Showing papers in "Infection and Immunity in 1989"

Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli.

Abstract: Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) adhere to the intestinal mucosa and produce an attaching and effacing (AE) lesion in the brush border microvillous membrane; the AE lesion is characterized by localized destruction of microvilli and intimate attachment of bacteria to the apical enterocyte membrane. A similar lesion is seen when bacteria adhere in vitro to a variety of human tissue culture cell lines. In both cases, dense concentrations of microfilaments are present in the apical cytoplasm beneath attached bacteria. Using a fluorescein-labeled phallotoxin, we have shown that these microfilaments are composed of actin. Cells infected with EPEC and EHEC strains known from electron microscopic studies to produce the AE lesion all exhibited intense spots of fluorescence which corresponded in size and position with each adherent bacterium; cells infected with adherent E. coli strains known not to produce the AE lesion did not produce this striking pattern of fluorescence and were indistinguishable from uninfected control cells. These results indicate that such site-specific concentrations of cytoskeletal actin are characteristic of the AE membrane lesion and can form the basis of a simple, highly sensitive diagnostic test for EPEC and EHEC. Images

Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation.

Abstract: The role of listeriolysin O in the intracellular multiplication of Listeria monocytogenes and, therefore, its pathogenicity was questioned through a genetic complementation study. A nonhemolytic mutant was generated by inserting a single copy of transposon Tn917 in the bacterial chromosome. This insertion was localized by DNA sequence analysis in hlyA, the gene coding for listeriolysin O. As was another mutant that we previously characterized, this mutant was avirulent in the mouse. It was transformed with a plasmid carrying only hlyA, able to replicate in L. monocytogenes, and stably maintained in vitro and in vivo. The complemented strain displayed a hemolytic phenotype identical to that of the wild-type strain and was fully virulent, therefore attributing a crucial role to listeriolysin O in virulence and excluding the hypothesis of a polar effect of the transposon insertion on genes adjacent to hlyA and possibly involved in virulence.

Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae.

Abstract: Insertion-duplication mutagenesis was used to construct a pneumolysin-negative derivative of Streptococcus pneumoniae. This was achieved by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 690-base-pair DNA fragment from the middle of the pneumolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, pneumolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these produced pneumolysin. Southern blot analysis confirmed that the pneumolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The pneumolysin-negative mutants showed reduced virulence for mice compared with D39, as judged by survival time after intranasal challenge, intraperitoneal 50% lethal dose, and blood clearance studies. Pneumolysin production was reinstated in one of the mutants by transformation with the cloned pneumolysin gene, with the concomitant loss of erythromycin resistance; the virulence in mice of this isolate was indistinguishable from that of D39. These results confirm the involvement of pneumolysin in pneumococcal pathogenesis.

Toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production.

Abstract: Toxic shock syndrome-associated staphylococcal and streptococcal exotoxins were tested for an ability to induce the production of tumor necrosis factor (TNF). Staphylococcal enterotoxins B and C1, along with streptococcal pyrogenic exotoxin A, all induced TNF production in a dose-dependent manner, with production peaking on the average at 3 days but continuing over the 6 days tested. This time course of exotoxin-induced TNF production contrasts with the 1-day peak-2-day duration observed with endotoxin as the stimulus and may be significant to development of toxic shock syndrome.

Endotoxin-induced serum factor that stimulates gamma interferon production.

Abstract: Serum from Mycobacterium bovis BCG-infected mice that had been challenged with lipopolysaccharide (LPS) exhibited a marked ability to induce gamma interferon (IFN-gamma) in cultures of spleen cells of normal mice in the presence of interleukin-2 (IL-2). The inducing activity became detectable in the circulatory system about 90 min after LPS challenge, disappeared at around 5 h, and was observable upon 640x dilution of the serum. Addition of monoclonal anti-IL-2 receptor antibody to the culture inhibited the induction by the serum. The activity induced high levels of IFN-gamma even in nylon wool-nonadherent cells, while concanavalin A failed to do so. Serum from uninfected mice challenged with LPS contained no such activity. The molecular weight of the active substance, estimated by gel filtration, was about 70,000. There were pronounced differences among mouse strains in the activities of the sera prepared, which paralleled the amounts of IFN-gamma produced in vivo. However, the levels of IFN-gamma produced in whole spleen cells and in nylon wool-nonadherent cells from mice of various strains were the same when stimulated with competent serum. These results indicate the existence of an unidentified factor that induces IFN-gamma in cooperation with IL-2 in macrophage-depleted splenocytes. They also suggest that IFN-gamma production in vivo is not genetically controlled at the lymphocyte level but, rather, at the level of synthesis of the unknown factor.

Purification, composition, and activity of two bactenecins, antibacterial peptides of bovine neutrophils.

Abstract: Extracts of granules of bovine neutrophils are known to exhibit a marked antibacterial activity in vitro. By a simple, two-step chromatographic procedure, we have resolved two peptide components of the antibacterial system. They were named Bac-5 and Bac-7 from the general term bactenecin and had molecular masses of about 5 and 7 kilodaltons, respectively. Over 45 and 20% of the amino acid residues in the two bactenecins are proline and arginine, respectively. The remaining amino acids are mainly hydrophobic (isoleucine, leucine, and phenylalanine). Both Bac-5 and Bac-7 efficiently kill Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae. They also arrest the growth of Enterobacter cloacae (MICs, 25 to 200 micrograms/ml) but not of Proteus vulgaris, Staphylococcus aureus, and Streptococcus agalactiae (MIC, greater than 200 micrograms/ml). Finally, Bac-7 but not Bac-5 has MICs of less than or equal to 200 micrograms/ml for Pseudomonas aeruginosa and Staphylococcus epidermidis. From the comparison between the efficient bactericidal concentrations in vitro and the estimated content of bactenecins in neutrophils (125 ng of Bac-5 and Bac-7 each per 10(6) cells), it is reasonable to conclude that the two cationic peptides may exert a major role in host defense against at least some microorganisms.

Identification of an extracellular protein of Listeria monocytogenes possibly involved in intracellular uptake by mammalian cells.

Abstract: Mutants of Listeria monocytogenes were recently isolated which are impaired in the synthesis of a major extracellular protein (p60). As shown in this investigation, the p60 mutants have lost the capability of invading nonprofessional phagocytic 3T6 mouse fibroblast cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant proteins of these mutants indicated that no other extracellular protein was altered in these mutants. The p60 mutants formed long cell chains which disaggregated to normal-sized single bacteria upon treatment with partially purified p60. These disaggregated bacterial cells were able to invade 3T6 cells. Physical disruption of the cell chains by ultrasonication produced similar single cells which were, however, noninvasive. Treatment of these ultrasonicated mutant cells with wild-type p60 restored their ability to invade 3T6 cells.

Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis serovars.

Abstract: The amino acid sequences of major outer membrane proteins (MOMPs) from Chlamydia trachomatis serovars A, B, C, L1, and L2 are predominantly conserved but have four variable domains (VDs) in which major neutralizing and serotyping antigenic determinants are located. Because these MOMP VDs are primarily responsible for antigenic differences between serovars and are associated with important immunological and biological properties, we undertook studies focused on defining these sequences within the MOMPs of all 15 C. trachomatis serovars. We used oligonucleotide primer extension sequencing of MOMP mRNA to determine the nucleotide and deduced amino acid sequences of the four MOMP VDs of the 15 C. trachomatis serovars. Comparative amino acid sequence homologies of all four domains separated the serovars into three groups: group 1, serovars B, Ba, D, E, L1, and L2; group 2, serovars G and F; and group 3, serovars A, C, H, I, J, K, and L3. Hydrophilicity and charge values for each domain were determined. The MOMP VDs of given serovars with the greatest total hydrophilicity and charge values were found to be the location of antigenic determinants recognized by MOMP-specific monoclonal antibodies. These findings should be useful for predicting MOMP antigenic determinants and testing the antigenic properties of these VDs by using synthetic peptides corresponding to each MOMP VD. The potential usefulness of the VD sequence information is discussed in relation to the development of defined synthetic peptides and oligonucleotides that may be used to develop new serological and diagnostic assays for C. trachomatis infections.

Role of M cells in initial antigen uptake and in ulcer formation in the rabbit intestinal loop model of shigellosis.

Abstract: Strains of Shigella flexneri with different invasive and pathogenic potentials were inoculated into the intestinal lumen of acutely ligated loops in nonimmune rabbits. After 90 min, tissues processed for ultrastructural as well as light microscopy showed that the bacilli were phagocytosed by M cells over lymphoid follicles of Peyer9s patches and carried in vacuoles into the epithelium. Nonpathogenic as well as pathogenic strains were readily taken up regardless of the presence of the 140-megadalton virulence plasmid. More virulent than avirulent shigellae were found in M cells at 90 min, reflecting replication or preferential uptake of the virulent strains. Heat-killed shigellae of the virulent strain were taken up by M cells to the same degree as the avirulent strains. Incubation of the bacteria for 18 h resulted in surface ulceration which was limited to epithelium overlying lymphoid follicles (M cell areas) in acute loops exposed to the virulent shigellae. Villus epithelium adjacent to the ulcerated follicular domes was intact, although there was mucus depletion. In the present study, we found that pathogenic shigellae appear to replicate in the M cells, escape from the phagocytic vesicles, and thereby initiate the ulcerations in this experimental model of dysentery. While initial antigen processing in the gut for a mucosal immune response may require uptake of luminal microorganisms by M cells, this may pose a threat under some circumstances. Images

Contribution of autolysin to virulence of Streptococcus pneumoniae.

Abstract: Insertion-duplication mutagenesis was used to construct an autolysin-negative derivative of Streptococcus pneumoniae. This derivative was obtained by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 375-base-pair TaqI DNA fragment from the middle of the autolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, autolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these transformants produced autolysin. Southern blot analysis confirmed that the autolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The autolysin-negative mutants showed markedly reduced virulence for mice compared with that of strain D39; intranasal and intraperitoneal 50% lethal doses were increased 10(2)- and 10(5)-fold, respectively. Autolysin production was reinstated in one of the mutants by back-transformation with the cloned autolysin gene, with the concomitant loss of erythromycin resistance; the virulence of this isolate for mice was indistinguishable from that of D39. The importance of autolysin in pathogenesis was confirmed by immunization-challenge studies. Mice immunized with purified autolysin survived significantly longer than did control mice after intranasal challenge with strain D39. This study provides direct evidence that the pneumococcal autolysin contributes to virulence and identifies it as a potential vaccine antigen.

Coaggregation of Fusobacterium nucleatum, Selenomonas flueggei, Selenomonas infelix, Selenomonas noxia, and Selenomonas sputigena with strains from 11 genera of oral bacteria.

Abstract: Twenty-eight strains of Fusobacterium nucleatum and 41 Selenomonas strains, including S. sputigena (24 strains), S. flueggei (10 strains), S. infelix (5 strains), and S. noxia (2 strains), were tested for their ability to coaggregate with each other and with 49 other strains of oral bacteria representing Actinobacillus, Actinomyces, Bacteroides, Capnocytophaga, Gemella, Peptostreptococcus, Porphyromonas, Propionibacterium, Rothia, Streptococcus, and Veillonella species. Selenomonads coaggregated with fusobacteria and with Actinomyces naeslundii PK984 but not with any of the other bacteria, including other selenomonads. In contrast, fusobacteria coaggregated with members of all genera, although not with all strains of each species tested. Each fusobacterium strain appeared to have its own set of partners and coaggregation properties, unlike their partners, whose coaggregation properties in earlier surveys delineated distinct coaggregation groups. Coaggregations of fusobacteria with the 63 gram-negative strains were usually inhibited by EDTA, whereas those with the 27 gram-positive strains were usually not inhibited. Likewise, lactose-inhibitable coaggregations were common among some strains of fusobacteria and some strains from each of the genera containing gram-negative partners but were rarely observed with gram-positive partners. Heating the fusobacteria at 85 degrees C for 30 min completely prevented coaggregation with most partners, suggesting the involvement of a protein on the fusobacteria. Heat treatment of many of the gram-negative partners not only enhanced their coaggregation with the fusobacteria but also changed lactose-sensitive coaggregations to lactose-insensitive coaggregations. Although fusobacteria coaggregated with a broader variety of oral partner strains than any other group of oral bacteria tested to date, each fusobacterium exhibited coaggregation with only a certain set of partner strains, and none of the fusobacteria adhered to other strains of fusobacteria, indicating that recognition of partner cell surfaces is selective. The strains of F. nucleatum are heterogeneous and cannot be clustered into distinct coaggregation groups. Collectively, these results indicate that coaggregation between fusobacteria and many gram-negative partners is significantly different from their coaggregation with gram-positive partners. The contrasting variety of partners for fusobacteria and selenomonads supports the concept of coaggregation partner specificity that has been observed with every genus of oral bacteria so far examined.

Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo.

Abstract: The ompC, ompD, and ompF genes encode the three major porins of Salmonella typhimurium. ompR encodes a positive regulator required for the expression of ompC and ompF. Transposon-generated mutations in ompC, ompD, ompF, and ompR were introduced into the S. typhimurium mouse virulent strain SL1344 by P22-mediated transduction. Following preliminary characterization in vitro, the strains were used to challenge BALB/c mice by using the oral or intravenous route. Strains harboring ompC or ompF mutations were as virulent as SL1344 after oral challenge. Strains harboring ompD mutations had a slight reduction in virulence. In contrast, ompR mutants failed to kill BALB/c mice after oral challenge and the intravenous 50% lethal dose was reduced by approximately 10(5). The ompR mutants persisted in murine tissues for several weeks following oral or intravenous challenge. Furthermore, mice orally immunized with these ompR mutant strains were well protected against challenge with virulent SL1344.

Oral administration of a streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody responses in salivary glands and extramucosal tissues.

Abstract: Generation of local and systemic immune responses by the oral administration of antigens is frequently inefficient, requiring large quantities of immunogens and yielding only modest antibody responses. In this study, we have demonstrated that oral administration of microgram amounts of Streptococcus mutans protein antigen I/II covalently coupled to the B subunit of cholera toxin elicits vigorous mucosal as well as extramucosal immunoglobulin A and G antistreptococcal antibody responses in mice. These responses were manifested by the presence of large numbers of antibody-secreting cells in salivary glands, mesenteric lymph nodes, and spleens and by the development of high levels of circulating antibodies. This novel immunization strategy may find broad application in the construction of oral vaccines for the control of infectious diseases caused by pathogens encountered at mucosal and extramucosal sites.

A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection.

Abstract: To investigate the pathogenesis of Legionnaires disease at a molecular level, we mutated by directed allelic exchange a gene encoding a Legionella pneumophila-specific 24,000-dalton (Da) surface protein. Southern hybridization and immunoblot analyses demonstrated that the predicted DNA rearrangement occurred in L. pneumophila with a specific loss of 24-kDa antigen expression. Compared with its isogenic parent, the mutant was significantly impaired in its ability to infect transformed U937 cells, a human macrophagelike cell line; i.e., the bacterial inoculum of the mutant strain that was required to initiate infection of the macrophage monolayer was ca. 80-fold greater than that of the isogenic parent strain. The mutant strain regained full infectivity on reintroduction of a cloned 24-kDa protein gene, indicating that the reduced infectivity was due specifically to the mutation in that gene. Compared with the parent strain, the mutant strain was recovered at titers that were ca. 10-fold lower shortly after infection, but it exhibited a similar intracellular growth rate over the next 40 h, indicating that the mutant was defective in its ability to initiate macrophage infection rather than in its ability to replicate intracellularly. When opsonized, the mutant strain was still significantly less infectious than the parent strain, despite equivalent macrophage association, suggesting that the mutant was not merely missing a ligand for macrophage attachment. The mutant also exhibited reduced infectivity in explanted human alveolar macrophages, demonstrating the relevance of the U937 cell model for analyzing this mutant phenotype. These results represent the first identification of a cloned L. pneumophila gene that is necessary for optimal intracellular infection; we designate this gene mip, for macrophage infectivity potentiator. Images

Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.

Abstract: The adherence of enteropathogenic Escherichia coli (EPEC) to the small bowel mucosa is an important step in the pathogenesis of diarrheal diseases. It has been shown that many EPEC strains adhere to HEp-2 and especially HeLa cells in characteristic patterns termed localized adherence (LA) and diffuse adherence (DA). A plasmid-derived DNA fragment encoding a factor specific for LA hybridized only to EPEC strains expressing LA, which demonstrated that LA and DA are mediated by two genetically distinct adhesins. EPEC strain 2787 (O127:H27), isolated from a case of infantile diarrhea, exhibited three major properties: (i) it showed DA to HeLa cells, (ii) it carried two large (ca. 100-kilobase [kb]) plasmids and one small plasmid of about 3 kb, and (iii) no fimbriae could be detected by electron microscopy in organisms grown on agar plates or in liquid cultures. Whole isolated plasmid DNA was partially digested with EcoRI and cloned into the vector pBR322. One recombinant clone (pIB6) was found to exhibit the same DA pattern on HeLa cells as did the parent strain. This clone contained an 11-kb DNA fragment derived from the largest of the three plasmids, as shown by Southern hybridization. By deletion analysis, a 6.0-kb DNA fragment was shown to be sufficient for expression of the DA phenotype. This insert encoded the production of a 100,000-dalton protein mediating adhesion to HeLa cells.

The ail locus is found uniquely in Yersinia enterocolitica serotypes commonly associated with disease.

Abstract: Yersinia enterocolitica is a heterogeneous group of organisms with more than 50 serotypes and several biotypes. Only a few of these serotypes cause gastrointestinal disease in otherwise healthy hosts; these serotypes are the pathogenic serotypes. Although Y. enterocolitica requires a high-molecular-weight plasmid to cause disease, chromosome-encoded determinants are required for the full expression of virulence. The ability of Yersinia spp. to invade eucaryotic cells is thought to be a virulence factor, because nonpathogenic serotypes are noninvasive in animals and in tissue culture cell models. Current evidence indicates that invasion ability is chromosome encoded. We recently reported cloning two loci, inv and ail, from Y. enterocolitica O8 strain 8081c that allow Escherichia coli to invade tissue culture cells. We investigated the link between invasion in an in vitro tissue culture invasion (TCI) model and hybridization to probes derived from the two invasion loci, inv and ail. We examined 177 Yersinia strains. Strains of serotypes and species associated with disease were TCI+, whereas strains of serotypes and species not associated with disease were TCI-. Only TCI+ strains had DNA homologous to probes derived from ail. All strains (TCI+ and TCI-) had DNA homologous to probes derived from inv, but there were certain restriction fragment-linked polymorphisms that were associated primarily with TCI+ strains. These observations held true for strains epidemiologically associated with disease. Both the inv and ail loci were found to be clearly located on the chromosome. No other genera, including other invasive organisms, had DNA homologous to inv or ail. These data support the hypothesis that the ail locus encodes a Y. enterocolitica invasion factor that may be involved in pathogenesis. Images

Intracellular survival of wild-type Salmonella typhimurium and macrophage-sensitive mutants in diverse populations of macrophages.

Abstract: Salmonella typhimurium survives within macrophages and causes a fatal infection in susceptible strains of mice. A number of S. typhimurium mutants that contain Tn10 insertions in genes which are necessary for survival within the macrophage have been isolated. To demonstrate the importance of each gene in intracellular survival, the mutations were transduced into a smooth-strain background and the ability to survive intracellularly was assayed in five different populations of macrophages. The majority of the original macrophage-sensitive mutants retained the macrophage-sensitive phenotype in the smooth-strain background. The ability to survive or grow within macrophages varied with both the source of macrophages and the individual mutants. S. typhimurium grew best in the macrophage-like cell line J774, survived at moderate levels in splenic and bone marrow-derived macrophages, and was killed most efficiently in peritoneal macrophages. Macrophage-sensitive mutants transduced into a smooth background were also less virulent than the parent, with a 50% lethal dose of 2 to 5 logs greater than that of the parental strain. These experiments demonstrate that survival of S. typhimurium within macrophages varies with the source of cells, with a distinct ability to survive in macrophages from mouse spleens, where S. typhimurium grows rapidly. These experiments also demonstrate the heterogeneity in intracellular survival among the various macrophage-sensitive mutants, which may reflect the relative importance of the individual mutated genes in survival within macrophages.

Mucoid phenotype of Klebsiella pneumoniae is a plasmid-encoded virulence factor.

Abstract: We have previously reported that the presence of a 180-kilobase plasmid encoding production of aerobactin was correlated with the virulence of Klebsiella pneumoniae K1 and K2 isolates. This work demonstrates that a variant of a K2 strain which has lost this plasmid, pKP100, becomes avirulent. Labeling of this plasmid with the mobilizable, replication-defective element pME28, used here as a mobilizable transposon, allowed the transfer of this plasmid into a plasmidless derivative. Virulence was restored upon reacquisition of this tagged plasmid, pKP101. In addition to aerobactin production, another phenotype could be correlated with the presence of this virulence plasmid: the mucoid phenotype of the bacterial colonies. Both wild-type and plasmidless strains are encapsulated, but only the former presented mucoid colonies. Participation of this phenotype in the virulence of K. pneumoniae was demonstrated by constructing a mutant altered in the plasmid gene encoding this phenotype. The resulting strain demonstrated a 1,000-fold decrease in virulence. Introduction of the recombinant plasmid pKP200 carrying the gene encoding this mucoid phenotype into Escherichia coli HB101 also led to the production of a mucoid phenotype. Rocket immunoelectrophoresis demonstrated that in E. coli this phenotype was due to the production of colanic acid. On the other hand, neither the overproduction of K2 capsular polysaccharide nor the presence of colanic acid was detected in mucoid strains of K. pneumoniae. We conclude that this mucoid phenotype is definitely an important virulence factor of K. pneumoniae. It is due to the plasmid-encoded production of a substance which is different from colanic acid and the capsular polysaccharide of K. pneumoniae.

Isolation and characterization of the Streptococcus mutans gtfD gene, coding for primer-dependent soluble glucan synthesis.

Abstract: Two glucosyltransferase genes from Streptococcus mutans GS-5, gtfB and gtfC, have been previously isolated and sequenced in this laboratory. In the present communication a third gtf gene, gtfD, was isolated and characterized. Isolation of the gene involved a novel procedure utilizing the integration plasmid pVA891. A peptide expressed by the 1.7-kilobase DNA fragment from strain NHS1 (containing deletions in both the gtfB and gtfC genes) was initially identified in a pUC18 clone bank with antiglucosyltransferase antibodies. This fragment was integrated into the GS-5 chromosome following ligation into pVA891 and transformation, yielding strain DP2. The vector together with one complete and one incomplete copy of the gtfD gene was removed from the chromosome of strain DP2 following EcoRI digestion, religation, and transformation of E. coli HB101. The resultant plasmid, pNH4, expressed glucosyltransferase S (GTF-S) activity. The enzyme was purified to near homogeneity and was shown to synthesize water-soluble glucan exclusively in a primer-dependent manner. The molecular mass (155 kilodaltons) and the kinetic parameters of the purified enzyme were similar to those observed for the GTF-S enzyme previously purified from culture fluids of strain GS-5. Insertional inactivation of the gtfD gene indicated that this gene is not required for in vitro sucrose-dependent adherence to smooth surfaces. Furthermore, inactivation of the gtfD gene in a gtfC gtfB mutant indicated that three distinct gtf genes involved in glucan formation are present on the S. mutans GS-5 chromosome. Southern blot analysis further suggested that the gtfD gene does not share demonstrable homology with the gtf genes from Streptococcus sanguis or Streptococcus sobrinus.

DNA sequence of mip, a Legionella pneumophila gene associated with macrophage infectivity.

Abstract: In a previous study, a 24-kilodalton (kDa) protein surface antigen of Legionella pneumophila was cloned into Escherichia coli and found to be expressed on the host cell surface. Subsequently, a site-directed mutation in this gene (designated mip) in L. pneumophila was found to impair the capacity of this bacterium to initiate intracellular infection in human macrophages. The work presented here indicates that the antigenic gene product is distinct from the 24- to 29-kDa major outer membrane protein of L. pneumophila. In addition, the antigen was identified as a highly basic protein on two-dimensional nonequilibrium polyacrylamide gels and on two-dimensional monoclonal antibody immunoblots. When the DNA fragment encoding this protein was sequenced, a long open reading frame of 699 base pairs was identified within a region to which antigen expression was previously mapped. mip mRNA isolated from both L. pneumophila and transformed E. coli had the same 5' end, as determined by primer extension analysis, indicating that the same promoter sequences are used in both species. A likely factor-independent transcriptional terminator was found 20 residues downstream of the stop codon, suggesting that mip is encoded on a monocistronic message. The inferred polypeptide began with a possible 20- to 24-residue signal sequence, and, as predicted by two-dimensional electrophoresis, had a molecular weight of 24,868 and was a potent polycation with an estimated pI of 9.8.

Campylobacter pylori virulence factors in gnotobiotic piglets.

Abstract: Thirty-three gnotobiotic piglets from four litters were challenged with motile and nonmotile strains of Campylobacter pylori. The most motile strain, 26695, was the most virulent, with a 100% infection rate. The least motile strain, Tx30a, was the least virulent, with an infection rate of only 17%. Strain 60190 was weakly motile and had intermediate virulence, with an infection rate of 40%. Strains recovered from piglets were more motile than the challenge strains. The challenge strains also differed in cytotoxin production. The least virulent strain, Tx30a, was nontoxigenic, while the other two strains produced high levels of cytotoxin. Thus, virulence of C. pylori for gnotobiotic piglets correlated very well with motility and not as well with cytotoxin production. Images

Construction and characterization of isogenic mutants of Streptococcus mutans deficient in major surface protein antigen P1 (I/II).

Abstract: The gene (spaP) coding for the Streptococcus mutans major surface protein antigen P1 (or I/II) has been cloned into Escherichia coli (S. F. Lee, A. Progulske-Fox, and A. S. Bleiweis, Infect. Immun. 56:2114-2119, 1988). In the present study, this gene has been disrupted in vitro by insertional inactivation with pVA981, which carries a Tcr marker, and transformed into S. mutans NG8 (serotype c) by electroporation. Upon homologous recombination, the defective spaP was integrated into the genome as demonstrated by Southern hybridization analysis. One Tcr mutant, designated 834, selected by its nonreactivity with anti-P1 monoclonal antibodies, was found to lack the cell surface fuzzy layer which was clearly present on the parent cells. Analysis of extracellular fluids, sodium dodecyl sulfate-solubilized membranes, and cytoplasmic fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 834 had protein profiles identical to the parent. However, a 185-kilodalton protein which reacts with anti-P1 antibodies was missing from the wall of 834, suggesting that spaP has been specifically inactivated. This mutant displayed levels of glucosyltransferase and fructosyltransferase activities similar to those of the parent. It was much less hydrophobic than the parent. S. mutans NG8 aggregated readily in the presence of clarified whole saliva or a high-molecular-weight salivary agglutinin. This strain also adhered to agglutinin-coated hydroxyapatite. The P1-negative mutants, however, did not display these two properties, suggesting that P1 may play a role in saliva-mediated aggregation and adherence.

Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular-weight protein of Mycobacterium tuberculosis

Abstract: Only a limited number of proteins from Mycobacterium tuberculosis have so far been shown to possess species-specific epitopes as defined by monoclonal antibodies. One such protein is protein antigen b (Pab) of molecular weight 38,000, which binds the monoclonal antibodies HYT 28, HAT 2, HBT 12, HGT 3, TB 71, and TB 72. The gene encoding this protein was isolated from a lambda gt11 M. tuberculosis DNA library. The nucleotide sequence of the recombinant mycobacterial insert was determined, and an open reading frame of 374 amino acids was identified. The amino acid sequence exhibited 30% homology to a phosphate-binding protein, PstS, from Escherichia coli. The pab gene was subcloned into pBR322 in conjunction with the lacZ gene, and deletions were obtained from the 3' end. The anti-Pab monoclonal antibodies were used to probe crude protein lysates of E. coli transformed with the deletion plasmids. The monoclonal antibodies showed two reactivity patterns; one group of antibodies were dependent on the presence of the ultimate 91 amino acids of the protein, whereas another group of antibodies recognized an antigenic domain located on the middle portion of the molecule. None of the antibodies bound to the N-terminal 117-amino-acid peptide.

Role of tryptophan degradation in respiratory burst-independent antimicrobial activity of gamma interferon-stimulated human macrophages.

Abstract: To determine whether extracellular tryptophan degradation represents an oxygen-independent antimicrobial mechanism, we examined the effect of exogenous tryptophan on the intracellular antimicrobial activity of gamma interferon (IFN-gamma)-stimulated human macrophages. IFN-gamma readily induced normal monocyte-derived macrophages (MDM) to express indoleamine 2,3-dioxygenase (IDO) activity and stimulated MDM, alveolar macrophages, and oxidatively deficient chronic granulomatous disease MDM to degrade tryptophan. All IFN-gamma-activated, tryptophan-degrading macrophages killed or inhibited Toxoplasma gondii, Chlamydia psittaci, and Leishmania donovani. Although exogenous tryptophan partially reversed this activity, the increases in intracellular replication were variable for normal MDM (T. gondii [5-fold], C. psittaci [3-fold], L. donovani [2-fold]), chronic granulomatous disease MDM (T. gondii [2.5-fold], C. psittaci [5-fold]), and alveolar macrophages (T. gondii [1.5-fold], C. psittaci [1.5-fold]). In addition, IFN-alpha and IFN-beta also stimulated normal MDM to express IDO and degrade tryptophan but failed to induce antimicrobial activity, and IFN-gamma-treated mouse macrophages showed neither IDO activity nor tryptophan degradation but killed T. gondii and L. donovani. These results suggest that while tryptophan depletion contributes to the oxygen-independent antimicrobial effects of the activated human macrophage, in certain cytokine-stimulated cells, tryptophan degradation may be neither sufficient nor required for antimicrobial activity.

Roles of alpha-toxin and beta-toxin in virulence of Staphylococcus aureus for the mouse mammary gland.

Abstract: Mutants of Staphylococcus aureus which fail to express alpha-toxin (Hly), beta-toxin (Hlb), or both have been constructed by site-specific mutagenesis. The virulence of the mutants was compared with that of wild-type toxigenic strains by intramammary inoculation of lactating mice. A bovine strain, M60, and a laboratory strain, 8325-4, caused acute mastitis and death within 48 h for 60% of the mice inoculated. Animals inoculated with Hly mutants also developed acute mastitis, but no deaths occurred. Comparisons of Hly- or Hlb-positive strains with the double mutation Hly Hlb showed that both toxins led to a significantly higher recovery of S. aureus from the gland 48 h postinfection. Histopathological examination of mammary glands showed that phagocytosis of bacteria occurred irrespective of toxigenicity, but toxigenic strains, particularly those which were Hly+, continued to multiply, invaded the interalveolar tissues, and produced severe lesions. Stimulation of an inflammatory response by inoculation of the mammary gland with endotoxin prior to challenge with S. aureus reduced recovery of the bacteria 10- to 100-fold and, under these conditions, neither alpha-toxin nor beta-toxin contributed significantly to growth and survival.

Immunohistochemical and electron microscopic study of interaction of Yersinia enterocolitica serotype O8 with intestinal mucosa during experimental enteritis.

Abstract: The experimental infection of mice with Yersinia enterocolitica serotype O8 was investigated in a quantitative and histological study. The course of bacterial penetration and spreading was precisely determined by immunohistochemical staining. After oral administration, the bacteria passed the epithelial barrier of the ileum and spread into the lamina propria. By preference they entered Peyer's patches, which were about 1,000 times more heavily colonized than the surrounding epithelium of a comparable surface area. The bacteria proliferated in the follicles, from which they spread into the lamina propria of the villi. At either site most of the bacteria multiplied extracellularly, with only a small percentage observed to be present within the phagocytes. The bacteria did not appear to be able to pass the intact basement membrane; hence, the integrity of the basement membrane is likely to play a role in determining the route of entry and limit of spread of Y. enterocolitica infection.

Effects of magainins and cecropins on the sporogonic development of malaria parasites in mosquitoes.

Abstract: Magainins and cecropins are families of peptides with broad antimicrobial and antiparasitic activities derived respectively from the skin of frogs or from giant silk moths. In insects, cecropins function as part of an inducible immune system against a number of bacterial infections. When injected into anopheline mosquitoes previously infected with a variety of Plasmodium species, both magainins and cecropins disrupt sporogonic development by aborting the normal development of oocysts; sporozoites are not formed and the vector cannot transmit the parasite to another host. It may be possible to induce effective transmission-blocking immunity in the mosquito vector by the introduction and expression of genes coding for magainins, cecropins, or similarly acting parasiticidal peptides into the mosquito genome.

Reduced adherence to traumatized rat heart valves by a low-fibronectin-binding mutant of Staphylococcus aureus.

Abstract: Isogenic strains of Staphylococcus aureus, differing in fibronectin binding, were constructed for studies of the contribution of fibronectin binding to the in vivo pathogenesis of staphylococcal disease. Mutagenesis of S. aureus 879R4S was accomplished by mating with Enterococcus faecalis FA378 that carried the transposon Tn918. Four low-fibronectin-binding mutants were identified that bound 24- to 35-fold less fibronectin than the parent strain did. A spectinomycin-resistant strain, R4SSp, was transduced by a bacteriophage (80 alpha) lysate propagated on a low-binding mutant of 879R4S to produce R4SSp/1536, which bound less fibronectin, contained a single copy of the transposon, and grew on spectinomycin-containing medium. Using a rat model of endocarditis, we determined the distribution of S. aureus R4SSp and its transductant in normal and cardiac catheterized rats. Cultures of heart tissue showed that catheterized rats challenged with the fibronectin-binding parent strain had over 250-fold more organisms in the left heart than did rats challenged with the low-binding transductant. The ability to bind fibronectin had no effect on the number of S. aureus cells cultured from other tissues. These data suggest that the ability of S. aureus to bind fibronectin is an important factor in establishing adherence to damaged heart valves in vivo.

Adhesion to and invasion of HEp-2 cells by Campylobacter spp.

Abstract: Twenty-one isolates were tested for their ability to adhere to and invade HEp-2 cells in vitro. Of the 21 organisms tested, 2 did not invade the HEp-2 cells, and 1 of these did not adhere to the epithelial cells. Campylobacter jejuni clinical isolates were more invasive than the nonclinical strains that were tested. When HEp-2 cells were treated with cytochalasin B, the invasiveness of C. jejuni was reduced, indicating active participation of the host cell in the uptake of these organisms. The number of intracellular C. jejuni isolates decreased when Campylobacter whole-cell lysates were absorbed onto HEp-2 cell monolayers. Experiments were also conducted to identify the functional sites of the antigens responsible for expression of Campylobacter invasion. Oxidation of lysates with sodium meta-periodate significantly affected its inhibitory capacity. This implies that the Campylobacter invasive ligand appears to be dependent upon an intact carbohydrate moiety.

Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens.

Abstract: A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa. Images