bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4+ T cells play a significant role in adoptive immunity to Chlamydia trachomatis infection of the mouse genital tract
Authors: Su H , Caldwell H D
Published: January 2020

The ability of CD4+ and CD8+ T cells to adoptively immunize mice against Chlamydia trachomatis infection of the mouse genital tract was studied. Adoptive transfer experiments were performed with splenic CD4+ or CD8+ T cells obtained from mice following resolution of a primary genital tract infection and after a secondary chlamydial challenge. The results show that donor CD4+ T cells, but not CD8+ T cells, obtained from mice following resolution of a primary infection or after secondary challenge were effective in transferring significant antichlamydial immunity to the genital tracts of naive animals. The lymphokine profiles in the culture supernatants of proliferating Chlamydia-specific CD4+ T cells obtained from mice following resolution of a primary infection and after secondary challenge were assayed by an enzyme-linked immunoadsorbent assay. Protective CD4+ T cells restimulated in vitro secreted interleukin 2, gamma interferon, and interleukin 6, lymphokine profiles characteristic of both Th1- and Th2-like responses. Resting CD4+ T cells obtained from mice 4 months following resolution of a primary infection were also capable of conferring significant levels of adoptive protective immunity to naive mice. These findings support an important role for CD4+ T cells in acquired immunity to chlamydial infection of the genital tract and indicate that protective CD4+ immune responses in this model are relatively long lived.
Molecular cloning and immunological characterization of a novel linear-plasmid-encoded gene, pG, of Borrelia burgdorferi expressed only in vivo
Authors: Wallich R , Brenner C , Kramer M D , Simon M M
Published: January 2020

Previously we have found that sera from immunocompetent mice infected either naturally by ticks or experimentally with low numbers of Borrelia burgdorferi ZS7 bacteria lack OspA- and OspB-specific antibodies but confer optimal protection on severe combined immunodeficiency mice against challenge with spirochetes (U.E. Schaible, L. Gern, R. Wallich, M. D. Kramer, M. Prester, and M. M. Simon, Immunol. Lett. 36:219-226, 1993). We have now used the latter immune sera to identify new spirochetal structures with relevance for protection from an expression library of the virulent European strain B. burgdorferi ZS7. Here we report the cloning and characterization of a novel lipoprotein, designated pG, the gene for which is located on a 48-kb linear plasmid. Sequence analysis of the pG gene revealed an open reading frame encoding a putative lipoprotein of 196 amino acids with a calculated molecular mass of 22 kDa and a consensus cleavage sequence (Leu-X-Y-Z-Cys) recognized by signal peptidase II. Restriction fragment length polymorphism analyses of pG derived from independent B. burgdorferi isolates from different geographic areas revealed that the gene is species specific, with, however, extensive genotypic heterogeneity. Comparison of the protein sequence of pG with those of other known B. burgdorferi outer surface lipoproteins (OspA to OspF and P27) demonstrated that pG is most related to OspF. Furthermore, the upstream region of pG exhibited extensive sequence homology (> 94%) with the ospEF promoter region. Mouse immune sera to recombinant pG did not recognize a corresponding molecule in lysates of in vitro-propagated ZS7 spirochetes. However, experimental or natural infection of mice with ZS7 resulted in the induction of antibodies with reactivity for pG and the potential to delay the development of clinical arthritis. Together with the finding that sera from Lyme disease patients also contain antibodies to pG, our data suggest that the pG gene is preferentially expressed in the mammal environment.
Initial serum antibody titer to Porphyromonas gingivalis influences development of antibody avidity and success of therapy for chronic periodontitis
Authors: Mooney J , Adonogianaki E , Riggio M P , Takahashi K , Haerian A , Kinane D F
Published: January 2020

This study assessed the effect of periodontal therapy on specific serum antibody concentration, expressed as titer, and antibody binding strength, expressed as relative avidity. The immune responses to Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were investigated. Antibody titer was assayed by enzyme-linked immunosorbent assay (ELISA) and relative avidity was measured by thiocyanate elution in 17 adult periodontitis patients before and after therapy. Immunoglobulin G (IgG) avidities (expressed as thiocyanate molarity) to P. gingivalis increased from 1.01 to 1.38 M (P = 0.05) and IgA titers (expressed as ELISA units [EU]) increased from 89 to 237 EU (P = 0.012). There were no significant changes in avidity to A. actinomycetemcomitans, but the titer of all three immunoglobulin classes increased significantly (P < 0.03). More specifically, when patients were divided into subgroups which had originally been either IgG seropositive (i.e., having an IgG titer to this organism > 2 times the control median) or seronegative for P. gingivalis, only patients who were initially seropositive showed a significant increase in antibody avidity (P = 0.026; mean difference, 0.69 M). Patients who were originally seropositive in terms of IgG and IgA titer to P. gingivalis had demonstrably better treatment outcomes in terms of a reduced number of deep pockets and sites which bled on probing (P < 0.05). These findings suggest that periodontal therapy affects the magnitude and quality of the humoral immune response to suspected periodontopathogens, that this effect is dependent on initial serostatus, and that initial serostatus may have a bearing on treatment outcome.
The tetrasaccharide L-alpha-D-heptose1-->2-L-alpha-D-heptose1--> 3-L-alpha-D-heptose1-->(3-deoxy-D-manno-octulosonic acid) and phosphate in lipid A define the conserved epitope in Haemophilus lipopolysaccharides recognized by a monoclonal antibody
Authors: Borrelli S , Hegedus O , Shaw D H , Jansson P E , Lindberg A A
Published: January 2020

A murine monoclonal antibody, MAHI 3 (immunoglobulin G2b), that is broadly reactive with Haemophilus influenzae lipopolysaccharides (LPSs) but nonreactive with all enterobacterial LPSs tested was generated by fusing mouse myeloma cells with spleen cells of BALB/c mice immunized with azide-killed H. influenzae RM.7004. MAHI 3 bound to all H. influenzae, all other human Haemophilus spp., all Bordetella pertussis and Bordetella parapertussis, and all Aeromonas spp. tested but not to any Neisseria or Moraxella catarrhalis strains, as determined by enzyme immunoassay, colony dot immunoblotting, and immunoblotting. In an inhibition enzyme immunoassay, MAHI 3 reacted with all 45 H. influenzae LPSs tested but not with the LPS from the rough mutant I69 Rd-/b+, which has only 3-deoxy-D-manno-octulosonic acid (P) [Kdo(P)] and lipid A. The antibody was not inhibited by H. influenzae lipid A or lipid-free polysaccharide isolated after mild acid hydrolysis. Only native LPSs show positive inhibitory activity, indicating that part of lipid A is involved in the binding of MAHI 3. From the results, it is indicated that the structural element recognized by MAHI 3 is Hep alpha 1-->2Hep alpha 1-->3Hep alpha 1-->Kdo together with part of lipid A, including the phosphate.
The Salmonella dublin virulence plasmid does not modulate early T-cell responses in mice
Authors: Guilloteau L A , Lax A J , MacIntyre S , Wallis T S
Published: January 2020

The virulence plasmid in Salmonella dublin mediates systemic infection in mice and cattle. The role of gamma delta T cells or hepatic extrathymic T cells has recently been reported to be important in the control of the early stage of Salmonella choleraesuis infections of mice. Here, we report on T-cell responses in conventional mice after challenge with a virulent strain of S. dublin carrying a virulence plasmid or with a strain cured of the plasmid. Over a period of 4 days postinfection, when both strains could be compared, similar changes in alpha beta and gamma delta T-cell subsets in peritoneal cavities, livers, and spleens were recorded, demonstrating no clear role of the virulence plasmid in modulation of early T-cell responses. To investigate further the role of the virulence plasmid in pathogenesis, the growth of the plasmid-cured strain was assessed in SCID, SCID bg, and irradiated mice. During the first 6 days after infection, there was no statistically difference in the net growth of Salmonella cells in the livers and spleens of SCID and SCID bg mice compared with conventional BALB/mice. This observation excludes a key role for a T- or B-cell-mediated immune response in controlling the initial growth of the plasmid-cured S. dublin strain. Thereafter, the immunocompromised mice were no longer able to control infection, although SCID mice were more efficient at controlling net bacterial multiplication than SCID bg mice, potentially implicating NK cells in the control of infection in SCID mice. The early control of net bacterial multiplication in the spleens and livers of BALB/c mice was ablated by whole-body X-irradiation. Both wild-type and plasmid-cured strains multiplied significantly more rapidly in irradiated than in conventional BALB/c mice. However, the numbers of wild-type bacterial still increased more rapidly than in the numbers of the cured strains. These results are consistent with a role of the S. dublin virulence plasmid in promoting in vivo growth of Salmonella cells.
Characterization of 20K fimbria, a new adhesin of septicemic and diarrhea-associated Escherichia coli strains, that belongs to a family of adhesins with N-acetyl-D-glucosamine recognition
Authors: Bertin Y , Girardeau J P , Darfeuille-Michaud A , Contrepois M
Published: January 2020

Bovine septicemic Escherichia coli 31A agglutinates bovine, rabbit, and human erythrocytes and adheres in vitro to the brush border of bovine or ovine intestinal epithelial cells and to the human colon carcinoma Caco-2 cell line. The adhesion and hemagglutination of E. coli 31A are mediated by a chromosome-encoded fimbrial adhesin serologically distinct from known fimbrial adhesins found in enterotoxigenic and septicemic bovine E. coli strains. By electron microscopy studies the fimbriae designated 20K were observed as fine flexible filaments (diameter, 3 nm) and the purified major fimbrial subunit appeared with an apparent molecular mass of 20,000 Da. Western blot (immunoblot) analysis, N-terminal sequence alignment, and amino acid composition revealed a high homology with the N-acetyl-D-glcosamine-specific G fimbria of human uropathogenic E. coli and with fimbriae belonging to the F17 family produced by bovine enterotoxigenic and invasive E. coli strains. Immunological study revealed that 20K fimbria was closely related to G fimbria and represents a serological variant of F17 fimbria. Hemagglutination and adhesion inhibition assays demonstrated that 20K, G, and F17 fimbriae bind to an N-acetyl-D-glucosamine-containing receptor, but each probably binds to different oligosaccharide sequences or different receptors on host tissues. 20K fimbriae were produced by a limited group of clonally related strains with the unusual m-inositol-positive phenotype and appeared highly associated with the plasmid-mediated virulence factor. An examination of natural occurrence of 20K fimbriae among a large collection of human and animal pathogenic E. coli showed that 20K fimbria is the prominent adhesin among bovine septicemic E. coli isolated from European countries.
P55, an immunogenic but nonprotective 55-kilodalton Borrelia burgdorferi protein in murine Lyme disease
Authors: Feng S , Barthold S W , Telford S R , Fikrig E
Published: January 2020

Immunization of C3H mice with P55 (previously called S1), a 55-kDa Borrelia burgdorferi antigen that is immunogenic after infection, elicited a strong antibody response but did not protect mice against B. burgdorferi challenge. Mice immunized with a P55 fusion protein in complete Freund's adjuvant developed anti-P55 antibodies, detectable at a titer of 1:10,000 by immunoblotting. To determine, if a protective response had been elicited, P55-vaccinated mice were fed upon by ticks infected with B. burgdorferi. The frequency of B. burgdorferi infection was similar in P55-immunized and control mice, and spirochetes were not destroyed within ticks that fed on P55-vaccinated mice. P55 is an immunogenic antigen that does not induce a protective response in the vertebrate or invertebrate host.
Immunosuppressive factor from Actinobacillus actinomycetemcomitans down regulates cytokine production
Authors: Kurita-Ochiai T , Ochiai K
Published: January 2020

A cytoplasmic soluble fraction of Actinobacillus actinomycetemcomitans Y4 was isolated and characterized as suppressing mitogen-stimulated proliferation of and cytokine production by C3H/HeN mouse splenic T cells. This factor, designated suppressive factor 1 (SF1), was isolated from the supernatant of sonicated whole bacteria and purified by Q-Sepharose Fast Flow column chromatography, DEAE-Sepharose Fast Flow column chromatography, hydroxyapatite high-pressure liquid chromatography (HPLC), and Protein Pack 300 & 125 gel filtration HPLC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the purified SF1 migrated as a single band corresponding to a molecular mass of 14 kDa. This molecule was protease labile, heat resistant, and noncytotoxic. N'-terminal sequence analysis revealed no homology with any known peptides of periodontopathic bacteria or with any host-derived growth factors. Purified SF1 suppressed the proliferation of mouse splenic T cells which had been stimulated with concanavalin A, as well as suppressing the production of interleukin-2 (IL-2), gamma interferon, IL-4, and IL-5 from CD4+ T cells as 0.1 microgram/ml or more. These data suggest that SF1 produced by the periodontal pathogen A. actinomycetemcomitans functions as a virulence factor by down regulating T-cell proliferation and cytokine production at local defense sites.
Resistance to Cryptococcus neoformans is associated with an inflammatory response to Toxoplasma gondii in the central nervous system of mice
Authors: Aguirre K M , Sayles P C , Gibson G W , Johnson L L
Published: January 2020

We have studied the resistance of Toxoplasma gondii-infected mice to subsequent infection with Cryptococcus neoformans. Mice infected with the moderately virulent ME49 strain of T. gondii are resistant to proliferation of yeast cells in their brains after intravenous inoculation of the serotype A C. neoformans strain 184. The resistance serves to limit proliferation of yeast cells that colonize the brain. Maximal levels of resistance correlate not with maximal systemic specific anti-Toxoplasma resistance but rather with high levels of inflammatory response, presumably to parasites released from cysts in the brain. Resistance is localized, as mice infected with ME49 show only limited resistance in their lungs after intratracheal instillation of yeast cells, but there is substantial protection against development of cerebral cryptococcosis.
Identification, cloning, and sequencing of the immunoglobulin A1 protease gene of Streptococcus pneumoniae
Authors: Wani J H , Gilbert J V , Plaut A G , Weiser J N
Published: January 2020

The pneumococcus expresses a protease that hydrolyzes human immunoglobulin A1 (IgA1). A gene for IgA1 protease was identified from a plasmid library of pneumococcal DNA because of the effect of its overexpression on the colony morphology of Streptococcus pneumoniae. The deduced 1,964-amino-acid sequence is highly homologous to that of the IgA1 protease from Streptococcus sanguis. The similarity to the S. sanguis enzyme and the presence of a putative zinc-binding site suggest that the pneumococcal enzyme is a metalloprotease. The two streptococcal sequences differ in a hydrophilic region with 10 tandem repeats of a 20-mer in S. sanguis, which is replaced by a similar but less repetitive sequence in S. pneumoniae. Antiserum reactive with the pneumococcal IgA1 protease was used to demonstrate that the majority of the protein is cell associated. The expression and function of this gene were confirmed by insertional mutagenesis. Interruption of the chromosomal gene resulted in loss of expression of an approximately 200-kDa protein and complete elimination of detectable IgA1 protease activity.
Cellular immune responses to recombinant heat shock protein 70 from Histoplasma capsulatum
Authors: Allendoerfer R , Maresca B , Deepe G S
Published: January 2020

Heat shock protein (hsp) 70 from several microbes is antigenic in mammals. In this study we sequenced and expressed the gene encoding this protein from Histoplasma capsulatum to study its immunological activity. The deduced amino acid sequence of the gene demonstrated 71 and 76% identity to hsp7O from humans and Saccharomyces cerevisiae, respectively. A cDNA was synthesized by reverse transcription-PCR and was expressed in Escherichia coli. Recombinant protein reacted with a mouse monoclonal antibody raised against human hsp7O. Splenocytes from C57BL/6 mice immunized with recombinant hsp7O emulsified in adjuvant, but not yeast cells, reacted in vitro to the antigen. Recombinant hsp7O elicited a cutaneous delayed-type hypersensitivity response in mice immunized with protein or with viable yeast cells. Mice were injected with recombinant hsp7O and challenged intranasally with a sublethal inoculum of yeast cells. Vaccination did not confer protection in this model. Thus, recombinant hsp7O can induce a cell-mediated immune response but does not induce a protective response.
Mechanisms of interferon-induced inhibition of Toxoplasma gondii replication in human retinal pigment epithelial cells
Authors: Nagineni C N , Pardhasaradhi K , Martins M C , Detrick B , Hooks J J
Published: January 2020

Inflammation associated with retinochoroiditis is a major complication of ocular toxoplasmosis in infants and immunocompetent individuals. Moreover, Toxoplasma gondii-induced retinal disease causes serious complications in patients with AIDS and transplant patients. The retinal pigment epithelial (RPE) cell is an important regulatory cell within the retina and is one of the cells infected with T. gondii in in vivo. We have developed a human RPE (HRPE) cell in vitro model system to evaluate T. gondii replication and the regulation of this replication by cytokines. T. gondii replication was quantitated by counting the foci of infection (plaque formation) and the numbers of tachyzoites released into the supernatant fluids. Pretreatment of cultures with recombinant human tumor necrosis factor alpha, alpha interferon (IFN-alpha), IFN-beta, or IFN-gamma for 24 h prior to inoculation inhibited T. gondii replication in a dose-dependent manner. Of these cytokines, IFN-gamma was the most potent, and T. gondii replication was completely inhibited at a concentration of 100 U/ml. The anti-toxoplasmotic activity of IFN-gamma was significantly blocked by monoclonal antibody to IFN-gamma. Treatment of the cultures with IFN-gamma from day 1 or 2 postinoculation with T. gondii also offered protection against the parasite. The anti-toxoplasmotic activity of tumor necrosis factor alpha or IFN-alpha, -beta, or -gamma in these cultures was found to be independent of the nitric oxide (NO) pathway, since NO production was not found in HRPE cells treated with these cytokines. However, addition of tryptophan to IFN-gamma-treated cells significantly reversed the inhibitory effects of IFN-gamma, suggesting that IFN-gamma acts by depleting cellular tryptophan. This effect was further confirmed by reverse transcription-PCR and Northern (RNA) blot analysis, which indicated induction of indoleamine 2,3-dioxygenase (IDO), an enzyme that converts tryptophan to kynurenine. These results indicated that interferons inhibited T. gondii replication in HRPE by NO-independent but IDO-dependent mechanisms. This in vitro model of T. gondii replication in HRPE may be useful in evaluating the effects of cytokines and drugs on T. gondii replication within the retina.
Immunoglobulin allotypes and immunoglobulin G subclass responses to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in early-onset periodontitis
Authors: Choi J I , Ha M H , Kim J H , Kim S J
Published: January 2020

The present study was performed to estimate the observed frequencies of the immunoglobulin heavy-chain (Gm) and light-chain (Km) allotypes among patients with early-onset periodontitis (EOP) and their effect on the IgG2 subclass responses against Actinobacillus actinomycetemcomitans Y4 and Porphyromonas gingivalis 381, respectively. Sixty-nine EOP patients, including 11 with localized juvenile periodontitis (LJP), 19 who had LJP, 15 with LJP-rapidly progressing periodontitis (RPP), and 24 with RPP, were examined for the Gm and Km allotypes by a hemagglutination inhibition test. Levels of immunoglobulin G2 (IgG2) antibodies against the two organisms were determined by enzyme-linked immunosorbent assay. Fifty race- and age-matched, periodontally healthy subjects were also included as a control group. The observed frequencies of the Gm haplotype afnb and Km(1) were significantly higher in the RPP and LJP groups, respectively. The G2m(n)+ group of those with RPP and the Km(1)+ group of those with LJP had significantly higher levels of IgG2 antibodies to A. actinomycetemcomitans and P. gingivalis, respectively. The results indicate that linkage disequilibrium of the G2m(n) locus in RPP patients or the Km(1) locus in LJP patients may be associated with high IgG2 antibody responses to the respective bacteria. It was reasoned that the IgG2 antibody responses are associated with the immunoglobulin allotypes. The function of IgG2 antibodies in their reaction to different bacterial antigens may be interpreted as either protective or nonprotective in the two different types of EOP (i.e., LJP and RPP).
Development of a germfree mouse model of Vibrio cholerae infection
Authors: Butterton J R , Ryan E T , Shahin R A , Calderwood S B
Published: January 2020

A mouse model of Vibrio cholerae infection was successfully developed with germfree mice. Three- to four-week-old germfree mice were orally inoculated with strains of V. cholerae to be tested and then moved to normal housing after inoculation. Stool culture, measurement of serum vibriocidal antibody titers, and determination of immune responses to the cholera toxin B subunit demonstrated that germfree mice are readily colonized by V cholerae and develop systemic and mucosal immune responses to antigens expressed by these organisms. Immune responses to the B subunit of Shiga toxin 1, which was expressed from a V. cholerae vaccine vector, were less pronounced. This model should be valuable for studying immune responses to V. cholerae infection and immunization, including responses to heterologous antigens expressed by cholera vector strains.
Attenuated virulence of uridine-uracil auxotrophs of Aspergillus fumigatus
Authors: D'Enfert C , Diaquin M , Delit A , Wuscher N , Debeaupuis J P , Huerre M , Latge J P
Published: January 2020

Aspergillus fumigatus mutants that are deficient in the de novo UMP biosynthesis pathway because of a mutation in the pyrG gene encoding orotidine-5'-phosphate decarboxylase (and therefore auxotrophic for uridine or uracil) were evaluated in a murine model of invasive aspergillosis. These mutants were entirely nonpathogenic, and mutant conidia remained ungerminated in alveolar macrophages. Both the germination and virulence defects could be restored by supplementing the drinking water of the animals with uridine. DNA-mediated transformation of one of the pyrG mutants with the Aspergillus niger pyrG gene also restored virulence. These results suggest that uridine and uracil are limiting in the lung environment, thus preventing conidium germination and hence virulence of the pyrG mutants.
Cellular basis of decreased immune responses to pneumococcal vaccines in aged mice
Authors: Garg M , Luo W , Kaplan A M , Bondada S
Published: January 2020

Previously, model systems were developed in our laboratory to study murine immune responses to the 23-valent pneumococcal polysaccharide vaccine Pnu-Imune, both in vivo and in vitro (M. Garg and B. Subbarao, Infect. Immun. 60:2329-2336, 1992; M. Garg, A. M. Kaplan, and S. Bondada, J. Immunol. 152: 1589-1596, 1994). Using these systems, we found that aged mice did not respond to the vaccine in vivo or in vitro. Cell separation studies showed that the unresponsiveness of the aged spleen cells to the vaccine was not due to an intrinsic B-cell defect or to T-cell-mediated immunosuppression but resulted from an accessory cell deficiency. Irradiated spleen cells from young mice enabled the old mouse spleen cells to respond to the vaccine. Interestingly, irradiated spleen cells from old mice also restored the vaccine responsiveness in old mice but were required in greater numbers than the young mouse spleen cells to induce similar levels of response. The accessory cell was an adherent cell that could be removed by passage through Sephadex G-10 and thus may be a macrophage. Accessory function could also be provided by the cytokine interleukin-1 (IL-1), IL-4, or IL-5 but not IL-2 or IL-6. Thus, one reason for the deficient immune response to pneumococcal vaccine in aged mice is a quantitative defect in adherent accessory cells.
Identification, characterization, and expression of the BiP endoplasmic reticulum resident chaperonins in Pneumocystis carinii
Authors: Stedman T T , Buck G A
Published: January 2020

We have isolated, characterized, and examined the expression of the genes encoding BiP endoplasmic reticulum (ER) resident chaperonins responsible for transport, maturation, and proper folding of membrane and secreted proteins from two divergent strains of Pneumocystis carinii. The BiP genes, Pcbip and Prbip, from the P. c. carinii (prototype) strain and the P. c. rattus (variant) strain, respectively, are single-copy genes that reside on chromosomes of approximately 330 and approximately 350 kbp. Both genes encode approximately 72.5-kDa proteins that are most homologous to BiP genes from other organisms and exhibit the amino-terminal signal peptides and carboxyl-terminal ER retention sequences that are hallmarks of BiP proteins. We established short-term P. carinii cultures to examine expression and induction of Pcbip in response to heat shock, glucose starvation, inhibition of protein transport or N-linked glycosylation, and other conditions known to affect proper transport, glycosylation, and maturation of membrane and secreted proteins. These studies indicated that Pcbip mRNA is constitutively expressed but induced under conditions known to induce BiP expression in other organisms. In contrast to mammalian BiP genes but like other fungal BiP genes, P. carinii BiP mRNA levels are induced by heat shock. Finally, the Prbip and Pcbip coding sequences surprisingly exhibit only approximately 83% DNA and approximately 90% amino acid sequence identity and show only limited conservation in noncoding flanking and intron sequences. Analyses of the P. carinii BiP gene sequences support inclusion of P. carinii among the fungi but suggest a large divergence and possible speciation among P. carinii strains infecting a given host.
Temperature- and medium-dependent secretion of proteins by Shiga toxin-producing Escherichia coli
Authors: Ebel F , Deibel C , Kresse A U , Guzmán C A , Chakraborty T
Published: January 2020

Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal disease in humans and livestock, and these bacteria have recently emerged as a leading cause of renal failure in children. In this study, we have examined medium- and temperature-dependent production of secreted proteins from a STEC O26 serotype strain. Growth of bacteria in Luria broth led to the detection of secreted polypeptides of 104, 55, 54, and 37 kDa (p104, p55, p54, and p37, respectively). When grown in serum-free tissue culture medium, only p104, p37 and two additional polypeptides of 25 and 22 kDa (p25 and p22) were present in supernatant fluids. Production of these polypeptides was growth temperature dependent and induced in cultures grown at 37 degrees C. N-terminal amino acid sequencing revealed that p104 was homologous to the secreted p110 of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a family of secreted proteins in pathogenic bacteria of which the immunoglobulin A protease of Neisseria gonorrhoeae is the prototype. The N-terminal amino acid sequences of p55 and p54 were unique to the STEC strain, while p37 and p25 were found to be highly homologous to the similarly sized EspA and EspB proteins, previously detected in culture supernatants of EPEC. Molecular cloning and sequencing of STEC espB alleles from two different serotypes showed that the encoded polypeptides were about 80% homologous. A monoclonal antibody raised against STEC EspB also cross-reacted with its EPEC analog and allowed us to demonstrate medium- and temperature-dependent production of this important virulence factor in STEC and EPEC strains of differing serotypes.
Effect of growth phase and acid shock on Helicobacter pylori cagA expression
Authors: Karita M , Tummuru M K , Wirth H P , Blaser M J
Published: January 2020

Helicobacter pylori strains possessing cagA are associated with peptic ulceration. To understand the regulation of expression of cagA, picB, associated with interleukin-8 induction, and ureA, encoding the small urease subunit, we created gene fusions of cagA, ureA, and picB of strain 3401, using a promoterless reporter (xylE). Expression of XylE after growth in broth culture revealed that basal levels of expression of cagA and urea in H. pylori were substantially greater than for picB. For cagA expression in stationary-phase cells, brief exposure to acid pH caused a significant increase in xylE expression compared with neutral pH. In contrast, expression of xylE in urea or picB decreased after parallel exposure to acid pH (pH 7 > 6 > 5 > 4), regardless of the growth phase. Expression of the CagA protein varied with growth phase and pH exposure in parallel with the observed transcriptional variation. The concentration of CagA in a cell membrane-enriched fraction after growth at pH 6 was significantly higher than after growth at pH 5 or 7. We conclude that the promoterless reporter xylE is useful for studying the regulation of gene expression in H. pylori and that regulation of CagA production occurs mainly at the transcriptional level.
Gamma interferon protects endothelial cells from damage by Candida albicans by inhibiting endothelial cell phagocytosis
Authors: Fratti R A , Ghannoum M A , Edwards J E , Filler S G
Published: January 2020

Once Candida albicans comes in contact with endothelial cells, it induces cellular injury. This endothelial cell injury may be a mechanism by which blood-borne organisms escape from the intravascular compartment and invade the tissue parenchyma during hematogenous infection. We have been investigating the ability of cytokines to modulate endothelial cell injury caused by C. albicans. Previously we reported that pretreatment of endothelial cells with gamma interferon (IFN-gamma) protects these cells from candidal injury in vitro. In the current study, we examined potential mechanisms of the cytoprotective effects of IFN-gamma. Time course experiments demonstrated that maximal reduction in candidal injury of endothelial cells occurred after the endothelial cells had been exposed to IFN-gamma for at least 72 h. In other studies, we determined that IFN-gamma reduced endothelial cell phagocytosis of C. albicans by 41.3% compared with that of untreated endothelial cells (P < 0.01). Since endothelial cell phagocytosis of C. albicans is required for damage to occur, inhibition of phagocytosis is likely a mechanism by which IFN-gamma protects endothelial cells from candidal injury. We also found that the cytoprotective effect of IFN-gamma is not mediated by reducing access of the organisms to intracellular endothelial cell iron or by upregulating the synthesis of reactive oxygen intermediates (which could potentially reduce the ability of C. albicans to injure endothelial cells). Thus, inhibiting endothelial cell phagocytosis of C. albicans may be a mechanism by which IFN-gamma augments the host defense against hematogenously disseminated candidal infections.
Expression of Salmonella typhimurium rpoS and rpoS-dependent genes in the intracellular environment of eukaryotic cells
Authors: Chen C Y , Eckmann L , Libby S J , Fang F C , Okamoto S , Kagnoff M F , Fierer J , Guiney D G
Published: January 2020

Adaptation to the intracellular environment of host cells is crucial for the pathogenesis of Salmonella infections. The alternative sigma factor RpoS is a global regulator of gene expression during starvation and stress conditions and is required for virulence in Salmonella spp. We have used lacZ reporter fusions to rpoS and rpoS-dependent genes to study rpoS regulation after entry of Salmonella typhimurium into macrophages and epithelial cells. The results demonstrate that expression of an rpoS::lacZ translational fusion increases rapidly in S. typhimurium after phagocytosis. Activity of RpoS also increases after bacterial entry into both macrophages and epithelial cells, as demonstrated by the induction of the rpoS-regulated genes katE and spvB. A control rpoS-independent promoter for neomycin resistance does not show significant induction after cell entry. These results demonstrate that the regulatory system mediated by RpoS in S. typhimurium is activated by the intracellular environment of eukaryotic cells.
Effect of amplification of the Cap b locus on complement-mediated bacteriolysis and opsonization of type b Haemophilus influenzae
Authors: Noel G J , Brittingham A , Granato A A , Mosser D M
Published: January 2020

Amplification of the Cap b locus of Haemophilus influenzae occurs frequently in clinical isolates and has been proposed to be a mechanism by which this organism evades host defense. To determine if amplification of this locus affected complement fixation, in vitro studies to determine complement-mediated bacteriolysis and complement-mediated opsonization of an isogenic set of organisms containing 2, 3, and 4 copies of the Cap b locus were performed. Organisms containing 4 copies of the Cap b locus were significantly more resistant to antibody-dependent, classical complement pathway-directed bacteriolysis than were organisms containing 2 copies. Organisms containing 3 copies of this locus exhibited intermediate susceptibility to lysis. Complement-mediated opsonization of these organisms was assessed by determining the degree of binding of bacteria to murine or human macrophages or to nonphagocytic cells transfected with the genes for human Mac-1, the complement receptor type 3. In all three assay systems, organisms containing 4 copies of the Cap b locus bound less well than did organisms containing 2 copies of this locus. Consistent with their decreased susceptibility to lysis and opsonization, organisms with 4 copies of the Cap b locus fixed less C3 than did organisms containing 2 copies. These data demonstrate that amplification of the Cap b locus is associated with decreased susceptibility to complement-mediated lysis and decreased complement-mediated opsonization and suggest that amplification is used by these pathogens to increase their resistance to complement-dependent host defense mechanisms [correction of mecanisms].
Initial route of antigen administration alters the T-cell cytokine profile produced in response to the mouse pneumonitis biovar of Chlamydia trachomatis following genital infection
Authors: Kelly K A , Robinson E A , Rank R G
Published: January 2020

A Th1-type response develops following vaginal infection with the mouse pneumonitis biovar of Chlamydia trachomatis (MoPn). Since the type of response, i.e., Th1 versus Th2, can be influenced by factors present during T-cell activation, we examined the effects of different routes of MoPn administration on the cytokine profile and resistance against infection following a MoPn vaginal challenge. A dominant Th1-type cytokine profile developed in mice given live MoPn via the intranasal, oral, and vaginal routes with ratios of gamma interferon-secreting cells to interleukin 4-secreting cells greater than 10. In contrast, mice injected subcutaneously produced a Th2-type profile with a gamma interferon/interleukin 4 ratio of only 0.7. These mice also had significantly higher anti-MoPn immunoglobulin G1 serum titers, confirming a Th2-type cytokine profile. Exposure of mice to live MoPn, by any route prior to vaginal challenge, resulted in a shortened course of infection. However, the subcutaneous group resolved the vaginal infection more slowly, with 60% (6 of 10 mice) of the mice still isolation positive 12 days after challenge compared with only 20% of mice given live MoPn by other routes. Administration of UV-inactivated MoPn did not provide protection against a vaginal challenge. The decreased ability to clear infection was not associated with a shift in the cytokine profile, since intranasal and oral administration of UV-inactivated MoPn resulted in a predominant Th1-type response. Taken together, these data indicate that the initial route of MoPn administration can direct the type of response produced after a local MoPn infection and thus influence the ability of the immune response to protect against subsequent infection.
Biphasic, organ-specific, and strain-specific accumulation of platelets induced in mice by a lipopolysaccharide from Escherichia coli and its possible involvement in shock
Authors: Shibazaki M , Nakamura M , Endo Y
Published: January 2020

Platelets contain a large amount of 5-hydroxytryptamine (5HT, serotonin). Intravenous injection into BALB/c mice of a Boivin's preparation of lipopolysaccharide (LPS) from Escherichia coli induced rapid 5HT accumulation in the lung (within 5 min) and slow 5HT accumulation in the liver (2 to 5 h later). The rapid response required high doses of LPS (more than 0.1 mg/kg). On the basis of 5HT measurements, 70% or more of the platelets which disappeared from the blood appeared to have accumulated rapidly in the lung, and a large number of platelets were found there by electron microscopy. A shock, which was manifested by crawling, convulsion, or prostration, followed shortly after the rapid accumulation of 5HT in the lung. On the other hand, the slow accumulation of 5HT in the liver could be induced by much lower doses of LPS (1 microg/kg or less), even when given by intraperitoneal injection. This 5HT accumulation appears to be a reflection of platelet accumulation in the liver (Y. Endo and M. Nakamura, Br. J. Pharmacol. 105:613-619, 1992). The combination of a low dose of LPS with D-galactosamine amplified the hepatic accumulation of 5HT, and the mice developed a severe hepatic congestion resulting in death. The rapid response was not induced at all in C3H/HeN mice. These results and comparison with other LPS preparations indicate that some component(s) of LPS from E. coli induces a biphasic, organ-specific and strain-specific accumulation of platelets, and it is proposed that this effect is involved in the development of shock.
Cloning and sequence analysis of the pfl gene encoding pyruvate formate-lyase from Streptococcus mutans
Authors: Yamamoto Y , Sato Y , Takahashi-Abbe S , Abbe K , Yamada T , Kizaki H
Published: January 2020

We have isolated a sorbitol-negative mutant of Streptococcus mutans GS-5 following random mutagenesis with plasmid pVA891 clone banks. This mutant did not metabolize sorbitol anaerobically but did so aerobically. A 10-kb chromosomal DNA fragment flanking the pVA891 insertion was deleted in this mutant. The corresponding region from the parental strain GS-5 was then recovered by a marker rescue method with Escherichia coli. The pyruvate formate-lyase gene, pfl, was identified within a 3-kb PstI-XbaI fragment located in the middle of the deleted region of the chromosome, and its inactivation in S. mutans produced the same sorbitol-negative phenotype. Nucleotide sequence analysis of the pfl gene revealed a 2.3-kb open reading frame (ORF) preceded by potential ribosome-binding and promoter-like sequences. The ORF specified a putative protein of 775 amino acid residues with a calculated molecular weight of 87,533. The amino acid sequence deduced from the ORF exhibited significant similarity to that of the E. coli pfl gene.
Membrane protein variations associated with in vitro passage of Borrelia burgdorferi
Authors: Carroll J A , Gherardini F C
Published: January 2020

Borrelia burgdorferi, the causative agent of Lyme disease, undergoes a loss in virulence with repeated passage in vitro. Defining the changes which occur after conversion to avirulence may assist in identifying virulence factors and mechanisms of pathogenesis. We have used a cross-adsorption technique and two-dimensional nonequilibrium pH gradient electrophoresis to compare virulent (low-passage) and avirulent (high-passage) variants of B. Burgdorferi B31. Using cross-adsorbed rabbit sera to probe immunoblots, we identified 10 low-passage-associated proteins (relative molecular masses of 78, 58, 49, 34, 33, 28, 24, 20, and 16 kDa) unique to the virulent strain B31. Cross-adsorbed human serum detected five proteins of similar sizes (78, 58, 34, 28, and 20 kDa), suggesting that several of of these proteins were expressed during human infection. By probing inner and outer membranes, two proteins (58 and 33 kDa) that localized specifically to the outer membrane were observed. An additional low-passage-associated protein (28 kDa) was identified when outer membranes from low- and high-pressure variants of strain B31 were compared by two-dimensional nonequilibrium pH gradient electrophoresis.
Selective induction of transforming growth factor beta in human monocytes by lipoarabinomannan of Mycobacterium tuberculosis
Authors: Dahl K E , Shiratsuchi H , Hamilton B D , Ellner J J , Toossi Z
Published: January 2020

The induction of macrophage-deactivating (interleukin-10 [IL-10] and transforming growth factor beta [TGF-beta] and macrophage-activating (IL-1, IL-6, and tumor necrosis factor alpha [TNF-alpha] cytokines by lipoarabinomannan (LAM) from pathogenic Mycobacterium tuberculosis Erdman and H37Rv strains (ManLAM) and nonpathogenic mycobacteria (AraLAM) in human blood monocytes was examined. ManLAM was significantly less potent in induction of TNF-alpha, IL-1, IL-6, and IL-10 protein and mRNA, whereas its ability to induce TGF-beta was similar to that of AraLAM. Differences in induction of TNF-alpha mRNA by the two LAM preparations only became apparent at late time points of culture (24 h). The induction of TNF-alpha and IL-1 by purified protein derivative of M. tuberculosis was significantly stronger than that by ManLAM. Pretreatment of monocytes with ManLAM did not, however, interfere with cytokine induction by lipopolysaccharide or AraLAM. The extensive mannosyl capping of arabinose termini of ManLAM may underlie the lack of ability to induce some cytokines (IL-1, TNF-alpha, and IL-10) and the retained ability to induce TGF-beta. The latter may have a role in shifting the cytokine milieu in favor of survival of M. tuberculosis.
An oxygen-induced but protein F-independent fibronectin-binding pathway in Streptococcus pyogenes
Authors: Lee J Y , Caparon M
Published: January 2020

Protein F is an important fibronectin-binding adhesin of Streptococcus pyogenes (group A streptococcus). However, all previous analyses of protein F have been conducted in a mutant strain which expresses protein F under anaerobic conditions nonpermissive for expression in other strains. In this study, we have examined the fibronectin-binding properties of several protein F-deficient mutants cultured under aerobic conditions and have identified a second pathway for binding fibronectin. Unlike the case with protein F, exposure to an aerobic environment does not induce transcription of a new gene product. Rather, O2 is apparently required for the modification of a protease-resistant cell surface component into a binding-component form. Modification occurred preferentially at a pH of 6.0 or less, and the binding of the modified component to fibronectin required Zn2+. The oxidizing agent Fe(CN)6 could be substituted for O2 and stimulated expression of binding activity under O2-limiting conditions. Streptococcal fibronectin binding mediated by this pathway but not by protein F could be inhibited by laminin and by streptococcal lipoteichoic acid, a molecule previously implicated as the streptococcal adhesin for fibronectin. The non-protein F-binding activity could also substantially enhance the binding of the organism for fibronectin. The non-protein F-binding activity could also substantially enhance the binding of the organism to basement membrane. By using differential inhibition, analyses of binding to non-protein F mutant strains demonstrated that the total level of fibronectin bound under aerobic conditions reflects contributions from both pathways. Because of its dependence on Zn2+, an oxidant, and pH, this binding activity has been designated the ZOP binding pathway.
Mycoplasma arthritidis mitogen up-regulates human NK cell activity
Authors: D'Orazio J A , Cole B C , Stein-Streilein J
Published: January 2020

While the effects of superantigens on T lymphocytes are well characterized, how superantigens interact with other immune cells is less clear. This report examines the effects of Mycoplasma arthritidis mitogen (MAM) on human natural killer (NK) cell activity. Incubation of peripheral blood mononuclear cells (PBMC) with MAM for 16 to 20 h augmented NK cytotoxicity (against K562) in a dose-dependent manner (P < or = 0.05). Superantigen-dependent cellular cytotoxicity, an activity of superantigen-activated cytotoxic T cells, was not involved in lysis of K562 cells because the erythroleukemic tumor target cells expressed no class II major histocompatibility complex by fluorescence-activated cell sorter analysis. Kinetic experiments showed that the largest increase in NK activity induced by MAM occurred within 48 h. Incubation with MAM caused a portion of NK cells to become adherent to tissue culture flasks, a quality associated with activation, and augmented NK activity was found in both adherent and nonadherent subpopulations. Experiments using cytokine-specific neutralizing antibodies showed that interleukin-2 contributed to enhancement of the NK activity observed in superantigen-stimulated PBMC. Interestingly, MAM was able to augment NK lysis of highly purified NK (CD56+) cells in the absence of other immune cells in 9 of 12 blood specimens, with the augmented lytic activity ranging from 110 to 170% of unstimulated NK activity. In summary, data presented in this report show for the first time that MAM affects human NK cells directly by increasing their lytic capacity and indirectly in PBMC as a consequence of cytokines produced by T cells. Results of this work suggest that, in vivo, one consequence of interaction with superantigen-secreting microorganisms may be up-regulation of NK lytic activity. These findings may have clinical application as a means of generating augmented NK effector cells useful in the immunotherapy of parasitic infections or neoplasms.