bluuebunny/crossref_metadata_2025 huggingface dataset:
Variation of Brucella abortus 2308 infection in BALB/c mice induced by prior vaccination with salt-extractable periplasmic proteins from Brucella abortus 19
Authors: Pugh G W , Tabatabai L B
Published: January 2020

The study compared the immune and protective responses induced in BALB/c mice vaccinated with six salt-extractable periplasmic protein fractions (Brucella cell surface proteins [BCSP]) of Brucella abortus 19 and later challenge exposed with B. abortus 2308. BCSP70 was precipitated with ammonium sulfate at 70% saturation, and BCSP100 was precipitated with ammonium sulfate at 100% saturation by use of supernatant fluid of BCSP70 that had been precipitated with 70% ammonium sulfate. Four subfractions were separated from BCSP100 by anion-exchange high-performance liquid chromatography (HPLC). Monophosphoryl lipid A (MPL) from Salmonella typhimurium Re mutant strain was used as a potential immune response modifier in some vaccines. Reduced or increased numbers of CFU and increased spleen size in the principal groups of mice relative to that of the nonvaccinated control group were considered protectiveness or virulence (survival) criteria. Results indicated that vaccines prepared from BCSP70 and BCSP100 were moderately protective and immunogenic. The subfractions designated BCSP100-A through BCSP100-D purified by anion-exchange HPLC were not protective when MPL was not used as an immune response modifier. However, two subfractions were associated with significant (P < 0.05) increases in CFU per spleen and splenomegaly in vaccinated mice compared with those in nonvaccinated challenge-exposed mice. MPL enhanced protection or was neutral when used with BCSP70, BCSP100, BCSP100-C, and BCSP100-D. Serologic results of an enzyme-linked immunosorbent assay indicated that MPL modulated the immunoglobulin G responses induced by BCSP70, BCSP100, and subfraction BCSP100-B vaccines only. The overall results suggest that certain proteinaceous periplasmic fractions might serve as virulence or survival factors in B. abortus infections.
Influence of beta 2-microglobulin expression on gamma interferon secretion and target cell lysis by intraepithelial lymphocytes during intestinal Listeria monocytogenes infection
Authors: Emoto M , Neuhaus O , Emoto Y , Kaufmann S H
Published: January 2020

Numerous microbial pathogens, including Listeria monocytogenes, enter the host through the intestine. Although relatively little is known about the biological functions of intestinal intraepithelial lymphocytes (i-IEL), they are generally considered a first line of defense against intestinal infections. In the mouse, the vast majority of i-IEL express the CD8 coreceptor either as a CD8 alpha/alpha homodimer or as a CD8 alpha/beta heterodimer. The CD8 receptor of T-cell receptor TcR gamma/delta i-IEL is exclusively homodimeric, whereas the CD8-expressing TcR alpha/beta i-IEL segregate into equal fractions of CD8 alpha/alpha and CD8 alpha/beta cells. We infected beta 2-microglobulin (beta 2m)+/- mice (possessing all i-IEL populations) and beta 2m -/- mutant mice (lacking all CD8 alpha/beta + i-IEL and having few CD8 alpha/alpha + TcR alpha/beta i-IEL) with L. monocytogenes per os and determined their biological functions after TcR ligation with monoclonal antibodies. Cytolytic activities of TcR alpha/beta and TcR gamma/delta i-IEL from beta 2m +/- mice were not influenced by intestinal listeriosis. Cytolytic activities of TcR alpha/beta i-IEL were impaired in uninfected beta 2m -/- mice, but this reduction was reestablished as a consequence of intestinal listeriosis. Frequencies of gamma interferon (IFN-gamma)-producing TcR alpha/beta i-IEL in uninfected beta 2m -/- mice were reduced, compared with that in their heterozygous controls. Equally low frequencies of IFN-gamma-producing TcR gamma/delta i-IEL in beta 2M +/- and beta 2m-/- mutants were found. Listeriosis increased frequencies of INF-gamma-producing TcR alpha/beta and TcR gamma/delta i-IEL in both mouse strains. Most remarkably, the proportion of IFN-gamma-producing TcR gamma/delta i-IEL was elevated 10-fold in listeria-infected beta 2M -/- mice. Our findings show that the beta 2m-independent CD8 beta- i-IEL expressing either TcR alpha/beta or TcR gamma/delta are stimulated by intestinal listeriosis independent of regional beta 2m expression. We conclude that the three major CD8+ i-IEL populations are stimulated by intestinal listeriosis and that CD8 beta- i-IEL compensate for the total lack of CD8 beta+ i-IEL in beta 2M -/- mutant mice. Hence, in contrast to the peripheral immune system, which crucially depends on CD8 alpha/beta + TcR alpha/beta lymphocytes, the mucosal immune system can rely on additional lymphocytes expressing the CD8 alpha/alpha homodimer.
Assembly and antigenicity of the Neisseria gonorrhoeae pilus mapped with antibodies
Authors: Forest K T , Bernstein S L , Getzoff E D , So M , Tribbick G , Geysen H M , Deal C D , Tainer J A
Published: January 2020

The relationship between the sequence of Neisseria gonorrhoeae pilin and its quaternary assembly into pilus fibers was studied with a set of site-directed antibody probes and by mapping the specificities of antipilus antisera with peptides. Buried and exposed peptides in assembled pili were identified by competitive immunoassays and immunoelectron microscopy with polyclonal antibodies raised against 11 peptides spanning the pilin sequence. Pili did not compete significantly with pilin subunits for binding to antibodies against residues 13 to 31 (13-31) and 18-36. Pilus fibers competed well with pilin protein subunits for binding to antibodies raised against peptides 37-56, 58-78, 110-120, 115-127, 122-139, and 140-159 and competed weakly for antibodies against residues 79-93 and 94-108. Antibodies to sequence-conserved residues 37-56 and to semiconserved residues 94-108 preferentially bound pilus ends as shown by immunoelectron microscopy. The exposure of pilus regions to the immune system was tested by peptide mapping of antiserum specificities against sets of overlapping peptides representing all possible hexameric or octameric peptides from the N. gonorrhoeae MS11 pilin sequence. The immunogenicity of exposed peptides incorporating semiconserved residues 49-56 and 121-126 was revealed by strong, consistent antigenic reactivity to these regions measured in antipilus sera from rabbits, mice, and human and in sera from human volunteers with gonorrhea. The conservation and variation of antigenic responses among these three species clarify the relevance of immunological studies of other species to the human immune response against pathogens. Overall, our results explain the extreme conservation of the entire N-terminal one-third of the pilin protein by its dominant role in pilus assembly: hydrophobic residues 1-36 are implicated in buried lateral contacts, and polar residues 37-56 are implicated in longitudinal contacts within the pilus fiber.
Alteration of gonococcal protein expression in acidic culture
Authors: Pettit R K , Filiatrault M J , Martin E S
Published: January 2020

We grew Neisseria gonorrhoeae under acidic, neutral, and alkaline conditions and noted altered expression of at least 12 outer membrane proteins between 31 and 100 kDa in size. One protein whose expression was upregulated under acidic conditions was gonococcal heat shock protein 63. These proteins may contribute to the pathogenesis of gonorrhea in the urogenital tract.
Neutrophils prevent extracellular colonization of the liver microvasculature by Salmonella typhimurium
Authors: Conlan J W
Published: January 2020

The early course of hepatic infection with the facultative intracellular bacterial pathogen Salmonella typhimurium was examined in control mice and in mice selectively depleted of neutrophils by treatment with a granulocyte-specific monoclonal antibody. The results show that >200-fold more salmonellae were recovered in livers of the latter group of mice than in livers of the former group by 24 h of parenterally initiated infection. Comparative histological examination of the livers from both groups of mice indicated that neutrophils participate in early anti-Salmonella defense in the liver in part by aborting infection in permissive hepatocytes and by inhibiting extracellular bacterial colonization of the hepatic microvasculature. It is shown in addition that systemic salmonellosis was also severely exacerbated in neutropenic mice infected intragastrically with the pathogen.
Sporadic invasion of cultured epithelial cells by Haemophilus influenzae type b
Authors: Johnson P D , Oppedisano F , Bennett-Wood V , Gilbert G L , Robins-Browne R M
Published: January 2020

While working with an in vitro invasion assay, we observed that Haemophilus influenzae type b occasionally exhibits highly invasive behavior. The phenomenon is not inhibited by colchicine or cytochalasin but is dependent on the presence of supplemental CO2. We propose that sporadic invasiveness may correlate with the unknown events that precede Haemophilus influenzae type b bacteremia.
A classical strain of Vibrio cholerae with diminished ability to process the proteolytically sensitive site in the A subunit of cholera toxin
Authors: Ichinose Y , Tsuji T , Kato M , Neves B C , Morita K , Ehara M , Hirayama T
Published: January 2020

Vibrio cholerae O1, No. 31, a strain isolated from a patient with mild diarrhea, produced mainly the unnicked cholera toxin. The amount of toxin that had accumulated in the cells was approximately 200 times lower than that secreted into the culture medium. When the unnicked toxin was purified by three successive column chromatographies and then extracted from the polyacrylamide gel, the unnicked toxin showed two bands corresponding to the A and B subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the A1 fragment was detected by trypsinization. Biological and enzymatic activities of the purified toxin with trypsinization were identical to those of cholera toxin from V. cholerae 569B as seen in the rabbit skin permeability test and the NAD:agmatine ADP-ribosyltransferase assay. DNA sequences of the A and B subunits were identical to those of the A- and B-subunit genes from the El Tor 2125 and classical 0395 strains, respectively. These data suggest that the wild V. cholerae strain, No. 31, produces a toxin identical to toxins previously reported in the literature and secretes it without accumulation in the cell, as is the case with other strains. However, strain No. 31's ability to nick the toxin is diminished compared with such abilities of other strains.
Proline iminopeptidase from the outer cell envelope of the human oral spirochete Treponema denticola ATCC 35405
Authors: Mäkinen K K , Chen C Y , Mäkinen P L
Published: January 2020

Certain periodontopathic organisms have been shown to exhibit high activity of proline iminopeptidase (PIPase). The human oral spirochete Treponema denticola ATCC 35405 was found to contain an easily extractable, novel PIPase (EC 3.4.11.5), which was purified to a sodium dodecyl sulfate- polyacrylamide gel electrophoresis-pure form by means of fast protein liquid chromatographic procedures. The range of the minimum monomeric molecular mass (280 amino acid residues) of the PIPase, based on amino acid analysis, was 30.35 to 30.39 kDa, but the likely in vivo form of the enzyme is a tetramer (minimum mass, 120.2 to 120.4 kDa). The molecular masses based on laser desorption mass spectrometry were 36.058 kDa for the monomer and 72.596 kDa for a dimer. The PIPase cleaves specifically the Pro-Y bond in dipeptides where Y is preferably Arg or Lys. Pro-Gln, Pro-Asn, and Pro-Ala were also good substrates, while Pro-Glu was hydrolyzed slowly and Pro-Asp was not hydrolyzed at all. Tripeptides were poor substrates or were not hydrolyzed (an exception was Pro-Gly-Gly, which cleaved at a moderate rate). Larger molecules, such as poly-L-Pro, were not hydrolyzed. The T. denticola enzyme can be regarded as a true PIPase, since replacing Pro in Pro-Y with other amino acid residues resulted in no hydrolysis. The activity of the PIPase may depend on an active carboxyl group and on an active seryl residue but not on metal cations. Diethylpyrocarbonate inactivated the enzyme in a reaction that was not reversible upon addition of NH2OH. The enzyme contains a relatively large percentage (ca. 15%) of proline residues. The dominance of the PIPase activity among aminopeptidase activities present in T. denticola and the proposed location of the enzyme in the outer cell envelope suggest that it has a vital function in the propagation of the cells within their biological niche (inflamed human periodontal tissues). The biologic role of the PIPase may be envisaged as in the termination of the overall peptidolytic cascade (liberating free proline and other amino acids), whereby host tissue proteins and peptides are first processed and inactivated by other peptidases possibly present within the same confines as the PIPase.
Antibody-mediated inhibition of Aedes aegypti midgut trypsins blocks sporogonic development of Plasmodium gallinaceum
Authors: Shahabuddin M , Lemos F J , Kaslow D C , Jacobs-Lorena M
Published: January 2020

The peritrophic matrix (PM) that forms around a blood meal is a potential barrier of Plasmodium development in mosquitoes. Previously, we have shown that to traverse the PM, Plasmodium ookinetes secrete a prochitinase and that an inhibitor of chitinase blocks further parasite development. Here we report that it is the mosquito trypsin that activates the Plasmodium prochitinase. Trypsin was identified as the chitinase-activating enzyme by two criteria: (i) trypsin activity and activating activity comigrated on one-dimensional gels, and (ii) activating activity and penetration of the PM by Plasmodium parasites were both hindered by trypsin-specific inhibitors. Subsequently, we examined the effect of antitrypsin antibodies on the parasite life cycle. Antibodies prepared against a recombinant blackfly trypsin effectively and specifically inhibited mosquito trypsin activity. Moreover, when incorporated into an infective blood meal, the antitrypsin antibodies blocked infectivity of Aedes aegypti mosquitoes by Plasmodium gallinaceum. This block of infectivity could be reversed by exogenously provided chitinase, strongly suggesting that the antibodies act by inhibiting prochitinase activation and not on the parasite itself. This work led to the identification of a mosquito antigen, i.e., midgut trypsin, as a novel target for blocking malaria transmission.
Expression of major surface protein 2 antigenic variants during acute Anaplasma marginale rickettsemia
Authors: Eid G , French D M , Lundgren A M , Barbet A F , McElwain T F , Palmer G H
Published: January 2020

Antigenic variants of Anaplasma marginale major surface protein 2 (MSP-2), a target of protective immune responses, have been detected by use of copy-specific monoclonal antibodies reactive with some, but not all, organisms during acute rickettsemia. The presence of polymorphic msp-2 genes was confirmed by cloning and sequencing two gene copies, 11.2 and DF5, each of which encodes a full-length MSP-2 with a unique amino acid sequence. Transcription of msp-2 genes during acute rickettsemia was analyzed by use of cDNA cloning of hybrid-selected msp-2 mRNA. Sequencing of cDNA clones, designated AR1 to AR14, indicated that DF5 msp-2 was transcribed during acute rickettsemia. Two classes of variant msp-2 genes were also transcribed during acute rickettsemia. The first class of variant transcripts, typified by clones AR3, AR4, AR7, and AR14, each encoded a single or small number of amino acid substitutions relative to DF5. The second type, AR5, encoded a large region of amino acid polymorphism, including additions, deletions, and substitutions, as compared to DF5. Specific antibody directed against the AR5 polymorphic region bound a unique MSP-2 expressed on A. marginale that was not recognized by antibody generated against DF5. Similarly, anti-AR5 peptide antibody reacted with a different MSP-2 that was not bound by anti-DF5 antibody. This expression confirmed that variant msp-2 transcripts encode structurally distinct MSP-2 molecules which bear unique B-cell epitopes. These results support the hypothesis that the large msp-2 gene family, which constitutes a minimum of 1% of the genome, encodes antigenic variants critical to evasion of protective immune response directed against surface MSP-2 epitopes.
Analysis of the specificity of bacterial immunoglobulin A (IgA) proteases by a comparative study of ape serum IgAs as substrates
Authors: Qiu J , Brackee G P , Plaut A G
Published: January 2020

Immunoglobulin A (IgA) proteases are bacterial enzymes with substrate specificity for human serum and secretory IgAs. To further define the basis of this specificity, we examined the ability of IgA proteases of Clostridium ramosum, Streptococcus pneumoniae (EC 3.4.24.13), Neisseria meningitidis (EC 3.4.21.72), and Haemophilus influenzae (EC 3.4.21.72) to cleave serum IgAs of gorillas, chimpanzees, and orangutans. All enzymes cleaved the IgAs of the three apes despite differences in ape IgA1 hinge sequence relative to the human prototype. To directly compare the ape and human hinge cleavage sites, the sites were identified in eight ape IgA digests. This analysis confirmed that ape proteins were all cleaved in the IgA hinge region, in all but one case after proline residues. The exception, C. ramosum protease, cleaved gorilla and chimpanzee IgAs at peptide bonds having no proline, but the scissile bonds were in the same hinge location as the Pro-221-Val-222 cleaved in human IgA1. These data indicate that proline is not an invariant substrate requirement for all IgA proteases and that the location of the scissile bond, in addition to its composition, is a critical determinant of cleavage specificity.
Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant
Authors: Di Tommaso A , Saletti G , Pizza M , Rappuoli R , Dougan G , Abrignani S , Douce G , De Magistris M T
Published: January 2020

Immunization of the female reproductive tract is important for protection against sexually transmitted diseases and other pathogens of the reproductive tract. However, intravaginal immunization with soluble antigens generally does not induce high levels of secretory immunoglobulin A (IgA). We recently developed safe mucosal adjuvants by genetically detoxifying Escherichia coli heat-labile enterotoxin, a molecule with a strong mucosal adjuvant activity, and here we describe the use of the nontoxic mutant LTK63 to induce a response in the mouse vagina against ovalbumin (Ova). We compared intravaginal and intranasal routes of immunization for induction of systemic and vaginal responses against LTK63 and Ova. We found that LTK63 is a potent mucosal immunogen when given by either the intravaginal or intranasal route. It induces a strong systemic antibody response and IgG and long-lasting IgA in the vagina. The appearance of vaginal IgA is delayed in the intranasally immunized mice, but the levels of vaginal anti-LTK63 IgA after repeated immunizations are higher in the intranasally immunized mice than in the intravaginally immunized mice. LTK63 also acts as a mucosal adjuvant, inducing a serum response against Ova, when given by both the intravaginal and intranasal routes. However, vaginal IgA against Ova is stimulated more efficiently when LTK63 and antigen are given intranasally. In conclusion, our results demonstrate that LTK63 can be used as a mucosal adjuvant to induce antigen-specific antibodies in vaginal secretions and show that the intranasal route of immunization is the most effective for this purpose.
Lipoteichoic acid and interleukin 1 stimulate synergistically production of hepatocyte growth factor (scatter factor) in human gingival fibroblasts in culture
Authors: Sugiyama A , Arakaki R , Ohnishi T , Arakaki N , Daikuhara Y , Takada H
Published: January 2020

Lipoteichoic acids (LTA) from various gram-positive bacteria, including oral streptococci such as Streptococcus sanguis, enhanced the production of hepatocyte growth factor (HGF) (scatter factor) by human gingival fibroblasts in culture, whereas lipopolysaccharides (LPS) from various gram-negative bacteria did not. In contrast, LPS induced interleukin 1 activity in human gingival epithelial cells in culture, while LTA had little effect. LTA and recombinant human interleukin 1 alpha enhanced synergistically the production of HGF/SF in human gingival fibroblast cultures. Recombinant human HGF, in turn, enhanced the proliferation of human gingival epithelial cells in culture.
Dominance of conserved B-cell epitopes of the Plasmodium falciparum merozoite surface protein, MSP1, in blood-stage infections of naive Aotus monkeys
Authors: Hui G S , Nikaido C , Hashiro C , Kaslow D C , Collins W E
Published: January 2020

We have shown that conserved B epitopes were immunodominant in animals hyperimmunized with parasite-purified or recombinant merozoite surface protein MSP1 of Plasmodium falciparum. Cross-priming studies also suggested that a conserved T-helper epitope(s) is efficient in inducing the anti-MSP1 antibody response. In this study, we determined whether a similar profile of immune responses was induced during live P. falciparum infections. Naive Aotus monkeys were infected by blood-stage challenge with either one of the two dimorphic MSP1 alleles represented by the FUP and FVO parasites. Sera collected after parasite clearance were analyzed by enzyme-linked immunosorbent assays (ELISAs). Monkeys infected with parasites carrying one allelic form of MSP1 had antibodies that were equally reactive with homologous or heterologous MSP1s. This preferential recognition of conserved epitopes of MSP1 was confirmed by competitive binding ELISAs. Studies with Plasmodium yoelii and P. falciparum show that the C-terminal 19-kDa fragment of MSP1, MSP1(19), is the target of protective immunity. Thus, monkey sera were assayed for recognition with recombinant MSP1(19)s expressing variant and conserved B epitopes. Results of direct and competitive binding ELISAs showed that the anti-MSP1(19) antibodies were also directed primarily against conserved determinants. The similarities between vaccine- or infection-induced antibody responses suggest a possible reciprocal enhancement of the two populations of anti-MSP1 antibodies when a subunit MSP1 vaccine is introduced into populations living in areas where malaria is endemic. This together with previous observations that conserved determinants are important in MSP1-mediated immunity provides an optimistic outlook that a subunit MSP1 vaccine may be effective and practical for field applications in malaria-exposed populations.
Trypanosoma cruzi-induced immunosuppression: selective triggering of CD4+ T-cell death by the T-cell receptor-CD3 pathway and not by the CD69 or Ly-6 activation pathway
Authors: Lopes M F , DosReis G A
Published: January 2020

In a model of experimental Chagas' disease induced with metacyclic forms of Trypanosoma cruzi, CD4+ but not CD8+ T cells undergo T-cell receptor (TCR)-CD3-mediated activation-induced cell death (AICD) in vitro. CD4+ T cells from T. cruzi-infected mice also developed unresponsiveness in proliferative responses to TCR-CD3-mediated stimulation. A linear correlation was found between extent of proliferative unresponsiveness and loss of CD4+ T-cell viability. CD4+ T-cell activation through the CD69 or Ly-6 A/E pathway, on the other hand, did not result in proliferative unresponsiveness compared with controls. Lack of suppression in proliferation assays correlated with lack of AICD by cells stimulated through the CD69 or Ly-6 A/E pathway. Concomitant stimulation through CD69, however, did not rescue CD4+ T cells from CD3-induced death. Flow cytometry study of cells stimulated in vitro showed no defect in interleukin-2 receptor expression by CD4+ T cells from infected donors, which escaped TCR-mediated AICD. In vivo injection of anti-CD3 into acutely infected mice, but not into control mice, led to splenocyte DNA fragmentation and failed to increase splenic CD4+ T-cell numbers. These results show that TCR-CD3-mediated AICD is involved in CD4+ T-cell unresponsiveness in vitro following infection with T. cruzi. In addition, successful activation of these cells through the CD69 and Ly-6 pathways is due to differences in the inability of these stimuli to trigger AICD. Since TCR-CD3-mediated AICD can be induced in vivo in infected mice, these findings may be relevant for the onset of immunological disturbances in the host.
Coordinate induction of two antibiotic genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and tumor necrosis factor alpha
Authors: Russell J P , Diamond G , Tarver A P , Scanlin T F , Bevins C L
Published: January 2020

Peptides with potent broad-spectrum antibiotic activity have been identified in many animal species. Recent investigations have demonstrated that epithelial cells are a site of antibiotic peptide expression, suggesting that these peptides contribute to host defense at mucosal surfaces. Expression of tracheal antimicrobial peptide (TAP), a member of the beta-defensin family of peptides, is inducible in cultured tracheal epithelial cells (TEC) upon challenge with bacterial lipopolysaccharide (LPS) (G. Diamond, J.P. Russell, and C.L. Bevins, Proc. Natl. Acad. Sci. USA, in press). In this study, an anchored reverse transcriptase PCR strategy was used to determine if TAP was the sole beta-defensin isoform expressed upon stimulation of the cells with LPS. In addition to TAP, a second class of cDNA clones which encoded lingual antimicrobial peptide (LAP), a beta-defensin peptide recently isolated from a different mucosal site, the bovine tongue, was identified (B.S. Schonwetter, E.D. Stolzenberg, and M. Zasloff, Science 267:1645-1648, 1995). Northern (RNA) blot analysis demonstrated in vivo expression of LAP mRNA in tracheal mucosa. Levels of LAP mRNA were higher in cultured TEC challenged with either LPS or tumor necrosis factor alpha than in control cells. Thus, a response of TEC exposed to inflammatory mediators is induction of antibiotic-encoding genes, including both TAP and LAP. This work complements the in vivo studies of Schonwetter et al. (cited above), which showed elevated levels of LAP mRNA in squamous epithelial cells of the tongue near sites of tissue injury and inflammation, by suggesting possible mediators of the in vivo observation. Together these lines of investigations support the hypothesis that inducible expression of endogenous antibiotic peptides by inflammatory mediators characterizes local defense of mammalian mucosal surfaces.
A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge
Authors: Fattom A I , Sarwar J , Ortiz A , Naso R
Published: January 2020

The efficacy of capsular polysaccharide (CP)-specific antibodies elicited by active immunization with vaccines composed of Staphylococcus aureus types 5 and 8 CP linked to Pseudomonas aeruginosa exoprotein A or with immune immunoglobulin G (I-IgG) obtained from vaccinated plasma donors was tested in lethal and sublethal bacterial mouse challenge models. A dose of 2 x 10(5) CFU of S. aureus type 5 CP per mouse administered intraperitoneally (i.p.) with 5% hog mucin was found to cause 80 to 100% mortality in BALB/c mice within 2 to 5 days. Mice passively immunized i.p. 24 h earlier or subcutaneously 48 h earlier with 0.5 ml of I-IgG showed significantly higher average survival rates than animals receiving standard IgG or saline (P < 0.01) following the bacterial challenge. Animals actively immunized with the monovalent type 5 CP-P. aeruginosa exoprotein A conjugate showed a survival rate of 73% compared with 13% in phosphate-buffered saline-immunized animals. The prechallenge geometric mean titer of type 5 CP antibodies in animals that died was significantly (P < 0.05) lower than that of animals which survived the challenge (95.7 versus 223.6 micrograms/ml, respectively). The IgG was further evaluated in mice challenged i.p. with a sublethal dose of 5 x 10(4) CFU per mouse. Serial blood counts were performed on surviving animals at 6, 12, 24, and 48 h. Surviving animals were sacrificed at 72 h, and bacterial counts were performed on their kidneys, livers, and peritoneal lavage fluids. Animals receiving I-IgG had lower bacterial counts in blood samples and lower bacterial densities in kidneys, livers, and peritoneal lavage samples than mice immunized with standard IgG (P < 0.05). These data suggest that S. aureus type 5 CP antibodies induced by active immunization or administered by passive immunization confer protection against S. aureus infections.
A recombinant minigene vaccine containing a nonameric cytotoxic-T-lymphocyte epitope confers limited protection against Listeria monocytogenes infection
Authors: An L L , Pamer E , Whitton J L
Published: January 2020

We have previously shown that vaccines expressing virus-derived cytotoxic-T-lymphocyte (CTL) epitopes as short minigenes can confer effective protection against virus challenges, and here we extend these studies to the bacterium Listeria monocytogenes. Host defense against this important human pathogen appears largely T cell mediated, and a nonamer CTL epitope from the listeriolysin O (LLO) protein has been identified in BALB/c mice. We have synthesized this nonamer as a minigene, expressed it in a recombinant vaccinia virus (VV-list), and used this to immunize mice. Memory CTLs cultured from VV-list-immunized mice specifically lyse target cells pulsed with a nonamer peptide identified at LLO amino acid residues 91 to 99. Four weeks postimmunization, mice were challenged with L. monocytogenes. By day 6 following challenge with a sublethal dose of L. monocytogenes, mice immunized with VV-list showed a approximately 2,000- to 6,000-fold reduction in bacteria CFU in the spleen and liver. At this time point, with control mice, bacterial were readily detectable by Gram stain of the liver but were undetectable in the VV-list-immunized animals. Additionally, when a normally lethal dose of bacteria was given, death was delayed in VV-list-immunized animals. This study has demonstrated that a single immunization with a recombinant vaccinia virus bearing only nine amino acids from a bacterial pathogen can induce specific CTLs able to confer partial protection against bacterial challenge.
Intranasal and intramuscular proteosome-staphylococcal enterotoxin B (SEB) toxoid vaccines: immunogenicity and efficacy against lethal SEB intoxication in mice
Authors: Lowell G H , Kaminski R W , Grate S , Hunt R E , Charney C , Zimmer S , Colleton C
Published: January 2020

Intranasal or intramuscular (i.m.) immunization of mice and i.m. immunization of rabbits with formalinized staphylococcal enterotoxin B (SEB) toxoid in saline elicited higher anti-SEB serum immunoglobulin G (IgG) titers when the toxoid was formulated with proteosomes. In addition, intranasal immunization of mice with this proteosome-toxoid vaccine elicited high levels of anti-SEB IgA in lung and intestinal secretions, whereas the toxoid without proteosomes did not. Two i.m. immunizations with proteosome-toxoid plus alum also induced higher murine serum responses than alum-adjuvanted toxoid without proteosomes. Furthermore, proteosome-toxoid delivered intranasally in saline or i.m. with either saline or alum afforded significant protection against lethal SEB challenge in two D-galactosamine-sensitized murine models of SEB intoxication, i.e., the previously described i.m. challenge model and a new respiratory challenge model of mucosal SEB exposure. Efficacy correlated with the induction of high serum levels of anti-SEB IgG. In contrast, intranasal or i.m. immunization with toxoid in saline without proteosomes was not significantly protective in either challenge model. Proteosome-toxoid plus alum given i.m. also elicited more significant protection against respiratory challenge than the alum-adjuvanted toxoid alone. The capacity of proteosomes to enhance both i.m. and intranasal immunogenicity and efficacy of SEB toxoid indicates that testing such proteosome-SEB toxoid vaccines in the nonhuman primate aerosol challenge model of SEB intoxication prior to immunogenicity trials in humans is warranted. These data expand the applicability of the proteosome mucosal vaccine delivery system to protein toxoids and suggest that respiratory delivery of proteosome vaccines may be practical for enhancement of both mucosal and systemic immunity against toxic or infectious diseases.
Leucine auxotrophy restricts growth of Mycobacterium bovis BCG in macrophages
Authors: Bange F C , Brown A M , Jacobs W R
Published: January 2020

The ability of slow-growing mycobacteria to replicate within host mononuclear phagocytes is thought to be central to the pathogenesis of mycobacterial infection. However, because of the lack of a mycobacterial mutant defective for intracellular replication, it has not been possible to test this hypothesis directly. Previously, we showed that a BCG leucine auxotroph with a transposon disruption of the leuD gene is unable to grow in mice. Here we demonstrate that this mutant is also incapable of replicating within cultured macrophages in vitro. Complementation of the leuD mutation with the leuCD genes of Escherichia coli restored wild-type levels of growth in macrophages, establishing that the defect for intracellular replication was due to leucine auxotrophy per se and not to a polar effect of the transposon insertion on an adjacent gene. These results suggest that the inability of the leucine auxotroph to grow in mice was due to its sequestration, after phagocytosis, in an intracellular compartment from which it could not obtain leucine.
Antigenic topology of the P29 surface lipoprotein of Mycoplasma fermentans: differential display of epitopes results in high-frequency phase variation
Authors: Theiss P , Karpas A , Wise K S
Published: January 2020

Antibodies to P29, a major lipid-modified surface protein of Mycoplasma fermentans, reveal phase variation of surface epitopes occurring with high frequency in clonal lineages of the organism. This occurs despite continuous expression of the entire epitope-bearing P29 product (detected by Western immunoblotting) and contrasts with phase variation of other surface antigens mediated by differential expression of proteins. To understand the structure and antigenic topology of P29, the single-copy p29 gene from strain PG18 was cloned and sequenced. The gene encodes a prolipoprotein containing a signal sequence predicted to be modified with lipid and cleaved at the N-terminal Cys-1 residue of the mature P29 lipoprotein. The remaining 218-residue hydrophilic sequence of P29 is predicted to be located external to the single plasma membrane. Additional Cys residues at positions 91 and 128 in the mature protein were shown to form a 36-residue disulfide loop by selectively labeling sulfhydryl groups that were liberated only after chemical reduction of monomeric P29. Two nearly identical charged amino acid sequences occurred in P29, within the disulfide loop and upstream of this structure. Two distinct epitopes binding different monoclonal antibodies were associated with opposite ends of the P29 protein, by mapping products expressed in Escherichia coli from PCR-generated 3' deletion mutations of the p29 gene. Each monoclonal antibody detected high-frequency and noncoordinate changes in accessibility of the corresponding epitopes in colony immunoblots of clonal variants, yet sequencing of the p29 gene from these variants and analysis of disulfide bonds revealed no associated changes in the primary sequence or disulfide loop structure of P29. These results suggest that P29 surface epitope variation may involve masking of selected regions of P29, possibly by other surface components undergoing phase variation by differential expression. Differential masking may be an important mechanism for altering the antigenic or functional surface topology of this mycoplasma and other wall-less mycoplasmas.
Pathogenesis of corneal infection: binding of Pseudomonas aeruginosa to specific phospholipids
Authors: Panjwani N , Zhao Z , Raizman M B , Jungalwala F
Published: January 2020

Clinical isolates of Pseudomonas aeruginosa were examined for binding interactions with phospholipids of corneal epithelium. Thin-layer chromatography (TLC) of lipids extracted from corneal epithelia followed by staining with an ammonium molybdate spray reagent revealed three phospholipid components, PL1, PL2, and PL3. The chromatographic mobility of PL1 was similar to that of the phospholipid standards phosphatidylinositol (PI) and phosphatidylserine (PS), which were not well resolved from one other; PL2 and PL3 comigrated with the standards phosphatidylcholine and phosphatidylethanolamine, respectively. By use of a TLC-bacterial overlay procedure, 35S-labeled P. aeruginosa organisms were shown to bind to PL1 but not to PL2 or PL3. P. aeruginosa binding to PL1 was concentration dependent. Alkaline methanolysis abolished the binding. PL1 was separated into two components, PL1-I and PL1-S, by chromatography on borate-treated TLC plates. Both PL1-I and PL1-S contained binding sites for P. aeruginosa. Mass spectral analysis identified PL1-I and PL1-S as PI and PS, respectively. Radiolabeled P. aeruginosa organisms were subsequently shown to bind to commercially available bovine PI and PS and synthetic dipalmitoyl-PS but not to other phospholipid standards, including bovine SM and PC or synthetic dioleoyl- and distearoyl-PC. A control Escherichia coli strain did not bind to either PS or PI. Tetramethylurea, a disrupter of hydrophobic associations, did not influence the binding of P. aeruginosa to PS or PI. P. aeruginosa bound to the monolayers of corneal epithelial cells. P. aeruginosa binding to the monolayer cultures as well as to rabbit corneas pretreated with exogenous PS and PI was significantly higher than that to those preincubated with PC or medium alone. The data suggest that phospholipids PS and PI present in mucus or on the cell surface may function as P. aeruginosa receptors and contribute to selective bacterium-host interactions responsible for initial colonization.
Lacto-N-fucopentaose III (Lewis x), a target of the antibody response in mice vaccinated with irradiated cercariae of Schistosoma mansoni
Authors: Richter D , Incani R N , Harn D A
Published: January 2020

Carbohydrates on soluble egg antigens are major epitopes for the antibody responses of patients and mice infected with Schistosoma mansoni. Recently, protective sera of mice vaccinated with irradiated cercariae were shown to recognize carbohydrate epitopes on schistosomal glutathione S-transferase. The present study demonstrates that carbohydrate epitopes are major targets of sera from C57BL/6J and CBA/J mice vaccinated with 15- or 50-kilorad-irradiated cercariae of S. mansoni. Antibody titers to carbohydrate epitopes increased with the number of vaccinations and were considerably higher in C57BL/6J mice than in CBA/J mice. The specificity of this anticarbohydrate response was determined by measuring antibody binding to defined oligosaccharide residues known to be present on the parasite. A predominant target of the humoral anticarbohydrate response of vaccinated mice was lacto-N-fucopentaose III, a molecule relevant for cell trafficking. We observed no binding to its nonfucosylated homolog, lacto-N-neotetraose, or to oligosaccharides present on keyhole limpet hemocyanin. The strongest antibody response to lacto-N-fucopentaose III was observed for C57BL/6J and CBA/J mice repeatedly vaccinated with 15-kilorad-irradiated cercariae, which also achieve the highest levels of protection. Immunoglobulin M was the predominant antibody class binding to lacto-N-fucopentaose III. We conclude that in the irradiated-cercariae vaccine model, C57BL/6J and CBA/J mice produce anticarbohydrate antibodies against various stages of S. mansoni and that the oligosaccharide lacto-N-fucopentaose III is one target of this response. Lacto-N-fucopentaose III and its specific antibodies may profoundly affect host resistance and parasite homing.
Iron starvation causes release from the group A streptococcus of the ADP-ribosylating protein called plasmin receptor or surface glyceraldehyde-3-phosphate-dehydrogenase
Authors: Eichenbaum Z , Green B D , Scott J R
Published: January 2020

In many pathogenic bacteria, iron starvation serves as an environmental signal that triggers the expression of virulence factors, many of which are found on the cell surface or secreted into the culture supernatant. Using the chelating agent nitrilotriacetic acid, we have established conditions for iron starvation of the important human pathogen Streptococcus pyogenes (the group A streptococcus) and determined that iron limitation results in the specific appearance of several new proteins in the culture supernatant. One of these supernatant proteins is the ADP-ribosylating protein known as streptococcal plasmin receptor (Plr) or as the streptococcal surface glyceraldehyde-3-phosphate-dehydrogenase because of its other activities. Upon iron starvation, Plr is specifically released into the culture supernatant in a time-dependent manner, and its appearance in the supernatant is not accompanied by induction of plr mRNA synthesis. Release of Plr from the bacteria may be important for the virulence of group A streptococci and the manifestation of diseases.
Fraction 1 capsular antigen (F1) purification from Yersinia pestis CO92 and from an Escherichia coli recombinant strain and efficacy against lethal plague challenge
Authors: Andrews G P , Heath D G , Anderson G W , Welkos S L , Friedlander A M
Published: January 2020

As a first step in formulating an improved plague vaccine, we developed a simple purification strategy that produced high yields of pure cell-associated and culture supernatant-derived fraction 1 capsular antigen (F1) from both avirulent Yersinia pestis C092 (Pgm- Lcr-) and an Escherichia coli F1-producing recombinant strain. Cell-associated F1 was partially purified by sequential ammonium sulfate precipitations of a sodium chloride extract of acetone-dried bacteria harvested from broth cultures. Cell-free F1 was precipitated directly from culture supernatants with a single application of 30% ammonium sulfate. By exploiting the aggregative property of F1, large quantities of purified high-molecular-weight F1 species from both cell extracts and supernatants were isolated in the void volume of a preparative gel filtration column. Highly purified, endotoxin-free F1, combined with two different adjuvants, induced very high F1 titers in mice and protected them against either subcutaneous (70 to 100% survival) or aerosol (65 to 84% survival) challenge with virulent organisms. This protection was independent of the source of the antigen and the adjuvant used. F1-induced protection against both subcutaneous and aerosol challenge was also significantly better than that conferred by immunization with the licensed killed whole-cell vaccine. Our results indicate that F1 antigen represents a major protective component of previously studied crude capsule preparations, and immunity to F1 antigen provides a primary means for the host to overcome plague infection by either the subcutaneous or respiratory route.
Hemin-induced modifications of the antigenicity and hemin-binding capacity of Porphyromonas gingivalis lipopolysaccharide
Authors: Cutler C W , Eke P I , Genco C A , Van Dyke T E , Arnold R R
Published: January 2020

Previous studies have shown that the physical, biochemical, and antigenic properties of the bacterial outer membrane are profoundly influenced by the growth environment. In the present study, the effects of growth in hemin-replete (H+) and hemin-depleted (H-) media on the lipopolysaccharide (LPS) of the oral pathogen Porphyromonas gingivalis were investigated. Our studies show that LPS from P. gingivalis cultured in H+ media (H+LPS) expressed additional low-molecular-mass antigens, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis. Particularly evident was a 26-kDa antigen (26 LPSC) that was lost from the LPS upon transfer of P. gingivalis into H- media. The loss of the 26 LPSC was accompanied by a marked reduction in the hemin-binding capacity of the LPS. The 26 LPSC was refractory to Coomassie blue staining and proteinase K digestion. H+LPS from strain W50/BE1, a nonpigmented pleiotropic strain, lacked the 26 LPSC and did not bind hemin. Polyclonal antiserum raised to whole-cell antigens of P. gingivalis A7436, W83, and HG405 grown in H+ media, but not in H- media, recognized the 26 LPSC in the purified H+LPS from any of the three strains. The immunoreactivities of sera from humans with (n = 24) or without (n = 25) periodontitis to the 26 LPSC and other H+LPS determinants were analyzed by Western blot. Overall, 75% of adult periodontitis patient sera reacted with multiple bands in the H+LPS stepladder, particularly in the range of 14 to 27 kDa. In contrast, only 20% of control sera reacted faintly with H+LPS bands in the range 27 to 34 kDa. The 26 LPSC was recognized by over 40% of sera from adult patients with periodontitis and none of the healthy control sera. Taken together, these results suggest that the antigenicity and hemin-binding properties of P. gingivalis LPS can be modified by growth in H+ media.
Experimental genital mycoplasmosis: time of infection influences pregnancy outcome
Authors: Brown M B , Steiner D A
Published: January 2020

Genital infection of rats with Mycoplasma pulmonis causes adverse pregnancy outcome and can result in in utero spread of infection to the fetus. The current study was designed to determine whether the stage of pregnancy when infection occurs influences pregnancy outcome. Rats were inoculated with 3 X 10(7) CFU of M. pulmonis at 10 days prior to breeding (-10) or at gestational day (gd) 11 or 14 and were necropsied at gd 11, 14, or 18 or within 24 h of parturition (term). Control rats received sterile broth. M. pulmonis was isolated from the placenta, amniotic fluid, or fetal tissues only from rats infected prior to breeding (P < 0.001). All infected rats had significantly more loss of pups than did control rats (P < 0.006), but rats infected prior to breeding or at the beginning of the third trimester (gd 14) were much more likely to have fetal losses. Rats infected in the early second trimester after implantation (gd 11) did not experience severe losses. Litter sizes, total litter weight, and individual pup weight from all infected rats, regardless of gestational stage when infected, were significantly smaller than those of control rats (P < 0.001). On the basis of the results of this study, we conclude that the time of infection plays a major role in determination of pregnancy outcome and spread of infection from the genital tract to the respiratory tract.
Phosphorylation of myosin light chain at distinct sites and its association with the cytoskeleton during enteropathogenic Escherichia coli infection
Authors: Manjarrez-Hernandez H A , Baldwin T J , Williams P H , Haigh R , Knutton S , Aitken A
Published: January 2020

Myosin light chain, the most prominent host cell phosphoprotein during adhesion of enteropathogenic Escherichia coli to cultured HEp-2 cells, was shown to be distributed between cytosolic and cytoskeletal cell fractions; its association with the cytoskeletal fraction increased with time of enteropathogenic E. coli incubation. Phosphopeptide mapping indicated cytosolic and cytoskeletal myosin light chain phosphorylation at different sites by protein kinase C and myosin light chain kinase.