the anti– 170-kD Gal lectin mAb, H85. The number of intracellular bacteria and percentage of invasion were obtained exactly as in Fig. 3.
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Related In: Results  -  Collection

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Figure 5: (A) Invasion of H. vermiformis by L. pneumophila in the presence of anti–170-kD Gal lectin mAbs. Amebas were preincubated with the 50 μg/ml of anti–170-kD Gal lectin mAbs, and infected with L. pneumophila for 2 h, followed by gentamicin treatment to kill extracellular bacteria. Intracellular bacteria were released by mild lysis of the amebas (0.04% Triton X-100), and plated for colony enumeration. The percentage of  invasion was derived from the relative number of intracellular bacteria in the presence of mAbs compared to untreated cultures. Values are the means of triplicate samples, and error bars represent standard deviations. (B) Invasion of H. vermiformis by L. pneumophila in the presence of increasing concentrations of the anti– 170-kD Gal lectin mAb, H85. The number of intracellular bacteria and percentage of invasion were obtained exactly as in Fig. 3.
Mentions: To test that the Gal/GalNAc lectin was used for L. pneumophila entry, H. vermiformis were preincubated with various antilectin mAbs prior to infection. The number of intracellular bacteria was determined after gentamicin treatment to kill extracellular bacteria, followed by lysis of amebas and plating of intracellular bacteria for colony enumeration. Three mAbs, BC6, H85, and 1G7, inhibited invasion by 62, 59, and 49%, respectively (Fig. 5 A). These results are similar to the blocking effect of various antilectin mAbs on the adherence of E. histolytica to epithelial cells and colonic mucins (32–34). In contrast, normal mouse IgG had no effect on invasion of H. vermiformis by L. pneumophila. The H85 and 1G7 mAbs have been shown to be specific for the surface-exposed cysteine-rich epitope of the 170-kD lectin, and both mAbs inhibit adherence of E. histolytica to human epithelial cells (28, 33). Moreover, the E. histolytica 170-kD–specific mAb 7F4, which does not inhibit adherence of E. histolytica to epithelial cells, did not have any detectable effect on invasion of H. vermiformis by L. pneumophila (Fig. 5 A). These data indicated a high level of antigenic conservation in the surface-exposed epitopes of the 170-kD Gal lectin in both protozoa. One of the mAbs (8C12), which inhibits adherence of E. histolytica to epithelial cells, did not have any detectable effect on the ability of L. pneumophila to invade H. vermiformis. These data indicated that although the 170-kD Gal lectin was antigenically conserved, certain epitopes may be different in the two protozoa.

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