bluuebunny/crossref_metadata_2025 huggingface dataset:
Conjugates of synthetic cyclic peptides elicit bactericidal antibodies against a conformational epitope on a class 1 outer membrane protein of Neisseria meningitidis
Authors: Hoogerhout P , Donders E M , van Gaans-van den Brink J A , Kuipers B , Brugghe H F , van Unen L M , Timmermans H A , ten Hove G J , de Jong A P , Peeters C C
Published: January 2020

Bactericidal antibodies directed against surface loops of class 1 outer membrane proteins play a crucial role in protection against meningitis and sepsis caused by Neisseria meningitidis. So far, all efforts to obtain protective antibodies against these apparently conformational epitopes by using linear peptide analogs have been in vain. In this study, conjugates of head-to-tail cyclic peptides encompassing the predicted top of a protective surface loop were used for immunization. A series of 18 cyclic peptides with a ring size ranging from 7 to 17 residues, conjugated to tetanus toxoid, was investigated. Antipeptide and anti-whole-cell immunoglobulin G (IgG) titers elicited by the conjugates were determined. Conjugates of three peptides, containing 14, 15, and 17 amino acid residues (peptides 7, 12, and 13, respectively), induced an anti-whole-cell titer when Quillaja saponin A was used as the adjuvant. When alum was used as the adjuvant, the conjugate of peptide 12 did not elicit an anti-whole-cell response. From the Quillaja saponin A group, some of the sera obtained with conjugates of peptides 7 and 12 and all sera obtained with the peptide 13 conjugate were bactericidal in vitro. None of the sera evoked with alum as the adjuvant showed bactericidal activity. Nonbactericidal sera contained IgG1 primarily, whereas bactericidal sera showed significant titers of IgG2a and IgG2b. Class 1 protein-derived synthetic cyclic peptides which are capable of eliciting bactericidal antibodies, such as peptide 13 derived from meningococcal strain H44/76, represent potential candidates for a (semi)synthetic vaccine against meningococcal disease.
Role of the 145-kilodalton surface protein in virulence of the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius for infant rats
Authors: Rubin L G
Published: January 2020

Brazilian purpuric fever (BPF) is a fulminant infection associated with bacteremia with clonally related strains of Haemophilus influenzae biogroup aegyptius. Case-associated clone strains are more virulent for infant rats than are non-BPF case-associated H. influenzae biogroup aegyptius isolates. I sought to determine the possible role of P145, a 145-kDa surface protein of BPF case H. influenzae biogroup aegyptius clone isolates, in virulence. First, I compared the virulence of two case-associated clone isolates from the blood of children with BPF from Serrana, Brazil, which differed in P145 expression but were identical in all other phenotypic and genotypic characteristics studied. Twenty-four hours after intraperitoneal inoculation of infant rats, there was a significantly higher incidence (51 versus 26%; P = 0.035) and magnitude (2.9 +/- 5.8 versus 0.7 +/- 2.0 CFU/0.01 ml; P = 0.024) of bacteremia in rats inoculated with the P145-expressing strain. I next compared the virulence of a P145-expressing case-associated clone strain with two P145-nonexpressing phase variants of this strain. One variant exhibited a lower mean magnitude of bacteremia and one displayed a similar magnitude of bacteremia compared with that displayed the P145-expressing parental strain. P145-expressing revertants of the P145-nonexpressing strains exhibited the same virulence as the P145-negative variants from which they were derived. Colonies grown from blood cultures maintained the P145 phenotype of the inoculated strain. These results suggest that P145 expression does not contribute to the virulence of the BPF case clone strain for infant rats following intraperitoneal inoculation.
Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis
Authors: Soong L , Duboise S M , Kima P , McMahon-Pratt D
Published: January 2020

In the search for a leishmaniasis vaccine, extensive studies have been carried out with promastigote (insect stage) molecules. Information in this regard on amastigote (mammalian host stage) molecules is limited. To investigate host immune responses to Leishmania amastigote antigens, we purified three stage-specific antigens (A2, P4, and P8) from in vitro-cultivated amastigotes of Leishmania pifanoi by using immunoaffinity chromatography. We found that with Corynebacterium parvum as an adjuvant, three intraperitoneal injections of 5 micrograms of P4 or P8 antigen provided partial to complete protection of BALB/c mice challenged with 10(5) to 10(7) L. pifanoi promastigotes. These immunized mice developed significantly smaller or no lesions and exhibited a 39- to 1.6 x 10(5)-fold reduction of lesion parasite burden after 15 to 20 weeks of infection. In addition, P8 immunization resulted in complete protection against L. amazonensis infection of CBA/J mice and partial protection of BALB/c mice, suggesting that this antigen provided cross-species protection of mice with different H-2 haplotypes. At different stages during infection, vaccinated mice exhibited profound proliferative responses to parasite antigens and increased levels of gamma interferon production, suggesting that a Th1 cell-mediated immune response is associated with the resistance in these mice. Taken together, the data in this report indicate the vaccine potential of amastigote-derived antigens.
Interaction of Listeria monocytogenes with mouse dendritic cells
Authors: Guzman C A , Rohde M , Chakraborty T , Domann E , Hudel M , Wehland J , Timmis K N
Published: January 2020

In this study, the interaction of murine dendritic cells with Listeria monocytogenes was investigated. Dendritic cells are efficient antigen-presenting cells, play a key role in the immune response, and are capable of migrating over substantial distances between sites of infection and lymphoid tissues. L. monocytogenes EGD invaded dendritic cells, escaped from phagosomes into the cytoplasm, and there directed actin nucleation, polymerization, and polarization in a typical fashion, thereby achieving intracellular movement and cell-to-cell spread. The internalization process appears to be independent of the inl locus. Interestingly, an intact microtubular function was essential for efficient uptake, whereas in a previous report, microtubule disruption did not affect bacterial spread in Caco-2 cells. The results obtained also suggest that L. monocytogenes binds to glycosylated receptors of dendritic cells. Uptake of Listeria cells was mediated by a protein kinase-dependent transducing phosphorylation signal that induces the actin polymerization-dependent phagocytic process. To achieve efficient uptake, de novo protein synthesis of eukaryotic and prokaryotic cells is also required. Despite the killing of dendritic cells, wild-type bacteria were found to persist in small numbers in some cells for at least 24 h. When different isogenic mutants of the EGD strain were analyzed for their capability to interact with dendritic cells, it was observed that some virulence-attenuated mutants (i.e., prfA and delta hly) persisted in large numbers for even longer times. Invasion of dendritic cells by L. monocytogenes, which in turn could result in either cell death or persistent infection, might have an important role in the pathogenesis of listeriosis, leading to impaired immune responses with inefficient bacterial clearance and/or promoting bacterial spread.
Inability of an isogenic urease-negative mutant stain of Helicobacter mustelae to colonize the ferret stomach
Authors: Andrutis K A , Fox J G , Schauer D B , Marini R P , Murphy J C , Yan L , Solnick J V
Published: January 2020

Eight ferrets specific-pathogen-free for Helicobacter mustelae were given, per dose, approximately 3.0 x 10(7) CFU of either the wild-type parent strain of H. mustelae (NCTC 12032) (two ferrets) the isogenic urease-negative mutant strain of H. mustelae (10::Tn3Km) (four ferrets), or sterile culture broth (two ferrets). Infection status was monitored by endoscopic gastric biopsy for urease activity, histopathology, and culture and by serology at 3, 6, 10, and 21 weeks. All ferrets were necropsied at 25 weeks. Both negative control ferrets remained uninfected, both ferrets receiving the H. mustelae wild-type parent strain became infected after two doses of the organism, and all four ferrets given two doses of the isogenic urease-negative mutant strain of H. mustelae remained uninfected throughout the 6-month study. Histopathology correlated with infection status. H. mustelae-infected ferrets exhibited diffuse mononuclear inflammation in the subglandular region and the lamina propria of the gastric mucosa, while uninfected ferrets showed no or minimal inflammation. These results suggest that urease activity is essential for colonization of the ferret stomach by H. mustelae.
A patient-derived cytotoxic T-lymphocyte clone and two peptide-dependent monoclonal antibodies recognize HLA-B27-peptide complexes with low stringency for peptide sequences
Authors: Huang F , Hermann E , Wang J , Cheng X K , Tsai W C , Wen J , Kuipers J G , Kellner H , Ackermann B , Roth G , Williams K M , Yu D K , Raybourne R B
Published: January 2020

HLA-B27 molecules expressed on the T2 mutant cell line do not have peptides. Such empty HLA-B27 molecules were not recognized by an HLA-B27-restricted cytotoxic T-lymphocyte (CTL) clone (auto-1) derived from synovial fluid. To test for peptide dependency of the clone, B27-T2 cells were incubated with a panel of 48 different peptides. This lack of stringency was compared with that of a peptide-dependent monoclonal antibody, B27.M2. Positive B27.M2 reactivity resulted when the B27-T2 cells were incubated with two peptides: RRKAMFEDI and RRMGPPVGHR, derived from Chlamydia HSP60 and human ribonucleoprotein, respectively. Because of the limited availability of CTL versus monoclonal antibody, the specificity of B27.M2 was studied in greater detail. The importance of the HLA-B27 heavy chain in antibody recognition of class I-peptide complexes was demonstrated by site-directed mutagenesis. The stringency of the peptide residues was tested by making analogs of each of the nine residues in RRKAMFEDI, creating a panel of 180 analogs. Although stringency was highest for the sixth position, as many as six different amino acids provided positive reactivity. These results indicate that immune recognition of HLA-B27-peptide complexes might have rather low stringency for the peptide sequences. In theory, then, pathogen-derived peptides which induce autoimmunity by generating autoreactive CTL might not share much sequence similarity with the responsible self peptides.
Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG
Authors: Harboe M , Oettinger T , Wiker H G , Rosenkrands I , Andersen P
Published: January 2020

ESAT-6 is a secreted protein present in the short-term culture filtrate of Mycobacterium tuberculosis after growth on a synthetic Sauton medium. ESAT-6 has recently been demonstrated to induce strong T-cell responses in a mouse model of memory immunity after infection with M. tuberculosis. In Western blotting (immunoblotting), the monoclonal antibody HYB76-8, reacting with ESAT-6, gave a 6-kDa region was observed in filtrates from four of eight substrains of M. bovis BCG that produced high levels of MPB64, while no band occurred in the 6-kDa region with any of these BCG substrains. Southern blotting and PCR experiments with genomic mycobacterial DNA showed the presence of the esat-6 gene in reference strains and clinical isolates of M. tuberculosis as well as in virulent M. bovis. The esat-6 gene could not be demonstrated in any of the eight substrains of M. bovis BCG tested by these techniques. Two gene deletions that distinguish M. bovis BCG from virulent M. bovis have thus now been demonstrated. Deletion of mpb64 affects four of the eight substrains tested; deletion of esat-6 affects all of them. The reaction of HYB76-8 AT 26 kDa with four of the BCG substrains was demonstrated to result from cross-reactivity with MPB64. HYB76-8 was also shown to cross-react with the A, B, and C components of the antigen 85 complex and MPT51.
Adherence, fibronectin binding, and induction of cytoskeleton reorganization in cultured human cells by Mycoplasma penetrans
Authors: Girón J A , Lange M , Baseman J B
Published: January 2020

Mycoplasma penetrans adhered to cultured human cells, forming clusters that localized to specific areas of the host cell surface. Adherence and cluster formation were inhibited by anti-M. penetrans antibodies, suggesting the involvement of specific adhesin-receptor interactions. Ultrastructural studies showed that after 2 h of infection, mycoplasmas attach to and penetrate the host cell surface. M. penetrans bound selectively to immobilized fibronectin, an interaction which was not inhibited by a 70-kDa fragment containing a heparin-gelatin-binding domain of fibronectin, other matrix glycoproteins, or an RGD tripeptide, suggesting the recognition of other specific binding sites on the fibronectin molecule. A ca. 65-kDa fibronectin-binding protein of M. penetrans was eluted following Sepharose-fibronectin affinity chromatography. Confocal, light, and immunofluorescence microscopy demonstrated that the interaction of M. penetrans with target cells triggers a signal that causes recruitment of several cytoskeletal components, including tubulin and alpha-actinin, and aggregation of phosphorylated proteins. Detergent-soluble mycoplasma proteins with apparent molecular masses of 18, 28, 32, 36, 39, and 41 kDa selectively bound to glutaraldehyde-fixed HEp-2 cells. Our findings offer new insights into understanding the interaction of this human mycoplasma with host target cells.
Chagas' disease is attenuated in mice lacking gamma delta T cells
Authors: Santos Lima E C , Minoprio P
Published: January 2020

The role of gamma delta T cells in the immunopathology of Chagas' disease is evaluated by monitoring the course of Trypanosoma cruzi infection in mice lacking gamma delta T cells after disruption of the T-cell receptor C delta locus. Levels of parasitemia, states of lymphocyte activation, and levels of lymphokine production as well as tissue pathology are compared in delta knockout mice and their littermates in acute and chronic phases of infection. Although the levels of circulating parasites do not significantly differ in the two groups, mortality scores and numbers of inflammatory lesions of skeletal and cardiac muscles are lower in gamma delta T cell-deficient m ice than in littermate controls. Furthermore, polyclonal lymphocyte activation, as measured by proliferative activities and numbers of B- and T-cell blasts in the spleen, are reduced in deficient mice in the acute and chronic phases of infection. Levels of gamma interferon mRNA obtained from total spleen cells, known to be a critical lymphokine in resistance to T. cruzi infection, are significantly higher in uninfected gamma delta T cell-deficient mice than in control animals and slightly above levels for littermates in the course of acute infection. Interestingly, however, in chronic phases, the levels of this lymphokine are not statistically different between the two groups of mice. These results indicate that gamma delta T cells do not play a crucial role in parasite clearance during the acute phase of the disease but contribute to the mechanisms leading to tissue damage and pathology.
A glyceraldehyde-3-phosphate dehydrogenase homolog in Borrelia burgdorferi and Borrelia hermsii
Authors: Anda P , Gebbia J A , Backenson P B , Coleman J L , Benach J L
Published: January 2020

A polyreactive monoclonal antibody recognized a 38.5-kDa polypeptide with amino-terminal sequence identity to conserved regions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Borrelia burgdorferi, the Lyme disease agent, and Borrelia hermsii, an agent of American relapsing fever. This monoclonal antibody also recognized GAPDH from other pathogenic spirochetes and other prokaryotes and eukaryotes as well. GAPDH activity was detected in sonicates of both B. burgdorferi and B. hermsii but not in live, intact organisms, indicating the possibility of a subsurface localization for the Borrelia GAPDH activity. Degenerate primers constructed from highly conserved regions of gapdh of other prokaryotes successfully amplified this gene homolog in both B. burgdorferi and B. hermsii. Nuclei acid and deduced amino acid sequence analysis of the 838-bp probes for each borrelia indicated 93.9% identity between B. burgdorferi and B. hermsii at the amino acid level. Amino acid identities of B. burgdorferi and B. hermsii with Bacillus stearothermophilus were 59.2% and 58.8% respectively. Southern hybridization studies indicated that the gene encoding GAPDH is located on the chromosome of each borrella. In other bacterial species, GAPDH has other functions in addition to its traditional enzymatic role in the glycolytic pathway. GAPDH may play a similar role in borrelias.
T-cell responses to outer membrane proteins of Neisseria meningitidis: comparative study of the Opa, Opc, and PorA proteins
Authors: Wiertz E J , Delvig A , Donders E M , Brugghe H F , van Unen L M , Timmermans H A , Achtman M , Hoogerhout P , Poolman J T
Published: January 2020

Former studies have shown that the class 5 outer membranes proteins (Opa and Opc proteins) of Neisseria meningitidis are at least as immunogenic as meningococcal porin proteins. High antibody titers to class 5 proteins have been observed in sera obtained during convalescence after meningococcal infection. A strong increase in anti-class 5 antibodies has also been observed in vaccinees who received a meningococcal outer membrane vesicle preparation. The enhanced B-cell response to class 5 proteins may be due to the presence of immunodominant helper T-cell epitopes in these proteins. In order to investigate this hypothesis, we tested purified Opa, Opc, and class 1 proteins for recognition by human T cells. a hierarchy of T-cell immunogenicity was observed among the outer membrane proteins, the Opa protein being more immunogenic than the other proteins. In most cases, the proliferative responses elicited by Opc were higher than the responses observed for the class 1 protein. The epitopes recognized by the immune T cells were identified by using overlapping synthetic peptides spanning the protein sequences of OpaB, Opa5d, and Opc.
Phospholipase C and perfringolysin O from Clostridium perfringens upregulate endothelial cell-leukocyte adherence molecule 1 and intercellular leukocyte adherence molecule 1 expression and induce interleukin-8 synthesis in cultured human umbilical vein endothelial cells
Authors: Bryant A E , Stevens D L
Published: January 2020

Clostridium perfringens phospholipase C (PLC) and perfringolysin O (PFO) differentially induced human umbilical vein endothelial cell expression and synthesis of endothelial cell-leukocyte adherence molecule-1 (ELAM-1), intracellular leukocyte adherence molecule-1 (ICAM-1), and interleukin-8 (IL-8). PLC strongly induced expression of ELAM-1, ICAM-1, and IL-8, while PFO stimulated early ICAM-1 expression but did not promote ELAM-1 expression or IL-8 synthesis. PLC caused human umbilical vein endothelial cells to assume a fibroblastoid morphology, whereas PFO, in high concentrations or after prolonged low-dose toxin exposure, caused cell death. The toxin-induced expression of proadhesive and activational proteins and direct cytopathic effects may contribute to the leukostasis, vascular compromise, and capillary leak characteristics of C. perfringens gas gangrene.
The pef fimbrial operon of Salmonella typhimurium mediates adhesion to murine small intestine and is necessary for fluid accumulation in the infant mouse
Authors: Bäumler A J , Tsolis R M , Bowe F A , Kusters J G , Hoffmann S , Heffron F
Published: January 2020

We investigated the role of the pef operon, containing the genes for plasmid-encoded (PE) fimbriae of Salmonella typhimurium, in adhesion to the murine small intestine. In an organ culture model, a mutant of S. typhimurium carrying a tetracycline resistance cassette inserted in pefC was found to be associated in lower numbers with murine small intestine than the wild-type. Similarly, heterologous expression of PE fimbriae in Escherichia coli increased the bacterial numbers recovered from the intestine in the organ culture model. Adhesion to villous intestine mediated by PE fimbriae was further demonstrated by binding of an E. coli strain expressing PE fimbriae to thin sections of mouse small intestine. The contribution of pef-mediated adhesion on fluid accumulation was investigated in infant mice. Intragastric injection of S. typhimurium 14028 and SR-11 caused fluid accumulation in infant mice. In contrast, pefC mutants of S. typhimurium 14028 and SR-11 were negative in the infant mouse assay. Introduction of a plasmid containing pefBACD and orf5, the first five genes of the pef operon, into the pefC mutant complemented for fluid accumulation in the infant mouse assay. However, heterologous expression of PE fimbriae in E. coli did not result in fluid accumulation in the infant mouse, suggesting that factors other than fimbriae are involved in causing fluid accumulation.
Detection and characterization by differential PCR of host eukaryotic cell genes differentially transcribed following uptake of intracellular bacteria
Authors: Schwan W R , Kügler S , Schüller S , Kopecko D J , Goebel W
Published: January 2020

Host eukaryotic cell genes that are differentially transcribed after phagocytosis of various pathogenic and nonpathogenic bacterial cells were identified by a differential PCR (DPCR) system. This DPCR procedure favors detection and isolation of host genes affected at the transcriptional level by selecting for poly(A) tails but differs substantially from reverse transcription-PCR. Several unidentified macrophage gene fragments from genes that were either transcriptionally activated or downregulated following uptake of Listeria monocytogenes into J774 mouse macrophage cells were initially defined by this DPCR procedure. Because of the sensitivity of the DPCR technique, all of the genes exhibited less than a 10-fold difference in transcription compared with noninfected cells as measured by limiting-dilution PCR. One of the gene fragments has a very high level of homology with a mitogen-activated protein kinase phosphatase (MKP-1), whereas the other affected fragments showed no homologies to known gene sequences. In addition, one of the gene fragments (WS30-B2/1) was specifically downregulated after L. monocytogenes uptake and another gene was repressed by uptake of either Shigella flexneri or L. monocytogenes, while transcription of the genes represented by fragment WS13-B9/9, and to some extent MKP-1, was activated following general phagocytosis (i.e., following uptake of any species of bacterium tested). Further characterization of the affected genes was conducted by using mutants of L. monocytogenes. A hemolysin-negative mutant of L. monocytogenes failed to elicit transcriptional regulation of gene fragment WS10-B4/14 or WS30-B2/1, and it elicited only minimal regulation of MKP-1, suggesting that escape from the phagosome may be required to initiate these responses. Furthermore, mutants with mutations in mpl and actA, two genes whose gene products are involved in actin polymerization and intrahost spread, also did not induce regulation of WS10-B4/14. These results demonstrate that (i) DPCR can identify specific host cell genes which are differentially transcribed after infection with certain microorganisms and (ii) some of these genes may be new or may never before have been linked to interactions between hosts and pathogens.
Nitric oxide synthesis is depressed in Bos indicus cattle infected with Trypanosoma congolense and Trypanosoma vivax and does not mediate T-cell suppression
Authors: Taylor K , Lutje V , Mertens B
Published: January 2020

Infection with African trypanosomes causes the diseases sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. Suppression of cellular immune responses is a feature of trypanosomiasis in bovine, human, and murine hosts. Some aspects of immunosuppression in the murine model are mediated by nitric oxide (NO) produced by gamma interferon (IFN-gamma)-activated macrophages. We have investigated whether a similar mechanism is responsible for T-cell unresponsiveness in bovine trypanosomiasis. Bovine monocytes and macrophages from uninfected cattle and activated in vitro with IFN-gamma produced NO; however, this response was down-regulated in infected cattle. Similarly, the expression of inducible NO synthase messenger RNA was depressed in macrophages of infected cattle. Proliferation of mononuclear cells of trypanosome-infected cattle cultured with mitogen or trypanosome antigens was unchanged by the addition of an NO synthase inhibitor. Lymphocytes of infected cattle secreted interleukins with T-cell growth factor activity after in vitro activation with mitogens but not after activation with trypanosome antigens. Although lymph node cells secreted IFN-gamma after in vitro activation, ex vivo expression of mRNA was depressed. In contrast, the level of expression of interleukin 10 mRNA was higher during infection. We conclude that NO is not involved in the loss of T-cell proliferative function associated with trypanosomiasis in cattle and that, in contrast to the mouse model, the capacity of monocytes and macrophages to produce NO is actually down-regulated in infected cattle.
Insertional inactivation of an intrageneric coaggregation-relevant adhesin locus from Streptococcus gordonii DL1 (Challis)
Authors: Whittaker C J , Clemans D L , Kolenbrander P E
Published: January 2020

Transposon Tn916 was used to insertionally inactivate a coaggregation-relevant locus of Streptococcus gordonii DL1 (Challis). One mutant (F11) was isolated that lost the ability to coaggregate with the streptococcal partners of DL1 but retained the ability to coaggregate with partners belonging to other genera. A probe specific for the region flanking the Tn916 insertion was used to isolate a locus-specific fragment from a chromosomal lambda library. Southern analysis of the resulting phagemids revealed that a 0.5-kb EcoRI fragment hybridized with the F11 probe. Cloning of the 0.5-kb EcoRI fragment into the E. coli-streptococcal insertion vector p(omega) yielded pCW4, which was used to insertionally inactivate the putative coaggregation-relevant gene in DL1. Insertion mutants showed altered coaggregation with streptococci but retained wild-type coaggregation properties with other genera of bacteria. Comparison of immunoblots of cell surface proteins showed a 100-kDa protein in DL1 which was not detected in the Tn916 and pCW4 insertion mutants. These results indicate that the 0.5-kb EcoRI fragment is part of an adhesin-relevant locus that is involved in the production of a 100-kDa protein at the cell surface.
Resistance to tick-borne spirochete challenge induced by Borrelia burgdorferi strains that differ in expression of outer surface proteins
Authors: Kurtti T J , Munderloh U G , Hughes C A , Engstrom S M , Johnson R C
Published: January 2020

Hamsters were immunized with thimerosal-killed Borrelia burgdorferi 297 or a mutant of 297 (M297) that lacks the 49-kb linear plasmid and expression of outer surface proteins A and B (OspA and OspB). Ixodes scapularis nymphs infected with either the B. burgdorferi sensu stricto strain 297 or JMNT, similar in OspA and OspB but differing in OspC expression, were used to evaluate protection. In a homologous challenge, 24 hamsters were vaccinated, 8 each with 297 or M297 and 8 sham (adjuvant)-vaccinated controls. Hamsters vaccinated with either bacterin were completely protected against a natural tick bite or subcutaneous (s.c.) inoculation of 297. Borreliae were effectively eliminated from 80 to 90% of the 297-infected ticks that fed on four hamsters immunized with the 297 bacterin. Cultures of spirochetes isolated from the ticks that remained infected were infectious and induced joint inflammation in naive hamsters. There was no reduction of strain 297 spirochetes in ticks that fed on four hamsters immunized with M297, but the hamsters were protected. Results with the M297 bacterin indicate that proteins other than OspA or OspB can protect hamsters against a tick challenge without eliminating B. burgdorferi in the tick. In a heterologous challenge, 36 hamsters were vaccinated, 12 with each bacterin and 12 controls. None of the hamsters immunized with either bacterin were protected from a challenge involving JMNT-infected ticks, while two of four were protected against an s.c. challenge. Hamsters challenged s.c. with strain 297 spirochetes were protected. There was partial elimination of JMNT spirochetes in ticks that fed on the group of four hamsters immunized with the 297 bacterin, and infection rates were reduced by 50 to 60%. JMNT spirochetes reisolated from the ticks that fed on 297-vaccinated hamsters also remained infectious for hamsters. In the JMNT-infected ticks that fed on four M297-immunized hamsters, there was no decline in the proportion of infected ticks. Destruction of spirochetes in ticks that fed on the hamsters vaccinated with the 297 bacterin suggests that antibodies to OspA and OspB may have been responsible, since the mutant did not induce this activity.
Cloning, expression, and molecular characterization of the dermonecrotic toxin gene of Bordetella spp
Authors: Pullinger G D , Adams T E , Mullan P B , Garrod T I , Lax A J
Published: January 2020

A cosmid library of random fragments of Bordetella bronchiseptica genomic DNA was prepared and screened with oligonucleotides designed from the sequence of the B. pertussis dermonecrotic toxin (DNT) gene. Two cosmid clones which apparently contained the complete B. bronchiseptica DNT gene were identified, but they did not express the toxin. A 5-kb fragment containing the DNT gene was subcloned from one of the cosmid clones onto a high-copy-number plasmid, and this resulted in low-level expression of the toxin. The expression level was increased by deletion of a small region upstream of the coding sequence. Assays for biological activity, including the infant mouse dermonecrosis assay, confirmed that the product of the cloned gene was DNT. The complete sequence of the B. bronchiseptica DNT gene was determined and was more than 99% homologous to the DNT gene of B. pertussis. A putative purine nucleotide-binding motif was shown to be important for toxic activity. Extracts containing the recombinant or the native toxin induced DNA synthesis in Swiss 3T3 cells but inhibited cell division leading to binucleation.
Apoptosis of monocytes and prolonged survival of granulocytes as a result of phagocytosis of bacteria
Authors: Baran J , Guzik K , Hryniewicz W , Ernst M , Flad H D , Pryjma J
Published: January 2020

Monocytes and granulocytes were incubated with suspensions of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or Salmonella enteritidis and, after being washed free of bacteria, cultured for up to 48 h. Every few hours, samples of cultured cells were taken for DNA isolation. Monocytes which phagocytosed bacteria showed features of apoptotic cells, as determined by light microscopy and DNA fragmentation detected by gel electrophoresis. The phenomenon was observed 2 to 4 h after phagocytosis, in contrast, control monocytes did not show signs of apoptosis until 48 h of culture. Intact control granulocytes spontaneously became apoptotic after 12 h of culture. In contrast, degradation of DNA in cells exposed to bacteria was delayed by 12 to 24 h. In conclusion, our observation suggests that granulocytes and monocytes react differently to phagocytosis of bacteria.
Binding sites of salivary statherin for Porphyromonas gingivalis recombinant fimbrillin
Authors: Amano A , Kataoka K , Raj P A , Genco R J , Shizukuishi S
Published: January 2020

We investigated the binding sites of salivary statherin involved in the interaction with Porphyromonas gingivalis recombinant fimbrillin (r-Fim). Synthetic peptides representing statherin analogs were used to localize the binding domains of statherin. Peptide F4 (residues 29 to 43) significantly bound to r-Fim and inhibited r-Fim binding to statherin-coated hydroxyapatite beads. Successive peptides in which pairs of amino acid residues were deleted starting at the N terminus of peptide F4 were synthesized. Peptide N1 without Leu-29-Tyr-30 had significantly reduced direct binding and inhibition ability. The deletions of residues 31 to 40 had little effect on interaction with r-Fim. The tripeptide N6 representing Tyr-41-Thr-42-Phe-43 retained significant binding to r-Fim. Another set of peptides was synthesized by deleting individual amino acid residues from the C and N termini of peptide F4 to identify functional residues among the five putative functional residues 29, 30, and 41 to 43. Peptide C1 missing Phe-43 lost over 50% of its binding ability. Binding ability was gradually reduced with deletions from the peptides. Peptide C5 (amino acids 31 to 40) weakly affected direct binding and inhibition. Collectively, the results of this study suggests that Leu-29-Tyr-30 and Tyr-41-Thr-42-Phe-43 are important binding regions that mediate the binding of statherin to P. gingivalis fimbrillin.
Clusterin, a putative complement regulator, binds to the cell surface of Staphylococcus aureus clinical isolates
Authors: Partridge S R , Baker M S , Walker M J , Wilson M R
Published: January 2020

The ability of Staphylococcus aureus Cowan I strain and a number of S. aureus clinical isolates to bind to the human blood glycoprotein clusterin was investigated. Binding of clusterin to these strains was tested by both enzyme-linked immunosorbent assay and flow cytometry. All of the S. aureus strains examined appeared to bind clusterin to some extent, while nonpathogenic control strains Bacillus subtilis BR151 and Escherichia coli JM109 did not. Three S. aureus isolates were selected for more detailed study; binding of labeled clusterin was saturable, inhibited in the presence of excess unlabeled clusterin, and prevented by pretreatment of bacteria with proteases. From the saturation binding studies, estimates of the affinity constants for the binding of clusterin to the bacteria ranged from 31 to 57 nM. Addition of clusterin to S. aureus cultures was also found to result in aggregation of the bacterial cells; aggregation was not detected when clusterin was added to B. subtilis BR151 or E. coli JM109 cultures. These results suggest that at least some S. aureus strains possess specific proteinaceous receptors for clusterin. Such receptors may be an important new bacterial virulence determinant for S. aureus, as clusterin has been proposed to have a role in the regulation of complement activity.
Induction of interleukin (IL)-1beta and IL-8 mRNA expression in porcine macrophages by lipopolysaccharide from Serpulina hyodysenteriae
Authors: Sacco R E , Nibbelink S K , Baarsch M J , Murtaugh M P , Wannemuehler M J
Published: January 2020

Lipopolysaccharide (LPS) is a classic inducer of inflammatory cytokines and is a key virulence factor for most gram-negative pathogens. The effect of phenol-water (LPS) and butanol-water (endotoxin) extracts from Serpulina hyodysenteriae on inflammatory cytokine mRNA expression from porcine alveolar macrophages was investigated. The LPS and endotoxin extracts from S. hyodysenteriae induced a dose-dependent expression of interleukin 1beta (IL-1beta) and IL-8 which was weak compared with the responses induced by Escherichia coli LPS. In addition, the spirochetal extracts induced no detectable upregulation of mRNA expression for either IL-6 or tumor necrosis factor alpha.
Infection of macrophage-like cells by Legionella species that have not been associated with disease
Authors: O'Connell W A , Dhand L , Cianciotto N P
Published: January 2020

We assessed the ability of Legionella species that have not been associated with disease to infect macrophage-like U937 cells. Two of fourteen species tested exhibited a 50% infective dose that was within I log unit of that of virulent L. pneumophila. Indeed, intracellular CFU of L. jamestowniensis and L. parisiensis increased 100-fold over a 72-h period. These data indicate that additional legionellae can flourish within phagocytes and therefore, can, if given the opportunity, cause disease.
Immunological response to the Brucella abortus GroEL homolog
Authors: Lin J , Adams L G , Ficht T A
Published: January 2020

Western blot (immunoblot) analysis of sera from cattle vaccinated with Brucella abortus S19 exhibit an elevated serologic response to Hsp62, the GroEL homolog (BaGroEL). Serologic screening of individual cows vaccinated with B. abortus S19 revealed no correlation between the immune response to BaGroEL and protection against a challenge with virulent organisms. The humoral immune response to BaGroEL was restricted to a region of the mature protein which mapped to amino acids 317 to 355 and may represent a useful diagnostic tool for monitoring exposure to B. abortus. Immunity to a challenge with virulent B. abortus S2308 was not observed in the BaGroEL vaccinated mouse model.
Role of the staphylococcal accessory gene regulator (sar) in septic arthritis
Authors: Nilsson I M , Bremell T , Rydén C , Cheung A L , Tarkowski A
Published: January 2020

Staphylococcus aureus arthritis is a highly erosive disease in which both host and bacterial factors are of importance for its induction and progression. At the transcriptional level, three known loci act in regulating production of exoproteins and expression of cell wall structures. The aim of our study was to assess the role of the sar locus as a virulence determinant in the pathogenesis of septic arthritis. A recently established murine model of hematogenously spread S. aureus arthritis was employed. S. aureus strains, isogenic for the sar locus, were inoculated intravenously into NMRI mice, and the clinical, bacteriological, serological, and histopathological progression of the disease was studied. Within 1 week after inoculation of bacteria, the frequency of arthritis was 79% in the group of mice inoculated with the sar+ strain, whereas the corresponding frequency in sar mutants was 21% (P < 0.01). Mice inoculated with the sar+ staphylococcal strain exhibited a more pronounced T- and B-lymphocyte activation than those inoculated with the sar mutant, evidenced by splenomegaly, polyclonal B-cell activation, and high serum levels of interleukin 6 and gamma interferon. Also, infection with sar+ staphylococci induced a pronounced weight loss. To assess the relationship between clinical signs and spread of bacteria, we analyzed the homing pattern and persistence of S. aureus in host tissues. Kidneys and joints from sar+-inoculated subjects displayed a higher degree of bacterial persistence than other organs. Our results suggest that molecules controlled by the sar locus are important virulence determinants in the induction and progression of septic arthritis.
Antibacterial activity of antileukoprotease
Authors: Hiemstra P S , Maassen R J , Stolk J , Heinzel-Wieland R , Steffens G J , Dijkman J H
Published: January 2020

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been identified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042-5047, 1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity. Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its antimicrobial activity may equip ALP to contribute to host defense against infection.
Comparison of antibody and cytokine responses to primary Giardia muris infection in H-2 congenic strains of mice
Authors: Venkatesan P , Finch R G , Wakelin D
Published: January 2020

The course of primary infections with Giardia muris differs between BALB and B10 H-2 congenic strains of mice. In the first 3 weeks of infection, there is a more rapid decline in intestinal trophozoite and fecal cyst counts in B10 strains than in BALB strains. To determine whether this difference could be explained by variation in specific antibody responses, both secretory immunoglobulin A (IgA) and serum antibody responses were compared between these strains. No significant differences in the timing, titer, or specificity of secretory or serum antibodies were found. However, on comparing specific anti-G. muris serum IgG subclass responses, we found that B10 strains produced IgG2a while BALB strains produced IgG1, suggesting differential involvement of T helper 1 and 2 subsets of lymphocytes. When cells harvested from mesenteric lymph nodes were stimulated with concanavalin A in vitro, both gamma interferon and interleukin-5 were secreted by cells from B10 mice, but only interleukin-5 was secreted by cells from BALB/c mice. Specific blockade of gamma interferon by monoclonal antibody administered to B10 mice resulted in an enhanced intensity of infection.
Recombinant V antigen protects mice against pneumonic and bubonic plague caused by F1-capsule-positive and -negative strains of Yersinia pestis
Authors: Anderson G W , Leary S E , Williamson E D , Titball R W , Welkos S L , Worsham P L , Friedlander A M
Published: January 2020

The purified recombinant V antigen from Yersinia pestis, expressed in Escherichia coli and adsorbed to aluminum hydroxide, an adjuvant approved for human use, was used to immunize outbred Hsd:ND4 mice subcutaneously. Immunization protected mice from lethal bubonic and pneumonic plague caused by CO92, a wild-type F1+ strain, or by the isogenic F1- strain C12. This work demonstrates that a subunit plague vaccine formulated for human use provides significant protection against bubonic plague caused by an F1- strain (C12) or against substantial aerosol challenges from either F1+ (CO92) or F1-(C12) Y. pestis.
Generation of gamma interferon responses in murine Peyer's patches following oral immunization
Authors: George A
Published: January 2020

To date, oral immunizations have been shown to generate only Th2 responses in murine Peyer's patches (PP), raising the possibility that T cells present in PP may be capable of mounting only Th2 responses or that the microenvironment of PP does not favor the generation of Th1 cells. However, it is also possible that antigens that can generate Th1 responses have not yet been used for oral immunizations. This study shows that T cells in PP of mice immunized orally with live Salmonella typhimurium secrete large amounts of gamma interferon (IFN-gamma) when they are stimulated with bacterial sonicate in vitro. Moreover, oral challenge of mice with live bacteria 4 months after immunization elicits a secondary IFN-gamma response in PP and mesenteric lymph nodes. Parenteral immunization does not generate an IFN-gamma T-cell response in PP, and parenteral challenge of orally immunized mice does not elicit a secondary response in PP. However, oral challenge of intraperitoneally immunized mice elicits a secondary IFN-gamma response in PP and mesenteric lymph nodes, and intraperitoneal challenge of orally immunized mice elicits a secondary response in the spleen. The data suggest that memory T cells recirculate between mucosal and nonmucosal compartments and that they may be recruited to the site of antigenic challenge.
Regulation of gonococcal sialyltransferase, lipooligosaccharide, and serum resistance by glucose, pyruvate, and lactate
Authors: McGee D J , Rest R F
Published: January 2020

Strain F62 of Neisseria gonorrhoeae gonococci (GC) is sensitive to normal human serum unless CMP-N-acetylneuraminic acid (CMP-NANA) is present. NANA is transferred primarily to a 4.5-kDa lipooligosaccharide (LOS) structure by a GC sialyltransferase (Stase). We investigated LOS and Stase expression and serum resistance in strain F62 grown in different carbon sources and growth conditions. Pyruvate-grown GC expressed 1.9- to 5.6-fold more Stase activity than did glucose-grown GC, whereas lactate-grown GC generally expressed intermediate Stase activities. Broth-grown GC expressed two- to fourfold more Stase activity than did plate-grown GC in all carbon sources. Pyruvate- or lactate-grown GC expressed significantly more of the sialylateable 4.5-kDa LOS species than did glucose-grown GC. Anaerobically, the 4.5-kDa LOS species was expressed in greater quantity than the 4.9-kDa N-acetyl galactosamine-terminating species in all carbon sources. Pyruvate-grown GC also incorporated up to threefold more radiolabelled CMP-NANA onto the 4.5-kDa LOS species than did glucose-grown GC. In serum resistance studies, pyruvate-grown GC were 6.5- to 16.1-fold more serum resistant than glucose-grown GC at limiting CMP-NANA concentrations (1.56 to 12.50 microg/ml). Taken together, these results indicate that gonococcal expression of Stase activity is up-regulated by growth in pyruvate or lactate, which correlates with enhanced expression of the sialylateable 4.5-kDa LOS and, for growth in pyruvate, correlates with enhanced sialylation of gonococcal LOS and greater serum resistance. In different in vivo niches, gonococcal LOS sialylation, serum resistance, and interaction with host cells can be highly regulated.
No apparent influence of immunoglobulins on indigenous oral and intestinal microbiota of mice
Authors: Marcotte H , Lavoie M C
Published: January 2020

The role of secretory immunoglobulin A (sIgA) in the control of the indigenous microbiota is not well understood. In this study, we compared the oral and intestinal microbiota of transgenic B-cell-deficient (microMT) mice with their heterozygous (microMT/+) normal littermates. The levels of salivary IgA and serum IgA and IgG were normal in microMT/+ mice, while no immunoglobulins were detected in microMT/microMT mice. The acquisition and proportions of the different species of the oral and intestinal indigenous bacterial populations were not significantly different between the two groups of mice. Our results thus suggest that secretory IgA does not play a major role in the regulation of the indigenous microbiota of mice.