Type III machines of pathogenic yersiniae secrete virulence factors into the extracellular milieu

Gram-negative bacteria use type III machines to inject toxic proteins into the cytosol of eukaryotic cells. Pathogenic Yersinia species export 14 Yop proteins by the type III pathway and some of these, named effector Yops, are targeted into macrophages, thereby preventing phagocytosis and allowing bacterial replication within lymphoid tissues. Hitherto, YopB/YopD were thought to insert into the plasma membrane of macrophages and to promote the import of effector Yops into the eukaryotic cytosol. We show here that the type III machines of yersiniae secrete three proteins into the extracellular milieu (YopB, YopD and YopR). Although intrabacterial YopD is required for the injection of toxins into eukaryotic cells, secreted YopB, YopD and YopR are dispensable for this process. Nevertheless, YopB, YopD and YopR are essential for the establishment of Yersinia infections in a mouse model system, suggesting that type III secretion machines function to deliver virulence factors into the extracellular milieu also.     

Genes encoding specific nickel transport systems flank the chromosomal urease locus of pathogenic yersiniae

The transition metal nickel is an essential cofactor for a number of bacterial enzymes, one of which is urease. Prior to its incorporation into metalloenzyme active sites, nickel must be imported into the cell. Here, we report identification of two loci corresponding to nickel-specific transport systems in the gram-negative, ureolytic bacterium Yersinia pseudotuberculosis. The loci are located on each side of the chromosomal urease gene cluster ureABCEFGD and have the same orientation as the latter. The yntABCDE locus upstream of the ure genes encodes five predicted products with sequence homology to ATP-binding cassette nickel permeases present in several gram-negative bacteria. The ureH gene, located downstream of ure, encodes a single-component carrier which displays homology to polypeptides of the nickel-cobalt transporter family. Transporters with homology to these two classes are also present (again in proximity to the urease locus) in the other two pathogenic yersiniae, Y. pestis and Y. enterocolitica. An Escherichia coli nikA insertion mutant recovered nickel uptake ability following heterologous complementation with either the ynt or the ureH plasmid-borne gene of Y. pseudotuberculosis, demonstrating that each carrier is necessary and sufficient for nickel transport. Deletion of ynt in Y. pseudotuberculosis almost completely abolished bacterial urease activity, whereas deletion of ureH had no effect. Nevertheless, rates of nickel transport were significantly altered in both ynt and ureH mutants. Furthermore, the ynt ureH double mutant was totally devoid of nickel uptake ability, thus indicating that Ynt and UreH constitute the only routes for nickel entry. Both Ynt and UreH show selectivity for Ni(2+) ions. This is the first reported identification of genes coding for both kinds of nickel-specific permeases situated adjacent to the urease gene cluster in the genome of a microorganism.     

Structural characterization of the lipid A component of pathogenic Neisseria meningitidis.

The lipid A component of meningococcal lipopolysaccharide was structurally characterized by using chemical modification methods, methylation analysis, 31P nuclear magnetic resonance, and laser desorption mass spectroscopy. It was shown that Neisseria meningitidis lipid A consists of a 1,4'-bisphosphorylated beta(1'----6)-linked D-glucosamine disaccharide (lipid A backbone), both phosphate groups being largely replaced by O-phosphorylethanolamine. This disaccharide harbors two nonsubstituted hydroxyl groups at positions 4 and 6', the latter representing the attachment site of the oligosaccharide portion in lipopolysaccharide. In addition, it is substituted by up to six fatty acid residues. In the major lipid A component, representing a hexaacyl species, the hydroxyl groups at positions 3 and 3' carry (R)-3-hydroxydodecanoic acid [12:0(3-OH)], whereas the amino groups at positions 2 and 2' are substituted by (R)-3-(dodecanoyloxy)tetradecanoic acid [3-O(12:0)-14:0]. A minor portion was present as a tetraacyl lipid A component lacking either dodecanoic acid (12:0) or 12:0 and 12:0(3-OH). N. meningitidis lipid A, therefore, significantly differs from Escherichia coli lipid A by the nature and locations of fatty acids and the substitution of O-phosphorylethanolamine for the nonglycosyl (4'-P) and glycosyl phosphate.     

Variation in skin surface lipid composition among the Equidae

Skin surface lipids from Equus caballus, E. przewalskii, E. asinus, E. grevyi, E. hemionus onager and a mule (E. asinus/E. caballus) were analyzed in detail. In all species the surface lipid mixtures consisted of giant-ring lactones, cholesterol, cholesteryl esters and minor amounts of wax diesters. In E. caballus, the lactone hydroxyacids were entirely branched chained, while in E. asinus and E. grevyi they were almost exclusively straight chained. In E. przewalskii, the onager and the mule there were both straight and branched chain hydroxyacid lactones. These results are in harmony with published interpretations of the evolutionary relationships among Equus species.     

An unusual lipid in the human pathogenic fungus Epidermophyton floccosum.

The dermatophyte Epidermophyton floccosum contains an unusual lipid characterized as 1(3),2-diacylglyceryl-3(1)-O-4'-(N,N,N-trimethyl)homoserine. Its concentration in E. floccosum is approx. 15% of the polar lipids. The role of this lipid in the biosynthesis of polar lipids and fatty acids is not yet known. However, it is interesting from a chemotaxonomic viewpoint that the lipids from two other genera of dermatophyte, Microsporum cookei and Trichophyton rubrum do not contain this novel lipid.     

Variation between pathogenic serovars within Salmonella pathogenicity islands

Although four of the five Salmonella pathogenicity islands (SPIs) have been characterized in detail for Salmonella enterica serovar Typhimurium, and the fifth has been characterized for Salmonella enterica serovar Dublin, there have been limited studies to examine them in detail in a range of pathogenic serovars of S. enterica. The aim of this study was to examine these regions, shown to be crucial in virulence, in pathogenic serovars to identify any major deletions or insertions that may explain variation in virulence and provide further understanding of the elements involved in the evolution of these regions. Multiple strains of each of the 13 serovars were compared by Southern blot hybridization using a series of probes that together encompassed the full length of all five SPIs. With the exception of serovar Typhimurium, all strains of the same serovar were identical in all five SPIs. Those serovars that differed from serovar Typhimurium in SPI-1 to SPI-4 and from serovar Dublin in SPI-5 were examined in more detail in the variant regions by PCR, and restriction endonuclease digestion and/or DNA sequencing. While most variation in hybridization patterns was attributable to loss or gain of single restriction endonuclease cleavage sites, three regions, in SPI-1, SPI-3, and SPI-5, had differences due to major insertions or deletions. In SPI-1 the avrA gene was replaced by a 200-base fragment in three serovars, as reported previously. In SPI-5, two serovars had acquired an insertion with similarity to the pagJ and pagK genes between pipC and pipD. In SPI-3 the genes sugR and rhuM were deleted in most serovars and in some were replaced by sequences that were very similar to either the Escherichia coli fimbrial operon, flanked by two distinct insertion sequence elements, or to the E. coli retron phage PhiR73. The distribution of these differences suggests that there have been a number of relatively recent horizontal transfers of genes into S. enterica and that in some cases the same event has occurred in multiple lineages of S. enterica. Thus, it seems that insertion sequences and retron phages are likely to be involved in continuing evolution of the pathogenicity islands of pathogenic Salmonella serovars.     

The structure of the lipid A component of Sphaerotilus natans

The lipopolysaccharide of Sphaerotilus natans afforded a ladder-like pattern of bands in sodium deoxycholate-polyacrylamide gel electrophoresis, indicating the presence of a S-form lipopolysaccharide. The chemical analysis showed neutral sugars (rhamnose, glucose, l-glycero-d-manno-heptose), 3-deoxy-octulosonic acid (Kdo), amino compounds (glucosamine, glucosamine phosphate, ethanolamine and ethanolamine phosphate), and phosphorus. The lipid A fraction contained saturated and unsaturated capric, lauric, and myristic acids, and 3-hydroxy capric acid (3-OH-10:0). Its chemical structure was consisting of a glucosamine disaccharide, glycosidically substituted by a phosphomonoester, and substituted at C-4 by a pyrophosphodiester esterified with ethanolamine. The amino groups of both glucosamines are acylated by 3-hydroxy capric acids and these in turn are substituted by saturated and unsaturated capric, lauric, and myristic acids. Hydroxyl groups of the backbone disaccharide at C-3 and C-3 were also esterified by 3-hydroxy capric acid, those at C-4 and C-6 were unsubstituted. The latter provides the attachment site for Kdo.Abbreviations Kdo 3-deoxy-d-manno-octulosonic acid - 3-OH-10:0 3-hydroxy capric acid - DOC-PAGE deoxycholate-polyacrylamide gel electrophoresis - GC-MS gas chromatography/mass spectrometry - LD-MS laser desorption mass spectrometry - LPS lipopolysaccharide - PS polysaccharide     

Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide curvacin A

The 3D structure of the membrane-permeabilizing 41-mer pediocin-like antimicrobial peptide curvacin A produced by lactic acid bacteria has been studied by NMR spectroscopy. In DPC micelles, the cationic and hydrophilic N-terminal half of the peptide forms an S-shaped beta-sheet-like domain stabilized by a disulfide bridge and a few hydrogen bonds. This domain is followed by two alpha-helices: a hydrophilic 6-mer helix between residues 19 and 24 and an amphiphilic/hydrophobic 11-mer helix between residues 29 and 39. There are two hinges in the peptide, one at residues 16-18 between the N-terminal S-shaped beta-sheet-like structure and the central 6-mer helix and one at residues 26-28 between the central helix and the 11-mer C-terminal helix. The latter helix is the only amphiphilic/hydrophobic part of the peptide and is thus presumably the part that penetrates into the hydrophobic phase of target-cell membranes. The hinge between the two helices may introduce the flexibility that allows the helix to dip into membranes. The helix-hinge-helix structure in the C-terminal half of curvacin A clearly distinguishes this peptide from the other pediocin-like peptides whose structures have been analyzed and suggests that curvacin A along with the structural homologues enterocin P and carnobacteriocin BM1 belong to a subgroup of the pediocin-like family of antimicrobial peptides.     

Variation of the lipid content of yeast cells during sporulation

Changes in the lipid content of yeast cells during sporulation were studied. The lipid content increases during sporulation. The unsaponifiable fraction represents the major part of lipids extracted at the moment sporulation sets in.     

Roles of V antigen in promoting virulence and immunity in yersiniae

It is established that yersiniae harboring an approximately 45-megadalton Vwa-plasmid can produce V and W antigens (Vwa+), and that sera containing anti-V provides passive protection to mice against Yersinia pestis. This observation was extended by the use of monospecific anti-V prepared by injecting rabbits with partially purified V, absorption of antisera with a Vwa- extract, and then separation of gamma-globulin by traditional processes of fractionation or by affinity chromatography. These preparations provided passive protection against 10 minimum lethal doses of virulent Y. pestis KIM, Yersinia pseudotuberculosis PB1, and Yersinia enterocolitica WA. Kinetics of elimination of these Vwa+ yersiniae from organs and blood of passively immunized mice closely resembled those of avirulent Vwa- mutants from normal mice. Injection into mice of sterile crude extracts of Y. pseudotuberculosis PB1 containing V promoted significant survival and retention of Vwa- mutants of Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. This effect was eliminated by the removal of V before injection by precipitation with monospecific antibody. These results indicate that V antigen per se is the major virulence factor mediated by Vwa-plasmids.     

Variation in the primary structure of Bacillus subtilis flagellins

The flagella derived from 18 strains Bacillus subtilis were tested for their reaction with antiflagellar filament antibody and antiflagellin antibody. On the basis of their reactivity, at least five serologically distinct classes could be identified. Peptide map analysis of tryptic digests of the subunit proteins were consistent with the immunochemical analysis. Large differences in sequence existed among proteins of the different classes; proteins within an antigenic group differed by only a few peptides. Furthermore, 9 of the 27 tryptic peptides resolved were common to flagellin proteins from all the classes examined. The relationship between antigenic specificity, variability in peptide pattern, and the conformation of the flagellin protein are discussed.     

Role of lipid rafts in the processing of the pathogenic prion and Alzheimer's amyloid-beta proteins

The conformational conversion of the cellular form of the prion protein (PrP C) into the infectious form (PrP Sc) and the proteolytic processing of the amyloid-beta (Abeta) peptide are central pathogenetic events in the prion diseases and Alzheimer's disease, respectively. Cholesterol- and sphingolipid-rich lipid rafts have emerged as important sites for the conversion of PrP C into PrP Sc, and for the proteolytic production, degradation and aggregation of Abeta. Here, we discuss these findings and their implications for our understanding of these disease processes. In addition, the potential for rafts as sites for therapeutic intervention in prion diseases and Alzheimer's disease is considered.     

Variation in acid resistance among shiga toxin-producing clones of pathogenic Escherichia coli

Pathogenic strains of Escherichia coli, such as E. coli O157:H7, have a low infectious dose and an ability to survive in acidic foods. These bacteria have evolved at least three distinct mechanisms of acid resistance (AR), including two amino acid decarboxylase-dependent systems (arginine and glutamate) and a glucose catabolite-repressed system. We quantified the survival rates for each AR mechanism separately in clinical isolates representing three groups of Shiga toxin-producing E. coli (STEC) clones (O157:H7, O26:H11/O111:H8, and O121:H19) and six commensal strains from ECOR group A. Members of the STEC clones were not significantly more acid resistant than the commensal strains when analyzed using any individual AR mechanism. The glutamate system provided the best protection in a highly acidic environment for all groups of isolates (<0.1 log reduction in CFU/ml per hour at pH 2.0). Under these conditions, there was notable variation in survival rates among the 30 O157:H7 strains, which depended in part on Mg(2+) concentration. The arginine system provided better protection at pH 2.5, with a range of 0.03 to 0.41 log reduction per hour, compared to the oxidative system, with a range of 0.13 to 0.64 log reduction per hour. The average survival rate for the O157:H7 clonal group was significantly less than that of the other STEC clones in the glutamate and arginine systems and significantly less than that of the O26/O111 clone in the oxidative system, indicating that this clonal group is not exceptionally acid resistant with these specific mechanisms.     

On the structure of the hemocyanin channel in lipid bilayers

Keyhole limpet hemocyanin has been shown by others (Alvarez, O., Diaz, E. and Latorre, R. (1975) Biochim. Biophys. Acta 389, 444-448) to form single conductance channels in black lipid membranes. In an attempt to visualize how the large (300 A) water-soluble hemocyanin molecule interacts with lipid bilayers, we have examined hemocyanin in the presence of lipids with the electron microscope. We find that incubation of lipids with keyhole limpet hemocyanin produces a characteristic 70 A in diameter, ring-shaped particle or annulus associated with the bilayer. This annulus, which appears to be quite distinct from previously observed aggregated and dissociated forms of hemocyanin, may be responsible for the channel formation in black lipid membranes.