The region(s) of hsp70 critical for sulfogalactolipid (SGL) recognition has been defined through deletion analysis and site-directed mutagenesis. Truncated polymerase chain reaction products of hsp70 generated N-terminal fragments of 43, 35, 29, and 22 kDa. The C terminus substrate-binding domain (28 kDa) was also expressed. The N-terminal ATPase domain (rP43) shared the binding specificity of hsp70, because only sulfogalactosyl ceramide and sulfogalactosyl glycerolipid were recognized by both TLC overlay and RELISA. The C-terminal domain showed no binding. SGL binding of rP29 and rP22 was severely reduced. The loss of SGL binding for rP35 by RELISA but not TLC overlay was considered as a function of receptor presentation. The truncation of rP43 to rP35 demonstrates that residues 318 -387 (the base of the ATP binding cleft) are critical for high affinity SGL binding. Mutagenesis showed that Arg 342 and Phe 198 are crucial for this process. SGL binding, mediated by these conserved residues within the ATPase domain of hsp70, implies that this binding specificity is evolutionarily conserved.Heat shock proteins of the 70-kDa family (hsp70) have traditionally been described as intracellular chaperones that facilitate protein folding (1), degradation (2), translocation across membranes (3), and disassembly of protein oligomers (4). These functions are driven by ATPase activity contained within the N-terminal domain of all hsp70 family members (5). Hsp70s have also been described on the surface of bacteria (6 -9), male germ cells (10), and carcinoma cell lines (11,12). The absence of extracellular ATP, however, likely renders the hsp70 chaperone function inoperative. Exogenous hsp70 has recently been shown to elicit a cytokine response after binding to the plasma membrane of monocytes (13) and to bind to the surface of antigen-presenting cells and undergo receptor-mediated endocytosis (14), consistent with a cell surface "receptor" for hsp70.We have previously described a novel function of hsp70 family members as cell surface-associated, SGL-specific adhesins. Anti-hsp70 antibodies prevent the attachment of mycoplasma (15), acid-stressed Helicobacter pylori (16), and temperaturestressed Hemophilus influenzae (17) to SGC.1 This SGL binding specificity was found to be shared by the bovine brain hsp70, recombinant mycoplasma hsp70s (15), and the recombinant testis-specific hsc70 (18).We have recently extended this survey to demonstrate that recombinant hsp70 family members from Chlamydia trachomatis (6), H. pylori (19), H. influenzae (17), Escherichia coli (20), and an hsp70-related extracellular domain from the egg receptor of the sea urchin, Strongylocentrotus purpuratus (21), all possess the same restricted "lectin" binding specificity for SGC and SGG in vitro. 2 We further found that heterogeneity within the lipid moiety of SGC can differentially modulate binding by prokaryote, as compared with eukaryote, hsp70s, which may reflect their different in vivo adhesin functions.Sulfogalactolipids are found in a variety of t...

Osmoregulatory transporters Prop and ProU mediate the use of betaines as osmoprotectants by Escherichia coli. Glycine betaine and proline betaine are present in mammalian urines. Betaine uptake may therefore facilitate the growth of Em coli in the urinary tract, an environment of fluctuating osmolality. Prop transporter activity was approximately threefold higher in a pyelonephritis isolate, E. coli HU734, than in E. coli K-12. The growth rate of E. coli HU734 in aerated minimal salts medium was reduced twofold by 0 2 M NaCl in the absence and by 055 M NaCl in the presence of glycine betaine.Maximal growth rate stimulation was achieved when glycine betaine was added a t a concentration as low as 25 pM. Deletion of the prop locus impaired the growth rate of E. coli HU734 in human urine but not in minimal medium supplemented with NaCl (04 M), with or without glycine betaine (0-1 mM). The expression of pyelonephritis-associated (P) pili was reduced when E. coli HU734 was cultured in a rich culture medium (LB) of elevated salinity. The prop lesion had no influence on P pilus expression in witm or on the recovery of bacteria from the kidneys of inoculated mice. However, it did reduce their recovery from the bladders of inoculated mice 100-fold. These data provide the first direct evidence that osmoprotective betaine accumulation and transporter Prop are pertinent to both growth in human urine and colonization of the murine urinary tract by uropathogenic E. coli.

Specific 3'-sulfogalactolipid [SGL-sulfogalactosyl ceramide (SGCer) and sulfogalactosylglycerolipid (SGG)] binding is compared for hsp70s cloned from Helicobacter pylori, Haemophilus influenzae, Chlamydia trachomatis serovar E, Escherichia coli, murine male germ cells, and the hsp70-like extracellular domain within the sperm receptor from Strongylocentrotus purpuratus. This lectin activity, conserved among the different hsp70 family members, is modulated by the SGL aglycone. This is shown by differential binding to both SGC fatty acid homologues and 3'-sulfogalactolipid neoglycoproteins generated by coupling bovine serum albumin (BSA) and glycosyl ceramide acids synthesized by oxidation of the double bond of sphingosine. Eukaryotic hsp70s preferentially bound the SGCer fatty acid homologues SG(24)Cer, SG(18)Cer, and SG(20:OH)Cer, while prokaryotic hsp70s bound SG(18:1)Cer and SG(20:OH)Cer. Eukaryotic hsp70s bound SGCer-BSA and SG(24)Cer-BSA conjugates where the latter is the main constituent in SGCer-BSA, while prokaryotic hsp70s bound SG(20:OH)Cer-BSA. None of the hsp70s bound sulfogalactosyl sphingosine (SGSph) or SGSph-BSA, further demonstrating the important role of the aglycone. Although the primary SGL recognition domain of all hsp70s is conserved, we propose that aglycone organization differentially influences the interaction with the sub-site. Heterogeneous SGCer aglycone isoforms in cells and the differential in vitro binding of eukaryotic and prokaryotic hsp70s may relate to their different adhesin roles in vivo as mediators of germ cell and bacterial/host interactions, respectively.