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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: 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.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.

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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Inhibition of invasion of H. vermiformis by L. pneumophila in  the presence of different sugar monomers. (A) Invasion of H. vermiformis  by L. pneumophila in the absence or presence of various sugars at 10 and  100 mM concentrations. Amebas were infected with L. pneumophila for 4 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  sugars compared to untreated cultures. Values are the means of triplicate  samples, and error bars represent standard deviations. (B) Growth kinetics  of L. pneumophila in cocultures with H. vermiformis in the absence or presence of sugars at 100 mM concentrations. At several time intervals of the  infection, the number of bacteria in the cocultures was determined following growth on agar plates. The bacteria do not replicate extracellularly in the  coculture and thus the increase in the number of bacteria is due to intracellular replication.
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Figure 4: Inhibition of invasion of H. vermiformis by L. pneumophila in the presence of different sugar monomers. (A) Invasion of H. vermiformis by L. pneumophila in the absence or presence of various sugars at 10 and 100 mM concentrations. Amebas were infected with L. pneumophila for 4 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 sugars compared to untreated cultures. Values are the means of triplicate samples, and error bars represent standard deviations. (B) Growth kinetics of L. pneumophila in cocultures with H. vermiformis in the absence or presence of sugars at 100 mM concentrations. At several time intervals of the infection, the number of bacteria in the cocultures was determined following growth on agar plates. The bacteria do not replicate extracellularly in the coculture and thus the increase in the number of bacteria is due to intracellular replication.

Mentions: Previous work with E. histolytica showed a decrease in 170-kD lectin-mediated adherence to colonic mucosa in the presence of the monovalent sugars, Gal and GalNAc (35, 36). To investigate the functional relevance of involvement of 170-kD lectin in the invasion process, we performed blocking experiments using various sugars, H. vermiformis cultures were infected with L. pneumophila in the presence of various sugars and the levels of invasion were measured using two different assays. First, we used a gentamicin protection invasion assay in which 4 h following infection the extracellular bacteria were killed by treatment with gentamicin and intracellular bacteria were plated on BCYE plates for colony enumeration. Our results showed that both Gal and GalNAc decreased invasion of amebas in a dose-dependent manner. While 100 mM of GalNAc and Gal inhibited invasion by 83 and 75%, respectively (Fig. 4 A), other sugars like glucose, mannose, and lactose, showed little or no effect on invasion of amebas by L. pneumophila at any concentration tested (Fig. 4 A). These data are similar to the inhibition of adherence of E. histolytica in the presence of identical concentrations of Gal and GalNAc (35, 36).


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)

Inhibition of invasion of H. vermiformis by L. pneumophila in  the presence of different sugar monomers. (A) Invasion of H. vermiformis  by L. pneumophila in the absence or presence of various sugars at 10 and  100 mM concentrations. Amebas were infected with L. pneumophila for 4 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  sugars compared to untreated cultures. Values are the means of triplicate  samples, and error bars represent standard deviations. (B) Growth kinetics  of L. pneumophila in cocultures with H. vermiformis in the absence or presence of sugars at 100 mM concentrations. At several time intervals of the  infection, the number of bacteria in the cocultures was determined following growth on agar plates. The bacteria do not replicate extracellularly in the  coculture and thus the increase in the number of bacteria is due to intracellular replication.
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Related In: Results  -  Collection

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

Figure 4: Inhibition of invasion of H. vermiformis by L. pneumophila in the presence of different sugar monomers. (A) Invasion of H. vermiformis by L. pneumophila in the absence or presence of various sugars at 10 and 100 mM concentrations. Amebas were infected with L. pneumophila for 4 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 sugars compared to untreated cultures. Values are the means of triplicate samples, and error bars represent standard deviations. (B) Growth kinetics of L. pneumophila in cocultures with H. vermiformis in the absence or presence of sugars at 100 mM concentrations. At several time intervals of the infection, the number of bacteria in the cocultures was determined following growth on agar plates. The bacteria do not replicate extracellularly in the coculture and thus the increase in the number of bacteria is due to intracellular replication.
Mentions: Previous work with E. histolytica showed a decrease in 170-kD lectin-mediated adherence to colonic mucosa in the presence of the monovalent sugars, Gal and GalNAc (35, 36). To investigate the functional relevance of involvement of 170-kD lectin in the invasion process, we performed blocking experiments using various sugars, H. vermiformis cultures were infected with L. pneumophila in the presence of various sugars and the levels of invasion were measured using two different assays. First, we used a gentamicin protection invasion assay in which 4 h following infection the extracellular bacteria were killed by treatment with gentamicin and intracellular bacteria were plated on BCYE plates for colony enumeration. Our results showed that both Gal and GalNAc decreased invasion of amebas in a dose-dependent manner. While 100 mM of GalNAc and Gal inhibited invasion by 83 and 75%, respectively (Fig. 4 A), other sugars like glucose, mannose, and lactose, showed little or no effect on invasion of amebas by L. pneumophila at any concentration tested (Fig. 4 A). These data are similar to the inhibition of adherence of E. histolytica in the presence of identical concentrations of Gal and GalNAc (35, 36).

Bottom Line: 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.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.

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