Showing papers in "Infection and Immunity in 1990"

Pyelonephritogenic Escherichia coli and killing of cultured human renal proximal tubular epithelial cells: role of hemolysin in some strains.

Abstract: Acute pyelonephritis, a complication of Escherichia coli bacteriuria, must represent a bacterial invasion through the kidney epithelium. To study this process, we overlaid bacterial suspensions onto monolayers of cultured human kidney proximal tubular epithelial cells and measured cytotoxicity by release of lactate dehydrogenase (LDH). Thirty-four isolates cultured from patients with acute pyelonephritis were screened for the ability to cause pyelonephritis in CBA mice by transurethral challenge. The eight most virulent strains (greater than or equal to 70% of mice challenged developed greater than or equal to 10(3) CFU/g of kidney after 48 h) were selected for study. Each strain displayed mannose-resistant hemagglutination of human O erythrocytes; three strains were phenotypically and genotypically hemolytic. Pyelonephritogenic strains were significantly more cytotoxic (30.1 +/- 9.5% LDH release after 18 h) than eight fecal control strains (13.5 +/- 11.5% LDH release; P = 0.0068). We selected the most cytotoxic strain, CFT073, for further study. Sterile filtrate from this hemolytic strain was significantly more cytotoxic than was the filtrate of the fecal control strain, FN414. Transposon mutagenesis of CFT073 with TnphoA abolished hemolytic activity and cytotoxicity by both whole cells and sterile filtrate. Southern blot analysis revealed that the Tnphoa insertion mapped to the E. coli chromosomal hly determinant within a 12-kilobase SalI restriction fragment. Transformation of a nonhemolytic strain, CPZ005 with plasmid pSF4000, which carries a cloned hemolysin determinant, resulted in highly elevated cytotoxicity. Light micrographs of proximal tubular epithelial cell cultures demonstrated cell damage by pyelonephritogenic strains that was not induced by a fecal strain or the hemolysin-deficient mutant. Results indicate that pyelonephritogenic E. coli strains are more frequently cytotoxic for a putative target, that is, human renal tubular epithelium, than are fecal isolates. Hemolysin, in some strains, is apparently responsible for this cytotoxicity. Images

Characterization of and human serologic response to proteins in Helicobacter pylori broth culture supernatants with vacuolizing cytotoxin activity.

Abstract: Helicobacter pylori infection is strongly associated with histologic gastritis and peptic ulcer disease. Broth culture supernatants from a subset of H. pylori strains induce vacuolization in cultured cells, a phenomenon that has been attributed to cytotoxin activity. Concentrated culture supernatants from 15 of 28 (53.6%) H. pylori strains tested induced vacuolization in HeLa cells in titers ranging from 1:10 to 1:180. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of supernatants from these 28 strains and 2 control strains demonstrated an 82-kilodalton (kDa) protein band in 3 of 16 supernatants with vacuolizing activity, but in none of 14 supernatants without vacuolizing activity. By immunoblotting with human sera, a 128-kDa band was recognized in all 16 supernatants with vacuolizing activity, compared with 9 of 14 (64%) supernatants without vacuolizing activity (P = 0.014). Serologic recognition of the 128-kDa band in H. pylori culture supernatants was more prevalent among persons infected with vacuolizing H. pylori strains than among persons infected with nonvacuolizing strains, but the difference was not statistically significant (80 versus 45%; P = 0.079); human serologic recognition of the 82-kDa band was less common. The 128-kDa band was recognized by 100% of 31 serum samples from H. pylori-infected patients with duodenal ulcer disease, compared with 60.8% of 74 serum samples from H. pylori-infected persons without peptic ulcer disease (P = 0.0001). These data indicate that antigenic 128- and 82-kDa proteins are present in H. pylori broth culture supernatants with vacuolizing activity and that serologic responses to the 128-kDa protein are more prevalent among H. pylori-infected persons with duodenal ulceration than among infected persons without peptic ulceration.

Secretion of Yop proteins by Yersiniae.

Abstract: Upon incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic strains of the genus Yersinia cease growing and produce large amounts of a series of plasmid-encoded proteins involved in pathogenicity. These proteins, called Yops (for Yersinia outer membrane proteins), are detected in both the outer membrane fraction and the culture supernatant. We present here the nucleotide sequence of genes yop20 and yop25 from Yersinia enterocolitica O:9. Protein Yop25 is very similar to YopE, the corresponding protein from Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica O:8 (A. Forsberg and H. Wolf-Watz, J. Bacteriol. 172:1547-1555, 1990). This is the first report of a yop20 sequence of yersiniae. We present evidences that Yops are not membrane proteins. Their detection in the membrane fraction results either from copurification of large aggregates of extracellular Yops with the membrane fraction or from the adsorption of released proteins to the cell surface. In contrast with Yops, protein P1 has characteristics of a true membrane protein. The release of Yops by Y. enterocolitica occurs by a novel secretion mechanism that does not involve the cleavage of a typical signal sequence or the recognition of a carboxy-terminal domain.

Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type 1 fimbriae.

Abstract: Type 1 fimbriae of Escherichia coli are surface organelles which mediate binding to D-mannose-containing structures. By direct binding of FimH to D-mannose attached to a carrier protein, we demonstrated that this protein was uniquely responsible for the receptor specificity. Furthermore, we show by receptor immunoelectron microscopy that the FimH protein is located laterally in the structure of the type 1 fimbriae.

Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling.

Abstract: The ability to enter intestinal epithelial cells is an essential virulence factor of salmonellae. We have previously cloned a group of genes (invA, B, C, and D) that allow S. typhimurium to penetrate tissue culture cells (J. E. Galan and R. Curtiss III, Proc. Natl. Acad. Sci. USA 86:6383-6387, 1989). Transcriptional and translational cat and phoA fusions to invA (the proximal gene in the invABC operon) were constructed, and their expression was studied by measuring the levels of alkaline phosphatase or chloramphenicol acetyltransferase activity in mutants grown under different conditions. It was found that when strains containing the fusions were grown on media with high osmolarity, a condition known to increase DNA superhelicity, the level of invA transcription was approximately eightfold higher than that in strains grown on media with low osmolarity. The osmoinducibility of invA was independent of ompR, which controls the osmoinducibility of other genes. Strains grown in high-osmolarity media in the presence of subinhibitory concentrations of gyrase inhibitors (novobiocin or coumermycin A1), which reduce the level of DNA supercoiling, showed reduced expression of invA. Nevertheless, invA was poorly expressed in topA mutants of S. typhimurium, which have increased DNA superhelicity. In all cases, the differential expression of the invasion genes was correlated with the ability of S. typhimurium to penetrate tissue culture cells. These results taken together indicate that expression of S. typhimurium invasion genes is affected by changes in DNA supercoiling and suggest that this may represent a way in which this organism regulates the expression of these genes.

Intracellular and cell-to-cell spread of Listeria monocytogenes involves interaction with F-actin in the enterocytelike cell line Caco-2.

Abstract: Listeria monocytogenes penetrates and multiplies within professional phagocytes and other cells such as the Caco-2 human enterocytelike cell line. Listeriolysin O, a membrane-damaging cytotoxin accounts for intracellular multiplication through lysis of the membrane-bound phagocytic vacuole. This work demonstrates that once released within the cytosol, L. monocytogenes acquires the capacity to spread intracellularly and infect adjacent cells by interacting with host cell microfilaments. Such evidence was obtained by using drugs which disrupt the cell cytoskeleton. Nocodazole, which blocks polymerization of microtubules, did not affect intracellular spread, whereas cytochalasin D, which blocks polymerization of G-actin, inhibited the intracellular motility of the bacteria. By using fluorescence staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin), transmission electron microscopy, and immunogold labeling, direct evidence was obtained that intracellular bacteria were enveloped with a thick layer of F-actin. Within 2 h after entry, it was demonstrated by confocal microscopy that bacteria were following highly organized routes corresponding to stress fibers. Four hours after entry, some bacteria presented random movements which could be seen by the presence of a large trail of F-actin. Such movements also caused protrusions which deeply penetrated adjacent cells and resulted in the formation of vacuoles limited by a double membrane. After subsequent lysis of these membranes, bacteria released within the cytoplasm were able to multiply and invade new cells. In contrast, an hly::Tn1545 mutant of the wild-type microorganism demonstrated almost no intracellular spread. Only a few bacteria displaying delayed lysis of the phagocytic vacuole behaved like the wild-type strain. Hemolysin-mediated lysis of the phagocytic vacuole and subsequent interaction with host cell microfilaments may represent a major virulence factor allowing tissue colonization during listeriosis.

Molecular characterization of the Clostridium difficile toxin A gene.

Abstract: The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs had a mol% G + C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Gal alpha 1-3Gal beta 1-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.

Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins.

Abstract: The pathogenic spirochete Borrelia burgdorferi contains a set of integral membrane proteins which were selectively extracted into the detergent phase upon solubilization of intact B. burgdorferi with the nonionic detergent Triton X-114. Virtually all of these hydrophobic proteins were recognized by antibodies in pooled sera from patients with chronic Lyme arthritis, demonstrating that proteins partitioning into the detergent phase of Triton X-114 encompass the major B. burgdorferi immunogens. Furthermore, most of these immunogenic proteins, including the previously characterized OspA and OspB membrane antigens, could be biosynthetically labeled when B. burgdorferi was incubated in vitro with [3H]palmitate. The OspA and OspB antigens were radioimmunoprecipitated from [3H]palmitate-labeled detergent-phase proteins with monoclonal antibodies, and [3H]palmitate was recovered unaltered from these proteins after sequential alkaline and acid hydrolyses. The combined results provide formal confirmation that the major B. burgdorferi immunogens extracted by Triton X-114 are lipoproteins. The demonstration that B. burgdorferi integral membrane antigens are lipoproteins may explain the basis of their immunogenicity and may help to improve our understanding of the surface topology of B. burgdorferi.

Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread.

Abstract: To dissect the regulatory and structural requirements for Listeria monocytogenes intracellular growth and cell-to-cell spread, we designed a protocol based on transposon mutagenesis and the isolation of mutants which form small plaques in monolayers of mouse L2 cell fibroblasts. Two different transposable elements were used to generate libraries of insertion mutants: Tn916 and a derivative of Tn917-lac, Tn917-LTV3. Ten classes of mutants were isolated and evaluated for growth and cell-to-cell spread in J774 mouse macrophagelike cells, Henle 407 human epithelial cells, and mouse bone marrow-derived macrophages. Mutants were also evaluated for secretion of hemolysin and phospholipase (assayed by egg yolk opacity) and association with F-actin in the cytoplasm of cells, using NBD-phallacidin staining. The ten classes of mutants included (i) mutants showing abortive intracellular and extracellular growth; (ii) mutants showing abortive intracellular growth; (iii) rough mutants; (iv) mutants showing greatly reduced hemolysin and phospholipase secretion but showing normal growth in cells and little or no association with F-actin; (v) mutants with mutations mapping to an open reading frame (ORF) adjacent to hlyA and referred to as ORF U, lacking phospholipase activity, and with 50% normal hemolysin activity; (vi) mutants with reduced secretion of both hemolysin and phospholipase; (vii) nonhemolytic mutants with mutations mapping to the structural gene, hlyA; (viii) mutants with 25% normal hemolysin secretion and absolutely no association with F-actin; (ix) mutants with mutations mapping to ORF U, lacking phospholipase activity, and with normal hemolysin activity; and (x) mutants showing a mixed-plaque morphology but normal for all other parameters.

Secretory immunoglobulin-a carries oligosaccharide receptors for escherichia-coli type-1 fimbrial lectin

Abstract: Type 1 fimbriae with mannose-specific lectins are widely distributed among members of the family Enterobacteriaceae and confer the ability to attach to a range of host cells, including colonic epithelial cells. The mucosal surfaces are protected by secretory immunoglobulin A (IgA), which agglutinates microorganisms and prevents their attachment to host epithelial cells. This action has been attributed to a specificity of the antigen-combining site of mucosal immunoglobulins for bacterial and viral surface components. Here, we report a novel mechanism for the antibacterial effect of secretory IgA. Secretory IgA and IgA myeloma proteins, especially those of the IgA2 subclass, were shown to possess carbohydrate receptors for the mannose-specific lectin of type 1-fimbriated Escherichia coli. The presence of the high-mannose oligosaccharide chain Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc correlated with binding activity. The interaction between bacterial mannose-specific lectins and IgA receptor oligosaccharide resulted in agglutination of the bacteria and in inhibition of bacterial attachment to colonic epithelial cells. Thus, this interaction could form the basis for a broad antibacterial function of secretory IgA against enterobacteria regardless of the specificity of antibody molecules.

Molecular epidemiologic evidence for association of thermostable direct hemolysin (TDH) and TDH-related hemolysin of Vibrio parahaemolyticus with gastroenteritis.

Abstract: The Kanagawa phenomenon induced by the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus is almost exclusively associated with clinical strains, and TDH has been considered an important virulence factor. However, Kanagawa phenomenon-negative strains isolated from patients with diarrhea have recently been shown to produce TDH-related hemolysin (TRH). We studied the distribution of the tdh gene encoding TDH and the trh gene encoding TRH in vibrios by hybridization analyses. The presence or absence of the tdh gene and the trh gene in 285 strains of V. parahaemolyticus was examined by the DNA colony hybridization test with a tdh gene-specific probe and a newly constructed trh gene-specific probe. For assessment of the importance of TRH, many Kanagawa phenomenon-negative clinical strains (35.4% of all strains) were included. Of 214 clinical strains of V. parahaemolyticus, 112 strains (52.3%) had the tdh gene only, 52 strains (24.3%) had the trh gene only, and 24 strains (11.2%) carried both the tdh and the trh gene. The coexistence of the tdh and trh genes in these 24 strains was confirmed by Southern blot hybridization analysis. Of 71 environmental strains, 5 strains (7.0%) hybridized very weakly with the trh gene probe and none hybridized with the tdh gene probe. These results suggest that TRH as well as TDH is an important virulence factor of V. parahaemolyticus. Among 118 strains of other Vibrio species examined for the trh gene, only 1 strain of Vibrio furnissii gave a very weak hybridization signal. Among 48 representative trh gene-positive strains of V. parahaemolyticus, only 18 strains (37.5%) were found to produce TRH in culture medium when examined by a sensitive enzyme-linked immunosorbent assay method.

Pneumococcal surface protein A (PspA) is serologically highly variable and is expressed by all clinically important capsular serotypes of Streptococcus pneumoniae

Abstract: Pneumococcal surface protein A (PspA) has been shown previously to elicit antibodies protective against pneumococcal infection and to be necessary for full pneumococcal virulence in mice. The protein was originally defined by the two mouse monoclonal antibodies Xi64 and Xi126, which together recognized PspA on 14% of pneumococcal isolates. Some PspA molecules reacted with both antibodies, but most reacted with only one or the other. In the present study we demonstrated that PspA is produced by all pneumococci, confirming our hypothesis that there are variants of PspA which are not detected by Xi64 and Xi126. We produced a rabbit antiserum and five additional monoclonal antibodies specific for PspA for these studies. The rabbit antiserum reacted with each of 95 pneumococcal isolated tested, comprising 16 capsular serotypes. One or more of the seven monoclonal anti-PspA antibodies reacted with 95% (53 of 57) of pneumococcal isolates tested. The specificity of the monoclonal and polyclonal antibodies to PspA was confirmed in two ways: (i) by detection of molecules on wild-type pneumococci that are identical in molecular weight to those detected in Western blots (immunoblots) with Xi64 and Xi126 and (ii) by the use of mutants of Streptococcus pneumoniae that fail to produce PspA or that produce a truncated form of PspA. By using the seven monoclonal antibodies, we observed 31 PspA types among the 57 isolates. When the 53 strains reactive with the monoclonal antibodies were analyzed by capsular type as well as by serologic type and molecular weight of PspA, we observed 50 different clonotypes of pneumococci.

Purification and N-terminal analysis of urease from Helicobacter pylori.

Abstract: Urease of Helicobacter pylori (formerly Campylobacter pylori) is believed to represent a critical virulence determinant for this species. Ammonia generated by hydrolysis of urea may protect the acid-sensitive bacterium as it colonizes human gastric mucosa. An H. pylori strain, cultured from a gastric biopsy of a patient with complaints of abdominal pain and a history of peptic ulcer disease, was isolated on selective medium and cultured in Mueller-Hinton broth supplemented with 4% fetal calf serum. Whole cells were ruptured by French pressure cell lysis, and soluble protein was chromatographed on DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 resins. Purified urease represented 6% of the soluble protein of crude extract, was estimated to have a native molecular size of 550 kilodaltons (kDa), and was composed of two distinct subunits of apparent molecular sizes of 66 and 29.5 kDa. On the basis of subunit size, a 1:1 subunit ratio as measured by scanning densitometry of Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels, and estimated native molecular size, the data are consistent with a stoichiometry of (29.5 kDa-66 kDa)6 for the structure of the native enzyme. Km for urea was estimated at 0.2 mM. By N-terminal analysis, the 29.5-kDa subunit of H. pylori urease was found to share significant amino acid sequence similarity with the smallest of three subunits of the Proteus mirabilis and Morganella morganii ureases, as well as to the amino terminus of the unique jack bean subunit. The 66-kDa subunit also shared up to 80% similarity with the largest of three subunits of P. mirabilis, M. morganii, and Klebsiella aerogenes ureases and to internal sequences (amino acids 271 to 285) of the jack bean urease subunit. Thus, the amino acid sequence is conserved among ureases with one, two, and three distinct subunits, suggesting a common ancestral urease gene. Also, urease subunits of M. morganii and jack bean were specifically recognized by antisera raised against the 66-kDa subunit of H. pylori urease, demonstrating that at least some antigenic determinants were conserved among ureases from different species.

Activation of tuberculostatic macrophage functions by gamma interferon, interleukin-4, and tumor necrosis factor.

Abstract: The capacity of the cytokines gamma interferon (IFN-gamma), interleukin-4 (IL-4), and tumor necrosis factor (TNF) to activate tuberculostatic functions in murine bone marrow-derived macrophages (BMM phi) was investigated. In confirmation of earlier findings, IFN-gamma rendered BMM phi capable of inhibiting subsequent infection with Mycobacterium bovis. In contrast, IL-4 and TNF failed to inhibit mycobacterial growth. However, in already infected BMM phi, tuberculostasis was induced by subsequent stimulation with IL-4. Although TNF alone was ineffective, it showed synergy with IFN-gamma in the stimulation of tuberculostasis. Our data suggest that not only IFN-gamma but also IL-4 and TNF participate in the control of mycobacterial growth.

Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7.

Abstract: Enterohemorrhagic Escherichia coli O157:H7 isolates produce Shiga-like toxins and carry a 60-megadalton plasmid which encodes an adhesin for Henle 407 intestinal cells. A streptomycin-treated mouse model was used to compare the intestinal colonizing capacity of E. coli O157:H7 strain 933 with that of its 60-megadalton plasmid-cured derivative, strain 933cu. When fed individually to mice, both 933 and 933cu maintained a stable number of organisms per gram of feces, and the greatest numbers of 933 or 933cu were isolated from cecal and proximal colonic epithelial cells. When 933 and 933cu were simultaneously fed to mice, 933cu was unable to maintain a stable level of colonization in about two-thirds of the mice tested. However, in one-third of the mice, the number of 933cu in feces began to increase rapidly until a stable level of co-colonization with 933 was attained. The isolate from these mice, 933cu-rev, was excreted in high numbers when fed alone to mice and was found on epithelial cells throughout the entire large bowel and distal small intestine. Moreover, 933cu-rev grew in mucus from all segments of the intestine and at higher levels than strain 933 or 933cu. Only mice fed strain 933cu-rev died. Histopathological studies confirmed that mice fed 933cu-rev died from bilateral renal cortical tubular necrosis consistent with toxic insult, perhaps due to Shiga-like toxins. The virulence of 933cu-rev may reflect its ability to grow well in mucus and colonize the small as well as large bowel.

Helicobacter pylori urease activity is toxic to human gastric epithelial cells

Abstract: A human gastric adenocarcinoma cell line was used to evaluate the contribution of urease from Helicobacter (formerly Campylobacter) pylori to its cytotoxicity. Gastric cells cultured in medium supplemented with 20 mM urea were exposed to 5 x 10(6) CFU of H. pylori per ml with or without the addition of a urease inhibitor, acetohydroxamic acid. Viabilities of cells exposed to H. pylori for 2, 24, and 48 h, assessed by incorporation of neutral red dye, were 60, 27, and 16%, respectively; however, the viabilities of cells exposed to both H. pylori and acetohydroxamic acid were 92, 46, and 20% after 2, 24, and 48 h, respectively, (P less than 0.001). Therefore, the urease activity of H. pylori may play an important role in its pathogenicity, and inhibition of this enzyme activity may have therapeutic potential.

Evolutionary genetic relationships of clones of Salmonella serovars that cause human typhoid and other enteric fevers.

Abstract: Multilocus enzyme electrophoresis was employed to measure chromosomal genotypic diversity and evolutionary relationships among 761 isolates of the serovars Salmonella typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C, and S. sendai, which are human-adapted agents of enteric fever, and S. miami and S. java, which are serotypically similar to S. sendai and S. paratyphi B, respectively, but cause gastroenteritis in both humans and animals. To determine the phylogenetic positions of the clones of these forms within the context of the salmonellae of subspecies I, comparative data for 22 other common serovars were utilized. Except for S. paratyphi A and S. sendai, the analysis revealed no close phylogenetic relationships among clones of different human-adapted serovars, which implies convergence in host adaptation and virulence factors. Clones of S. miami are not allied with those of S. sendai or S. paratyphi A, being, instead, closely related to strains of S. panama. Clones of S. paratyphi B and S. java belong to a large phylogenetic complex that includes clones of S. typhimurium, S. heidelberg, S. saintpaul, and S. muenchen. Most strains of S. paratyphi B belong to a globally distributed clone that is highly polymorphic in biotype, bacteriophage type, and several other characters, whereas strains of S. java represent seven diverse lineages. The flagellar monophasic forms of S. java are genotypically more similar to clones of S. typhimurium than to other clones of S. java or S. paratyphi B. Clones of S. paratyphi C are related to those of S. choleraesuis. DNA probing with a segment of the viaB region specific for the Vi capsular antigen genes indicated that the frequent failure of isolates of S. paratyphi C to express Vi antigen is almost entirely attributable to regulatory processes rather than to an absence of the structural determinant genes themselves. Two clones of S. typhisuis are related to those of S. choleraesuis and S. paratyphi C, but a third clone is not. Although the clones of S. decatur and S. choleraesuis are serologically and biochemically similar, they are genotypically very distinct. Two clones of S. typhi were distinguished, one globally distributed and another apparently confined to Africa; both clones are distantly related to those of all other serovars studied.

Anaerobiosis, type 1 fimbriae, and growth phase are factors that affect invasion of HEp-2 cells by Salmonella typhimurium.

Abstract: The invasion of HEp-2 cells by Salmonella typhimurium was studied under various conditions. Anaerobiosis was shown to markedly affect the internalization of bacterial cells by HEp-2 cells. Anaerobically grown bacteria incubated with HEp-2 cells under anaerobic conditions markedly stimulated the rate of invasion. Anaerobiosis may therefore be a controlling factor in the invasion process. Cells obtained during the logarithmic phase of growth invaded at much higher rates than cells obtained during the stationary phase of growth. The presence of mannose-sensitive type 1 fimbriae on the bacterial surface also promoted invasion, and these fimbriae appear to play a role as an accessory virulence factor.

Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus.

Abstract: Translucent, avirulent spontaneous phase variants of Vibrio vulnificus MO6-24 reverted back to the original opaque, encapsulated phenotype under both in vivo and in vitro conditions. Two translucent, acapsular mutants, which did not show phase variation, were constructed by using the transposon Tn5 IS50L::phoA (TnphoA). Loss of capsule was accompanied by decreases in virulence, hydrophilicity, and serum resistance. The ability to utilize transferrin-bound iron for growth was lost in only one of the two unencapsulated mutants. Our data emphasize the apparent importance of capsule in the virulence of V. vulnificus and indicate that utilization of transferrin-bound iron is independent of encapsulation. Images

Electron microscopic evidence for in vivo extracellular localization of Yersinia pseudotuberculosis harboring the pYV plasmid.

Abstract: Electron microscopic evidence is presented that bacteria harboring the virulence plasmid pYV from Yersinia pseudotuberculosis are localized in extracellular sites during the course of infection in mice, often unambiguously undergoing active replication. Virulent pYV+ bacteria, often seen adherent to platelets, severely restricted granuloma formation, creating necrotic microabscesses poorly populated with inflammatory cells. This contrasts with granulomas produced by pYV- bacteria, which appear to be composed mainly of polymorphonuclear and mononuclear cells. Our results therefore strongly suggest that active replication of pYV+ bacteria predominantly, if not exclusively, occurs in vivo in extracellular sites. Images

Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH.

Abstract: Leishmania amastigotes are intracellular protozoan parasites of mononuclear phagocytes which reside within parasitophorous vacuoles of phagolysosomal origin. The pH of these compartments was studied with the aim of elucidating strategies used by these microorganisms to evade the microbicidal mechanisms of their host cells. For this purpose, rat bone marrow-derived macrophages were infected with L. amazonensis amastigotes. Intracellular acidic compartments were localized by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine as a probe. This indicator, which can be detected by light microscopy by using immunocytochemical methods, mainly accumulated in perinuclear lysosomes of uninfected cells, whereas in infected cells, it was essentially localized in parasitophorous vacuoles, which thus appeared acidified. Phagolysosomal pH was estimated quantitatively in living cells loaded with the pH-sensitive endocytic tracer fluoresceinated dextran. After a 15- to 20-h exposure, the tracer was mainly detected in perinuclear lysosomes and parasitophorous vacuoles of uninfected and infected macrophages, respectively. Fluorescence intensities were determined from digitized video images of single cells after processing and automatic subtraction of background. We found statistically different mean pH values of 5.17 to 5.48 for lysosomes and 4.74 to 5.26 for parasitophorous vacuoles. As for lysosomes of monensin-treated cells, the pH gradient of parasitophorous vacuoles collapsed after monensin was added. This very likely indicates that these vacuoles maintain an acidic internal pH by an active process. These results show that L. amazonensis amastigotes are acidophilic and opportunistic organisms and suggest that these intracellular parasites have evolved means for survival under these harsh conditions and have acquired plasma membrane components compatible with the environment.

Coaggregation of Streptococcus sanguis and other streptococci with Candida albicans

Abstract: Thirteen strains of viridans group streptococci and two strains of other streptococci were tested for coaggregation with Candida albicans. Streptococcus sanguis strains generally exhibited low levels of adherence to 28 degrees C-grown exponential-phase yeast cells, but starvation of yeast cells for glucose at 37 degrees C (or at 28 degrees C) increased their coaggregating activity with these streptococci by at least tenfold. This was a property common to four C. albicans strains tested, two of which were able to form mycelia (6406 and MEN) and two of which were not (MM2002 and CA2). The expression of the coaggregation adhesin during yeast cell starvation was inhibited by addition of trichodermin or amphotericin B. The strains of S. sanguis, Streptococcus gordonii, and Streptococcus oralis tested for coaggregating activity encompassed a diverse range of physiological and morphological types, yet all exhibited saturable coaggregation with starved C. albicans cells. There was no correlation of cell surface hydrophobicity, of either yeast or streptococcal cells, with their abilities to coaggregate. Strains of Streptococcus anginosus also coaggregated with starved yeast cells; Streptococcus salivarius and Streptococcus pyogenes coaggregated to a lesser degree with C. albicans, and the coaggregation with S. pyogenes was not promoted by yeast cell starvation; Streptococcus mutans and Enterococcus faecalis did not coaggregate with yeast. The coaggregation reactions of S. sanguis and S. gordonii with C. albicans were inhibited by EDTA and by heat or protease treatment of the yeast cells and were not reversible by the addition of lactose or other simple sugars. These observations extend the range of intergeneric coaggregations that are known to occur between oral microbes and suggest that coaggregations of C. albicans with viridans group streptococci may be important for colonization of oral surfaces by the yeast.

Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin type II.

Abstract: Escherichia coli O157:H7 strains have been implicated as etiologic agents in food-borne outbreaks of hemorrhagic colitis and the hemolytic-uremic syndrome. A prototype E. coli O157:H7 strain, designated 933, produces Shiga-like toxin I (SLT-I) and SLT-II and harbors a 60-MDa plasmid. In a previous study, streptomycin-treated mice were fed 933 together with a derivative cured of the 60-MDa plasmid (designated 933cu). Strain 933cu colonized poorly, but in approximately one-third of the animals, an isolate of 933cu was obtained from the feces that had regained the ability to colonize well. This isolate, designated 933cu-rev, killed all of the animals when fed alone to mice. In this investigation, two types of experiments were done to assess whether SLT-I, SLT-II, or both contributed to the death of mice fed 933cu-rev. (i) Mice were pretreated with monoclonal antibodies to SLT-I, SLT-II, SLT-I and SLT-II, or cholera toxin (as a control) before infection with 933cu-rev. (ii) Mice were fed either an E. coli K-12 strain carrying cloned SLT-I genes or the same K-12 strain carrying cloned SLT-II genes. The results of both types of experiments indicated that the deaths of the orally infected mice were due solely to SLT-II. Extensive histological and selected electron microscopic examinations of various tissues from the infected animals suggested that death was due to acute renal cortical tubular necrosis consistent with toxic renal damage. These data indicate a critical role for SLT-II, but not SLT-I, in renal damage associated with E. coli O157:H7 infection of streptomycin-treated mice. Images

Haemophilus influenzae adheres to and enters cultured human epithelial cells.

Abstract: Haemophilus influenzae is a common commensal organism of the human respiratory tract that initiates infection by colonizing the nasopharyngeal epithelium. In some individuals, colonization is followed by localized respiratory tract or systemic disease. To gain insight into the mechanisms by which H. influenzae attaches to and persists within the nasopharynx, we examined the interactions between a nonpiliated clinical isolate of H. influenzae and human epithelial cells. We noted substantial adherence that occurred independently of pili and required viable bacteria capable of de novo protein synthesis. Comparison of profiles of outer membrane proteins synthesized during incubation with epithelial cells for adherent and nonadherent bacteria identified several candidate adhesin molecules. In addition, a small number of adherent bacteria were capable of entering epithelial cells in a process that was inhibited by cytochalasin D and colchicine. The suggestion from our studies is that one or more of several newly synthesized nonpilus bacterial proteins are required for maximal in vitro adherence and invasion. We speculate that H. influenzae entry into epithelial cells may provide a mechanism for evasion of host defenses, thereby allowing persistence in the nasopharynx.

Chlamydia trachomatis-host cell interactions: role of the chlamydial major outer membrane protein as an adhesin.

Abstract: The major outer membrane protein (MOMP) of Chlamydia trachomatis is characterized by four symmetrically spaced variable domains (VDs I to IV) whose sequences vary among serotypes. The surface-exposed portions of these VDs contain contiguous sequences that are both serotyping determinants and in vivo target sites for neutralizing antibodies. Previous studies using surface proteolysis of C. trachomatis B implicated VDs II and IV of the MOMP of this serotype in the attachment of chlamydiae to host cells. In this study, we used monoclonal antibodies (MAbs) specific to antigenic determinants located in VDs II and IV of the MOMP of serotype B to further investigate the role of the MOMP in the attachment of chlamydiae to host cells. MABs specific to serotype- and subspecies-specific epitopes located in exposed VDs II and IV, respectively, neutralized chlamydial infectivity for hamster kidney cells by blocking chlamydial attachment. We radioiodinated these MAbs and used them to determine the number and topology of the surface-exposed VDs II and IV epitopes on chlamydial elementary bodies. VDs II and IV each comprised approximately 2.86 x 10(4) negatively charged sites and were in proximity on the chlamydial cell surface. These studies suggest that the MAbs blocked chlamydial attachment by inhibiting electrostatic interactions with host cells. We examined the effects of thermal inactivation on both chlamydial attachment and conformation of the MOMP. Heat-inactivated chlamydiae failed to attach to host cells and exhibited a conformational change in an inaccessible invariant hydrophobic nonapeptide sequence located within VD IV of the MOMPs of C. trachomatis serotypes. These findings suggest that in addition to electrostatic interactions, a common hydrophobic component of the MOMP also contributes to the binding of chlamydiae to host cells. Thus, we propose that the MOMP functions as a chlamydial adhesin by promoting nonspecific (electrostatic and hydrophobic) interactions with host cells. Surface-accessible negatively charged VDs appear to be important in electrostatic binding, while the invariant region of VD IV may provide a subsurface hydrophobic depression which further promotes binding of chlamydiae to host cells through hydrophobic interactions.

Rapid membrane permeabilization and inhibition of vital functions of gram-negative bacteria by bactenecins.

Abstract: Bactenecins are a class of arginine-rich antibacterial peptides of bovine neutrophil granules. Two bactenecins with approximate molecular weights of 5,000 and 7,000 designated Bac5 and Bac7, respectively, exert in vitro a potent bactericidal activity toward several gram-negative bacteria (R. Gennaro, B. Skerlavaj, and D. Romeo, Infect. Immun. 57:3142-3146, 1989). We have now found that this activity shows an inverse relationship to the ionic strength of the medium and is inhibited by divalent cations and greatly potentiated by lactoferrin. Under conditions supporting marked bactericidal activity, the two peptides cause a rapid increase in the permeability of both the outer and inner membranes of Escherichia coli, as shown by unmasking of periplasmic beta-lactamase and of cytoplasmic beta-galactosidase. In addition, the two bactenecins inhibit the respiration of E. coli and Klebsiella pneumoniae but not of Bac5- and Bac7-resistant Staphylococcus aureus. Furthermore, they induce a drop in ATP content in E. coli, K. pneumoniae, and Salmonella typhimurium and a marked decrease in the rates of transport and incorporation of [3H]leucine and [3H]uridine into E. coli protein and RNA, respectively. In general, all these effects become evident within 1 to 2 min and reach their maximal expression within about 5 min. Overall, these data strongly suggest that the decrease in bacterial viability is causally related to the increase in membrane permeability and the subsequent fall in respiration-linked proton motive force, with the attendant loss of cellular metabolites and macromolecular biosynthesis ability.

Laminin receptors on Candida albicans germ tubes.

Abstract: Recent evidence for the role of laminin in cell adhesion and in the pathogenesis of several bacterial infections has led us to investigate the existence of receptors for this extracellular matrix component in Candida albicans. At first, immunofluorescence demonstrated the presence of laminin-binding sites at the surface of germ tubes. Electron microscopy confirmed this result and permitted precise localization of the binding sites on the outermost fibrillar layer of the germ tube cell wall. By using 125I-radiolabeled laminin, the binding was shown to be saturable and specific, hence demonstrating characteristics of true receptors. Analysis of the data by the Scatchard equation indicated that there were about 8,000 binding sites per cell, with a dissociation constant (Kd) of 1.3 x 10(-9) M. Binding was inhibited by prior heating or trypsinization of cells. Furthermore, of the different proteins and carbohydrates tested in competition experiments, only fibrinogen greatly reduced the laminin binding. Finally, dithiothreitol and iodoacetamide treatment of germ tubes allowed us to identify the laminin receptors through analysis of this extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. Two components, of 68 kilodaltons and a doublet of 60 and 62 kilodaltons, were detected. Thus, C. albicans possesses germ tube-specific surface receptors for laminin which could mediate its attachment to basement membranes and so contribute to the establishment of candidiasis.

The Dr hemagglutinin, afimbrial adhesins AFA-I and AFA-III, and F1845 fimbriae of uropathogenic and diarrhea-associated Escherichia coli belong to a family of hemagglutinins with Dr receptor recognition.

Abstract: The receptor specificities of four Escherichia coli cloned hemagglutinins, AFA-I, AFA-III, F1845 fimbriae, and the Dr hemagglutinin were studied. Evidence is provided that all four hemagglutinins recognize as their receptor the Dr blood group antigen. However, results of experiments using enzyme-treated erythrocytes and monoclonal antibodies indicate that the four adhesins recognize different epitopes on the Dr antigen and thus constitute a family of Dr receptor-recognizing bacterial adhesins. Furthermore, the same results suggest that the Dr antigen itself may be divided into subcomponents on the basis of bacterial adhesins.

YopM inhibits platelet aggregation and is necessary for virulence of Yersinia pestis in mice

Abstract: In Yersinia pestis KIM there are 11 Yops (yersinial outer membrane proteins) encoded by the low-Ca2+ response virulence plasmid pCD1. Only YopM and YopN are found in easily detectable amounts in the culture medium. In our previous work, we characterized the yopM gene. In the present study, we constructed a YopM- mutant to elucidate the role of YopM in the virulence of Y. pestis. A lacZYA sequence was inserted 126 base pairs downstream from the start codon of the yopM gene in pCD1. The YopM- mutant had the same growth properties as the parent, Y. pestis KIM5-3001. The inserted lacZ gene was regulated by the promoter of the yopM gene. Accordingly, it was expressed strongly at 37 degrees C in the absence of Ca2+ and was decreased in expression when Ca2+ was present. Northern blot (RNA blot) analysis revealed that the yopM gene was in a monocistronic operon, suggesting that the yopM insertion mutation was unlikely to have polar effects on other genes. The YopM- mutant had strongly decreased virulence in mice, with a 50% lethal dose of 3.4 x 10(5) CFU. Virulence was restored by the cloned yopM-containing 5.5-kilobase HindIII F fragment of pCD1. However, supplying a cloned 1.57-kilobase fragment containing little more than the yopM structural gene caused the yopM mutant to significantly overexpress YopM and failed to restore virulence. The infection kinetics of the YopM- mutant revealed growth in both spleens and livers from days 2 to 4 after infection, followed by a precipitous clearance of the bacteria. YopM-containing supernatant proteins of Y. pestis inhibited thrombin- or ristocetin-induced platelet aggregation, whereas there was no inhibition by supernatant proteins from the YopM- Y. pestis mutant. Accordingly, YopM may prevent platelet-mediated events and serve as an important strategy for the yersiniae in the initial stages of a plague infection.

Cytokine response by monocytes and macrophages to free and lipoprotein-bound lipopolysaccharide.

Abstract: Recent evidence suggests that bacterial lipopolysaccharide binds to serum lipoproteins in vitro and in vivo and that lipopolysaccharide in the form that is bound to lipoprotein is less biologically active in several experimental models. In order to study the mechanism of this apparent detoxification, we compared the ability of free and lipoprotein-bound lipopolysaccharide from Escherichia coli O18 to stimulate interleukin-1, interleukin-6, and tumor necrosis factor from elicited murine peritoneal macrophages and circulating human monocytes. Lipopolysaccharide bound to lipoprotein was 20- to 1,000-fold less active than the unbound form in inducing the release of each cytokine. We also studied the binding of each form of lipopolysaccharide to the macrophage surface. Lipopolysaccharide complexed to lipoprotein was unable to compete for the binding of radiolabeled heterologous lipopolysaccharide to murine macrophages, and radiolabeled lipopolysaccharide-lipoprotein complexes bound poorly compared with molar equivalents of free lipopolysaccharide. Our experiments suggest that in the process of binding to lipoproteins, lipopolysaccharide may be rendered less toxic through a mechanism of decreased ability to induce monocytes and macrophages to release cytokines, perhaps because of an altered interaction at the cell surface.