bluuebunny/crossref_metadata_2025 huggingface dataset:
Serial isolates of Pseudomonas aeruginosa from a cystic fibrosis patient have identical pilin sequences
Authors: Pasloske B L , Joffe A M , Sun Q , Volpel K , Paranchych W , Eftekhar F , Speert D P
Published: January 2020

Five isolates of Pseudomonas aeruginosa (CD2, CD3, CD4, CD5, and CD10) from a patient with cystic fibrosis were examined with regard to several genotypic and phenotypic characteristics to determine whether the patient was colonized with one or several distinct strains. Isolates CD2, CD3, and CD4 were obtained from a single sputum sample, and CD5 and CD10 were obtained 1 and 2 years later, respectively. On the basis of colonial morphology, serotyping, and antibiograms, the five isolates appeared to be different strains. However, Southern blot analysis with a 1.2-kilobase DNA probe containing the P. aeruginosa PAK pilin gene indicated that all five strains were identical at that genetic locus. The pilin genes of the five isolates were cloned and sequenced at the nucleotide level and found to be identical. Southern blot analysis with a probe from a separate region of the P. aeruginosa chromosome, a 741-base-pair PstI-NruI DNA fragment adjacent to the exotoxin A gene, also revealed genetic identity among these five clinical isolates. On this basis, it was concluded that this patient was colonized with a single strain of P. aeruginosa and that the strain had remained genetically stable over a period of 2 years. The predicted pilin sequence of the CD isolates was almost identical to that of strain PA103 (97% homology) and serologically related to PAO pilin, with which it shared 80% homology. No immunological cross-reactivity was detected between the CD and PAK pilins, which shared the least homology (62%) among the four pilins considered in this study. Although all five CD isolates contained identical pilin genes, three had acquired mutations which prevented normal expression of the pilus system. CD3 was a putative regulatory mutant which was unable to produce normal amounts of pilin, and CD4 and CD10 were putative assembly mutants which produced normal amounts of pilin but were unable to assemble the pilin subunit into intact pili.
Nutritional requirements of shigellae for growth in a minimal medium
Authors: Ahmed Z U , Sarker M R , Sack D A
Published: January 2020

Most (about 81%) of the clinical isolates of shigellae that were tested failed to grow in a minimal medium. Of the auxotrophic isolates belonging to the four Shigella species, 98% grew in a minimal medium containing methionine, nicotinic acid, and tryptophan. The combination of methionine and tryptophan appears to be an obligatory requirement for Shigella dysenteriae serotype 1 strains, while the combination of nicotinic acid and tryptophan appears to be obligatory for serotype 2. Requirements which varied in other isolates were, however, genetically stable, indicating that the auxotypes may be useful as epidemiological markers. Cultures of shigellae in liquid minimal medium containing the above three supplements showed rapid growth and gave reasonably high cell yields.
Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes
Authors: Mengaud J , Vicente M F , Chenevert J , Pereira J M , Geoffroy C , Gicquel-Sanzey B , Baquero F , Perez-Diaz J C , Cossart P
Published: January 2020

To evaluate the role of hemolysin production in the virulence of Listeria monocytogenes, we have undertaken the analysis of the chromosomal region containing hlyA, the gene coding for listeriolysin O. A recombinant cosmid, conferring a hemolytic phenotype to Escherichia coli, was shown to express listeriolysin O, by immunoblotting with a specific antiserum against listeriolysin O. The presence of hlyA on the cosmid was demonstrated by DNA hybridization with a probe previously shown to contain part of hlyA. The complete nucleotide sequence of hlyA has been determined. The deduced protein sequence reveals the presence of a putative 25-amino-acid signal sequence: the secreted form of listeriolysin O would have 504 amino acids, in agreement with the molecular weight of purified listeriolysin O (58,000). The protein sequence is highly homologous to those of streptolysin O and pneumolysin. A peptide of 11 amino acids conserved in the three proteins contains the unique cysteine known to be essential for lytic activity. By DNA-DNA hybridization, the listeriolysin O gene was detected in all L. monocytogenes strains tested, even in the nonhemolytic type strain. The gene was absent in other species of the genus Listeria.
Detection of a novel catalase in extracts of Mycobacterium avium and Mycobacterium intracellulare
Authors: Wayne L G , Diaz G A
Published: January 2020

A novel class of catalase, which differs from the previously described M- and T-catalases of mycobacteria, was detected in strains of Mycobacterium avium and M. intracellulare. Designated A-catalase, this enzyme resisted inactivation at 68 degrees C, was inactivated by 3-amino-1,2,4-triazole (aminotriazole), and exhibited no peroxidase activity. All of these properties distinguished the enzyme from T-catalase. The A-catalase exhibited a Km of 70 mM H2O2, which is between the upper and lower extremes of the ranges reported for T- and M-catalases, respectively. The A-catalase appeared to be more hydrophobic than M-catalase and did not react with antiserum to a representative sample of this class. The banding patterns of T- and M-catalases seen by polyacrylamide gel electrophoresis (PAGE) were essentially unaffected by the incorporation of sodium dodecyl sulfate (SDS) into the PAGE system, whereas the single band of A-catalase seen by PAGE without SDS resolved into as many as five bands in the presence of SDS; these bands were all of slower mobility than the original band. The banding pattern seen with SDS appeared to be related more to counterion charge effects than to molecular size increases that could be attributed to SDS complexed to the protein. It remains to be determined whether the multiple A-catalase bands reflect different proteins or different SDS micellar complexes of a single protein.
Effects of the bactericidal/permeability-increasing protein of polymorphonuclear leukocytes on isolated bacterial cytoplasmic membrane vesicles
Authors: in't Veld G , Mannion B , Weiss J , Elsbach P
Published: January 2020

The bactericidal/permeability-increasing protein (BPI) of polymorphonuclear leukocytes is a potent bactericidal agent specific for gram-negative bacteria. The protein blocks bacterial multiplication without substantially inhibiting the uptake and incorporation of macromolecular precursors, suggesting that the cytoplasmic membrane escapes early injury. Because greater than 90% of bound BPI can be removed from the bacterial surface sites after irreversible loss of viability, it was uncertain whether BPI reaches the cytoplasmic membrane and, if so, affects its functions. This study shows that BPI caused similar dose-dependent inhibition of O2 consumption and metabolic energy-dependent amino acid transport by cytoplasmic membrane vesicles of both gram-negative (Escherichia coli) and gram-positive (Bacillus subtilis) bacteria. Near maximal inhibition occurred at BPI doses that caused complete killing of an equivalent number of intact E. coli, with binding of BPI to membrane vesicles that was less than or equal to 10% of binding to intact (BPI-sensitive) bacteria. The effects of BPI and of the membrane-disruptive peptide antibiotic polymyxin B on membrane vesicles were distinctly different, indicating that the two agents affect membrane function by different mechanisms. BPI also rapidly inhibited O2 consumption by intact E. coli, with minimal impairment of bacterial protein synthesis. Thus, BPI is capable of damaging the cytoplasmic membrane of both gram-negative and gram-positive bacteria and of inhibiting at least one cytoplasmic membrane-associated function in intact E. coli. The relationship between these effects and the mechanism of bacterial killing by BPI remains to be established.
Experimental Escherichia coli endocarditis in rats: roles of serum bactericidal activity and duration of catheter placement
Authors: Yersin B , Glauser M P , Guze P A , Guze L B , Freedman L R
Published: January 2020

Studies were undertaken to investigate the relationship of the sensitivity of Escherichia coli to the bactericidal properties of serum and the ability of different strains to induce and sustain endocardial infection in rats. Strains of E. coli demonstrated different degrees of serum sensitivity, as determined by a method which employed concentrations of serum from 10 to 95% and periods of incubation as long as 24 h. The greater the serum sensitivity of the E. coli strain, the less able it was to initiate infection and the more rapidly it was spontaneously eliminated from established infections. Endocardial infection with E. coli was established by intravenous challenge in rats with polyethylene catheters passing through the aortic valve into the left ventricle. An E. coli strain of low serum sensitivity was used; the initiation of infection depended upon the length of time the catheter had been in place and, in addition, whether the catheter was in place at the time of bacterial challenge. Removal of the catheter permitted spontaneous sterilization of the endocardial vegetations. The time necessary for sterilization was in direct proportion to the length of time the catheter remained in place following bacterial challenge. If the catheter was not removed, sterilization of the endocardial vegetations did not take place. These studies suggest that serum bactericidal activity is an important host defense mechanism, acting to prevent the initiation of endocarditis in the case of highly serum-sensitive E. coli and to sterilize experimentally induced endocarditis in the case of less-serum-sensitive bacteria. The catheter used to induce nonbacterial endocardial vegetations favored the colonization of vegetations by E. coli, and it delayed the spontaneous sterilization of infected vegetations which occurred in relation to the susceptibility of the strain to the bactericidal properties of the serum. This effect of the catheter was not attributable to bacteria remaining viable in its lumen, nor was it attributable to inhibition of the bactericidal capacity of the serum as measured in vitro. Whatever the mechanism responsible for the catheter effect, experimental studies of the evolution of infections established with this technique must take into consideration the duration of catheter placement and whether and for how long it was present before or after inoculation with test bacteria.
Reversible inactivation of bladder surface glycosaminoglycan antibacterial activity by protamine sulfate
Authors: Parsons C L , Stauffer C W , Schmidt J D
Published: January 2020

Prior studies in our laboratory have shown that the bladder surface is lined with glycosaminoglycans which appear to be an important antibacterial defense mechanism that operates by resisting bacterial adherence and infection. The present study further implicates bladder surface glycosaminoglycans as the key antiadherent factor and also suggests a potential model for diseases (such as urinary tract infections) whereby the antiadherent surface of the bladder is inactivated biochemically. Protamine sulfate treatment of bladder tissue was found to significantly increase bacterial adherence to the urinary bladder by approximately 2.3-fold. This effect was reversed by a second treatment of the bladder with pentosanpolysulfate (a polysaccharide known to duplicate the surface antiadherent effect). Protamine sulfate had no effect on bacterial viability or bacterial adherence when bacteria were pretreated with it.
Monoclonal antibodies to Actinobacillus actinomycetemcomitans
Authors: Place D A , Scidmore N C , McArthur W P
Published: January 2020

Murine hybridoma cell lines were developed which synthesized monoclonal antibodies against Actinobacillus actinomycetemcomitans-associated antigens. Monoclonal antibodies specific for an antigen(s) common to all A. actinomycetemcomitans isolates tested but not detected on other gram-negative oral plaque microorganisms or other Actinobacillus species were identified. Monoclonal antibodies specific for each serotype group of A. actinomycetemcomitans which did not bind to other Actinobacillus species or oral plaque microorganisms were also identified.
Cloning of the gene, speB, for streptococcal pyrogenic exotoxin type B in Escherichia coli
Authors: Bohach G A , Hauser A R , Schlievert P M
Published: January 2020

The structural gene encoding streptococcal pyrogenic exotoxin type B, designated speB, was cloned in Escherichia coli and localized onto a 4.5-kilobase BamHI-BglII DNA fragment. Streptococcal pyrogenic exotoxin type B, partially purified from E. coli clones, was immunologically related to streptococcus-derived toxin. Also, toxin derived from either E. coli or Streptococcus pyogenes had similar lymphocyte mitogenic activity and molecular weight (29,300) and displayed comparable microheterogeneity when evaluated by isoelectric focusing.
Dermonecrotic toxin and tracheal cytotoxin, putative virulence factors of Bordetella avium
Authors: Gentry-Weeks C R , Cookson B T , Goldman W E , Rimler R B , Porter S B , Curtiss R
Published: January 2020

We examined Bordetella avium for virulence factors common to Bordetella pertussis, including pertussis toxin, filamentous hemagglutinin, adenylate cyclase, dermonecrotic toxin, and tracheal cytotoxin. B. avium produced a dermonecrotic toxin and a tracheal cytotoxin. The dermonecrotic toxin of B. avium is a 155,000-molecular-weight, heat-labile protein which was lethal for mice, guinea pigs, young chickens, and turkey poults and produced dermonecrosis when injected intradermally into guinea pigs, chickens, and turkey poults. High-pressure liquid chromatography of B. avium culture supernatant fluid revealed the presence of a tracheal cytotoxin chemically identical to that produced by B. pertussis. B. avium isolates were negative for B. pertussis-like filamentous hemagglutinin and pertussis toxin when assayed with antibody against B. pertussis filamentous hemagglutinin and pertussis toxin. Furthermore, B. avium failed to induce the clustered CHO cell morphology characteristic of pertussis toxin. Adenylate cyclase assays indicated that B. avium does not produce an extracytoplasmic adenylate cyclase, even after passage through embryonated turkey eggs. Since production of virulence proteins by B. pertussis is regulated by growth in media containing nicotinamide or MgSO4 or by growth at reduced temperatures, we determined the effect of these supplements and growth conditions on production of dermonecrotic toxin by B. avium. Production of dermonecrotic toxin in B. avium was not altered by growth in media containing 100 microM FeSO4 or 500 micrograms of nicotinamide per ml or by growth at 25 or 42 degrees C, but production was significantly decreased by growth in media containing 20 mM MgSO4 and slightly reduced by growth in media containing 500 micrograms of nicotinic acid per ml. These studies revealed that B. avium is similar to B. pertussis in that both species produce a dermonecrotic toxin and a tracheal cytotoxin and production of dermonecrotic toxin is regulated by nicotinamide and MgSO4. The presence of dermonecrotic toxin and tracheal cytotoxin in all Bordetella species indicates that these products may be important virulence factors in bordetellosis.
Impairment of cellular but not humoral immune responses in chronic pulmonary and disseminated paracoccidioidomycosis in mice
Authors: Castaneda E , Brummer E , Pappagianis D , Stevens D A
Published: January 2020

Humoral and cellular immune responses were measured during the progression of chronic pulmonary and disseminated paracoccidioidomycosis in mice. The chronic disease was established by pulmonary infection of mice with different doses of the yeast form of Paracoccidioides brasiliensis isolate GAP. Levels of antibodies to P. brasiliensis, detected in serum by immunodiffusion and enzyme-linked immunosorbent assay, directly correlated with the size of the infectious challenge. Significant delayed-type hypersensitivity (DTH) responses to antigen were largely restricted to week 1 after pulmonary infection with intranasally administered high doses (5.0 x 10(6) or 1.1 x 10(7) CFU per inoculum). In vitro lymphoproliferative responses of peripheral blood lymphocytes (PBL) to P. brasiliensis antigens were significant only at 2 weeks after infection with intranasally administered 1.1 x 10(7) CFU. Responses of PBL to concanavalin A were depressed (50% of control response) as early as 8 weeks and reached a nadir at 10 to 18 weeks after infection. Infected mice made antibodies to sheep erythrocytes (SRBC) (10(9) intravenously [i.v.]) normally at all times tested after infection. In contrast, infected mice sensitized to SRBC (10(6) i.v.) had significantly depressed DTH responses to SRBC at 9 and 20 weeks postinfection compared with noninfected mice. These results indicated that in this model, normal humoral responses developed to homologous and heterologous antigens. In contrast, the T cellular immune responses were depressed with progression and chronicity of the disease. Thus, this model closely mimics the immunological findings in human paracoccidioidomycosis.
Virulence properties and protective efficacy of the capsular polymer of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5
Authors: Inzana T J , Ma J , Workman T , Gogolewski R P , Anderson P
Published: January 2020

The role of the capsule of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 in bacterial virulence, and the protective efficacy of antibody to serotype 5 capsule was investigated. Encapsulated H. pleuropneumoniae serotype 5 were resistant to killing by complement and antibody to capsule or somatic antigens, whereas a noncapsulated mutant was sensitive to killing by the alternative complement pathway alone. Antiserum to whole H. pleuropneumoniae serotype 5 bacteria or monospecific antiserum to capsule was capable of opsonizing bacteria of the homologous serotype for phagocytosis by swine polymorphonuclear leukocytes but was not opsonic for a heterologous serotype. An immunoglobulin M monoclonal antibody to the serotype 5 capsule was not opsonic for any serotype. Mice were protected against lethal, intranasal challenge with the homologous or heterologous serotype after immunization with live encapsulated or noncapsulated bacteria, but not after immunization with killed bacteria, lipopolysaccharide, or a capsule-protein conjugate vaccine. The protection induced by immunization with live bacteria was transferred to nonimmune, syngeneic mice by serum but not by spleen cells. Nonimmune pigs passively immunized with monospecific swine serum to capsule were protected from lethal infection but not from development of hemorrhagic lung lesions, whereas pigs passively immunized with swine antiserum to live bacteria did not develop severe respiratory lesions. Thus, the capsule of H. pleuropneumoniae serotype 5 was inhibitory to the bactericidal activity of serum and was antiphagocytic. Antibody to the capsule was opsonic but was not fully protective.
Stimulation of neutrophil actin polymerization and degranulation by opsonized and unopsonized Candida albicans hyphae and zymosan
Authors: Kolotila M P , Diamond R D
Published: January 2020

We previously showed that unopsonized Candida albicans hyphae stimulated a delayed rise in the putative neutrophil second messengers Ca2+ and inositol 1,4,5-trisphosphate and subsequent O2- release, as compared with opsonized hyphae or zymosan. Therefore, cytoskeletal and degranulation temporal responses to these stimuli were examined. Unopsonized zymosan elicited no neutrophil responses under the experimental condition used. Neutrophil actin polymerization (quantitated by fluorescent measurements of NBD phallacidin) was rapid after stimulation by opsonized hyphae or zymosan (peaking at 1 and 2 min, respectively). This corresponded to observed changes in microscopic actin polymerization, measured with rhodamine phalloidin, which progressed from initially diffuse to collarlike to cylinderlike staining patterns surrounding the hyphae. Compared with opsonized hyphae, unopsonized hyphae resulted in a delayed appearance of the last two visible patterns (P less than 0.05) and in quantitative actin polymerization despite similarly rapid initial contact and spreading over the hyphae by neutrophils. Unlike other neutrophil responses, degranulation did not follow the delayed patterns of responses to stimulation with unopsonized hyphae. In the absence of the release of the cytoplasmic marker lactate dehydrogenase, the release of beta-glucuronidase, an azurophil granule marker, gradually and progressively rose in response to all of the stimuli but unopsonized zymosan. The low but significant levels observed were within a range consistent with published results for degranulation responses to particulate stimuli without cytochalasin B. A quantitative immunoassay of lactoferrin, a specific granule marker, detected no release into supernatants, and immunofluorescent staining indicated concomitant depletion of lactoferrin from neutrophil granules and binding to hyphal and neutrophil surfaces after stimulation by unopsonized hyphae. Thus, the delayed actin polymerization response to unopsonized hyphae occurred subsequent to neutrophil attachment and spreading and resembled the temporal sequence of other neutrophil responses linked to the respiratory burst. In contrast, the degranulation responses to all stimuli appeared to begin and progress gradually after observed attachment and spreading of the neutrophil over hyphal surfaces without a clear temporal relationship to rises in cytoplasmic Ca2+ or F-actin. In addition, the avid binding of released lactoferrin to cell surfaces eliminates its value as a quantitative marker of enzyme release but raises the possibility that it might participate in fungicidal activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Age-associated increase in the expression of T-cell antigen receptor gamma-chain gene in conventional and germfree mice
Authors: Yoshikai Y , Matsuzaki G , Kishihara K , Nomoto K , Yokokura T , Nomoto K
Published: January 2020

To determine whether environmental antigens such as intestinal microflora contribute to expansion of the T-cell repertoire, age-related changes in the expression level of T-cell antigen receptor genes encoding gamma, beta, and alpha chains were compared in the lymphoid tissues of conventional versus germfree mice. Irrespective of the conditions of maintenance, an age-associated increase in the expression of the gamma-chain gene was evident in the thymus and spleen. Both conventional and germfree old mice (age, 40 weeks) had a relatively high proportion of Thy1+ L3T4- Lyt2- cells but a reduced level of Thy1+ L3T4- Lyt2+ cells in the thymus compared with their counterparts (age 8 weeks). The thymic dysfunction but not the stimulation by intestinal microflora may contribute to this age-related increase in gamma-gene transcripts in these tissues. On the other hand, an age-associated increase in the expression of gamma RNA was not evident in the mesenteric lymph nodes of germfree mice, although a remarkable increase in the gamma-chain gene messages was detected in the lymph nodes of the aged conventional mice. These results suggest that the expression of gamma RNA in cells of gut-associated lymphoid tissue is partly influenced by intestinal microflora.
Systemic uptake and intestinal inflammatory effects of luminal bacterial cell wall polymers in rats with acute colonic injury
Authors: Sartor R B , Bond T M , Schwab J H
Published: January 2020

The systemic uptake and local intestinal inflammatory potential of luminal bacterial cell wall polymers in rats with normal and acutely inflamed colons were measured. Rats were injected intracecally with either 125I-labeled group A streptococcal peptidoglycan-polysaccharide complexes or equal amounts of Na125I, after either nonspecific colonic injury with 4% acetic acid or injection with buffer. The colons of rats injected with peptidoglycan-polysaccharide had higher inflammatory scores than Na125I-injected rats, a greater incidence of mucosal ulceration and transmural inflammation after acetic acid injury, and an increased frequency of focal accumulations of inflammatory cells in the lamina propria and submucosa after buffer injection. Radioactivity in the liver, spleen, and mesenteric lymph nodes was higher in the colon-injured rats that received peptidoglycan-polysaccharide 48 h before tissue collection than in the noninjured rats (P less than 0.002). Group A streptococcal polysaccharide antigen concentration within the liver, spleen, and mesenteric lymph nodes, measured by enzyme-linked immunosorbent assay, was significantly higher in the colon-injured rats that received cell wall polymers than in noninjured rats. These results indicate that luminal bacterial cell wall polymers with well-described inflammatory and immunoregulatory potential can cross injured colonic epithelia and are capable of initiating and potentiating intestinal inflammation.
Analysis of expression and thermoregulation of the Yersinia pseudotuberculosis inv gene with hybrid proteins
Authors: Isberg R R , Swain A , Falkow S
Published: January 2020

A series of translational fusions between the Yersinia pseudotuberculosis inv locus and lacZ was constructed. Each Lac+ fusion strain expressed a hybrid protein containing invasin, the product of the inv locus, at its amino-terminal end. Analysis of these gene fusions allowed determination of the direction of translation of the inv gene. Previous studies of Y. pseudotuberculosis invasion have shown that entry into animal cells is temperature regulated. It is shown here that control of expression of the inv gene is also temperature regulated. phoA gene fusions to inv, when present in Y. pseudotuberculosis, were expressed at lower levels when bacteria were grown at 37 degrees C rather than at 28 degrees C. Similar fusions, in contrast, were regulated in a temperature-independent fashion in Escherichia coli, as was the wild-type inv gene. This implies that Y. pseudotuberculosis has chromosomally encoded trans-acting functions that normally thermoregulate expression of inv.
Longitudinal study of antibody response to lipopolysaccharides during chronic Pseudomonas aeruginosa lung infection in cystic fibrosis
Authors: Fomsgaard A , Høiby N , Shand G H , Conrad R S , Galanos C
Published: January 2020

Antibodies to Pseudomonas aeruginosa from 10 cystic fibrosis patients with chronic P. aeruginosa lung infections were quantitatively and qualitatively analyzed. The development of specific antibodies in patient serum was evaluated in a longitudinal study (1972 to 1987). The concentrations and specificities of immunoglobulin G (IgG) and IgM antibodies to purified lipopolysaccharides (LPS) from clinical isolates of P. aeruginosa and to a variety of other gram-negative bacteria were studied by immunoblotting and enzyme-linked immunosorbent assay techniques. Results were compared with the number of immunoprecipitates to P. aeruginosa whole-cell extracts detected by crossed immunoelectrophoresis. IgG, but not IgM, anti-Pseudomonas LPS concentrations increased significantly at the onset of chronic infection and continued to increase during the course of the infection. There was a good positive correlation between the concentration of IgG anti-Pseudomonas LPS antibodies and the number of crossed-immunoelectrophoresis precipitins. The increases in IgG anti-LPS antibody concentrations were much higher to Pseudomonas LPS than to other LPSs. Binding studies demonstrated an increase in binding of IgG anti-Pseudomonas LPS during infection, whereas the binding of other anti-LPS antibodies decreased. Immunoblotting studies confirmed that antibodies reacted strongly with Pseudomonas LPS and weakly with Escherichia coli core-lipid A. The specificity of the reaction with Pseudomonas LPS increased with the duration of infection. It is concluded that anti-LPS response in cystic fibrosis patients during chronic P. aeruginosa infection demonstrates a marked increase in IgG anti-Pseudomonas LPS antibody concentration, specificity, and affinity. The anti-LPS enzyme-linked immunosorbent assay is proposed as a routine test to diagnose and to follow the course of chronic P. aeruginosa lung infection in patients with cystic fibrosis.
Dynamic expression of cell surface hydrophobicity during initial yeast cell growth and before germ tube formation of Candida albicans
Authors: Hazen B W , Hazen K C
Published: January 2020

Expression of cell surface hydrophobicity (CSH) during initial growth of Candida albicans was monitored. CSH of hydrophobic and hydrophilic yeast cells changed within 30 min upon subculture into fresh medium. Morphologic evidence of germination was preceded by expression of CSH. These results indicate that CSH expression is important in C. albicans growth.
Elevated environmental temperature enhances immunity in experimental Chagas' disease
Authors: Anderson K J , Kuhn R E
Published: January 2020

C3H mice are highly susceptible to the Brazil strain of Trypanosoma cruzi. These mice usually die during the acute phase of infection and develop a profound immunosuppression to heterologous and parasite antigen. In this study, we confirmed earlier reports that infected mice maintained at elevated environmental temperature (36 degrees C) are significantly more resistant to T. cruzi than are mice kept at 20 to 24 degrees C. To determine whether the benefits of increased environmental temperature were due to alterations in the host immune system, the production of antibody to heterologous antigen and the development of parasite-specific T-helper cells were examined in noninfected and T. cruzi-infected mice. Mice were immunized with either sheep erythrocytes (SRBC) or trinitrophenyl groups (TNP) conjugated to fixed culture forms of T. cruzi, and the splenic direct plaque-forming cell (DPFC) responses to SRBC and to TNP-conjugated SRBC were determined. The DPFC response to SRBC from infected mice maintained at elevated environmental temperature was much higher than the suppressed response of infected mice held at room temperature and slightly higher than the response of age-matched noninfected control mice. Likewise, maintaining infected mice at 36 degrees C significantly enhanced the parasite-specific responses of T-helper cells, as reflected by anti-TNP DPFC responses of mice immunized with TNP-conjugated TC.
Sequence homology between tetanus and botulinum toxins detected by an antipeptide antibody
Authors: Halpern J L , Smith L A , Seamon K B , Groover K A , Habig W H
Published: January 2020

The extent of immunological similarity between tetanus toxin and botulinum toxins A, B, C1, and E was studied by using 10 antibodies produced against synthetic peptides representing different sequences of tetanus toxin, mouse antitetanus serum, and human Tetanus Immune Globulin. Antibodies produced against the synthetic peptides recognized tetanus toxin in an enzyme-linked immunosorbent assay and on Western blots (immunoblots) but did not appear to recognize the native protein. One of the antitetanus peptide antibodies, which was produced against a peptide from the amino terminal, cross-reacted with three of the four botulinum toxins on immunoblots. This antibody, 1, reacted strongly with botulinum toxins B and C1 and weakly with E but did not recognize type A toxin. None of the other peptide antibodies cross-reacted with the botulinum toxins. Mouse antitetanus serum and human Tetanus Immune Globulin did not recognize any of the botulinum toxins on immunoblots. The amino-terminal region of the light chain of tetanus toxin and botulinum toxin types A, B, C1, and E are known to have sequence homology. Our data demonstrate that for tetanus toxin and botulinum toxin types B, C1, and E this region also has immunological homology. Type A, which has the least amount of homology with tetanus toxin in this region, does not share this immunological homology. These data also suggest that although the native structures of tetanus and botulinum toxins have relatively few common immunological determinants, the two toxins may contain short stretches of identical or very similar amino acid sequences.
Type 3 fimbriae among enterobacteria and the ability of spermidine to inhibit MR/K hemagglutination
Authors: Gerlach G F , Allen B L , Clegg S
Published: January 2020

The distribution of the gene cluster encoding type 3 fimbriae among various isolates of the family Enterobacteriaceae was investigated by using 112 clinical and nonclinical isolates. Closely related DNA sequences were detected in all Klebsiella strains, in most Enterobacter isolates, in a smaller number of Escherichia coli and Salmonella spp., and in a single isolate each of Yersinia enterocolitica and Serratia liquefaciens but not in isolates of Morganella or Providencia species or Serratia marcescens. Except for E. coli and Salmonella strains, the presence of gene sequences was correlated with the phenotypic expression of either the MR/K hemagglutinin or fimbriae that reacted with specific antibodies. In one isolate of Y. enterocolitica the expression of type 3 fimbriae was plasmid determined. The polyamine spermidine was identified as an inhibitor of MR/K hemagglutinating activity, exhibiting an MIC of 1.2 mM. Spermidine inhibited the hemagglutination of 37 MR/K-positive clinical isolates from various genera. However, one clinical isolate of Enterobacter cloacae and most (four of five) nonclinical Klebsiella isolates were not completely inhibited.
Determination by western blot (immunoblot) of seroconversions to toxic shock syndrome (TSS) toxin 1 and enterotoxin A, B, or C during infection with TSS- and non-TSS-associated Staphylococcus aureus
Authors: Whiting J L , Rosten P M , Chow A W
Published: January 2020

Serum antibody responses to toxic shock syndrome (TSS) toxin 1 (TSST-1) and staphylococcal enterotoxins A, B, and C were determined by western blot (immunoblot) analysis of acute- and convalescent-phase paired sera from 18 TSS- and 31 non-TSS-associated Staphylococcus aureus infections. Compared with non-TSS cases, seroconversion to TSST-1 was significantly more frequent among both menstrual (5 of 8 versus 1 of 31; P less than 0.001) and nonmenstrual (3 of 10; P less than 0.05) patients. Seroconversion to staphylococcal enterotoxin A was also more frequent among both menstrual (2 of 8 versus 0 of 31; P less than 0.05) and nonmenstrual (2 of 9; P less than 0.05) TSS patients. In general, patients with TSS associated with TSST-1-positive S. aureus were more likely to seroconvert exclusively to TSST-1 (4 of 12 versus 0 of 6; P = 0.16), whereas those associated with TSST-1-negative S. aureus were more likely to seroconvert exclusively to enterotoxins (3 of 6 versus 0 of 11; P less than 0.05). Concurrent seroconversions to multiple exoproteins were more frequent among both menstrual (3 of 8; P less than 0.05) and nonmenstrual (2 of 9; P less than 0.05) TSS patients compared with persons without TSS (0 of 31). These data suggest but do not prove that enterotoxins (especially staphylococcal enterotoxin A) in addition to TSST-1 may be involved in both menstrual and nonmenstrual TSS. Furthermore, since exposure to multiple exoproteins is more likely to occur during TSS-associated than non-TSS-associated S. aureus infections, the possibility of additive or synergistic effects of these putative toxins in the pathogenesis of TSS should be further explored.
Molecular cloning, expression, and primary sequence of outer membrane protein P2 of Haemophilus influenzae type b
Authors: Munson R , Tolan R W
Published: January 2020

The structural gene for the porin of Haemophilus influenzae type b, designated outer membrane protein P2, was cloned, and the DNA sequence was determined. An oligonucleotide probe generated by reverse translation of N-terminal amino acid sequence data from the purified protein was used to screen genomic DNA. The probe detected a single EcoRI fragment of approximately 1,700 base pairs which was cloned to lambda gt11 and then into M13 and partially sequenced. The derived amino acid sequence indicated that we had cloned the N-terminal portion of the P2 gene. An overlapping approximately 1,600-base-pair PvuII genomic fragment was cloned into M13, and the sequence of the remainder of the P2 gene was determined. The gene for P2 was then reconstructed under the control of the T7 promoter and expressed in Escherichia coli. The N-terminal sequence of the purified protein corresponds to residues 21 through 34 of the derived amino acid sequence. Thus, the protein is synthesized with a 20-amino-acid leader peptide. The Mr of the processed protein is 37,782, in good agreement with the estimate of 37,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Host response to Escherichia coli heat-labile enterotoxin via two microvillus membrane receptors in the rat intestine
Authors: Zemelman B V , Chu S H , Walker W A
Published: January 2020

The responsiveness of enterocytes to Escherichia coli heat-labile enterotoxin (LT) was studied in the small intestine of 6- to 7-week-old rats. Dose-effect analysis showed the dose required for a 50% maximal LT-induced secretory response to be at 8 nM. After the well-documented glycolipid GM1 receptor was blocked with the cholera toxin B subunit, LT still activated the second messenger cascade, measured in terms of heightened cellular adenylate cyclase activity, and caused fluid to be secreted into ligated intestinal loops. Furthermore, Scatchard analysis of binding kinetics suggested that LT bound to two receptor sites on the intestinal microvillus membrane. The toxin also bound to delipidated membrane but was competitively inhibited by a galactose-specific lectin, RCA60, suggesting that the additional receptor is a galactoglycoprotein. Western blot analysis of toxin binding to membrane proteins revealed a group of binding components around 85 to 150 kilodaltons. When measured at 2.2 nM LT, approximately 70% of LT-binding activity took place through a high-affinity (Kd1, 0.38 nM) GM1 receptor and 30% of LT-binding activity took place through a low-affinity (Kd2, 3.3 nM) glycoprotein receptor. These results suggest that LT functions through two microvillus membrane receptors in the mature rat small intestine.
Antichlamydial specificity of conjunctival lymphocytes during experimental ocular infection
Authors: Whittum-Hudson J A , Taylor H R
Published: January 2020

Antigen-specific responses to chlamydiae have been demonstrated with lymphocytes isolated from the conjunctiva after primary ocular infection and after topical challenge of chlamydia-immune cynomolgus monkeys with noninfectious, Triton X-100-extracted antigen. Proliferative to viable elementary bodies homologous to the original infecting serovar were demonstrated. In addition, in vitro production of antichlamydial antibody by conjunctival B cells was demonstrated by enzyme-linked immunosorbent assay of culture supernatants collected after 7 to 21 days of culture. These findings demonstrate that antigen-specific lymphocytes appear in the conjunctiva as a result of ocular chlamydial infection and that a noninfectious chlamydial antigen stimulates their reappearance or expansion at the site of original infection.
Virulence, persistence, and immunogenicity of Yersinia enterocolitica O:8 aroA mutants
Authors: Bowe F , O'Gaora P , Maskell D , Cafferkey M , Dougan G
Published: January 2020

The virulent Yersinia enterocolitica strain 8081 killed BALB/c mice within 5 days of oral infection with a 50% lethal dose of log10 7.1, whereas an aroA mutant of 8081, YAM.1, and the plasmidless variant 8081c failed to kill mice. Unlike 8081, YAM.1 and 8081c did not persist or grow in the Peyer's patches, mesenteric lymph nodes, livers, or spleens of mice. Mice immunized orally with single doses of live YAM.1 were poorly protected against a lethal 8081 challenge, whereas mice immunized with three doses of YAM.1 were moderately well protected.
Molecular characterization of the pathogen-specific, 34-kilodalton membrane immunogen of Treponema pallidum
Authors: Swancutt M A , Riley B S , Radolf J D , Norgard M V
Published: January 2020

The 34-kilodalton (kDa) antigen of Treponema pallidum subsp. pallidum (T. pallidum) is a pathogen-specific integral membrane protein. DNA sequence analysis of the cloned gene revealed an open reading frame encoding a primary product of 204 residues with a molecular mass of 22,087 daltons. Sequences that correspond to a consensus Escherichia coli promoter and a ribosome-binding site were found upstream from the AUG start codon that begins the open reading frame, suggesting that the cloned gene can use its own regulatory sequences for expression. Examination of the deduced amino acid sequence revealed the presence of a typical procaryotic leader peptide 19 amino acids long; processing results in a mature molecule with a molecular mass of 20,123 daltons. Pulse-chase experiments with E. coli minicells confirmed that the 34-kDa antigen is synthesized as a higher-molecular-weight precursor that is processed to a mature form with the electrophoretic mobility that is characteristic for this protein. The presence in the leader peptide of the sequence Phe-Ser-Ala-Cys suggested that the 34-kDa antigen is a proteolipid. Although hydropathy analysis of the deduced amino acid sequence of the mature 34-kDa antigen predicted that the molecule was primarily hydrophilic, both the native and recombinant 34-kDa molecules displayed hydrophobic biochemical behavior by fractionating into the detergent phase after extraction of intact organisms with Triton X-114. Cell fractionation experiments with E. coli showed that the 34-kDa molecule was localized in both the inner and outer membranes of the recombinant host. The combined data demonstrate that the 34-kDa antigen is an integral membrane protein that behaves in a biochemically consistent manner in both T. pallidum and E. coli.
Interactions between endogenous gamma interferon and tumor necrosis factor in host resistance against primary and secondary Listeria monocytogenes infections
Authors: Nakane A , Minagawa T , Kohanawa M , Chen Y , Sato H , Moriyama M , Tsuruoka N
Published: January 2020

Intravenous injection of rat anti-mouse gamma interferon (IFN-gamma) monoclonal antibody as well as rabbit anti-mouse tumor necrosis factor (TNF) antibody into mice which had received a sublethal infection with Listeria monocytogenes cells resulted in acceleration of listeriosis. Endogenous IFN-gamma seemed to be produced early in infection, because suppression of antilisterial resistance was significant when a single injection of anti-IFN-gamma monoclonal antibody was given on day 0 or day 1 of infection. Production of TNF but not of IFN-gamma in the bloodstream early in infection was inhibited by administration of anti-IFN-gamma monoclonal antibody. The suppressive effect of anti-IFN-gamma and anti-TNF antibodies on antilisterial resistance was not augmented by simultaneous administration of these antibodies. On the other hand, in the secondary infection, simultaneous administration of anti-IFN-gamma and anti-TNF antibodies, but not of either of these antibodies alone, into L. monocytogenes-immune mice resulted in high mortality and explosive multiplication of bacterial cells in the spleens and livers. These results suggest that endogenously produced IFN-gamma and TNF are both essential to the host defense against L. monocytogenes infection and that these cytokines might act by different modes between the primary infection and the secondary infection.
Evidence of T-cell recognition in mice of a purified lipophosphoglycan from Leishmania major
Authors: Moll H , Mitchell G F , McConville M J , Handman E
Published: January 2020

We have previously reported that a Leishmania major lipophosphoglycan (LPG), given with killed Corynebacterium parvum as an adjuvant, can vaccinate mice against cutaneous leishmaniasis. In order to analyze whether T cells are able to recognize this important parasite antigen, we have studied both humoral and cellular immune responses to L. major LPG that had been isolated from promastigotes by sequential solvent extraction and hydrophobic chromatography. The data show that immunization of mice with highly purified LPG induced an increase in frequency of L. major-reactive T cells and the production of immunoglobulin G antibodies to LPG. Furthermore, genetically resistant mice infected with L. major were able to develop a specific delayed-type hypersensitivity response in the ear to L. major LPG. These findings strongly suggest that T cells can recognize and respond to glycolipid antigens, in this case a host-protective Leishmania LPG, even though such antigens appear not to be potent T-cell stimulators in mice.
Interleukin-6 induced at mucosal surfaces by gram-negative bacterial infection
Authors: de Man P , van Kooten C , Aarden L , Engberg I , Linder H , Svanborg Edén C
Published: January 2020

Interleukin-6 (IL-6) was produced in response to mucosal and systemic infection of mice with gram-negative bacteria. The IL-6 response was controlled by the lipopolysaccharide gene, Lps; in C3H/HeN mice (Lpsn/Lpsn), the urinary IL-6 levels increased within 30 min after challenge with Escherichia coli, but no response occurred in C3H/HeJ mice (Lpsd/Lpsd). In lipopolysaccharide-responder mice, the levels of local and systemic IL-6 were related to the degree of infection. The urinary response dominated after intravesical challenge, and the serum response dominated after intraperitoneal challenge. The results demonstrate that IL-6 is activated as part of lipopolysaccharide-induced mucosal and systemic responses to gram-negative bacterial infections.
Release of interleukin-1 beta associated with potent cytocidal action of staphylococcal alpha-toxin on human monocytes
Authors: Bhakdi S , Muhly M , Korom S , Hugo F
Published: January 2020

The pathogenetic relevance of Staphylococcus aureus alpha-toxin in humans has been debated because human cells have been thought to display a natural resistance toward the cytotoxic action of this cytolysin. Following our previous demonstration that human platelets represent sensitive targets for toxin attack, we have now identified monocytes as a second, highly vulnerable human cell species that succumb to attack by low doses (20 ng/ml) of alpha-toxin. The cytotoxic action of alpha-toxin is reflected in a rapid depletion of cellular ATP that is essentially complete within 30 min. The presence of human plasma proteins affords some protection of monocytes against the action of the toxin. In 10% autologous serum, ATP depletion commences at 80 to 300 ng of toxin per ml. Subcytolytic doses stimulate the release of tumor necrosis factor alpha, a process that is slightly accentuated in the presence of 50% serum. Cytocidal toxin doses unfailingly cause the release of large amounts of interleukin-1 beta from cultured cells, with levels of this monokine generally exceeding 10 ng/ml in the cell supernatants 60 min after application of toxin. Initial evidence suggests that this is due to processing of intracellular interleukin-1 rather than to de novo synthesis of the cytokine. All noted effects are abrogated in the presence of a neutralizing monoclonal antibody against alpha-toxin. Through its capacity to provoke cytokine release from monocytes and its attack on platelets, alpha-toxin may initiate cellular events that are relevant to the pathogenesis of staphylococcal infection.