Surface localized glyceraldehyde‐3‐phosphate dehydrogenase of Mycoplasma genitalium binds mucin

Abstract

SummaryMycoplasma genitalium is the smallest known selfreplicating cell. It was first isolated from urethral specimens in individuals with non-gonococcal urethritis and, more recently, from respiratory and synovial sites. Our laboratory has been interested in defining the mechanisms by which M. genitalium adheres to and colonizes host cell surfaces. In order to determine potential targets of adherence, we examined the interaction of M. genitalium with a primary component of the mucosal epithelial lining, mucin (Mn). Three Mn-binding proteins (MnBPs) of M. genitalium were isolated by affinity chromatography. One of these proteins was identified by N-terminal sequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antiserum raised against recombinant GAPDH blocked binding of intact biosynthetically labelled mycoplasmas to mucin by approximately 70%. Whole cell radioimmunoprecipitation indicated that GAPDH was surface-accessible and surface localization of GAPDH was further verified by membrane fractionation and immunoelectron microscopy. The role of GAPDH as an adhesin to Mn not only provides insights into the organism's mechanisms of adherence and colonization but also into its ability to maximize its limited genome.

Selenium effects on the metabolism of a Se-metabolizingLactobacillus reuteri: analysis of envelope-enriched and extracellular proteomes

“…This glycolytic enzyme, when extracellularly-exposed or released can act as an adhesine, since it is able to bind extracellular matrix proteins. 54 It can be hypothesized that this enzyme could act as a selenium storage protein, as already suggested by Ogasawara and co-workers. 55 Both glyceraldehyde 3-phosphate dehydrogenase and phosphoketolase were already identified in the in toto proteome 28 as selenocysteine-containing proteins: this result corroborates once more the ability of the strain to fix selenium generating organic Se-forms and can explain the previously reported increase of Se concentration in the external medium after about 6 hours of growth.…”

Role of Mycoplasma pneumoniae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in mediating interactions with the human extracellular matrix

“…The results of a semiquantitative ELISA with recombinant GAPDH of known concentration confirmed that between 50 and 70 % of all GAPDH was found in the TX-insoluble protein fraction. Alvarez et al (2003) reported that 10 % of the total GAPDH of M. genitalium was membrane-associated, but used a different method for membrane purification from the one used in the present study. Nevertheless, the data confirm the occurrence of remarkable amounts of GAPDH in the membrane-associated protein fraction of the bacteria, especially in early stages of the culture (2 days).…”

“…During the further culture period, the relationship between surface and cytosolic GAPDH remains nearly constant, suggesting constant transport of the protein to the bacterial cell membrane. Despite the occurrence of more than 40 cleavage sites for trypsin (www.expasy.ch/ tools/peptidecutter/), proteolytic digestion experiments resulted in a lack of accessible surface-exposed M. pneumoniae GAPDH, whereas M. genitalium GAPDH (82 % identity) was described as trypsin-sensitive (Alvarez et al, 2003). A clear antibody response to recombinant GAPDH was not detected either in hyperimmune sera of (Dallo et al, 2002;Barbosa et al, 2006;Egea et al, 2007), which could influence basic aspects of pathogenesis, like tissue tropism.…”

Streptococcus agalactiae GAPDH Is a Virulence-Associated Immunomodulatory Protein

“…The K M values obtained were higher (23.8 mM and 32.6 mM, respectively, for F1,6bisP and NAD ϩ ) than the values reported for GAPDH of group A streptococci (33) (1.33 mM for G-3-P and 156.7 M for NAD ϩ ), indicating a weaker affinity of the recombinant GBS enzyme for its substrates. In addition to its presence in the cytosol, GAPDH has also been described as a surface (28,31) or a secreted (36) protein. Consistently, we demonstrated in this study by Western blot and sequencing analysis that NEM316 GAPDH was present in culture supernatants devoid of isocitrate dehydrogenase activity, a cytosolic protein used as a marker of bacterial lysis.…”

“…In several pathogenic bacteria, GAPDH has been described as a protein associated with virulence (28 -30) due to its ability to bind several host proteins (28,(31)(32)(33) or to confer resistance against reactive oxygen species produced by host phagocytic cells (34). In GBS, GADPH is localized in their surface and is highly homologous to the plasmin receptor of group A streptococci (35).…”