Legionella pneumophila is a facultative intracellular pathogen responsible for severe lung disease in humans, known as legionellosis or Legionnaires' disease. Previously, we reported on the ϳ60-kDa glucosyltransferase (Lgt1) from Legionella pneumophila, which modified eukaryotic elongation factor 1A. In the present study, using L. pneumophila Philadelphia-1, Lens, Paris, and Corby genome databases, we identified several genes coding for proteins with considerable sequence homology to Lgt1. These new enzymes form three subfamilies, termed Lgt1 to -3, glucosylate mammalian elongation factor eEF1A at serine-53, inhibit its activity, and subsequently kill target eukaryotic cells. Expression studies on L. pneumophila grown in broth medium or in Acanthamoeba castellanii revealed that production of Lgt1 was maximal at stationary phase of broth culture or during the late phase of Legionella-host cell interaction, respectively. In contrast, synthesis of Lgt3 peaked during the lag phase of liquid culture and at early steps of bacterium-amoeba interaction. Thus, the data indicate that members of the L. pneumophila glucosyltransferase family are differentially regulated, affect protein synthesis of host cells, and represent potential virulence factors of Legionella.The protein synthesis machinery of eukaryotic cells is a wellknown target for pathogenic microorganisms during hostpathogen interaction. Examples of bacterial protein toxins targeting host protein synthesis include Shiga-and Shiga-like toxins, which act as rRNA N-glycosidases, and diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A, which ADPribosylate the modified histidine residue diphthamide in eukaryotic elongation factor 2 (eEF2). Both types of enzymatic activities result in inhibition of protein synthesis and death of target cells (26,34).Recently, an ϳ60-kDa glucosyltransferase, referred to here as Lgt1 (Legionella pneumophila glucosyltransferase 1), was identified in L. pneumophila cultures. Well-studied examples of bacterial glucosylating enzymes targeting eukaryotic proteins are the large clostridial cytotoxins. They glucosylate 20-to 25-kDa small GTPases of the Rho family, thereby inhibiting the regulatory functions of these switch proteins (1, 16). In contrast, the Legionella enzyme modified an ϳ50-kDa component in mammalian cell extracts, which was identified subsequently as eEF1A. This elongation factor also represents a GTP-binding protein, possessing GTPase activity. Lgt1 modifies serine-53 of eEF1A, located in the GTPase domain near the switch 1 region of the GTPase. This modification results in inhibition of protein synthesis both in vitro and in vivo and causes death of intoxicated eukaryotic cells (3, 4).Many Legionella proteins occur in a set of redundant molecules, executing apparently closely related functions (6,12,18,27,28). Therefore, we screened Legionella genome databases for Lgt1 analogs. Here we report that L. pneumophila strains Philadelphia-1, Lens, Paris, and Corby (GenBank accession numbers NC_002942, NC_006369, NC_006368...
Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia.
Background: Staphylococcus aureus is a Gram-positive bacterium that causes severe illnesses in the human population. The capacity of S. aureus strains to form biofilms on biotic and abiotic surfaces creates serious problems for treatment of hospital infections and has stimulated efforts to develop new means of specific protection or immunotherapy.Material and Methods:We found that rabbit serum raised against crude concentrated S. aureus liquid culture significantly decreased the development of staphylococcal biofilm in vitro. To discover the corresponding staphylococcal antigen, we used mass-spectrometry and molecular cloning and identified three major immunodominant proteins. They included α-haemolysin, serine proteinase SplB and S. aureus surface protein G, known as adhesin SasG.Results:Although according to literature data, all these proteins represent virulence factors of S. aureus and play diverse and important roles in the pathogenesis of staphylococcal diseases, only SasG can be directly implicated into the biofilm formation because of its surface location on a staphylococcal cell. Indeed, rabbit serum directed against purified recombinant SasG, similar to serum against crude staphylococcal liquid culture, prevented the formation of a biofilm.Conclusion:SasG can be considered as a target in an anti-biofilm drug development and a component of the vaccine or immunotherapeutic preparations directed against staphylococcal infections in humans.
Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62–K70 segment of Rps26 and the 5′ untranslated region of mRNA. The data suggested a specific role of the Y62–K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62–K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5′ untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62–K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast.
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