bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cysteine-Cysteine Family of Chemokines RANTES, MIP-1α, and MIP-1β Induce Trypanocidal Activity in Human Macrophages via Nitric Oxide
Authors: Villalta Fernando , Zhang Yuan , Bibb Kartz E. , Kappes John C. , Lima Maria F.
Published: December 2019

This paper describes a new role for the cysteine-cysteine (CC) chemokines RANTES, MIP-1α, and MIP-1β on human macrophage function, which is the induction of nitric oxide (NO)-mediated trypanocidal activity. In a previous report, we showed that RANTES, MIP-1α and MIP-1β enhanceTrypanosoma cruziuptake and promote parasite killing by human macrophages (M. F. Lima, Y. Zhang, and F. Villalta, Cell. Mol. Biol. 43:1067–1076, 1997). Here we study the mechanism by which RANTES, MIP-1α, and MIP-1β activate human macrophages obtained from healthy individuals to killT. cruzi. Treatment of human macrophages with different concentrations of RANTES, MIP-1α, and MIP-1β enhancesT. cruzitrypomastigote phagocytosis in a dose peak response. The optimal response induced by the three CC chemokines is attained at 500 ng/ml. The macrophage trypanocidal activity induced by CC chemokines can be completely inhibited byl-N-monomethyl arginine (l-NMMA), a specific inhibitor of thel-arginine:NO pathway, but not by itsd-enantiomer. Culture supernatants of chemokine-treated human macrophages contain increased NO2−levels, and NO2−production is also specifically inhibited byl-NMMA. The amount of NO2−induced by these chemokines in human macrophages is comparable to the amount of NO2−induced by gamma interferon. The killing of trypomastigotes by NO in cell-free medium is blocked by an NO antagonist or a NO scavenger. This data supports the hypothesis that the CC chemokines RANTES, MIP-1α, and MIP-1β activate human macrophages to killT. cruzivia NO, which is an effective trypanocidal mechanism.
The
ica
Locus of
Staphylococcus epidermidis
Encodes Production of the Capsular Polysaccharide/Adhesin
Authors: McKenney David , Hübner Johannes , Muller Eugene , Wang Ying , Goldmann Donald A. , Pier Gerald B.
Published: December 2019

Clinical isolates of coagulase-negative staphylococci often elaborate a biofilm involved in adherence to medical devices and resistance to host defenses. The biofilm contains the capsular polysaccharide/adhesin (PS/A), which mediates cell adherence to biomaterials, and another antigen, termed polysaccharide intercellular adhesin (PIA), which is thought to mediate bacterial accumulation into cellular aggregates. PIA is a polymer of β-1,6-linked
N
-acetyl glucosamine residues with a molecular mass of <30,000 kDa. We found that recombinant
Staphylococcus carnosus
and
Staphylococcus aureus
carrying a plasmid with genes of the
ica
locus, which was reported to encode the biosynthetic proteins for production of PIA, were also able to synthesize PS/A. PS/A and a chemically and immunologically identical polysaccharide isolated from
S. carnosus
carrying the
ica
genes on plasmid pCN27 were found to be high-molecular-mass (>250,000 kDa), acid-stable polymers of β-1,6-linked glucosamine substituted on the amino group primarily with succinate, although some preparations also contained acetate. Moreover, all recombinant staphylococcal strains with the
ica
genes had the biologic properties previously attributed to PS/A.
ica
-positive strains readily formed an in vitro biofilm on plastic, adhered 3- to 10-fold more to catheters during a 30-min assay compared with control strains carrying only the cloning vector, adsorbed out antibodies to PS/A from immune serum, and elaborated a capsule visualized by immunoelectron microscopy with antisera to PS/A. These properties were also seen with PS/A-producing strains of
Staphylococcus epidermidis
, but not with transposon mutants lacking PS/A. An antiserum raised to PIA contained high-titer antibody to PS/A that was readily adsorbed out by PS/A-positive strains of
S. epidermidis
and recombinant strains of staphylococci carrying the
ica
genes. We conclude that the
ica
locus encodes production of PS/A and that the properties of
S. epidermidis
associated with initial bacterial adherence, biofilm formation, and intercellular adhesion can be correlated with elaboration of PS/A.
Urease Plays an Important Role in the Chemotactic Motility of
Helicobacter pylori
in a Viscous Environment
Authors: Nakamura Hiroki , Yoshiyama Hironori , Takeuchi Hiroaki , Mizote Tomoko , Okita Kiwamu , Nakazawa Teruko
Published: December 2019

Helicobacter pylori
exhibits chemotactic responses to urea, flurofamide, acetohydroxamic acid, and sodium bicarbonate. In buffer, the chemotactic activities of a urease-positive strain were higher than those of the isogenic urease-negative strain. Moreover, the chemotactic activities of the urease-positive strain were increased in a viscous solution containing 3% polyvinylpyrrolidone, whereas those of the urease-negative mutant were not. These results are in accordance with the fact that the mutant strain did not show swarming in motility agar regardless of having flagella. Incubation of the wild-type strain with flurofamide resulted in partial inhibition of the chemotactic activities in the viscous solution. In addition, incubation with acetohydroxamic acid, a low-molecular-weight, diffusible urease inhibitor, resulted in complete loss of chemotactic activity in the viscous solution. The inhibition of the chemotactic activity by urease inhibitors paralleled the inhibition of urease. The chemotactic activity of
H. pylori
was also inhibited by the proton carrier carbonyl cyanide
m
-chlorophenylhydrazone, showing that
H. pylori
utilizes proton motive force for motility. These results indicate that cytoplasmic urease plays an important role in the chemotactic motility of
H. pylori
under a condition that mimics the ecological niche of the bacterium, the gastric mucous layer.
Interleukin-12 Is Essential for a Protective Th1 Response in Mice Infected withCryptococcus neoformans
Authors: Decken Klaus , Köhler Gabriele , Palmer-Lehmann Kathrin , Wunderlin Andrea , Mattner Frank , Magram Jeanne , Gately Maurice K. , Alber Gottfried
Published: December 2019

To analyze the roles of interleukin-12 (IL-12) and the IL-12-dependent Th1 response in resistance toCryptococcus neoformans, we have established a chronic infection model in wild-type mice and in mice with targeted disruptions of the genes for the IL-12p35 and IL-12p40 subunits (IL-12p35−/−and IL-12p40−/−mice, respectively) as well as in mice with a targeted disruption of the IL-4 gene. Long-term application of exogenous IL-12 prevented death of infected wild-type mice for the entire period of the experiment (up to 180 days) but did not resolve the infection. Infected IL-12p35−/−and IL-12p40−/−mice died significantly earlier than infected wild-type mice, whereas infection of IL-4-deficient mice led to prolonged survival. Interestingly, infected IL-12p40−/−mice died earlier and developed higher organ burdens than IL-12p35−/−mice, which, for the first time in an infection model, suggests a protective role of the IL-12p40 subunit independent of the IL-12 heterodimer. The fungal organ burdens of IL-4-deficient mice and IL-12-treated wild-type mice were significantly reduced compared to those of untreated wild-type mice and IL-12-deficient mice. Histopathological analysis revealed reduction of the number of granulomatous lesions following treatment with IL-12. Susceptibility of both IL-12p35−/−and IL-12p40−/−mice was associated with marginal production of gamma interferon and elevated levels of IL-4 from CD4+T cells, which indicates Th2 polarization in the absence of IL-12, whereas wild-type mice developed a Th1 response. Taken together, our data emphasize the essential role of IL-12 for protective Th1 responses againstC. neoformans.
Infection of Epithelial Cells by Pathogenic Neisseriae Reduces the Levels of Multiple Lysosomal Constituents
Authors: Ayala Patricia , Lin Lan , Hopper Sylvia , Fukuda Minoru , So Magdalene
Published: December 2019

Members of our group reported recently that neisseria infection of human epithelial cells results in accelerated degradation of the major lysosomal integral membrane protein LAMP1 and that this is due to hydrolysis of this glycoprotein at its immunoglobulin A1 (IgA1)-like hinge by the neisseria type 2 IgA1 protease (L. Lin et al., Mol. Microbiol. 24:1083–1094, 1997). We also reported that the IgA1 protease plays a major role in the ability of the pathogenic neisseriae to survive within epithelial cells and hypothesized that this is due to alteration of lysosomes as a result of protease-mediated LAMP1 degradation. In this study, we tested the hypothesis that neisseria infection leads to multiple changes in lysosomes. Here, we report that neisseria infection also reduces the levels of three other lysosomal markers: LAMP2, lysosomal acid phosphatase (LAP), and CD63. In contrast, neither the epidermal growth factor receptor level nor the β-tubulin level is affected. A detailed examination of LAMP2 indicated that the reduced LAMP2 levels are not the result of an altered biosynthetic rate or of cleavage by the IgA1 protease. Nevertheless, the protease plays a role in reducing LAMP2 and LAP activity levels, as these are partially restored in cells infected with an
iga
mutant. We conclude that neisseria infection results in multiple changes to the lysosomes of infected epithelial cells and that these changes are likely an indirect result of IgA1 protease-mediated cleavage of LAMP1.
Pathogenicity of an Enterotoxigenic
Escherichia coli
Hemolysin (
hlyA
) Mutant in Gnotobiotic Piglets
Authors: Moxley Rodney A. , Berberov Emil M. , Francis David H. , Xing Jun , Moayeri Mahtab , Welch Rodney A. , Baker Diane R. , Barletta Raúl G.
Published: December 2019

Pigs infected with hemolytic F4
+
strains of enterotoxigenic
Escherichia coli
often develop septicemia secondary to intestinal infection. We tested the hypothesis that inactivation of hemolysin would reduce the ability of F4
+
enterotoxigenic
E. coli
to cause septicemia in swine following oral inoculation. Inactivation of the hemolysin structural gene (
hlyA
) did not decrease the incidence of septicemia in the gnotobiotic piglet model.
Activation of Rho GTPases byEscherichia coliCytotoxic Necrotizing Factor 1 Increases Intestinal Permeability in Caco-2 Cells
Authors: Gerhard Ralf , Schmidt Gudula , Hofmann Fred , Aktories Klaus
Published: December 2019

The cytotoxic necrotizing factor 1 (CNF1) activates Rho GTPases by deamidation of glutamine-63 and thereby induces redistribution of the actin cytoskeleton and formation of stress fibers. Here, we have studied the effects of CNF1 on the transepithelial resistance of Caco-2 cells, a human intestinal epithelial cell line, in comparison with the Rho-inactivating toxin B ofClostridium difficile. Whereas toxin B decreased the transepithelial resistance of Caco-2 cells by about 80% after 4 h, CNF1 reduced it by about 40%. Significant changes of the transepithelial resistance induced by CNF1 were detected after 3 h of incubation. Half-maximal effects were observed with 10 and 41 ng of CNF1 and toxin B per ml, respectively. Flux measurement revealed no CNF1-induced increase of fluorescein isothiocyanate-dextran permeation within the first 4 h of incubation and a 2.9-fold increase after 24 h of incubation. In contrast, toxin B induced a 28-fold increase of permeation after 24 h. As detected by rhodamine-phalloidin staining, CNF1 increased polymerization of F actin at focal contacts of adjacent cells and induced formation of stress fibers. The data indicate that not only depolymerization but also polymerization of actin and subsequent reorganization of the actin cytoskeleton alter the barrier function of intestinal tight junctions.
5-Lipoxygenase Reaction Products Modulate Alveolar Macrophage Phagocytosis ofKlebsiella pneumoniae
Authors: Mancuso Peter , Standiford Theodore J. , Marshall Teresa , Peters-Golden Marc
Published: December 2019

The leukotrienes are potent lipid mediators of inflammation formed by the 5-lipoxygenase-catalyzed oxidation of arachidonic acid. Although the effects of leukotrienes on neutrophil chemotaxis and activation have been established, their role in modulating innate host defense mechanisms is poorly understood. In a previous study (M. Bailie, T. Standiford, L. Laichalk, M. Coffey, R. Strieter, and M. Peters-Golden, J. Immunol. 157:5221–5224, 1996), we used 5-lipoxygenase knockout mice to establish a critical role for endogenous leukotrienes in pulmonary clearance and alveolar macrophage phagocytosis ofKlebsiella pneumoniae. In the present study, we investigated the role of specific endogenous leukotrienes in phagocytosis ofK. pneumoniaeand explored the possibility that exogenous leukotrienes could restore phagocytosis in alveolar macrophages with endogenous leukotriene synthesis inhibition and enhance this process in leukotriene-competent cells. Rat alveolar macrophages produced leukotriene B4(LTB4), LTC4, and 5-hydoxyeicosatetraenoic acid (5-HETE) during the process of phagocytosis, and the inhibition of endogenous leukotriene synthesis with zileuton and MK-886 dramatically attenuated phagocytosis. We also observed a reduction in phagocytosis when we treated alveolar macrophages with antagonists to the plasma membrane receptors for either LTB4, cysteinyl-leukotrienes, or both. In leukotriene-competent cells, LTC4augmented phagocytosis to the greatest extent, followed by 5-HETE and LTB4. These 5-lipoxygenase reaction products demonstrated similar relative abilities to reconstitute phagocytosis in zileuton-treated rat alveolar macrophages and in alveolar macrophages from 5-lipoxygenase knockout mice. We conclude that endogenous synthesis of all major 5-lipoxygenase reaction products plays an essential role in phagocytosis. The restorative and pharmacologic effects of LTC4, LTB4, and 5-HETE may provide a basis for their exogenous administration as an adjunctive treatment for patients with gram-negative bacterial pneumonia.
Role of Liver NK Cells and Peritoneal Macrophages in Gamma Interferon and Interleukin-10 Production in Experimental Bacterial Peritonitis in Mice
Authors: Seki Shuhji , Osada Shun-ichi , Ono Satoshi , Aosasa Suefumi , Habu Yoshiko , Nishikage Tetsuro , Mochizuki Hidetaka , Hiraide Hoshio
Published: December 2019

Gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) production by liver, spleen, lung, peripheral blood mononuclear cells (MNC), and peritoneal exudate cells (PEC) in experimental bacterial peritonitis was examined by cecum ligation and puncture (CLP) (with an 18-gauge needle) of BALB/c mice. MNC of organs were cultured for 18 h, and cytokine levels in supernatants were examined. Cytokines contained in peritoneal lavage fluid were regarded as those produced by PEC. Only liver MNC and PEC produced substantial amounts of IFN-γ, and PEC were the main source of IL-10, especially 12 h after CLP. As reflected by the cytokine production by liver MNC and PEC, serum IFN-γ and IL-10 levels were elevated after CLP. C57BL/6 (B6) mice and BALB/c nude mice showed a similar pattern of cytokine production. TNF-α levels in culture supernatants, peritoneal lavage fluid, and sera were not significantly elevated compared to those of sham-operated mice. In vivo depletion of NK cells of B6 mice with anti-asialo GM1 or anti-NK1.1 antibody greatly decreased IFN-γ levels in liver MNC culture supernatants and sera, suggesting that liver NK cells are IFN-γ producers. On the other hand, plastic-adherent PEC macrophages are the major IL-10 producers. Mice subjected to a cecum ligation and cut procedure (which have a more severe peritonitis) showed much higher IFN-γ and IL-10 levels than those subjected to CLP, while mice subjected to CLP with a smaller (22-gauge) needle showed low levels of these cytokines. These findings show that liver NK cells and PEC macrophages are important for the production of proinflammatory and anti-inflammatory cytokines in bacterial peritonitis.
Altered Expression of Selectable Marker
URA3
in Gene-Disrupted
Candida albicans
Strains Complicates Interpretation of Virulence Studies
Authors: Lay Jennifer , Henry L. Keith , Clifford Julie , Koltin Yigal , Bulawa Christine E. , Becker Jeffrey M.
Published: December 2019

The ura-blaster technique for the disruption of
Candida albicans
genes has been employed in a number of studies to identify possible genes encoding virulence factors of this fungal pathogen. In this study, the
URA3
-encoded orotidine 5′-monophosphate (OMP) decarboxylase enzyme activities of
C. albicans
strains with ura-blaster-mediated genetic disruptions were measured. All strains harboring genetic lesions via the ura-blaster construct showed reduced OMP decarboxylase activities compared to that of the wild type when assayed. The activity levels in different gene disruptions varied, suggesting a positional effect on the level of gene expression. Because the
URA3
gene of
C. albicans
has previously been identified as a virulence factor for this microorganism, our results suggest that decreased virulence observed in strains constructed with the ura-blaster cassette cannot accurately be attributed, in all cases, to the targeted genetic disruption. Although revised methods for validating a
URA3
-disrupted gene as a target for antifungal drug development could be devised, it is clearly desirable to replace
URA3
with a different selectable marker that does not influence virulence.
Pathways for Potentiation of Immunogenicity during Adjuvant-Assisted Immunizations withPlasmodium falciparumMajor Merozoite Surface Protein 1
Authors: Hui George S. N. , Hashimoto Caryn N.
Published: December 2019

Vaccine adjuvants exert critical and unique influences on the quality of immune responses induced during active immunizations. We investigated the mechanisms of action of immunological adjuvants in terms of their requirements for cytokine-mediated pathways for adjuvanticity. Antibody responses potentiated by several adjuvants to aPlasmodium falciparumMSP1-19 (C-terminal 19-kDa processing fragment of MSP1) vaccine were studied in gamma interferon (IFN-γ) or interleukin (IL-4) knockout mice. The levels of anti-MSP1-19 antibodies and the induction of Th1- and Th2-type antibodies were analyzed. Results revealed a spectrum of requirements for cytokine-mediated pathways in the potentiation of immunogenicity, and such requirements were influenced by interactions among individual components of the adjuvant formulations. One adjuvant strictly depended on IFN-γ to induce appreciable levels of anti-MSP1-19 antibodies, while some formulations required IFN-γ only for the induction of Th1-type antibodies. Other formulations induced exclusively Th2-type antibodies and were not affected by IFN-γ knockout. There were three patterns of requirements for IL-4 by various adjuvants in the induction of Th2-type anti-MSP1-19 antibodies. Moreover, the induction of Th1-type anti-MSP1-19 antibodies by adjuvants showed two distinct patterns of regulation by IL-4. The utilization of an IL-4 regulated pathway(s) for the induction of Th2-type antibodies by the same adjuvant differed between mouse strains, suggesting that animal species variability in responses to vaccine adjuvants may be due, at least in part, to differences in the utilization of immune system pathways by an adjuvant among animal hosts.
Invasion of Aortic and Heart Endothelial Cells by
Porphyromonas gingivalis
Authors: Deshpande Rajashri G. , Khan Mahfuz B. , Attardo Genco Caroline
Published: December 2019

Invasion of host cells is believed to be an important strategy utilized by a number of pathogens, which affords them protection from the host immune system. The connective tissues of the periodontium are extremely well vascularized, which allows invading microorganisms, such as the periodontal pathogen
Porphyromonas gingivalis
, to readily enter the bloodstream. However, the ability of
P. gingivalis
to actively invade endothelial cells has not been previously examined. In this study, we demonstrate that
P. gingivalis
can invade bovine and human endothelial cells as assessed by an antibiotic protection assay and by transmission and scanning electron microscopy.
P. gingivalis
A7436 was demonstrated to adhere to and to invade fetal bovine heart endothelial cells (FBHEC), bovine aortic endothelial cells (BAEC), and human umbilical vein endothelial cells (HUVEC). Invasion efficiencies of 0.1, 0.2, and 0.3% were obtained with BAEC, HUVEC, and FBHEC, respectively. Invasion of FBHEC and BAEC by
P. gingivalis
A7436 assessed by electron microscopy revealed the formation of microvillus-like extensions around adherent bacteria followed by the engulfment of the pathogen within vacuoles. Invasion of BAEC by
P. gingivalis
A7436 was inhibited by cytochalasin D, nocodazole, staurosporine, protease inhibitors, and sodium azide, indicating that cytoskeletal rearrangements, protein phosphorylation, energy metabolism, and
P. gingivalis
proteases are essential for invasion. In contrast, addition of rifampin, nalidixic acid, and chloramphenicol had little effect on invasion, indicating that bacterial RNA, DNA, and de novo protein synthesis are not required for
P. gingivalis
invasion of endothelial cells. Likewise de novo protein synthesis by endothelial cells was not required for invasion by
P. gingivalis. P. gingivalis
381 was demonstrated to adhere to and to invade BAEC (0.11 and 0.1% efficiency, respectively). However, adherence and invasion of the corresponding
fimA
mutant DPG3, which lacks the major fimbriae, was not detected. These results indicate that
P. gingivalis
can actively invade endothelial cells and that fimbriae are required for this process.
P. gingivalis
invasion of endothelial cells may represent another strategy utilized by this pathogen to thwart the host immune response.
The Repertoire ofAnaplasma marginaleAntigens Recognized by CD4+T-Lymphocyte Clones from Protectively Immunized Cattle Is Diverse and Includes Major Surface Protein 2 (MSP-2) and MSP-3
Authors: Brown Wendy C. , Zhu Daming , Shkap Varda , McGuire Travis C. , Blouin Edmour F. , Kocan Katherine M. , Palmer Guy H.
Published: December 2019

Major surface proteins ofAnaplasma marginaleare vaccine candidates. We recently demonstrated that immunization of calves with outer membranes of the Florida strain ofA. marginaleresulted in protective immunity that correlated with a memory CD4+T-lymphocyte response specific for major surface protein 1 (MSP-1), MSP-2, and MSP-3 (W. C. Brown, V. Shkap, D. Zhu, T. C. McGuire, W. Tuo, T. F. McElwain, and G. H. Palmer, Infect. Immun. 66:5406–5413, 1998). As immunogens, these proteins have been shown to induce complete or partial protection against homologous challenge. To further define the T helper (Th) cell response to these and otherA. marginaleantigens and to determine conservation of Th cell epitopes among genetically distinctA. marginalestrains, Th cell clones obtained prior to challenge from three immunized calves were characterized for antigen-specific responses. Nine distinct antigenic profiles were defined by 11 Th cell clones derived by stimulation with the Florida strain. Several clones responded to MSP-2, MSP-3, or both. All of these MSP-2- or MSP-3-specific clones and the majority of other clones that did not respond to MSPs recognized all bovine blood-passaged strains ofA. marginale. These results demonstrate conservation of certain Th cell epitopes between MSP-2 and MSP-3 and show that Th cell epitopes in MSP-2, MSP-3, and undefined antigens are conserved among strains ofA. marginale. Of seven clones that responded to the blood-passaged Virginia strain, two did not recognize antigen prepared from this strain cultured in tick cells, suggesting differences in the antigenic composition between these stages. Analysis of the cytokines expressed by the Th cells revealed that all clones expressed gamma interferon and tumor necrosis factor alpha, and most coexpressed interleukin-4. Our results provide a rationale for identifying Th cell epitopes conserved among different strains ofA. marginalefor inclusion in a nucleic acid or recombinant protein vaccine.
DNA and a CpG Oligonucleotide Derived fromBabesia bovisAre Mitogenic for Bovine B Cells
Authors: Brown Wendy C. , Estes D. Mark , Chantler Sue Ellen , Kegerreis Kimberly A. , Suarez Carlos E.
Published: December 2019

DNAs from bacteria and variety of nonvertebrate organisms, including nematodes, mollusks, yeasts, and insects, cause polyclonal activation of murine B lymphocytes. Similar studies have not been reported for bovine B cells, and to date no studies have reported mitogenic properties of protozoal DNA for any species. However, we and others have observed that protozoal parasite antigens can induce the proliferation of lymphocytes from nonexposed donors. Extending these studies, we now show that the mitogenic property of protozoal antigen preparations is in part attributable to parasite DNA and thatBabesia bovisDNA is directly mitogenic for bovine B cells. DNase treatment ofB. bovisextracts abrogatedB. bovis-induced proliferation of peripheral blood mononuclear cells from nonexposed cattle. Like DNAs from other organisms that were mitogenic for murine B cells,B. bovisDNA is largely nonmethylated and induced a dose-dependent proliferation of bovine B cells, which was reduced upon methylation. Furthermore,B. bovisandE. coliDNAs enhanced immunoglobulin secretion by cultured B cells, inducing moderate increases in immunoglobulin G1 and stronger increases in immunoglobulin G2. Because certain nonmethylated CpG motifs present in bacterial DNA are known to stimulate proliferation of murine and human B cells, an 11-kb fragment ofB. bovisDNA was analyzed for CG dinucleotide content and for the presence of known immunostimulatory sequences (ISS) centered on a CG motif. The frequency of CG dinucleotides was approximately one-half of the expected frequency, and several CpG hexameric sequences with known activity for murine B cells were identified. An oligodeoxynucleotide containing one of these ISS (AACGTT), which is present within the rhoptry-associated protein-1 (rap-1) open reading frame, was shown to stimulate B-cell proliferation. These ISS may be involved in host immune modulation during protozoal infection and may be useful as vaccine adjuvants.
Antrum- and Corpus Mucosa-Infiltrating CD4+Lymphocytes inHelicobacter pyloriGastritis Display a Th1 Phenotype
Authors: Sommer Frank , Faller Gerhard , Konturek Peter , Kirchner Thomas , Hahn Eckhart G. , Zeus Jürgen , Röllinghoff Martin , Lohoff Michael
Published: December 2019

In this study, cytokine patterns produced by CD4+T cells isolated from antrum or corpus gastral biopsy specimens of 10 patients withHelicobacter pylori-positive gastritis were compared. To this end, expression of intracellular cytokines (interleukin-4 [IL-4] and gamma interferon) and of CD4 was assessed by flow cytometry. Ten to 60% of the isolated CD4+T cells produced gamma interferon upon stimulation. With the exception of one patient, IL-4-positive CD4+cells were not detected. Therefore, CD4+cells infiltrating antrum and corpus stomach mucosa duringH. pyloriinfection show a Th1 phenotype. This polarized Th1-type response may contribute to the inability of the immune system to eradicateH. pyloriinfection.
The Type IV Leader Peptidase/ N -Methyltransferase of
Vibrio vulnificus
Controls Factors Required for Adherence to HEp-2 Cells and Virulence in Iron-Overloaded Mice
Authors: Paranjpye Rohinee N. , Lara J. Cano , Pepe Jeffrey C. , Pepe Cynthia M. , Strom Mark S.
Published: December 2019

Vibrio vulnificus
expresses a number of potential virulence determinants that may contribute to its ability to cause a severe and rapidly disseminating septicemia in susceptible hosts. We have cloned and characterized two genes encoding products related to components of the type IV pilus biogenesis and general secretory (type II) pathways by complementation of a type IV peptidase/ N -methyltransferase (PilD) mutant of
Pseudomonas aeruginosa
with a
V. vulnificus
genomic library. One of the genes (
vvpD
) encodes a protein homologous to PilD and other members of the type IV peptidase family that completely restores this activity in a
P. aeruginosa
mutant deficient in the expression of PilD. The other gene (
vvpC
) encodes a homolog of PilC from
P. aeruginosa
, where it is essential for assembly of type IV pili. Phenotypic characterization of a
V. vulnificus vvpD
mutant, constructed by allelic exchange, showed that VvpD is required for the expression of surface pili, suggesting that the pili observed on
V. vulnificus
are of the type IV class. This mutant was also unable to secrete at least three extracellular degradative enzymes, and the localization of one of these (the cytolysin/hemolysin) to the periplasmic space indicates that these proteins are normally exported via the type II secretion pathway. Loss of VvpD resulted in significant decreases in CHO cell cytotoxicity, adherence to HEp-2 cells, and virulence in a mouse model. Capsule formation and serum resistance were not affected in the
vvpD
mutant, indicating that in addition to capsule, virulence of
V. vulnificus
requires type IV pili and/or extracellular secretion of several exoenzymes.
Cloning and Sequencing ofyajCandsecDHomologs ofBrucella abortusand Demonstration of Immune Responses to YajC in Mice Vaccinated withB. abortusRB51
Authors: Vemulapalli Ramesh , Duncan A. Jane , Boyle Stephen M. , Sriranganathan Nammalwar , Toth Thomas E. , Schurig Gerhardt G.
Published: December 2019

To identifyBrucellaantigens that are potentially involved in stimulating a protective cell-mediated immune response, a gene library ofBrucella abortus2308 was screened for the expression of antigens reacting with immunoglobulin G2a antibodies from BALB/c mice vaccinated withB. abortusRB51. One selected positive clone (clone MCB68) contained an insert of 2.6 kb; nucleotide sequence analysis of this insert revealed two open reading frames (ORFs). The deduced amino acid sequences of the first and second ORFs had significant similarities with the YajC and SecD proteins, respectively, of several bacterial species. Both the YajC and SecD proteins were expressed inEscherichia colias fusion proteins with maltose binding protein (MBP). In Western blots, sera from mice vaccinated withB. abortusRB51 recognized YajC but not SecD. Further Western blot analysis with purified recombinant YajC protein indicated that mice inoculated withB. abortus19 or 2308 orB. melitensisRM1 also produced antibodies to YajC. In response to in vitro stimulation with recombinant MBP-YajC fusion protein, splenocytes from mice vaccinated withB. abortusRB51 were able to proliferate and produce gamma interferon but not interleukin-4. This study demonstrates, for the first time, the involvement of YajC protein in an immune response to an infectious agent.
Cloning, Expression, and Sequencing of a Cell Surface Antigen Containing a Leucine-Rich Repeat Motif from
Bacteroides forsythus
ATCC 43037
Authors: Sharma Ashu , Sojar Hakimuddin T. , Glurich Ingrid , Honma Kiyonobu , Kuramitsu Howard K. , Genco Robert J.
Published: December 2019

Bacteroides forsythus
is a recently recognized human periodontopathogen associated with advanced, as well as recurrent, periodontitis. However, very little is known about the mechanism of pathogenesis of this organism. The present study was undertaken to identify the surface molecules of this bacterium that may play roles in its adherence to oral tissues or triggering of a host immune response(s). The gene (
bspA
) encoding a cell surface-associated protein of
B. forsythus
with an apparent molecular mass of 98 kDa was isolated by immunoscreening of a
B. forsythus
gene library constructed in a lambda ZAP II vector. The encoded 98-kDa protein (BspA) contains 14 complete repeats of 23 amino acid residues that show partial homology to leucine-rich repeat motifs. A recombinant protein containing the repeat region was expressed in
Escherichia coli
, purified, and utilized for antibody production, as well as in vitro binding studies. The purified recombinant protein bound strongly to fibronectin and fibrinogen in a dose-dependent manner and further inhibited the binding of
B. forsythus
cells to these extracellular matrix (ECM) components. In addition, adult patients with
B. forsythus
-associated periodontitis expressed specific antibodies against the BspA protein. We report here the cloning and expression of an immunogenic cell surface-associated protein (BspA) of
B. forsythus
and speculate that it mediates the binding of bacteria to ECM components and clotting factors (fibronectin and fibrinogen, respectively), which may be important in the colonization of the oral cavity by this bacterium and is also a target for the host immune response.
Function and Protective Capacity ofTreponema pallidumsubsp.pallidumGlycerophosphodiester Phosphodiesterase
Authors: Cameron Caroline E. , Castro Christa , Lukehart Sheila A. , Van Voorhis Wesley C.
Published: December 2019

Infectious syphilis, caused by the spirochete bacteriumTreponema pallidumsubsp.pallidum, remains a public health concern worldwide. The immune-response evasion mechanisms employed byT. pallidumare poorly understood, and prior attempts to identify immunoprotective antigens for subsequent vaccine design have been unsuccessful. Previous investigations conducted in our laboratory identified theT. pallidumglycerophosphodiester phosphodiesterase as a potential immunoprotective antigen by using a differential immunologic expression library screen. In studies reported here, heterologous expression of theT. pallidumglycerophosphodiester phosphodiesterase inEscherichia coliyielded a full-length, enzymatically active protein. Characterization of the recombinant molecule showed it to be bifunctional, in that it exhibited specific binding to human immunoglobulin A (IgA), IgD, and IgG in addition to possessing enzymatic activity. IgG fractionation studies revealed specific binding of the recombinant enzyme to the Fc fragment of human IgG, a characteristic that may play a role in enabling the syphilis spirochete to evade the host immune response. In further investigations, immunization with the recombinant enzyme significantly protected rabbits from subsequentT. pallidumchallenge, altering lesion development at the sites of challenge. In all cases, animals immunized with the recombinant molecule developed atypical pale, flat, slightly indurated, and nonulcerative reactions at the challenge sites that resolved before lesions appeared in the control animals. Although protection in the immunized rabbits was incomplete, as demonstrated by the presence ofT. pallidumin the rabbit infectivity test, glycerophosphodiester phosphodiesterase nevertheless represents a significantly immunoprotectiveT. pallidumantigen and thus may be useful for inclusion in an antigen cocktail vaccine for syphilis.
Secreted Effector Proteins of
Salmonella dublin
Act in Concert To Induce Enteritis
Authors: Jones Michael A. , Wood Michael W. , Mullan Paul B. , Watson Patricia R. , Wallis Tim S. , Galyov Edouard E.
Published: December 2019

The ability of enteropathogenic salmonellae to recruit inflammatory cells and induce secretory responses in the infected ileum is considered to be a main feature in
Salmonella
-induced enteritis. Interactions between the pathogen and intestinal epithelial cells result in a variety of cellular responses mediating inflammation and fluid secretion. It is becoming apparent that proteins secreted by the Inv-Spa type III secretion system of
Salmonella
spp. play a key role in the induction of these responses. We have recently demonstrated that the SopB effector protein is translocated into eukaryotic cells via a Sip-dependent pathway and mediates inflammation and fluid secretion in infected ileal mucosa. However, SopB did not appear to be the only effector involved, as inactivation of the
sopB
gene only partially impaired enteropathogenicity. We suggested that at least some of such protein effectors are likely to be proteins of the same class as SopB, i.e., secreted effector proteins translocated into eukaroyotic cells via a Sip-dependent pathway. In this work, we identify SopD, another secreted protein belonging to the family of Sop effectors of
Salmonella dublin
. Using the
cya
reporter system we showed that SopD is translocated into eukaroyotic cells. We assessed the potential involvement of SopD in enteropathogenicity and found that inactivation of
sopD
has an additive effect in relation to the
sopB
mutation.
Analysis of Clinical and Environmental Strains of Nontoxigenic
Vibrio cholerae
for Susceptibility to CTXΦ: Molecular Basis for Origination of New Strains with Epidemic Potential
Authors: Faruque Shah M. , Asadulghani  , Saha Manujendra N. , Alim A. R. M. Abdul , Albert M. John , Islam K. M. Nasirul , Mekalanos John J.
Published: December 2019

Toxigenic
Vibrio cholerae
strains are lysogens of CTXΦ, a filamentous phage which encodes cholera toxin. The receptor for CTXΦ for invading
V. cholerae
cells is the toxin-coregulated pilus (TCP), the genes for which reside in a larger genetic element, the TCP pathogenicity island. We analyzed 146 CTX-negative strains of
V. cholerae
O1 or non-O1 isolated from patients or surface waters in five different countries for the presence of the TCP pathogenicity island, the regulatory gene
toxR
, and the CTXΦ attachment sequence
attRS
, as well as for susceptibility of the strains to CTXΦ, to investigate the molecular basis for the emergence of new clones of toxigenic
V. cholerae
. DNA probe or PCR assays for
tcpA
,
tcpI
,
acfB
,
toxR
, and
attRS
revealed that 6.85% of the strains, all of which belonged to the O1 serogroup, carried the TCP pathogenicity island,
toxR
, and multiple copies of
attRS
, whereas the remaining 93.15% of the strains were negative for TCP but positive for either one or both or neither of
toxR
and
attRS
. An analysis of the strains for susceptibility to CTXΦ, using a genetically marked derivative of the phage CTX-KmΦ, showed that all TCP-positive CTX-negative strains and 1 of 136 TCP-negative strains were infected by the phage either in vitro or in the intestines of infant mice. The phage genome integrated into the chromosome of infected
V. cholerae
O1 cells forming stable lysogens. Comparative analysis of rRNA gene restriction patterns revealed that the lysogens derived from nontoxigenic progenitors were either closely related to or distinctly different from previously described clones of toxigenic
V. cholerae
. To our knowledge, this is the first demonstration of lysogenic conversion of naturally occurring nontoxigenic
V. cholerae
strains by CTXΦ. The results of this study further indicated that strains belonging to the O1 serogroup of
V. cholerae
are more likely to possess the TCP pathogenicity island and hence to be infected by CTXΦ, leading to the origination of potential new epidemic clones.
Expression of the pMGA Genes of
Mycoplasma gallisepticum
Is Controlled by Variation in the GAA Trinucleotide Repeat Lengths within the 5′ Noncoding Regions
Authors: Glew Michelle D. , Baseggio Nina , Markham Philip F. , Browning Glenn F. , Walker Ian D.
Published: December 2019

We analyzed the segment of DNA which contains the expressed pMGA gene from one strain of
Mycoplasma gallisepticum
in normal (strain S6) cells and in cells in which pMGA1.1 gene expression had ceased as a consequence of in vitro culture in the presence of pMGA1.1-specific antibodies. Sequence analysis of isolates lacking pMGA1.1 expression revealed that this gene, which is typically expressed, exhibited sequence changes within a region 5′ to its promoter. Specifically, pMGA1.1
+
cells contained a (GAA)
12
motif upstream of the promoter, whereas in pMGA1.1
−
cells the corresponding region contained a (GAA)
10
motif; when such cells were grown in medium no longer containing pMGA-specific antibodies, pMGA1.1 was reexpressed and the 5′ (GAA)
12
motif was restored. Two other genes, pMGA1.9 and pMGA1.2, were also shown to acquire a (GAA)
12
motif in clones which expressed these genes. The results imply the evolution by the pMGA genes of
M. gallisepticum
of a novel transcriptional requirement which facilitates rapid and reversible switches in the pMGA expression pattern.
The Hemagglutination-Positive Phenotype ofMycoplasma synoviaeInduces Experimental Infectious Synovitis in Chickens More Frequently than Does the Hemagglutination-Negative Phenotype
Authors: Narat M. , Bencina D. , Kleven S. H. , Habe F.
Published: December 2019

Inoculation with hemagglutination-positive (HA+) cultures ofMycoplasma synoviaeAAY-4 induced acute synovitis significantly more frequently (P= 0.001) in chicken tibiotarsal-tarsometatarsal joints than did inoculation with HA-negative (HA−) cultures derived from the same clone of AAY-4. Immunoblotting analyses showed that HA+cultures abundantly expressed two phase-variable hemadherence-associated surface membrane proteins of 53 kDa and 48 to 50 kDa defined by monoclonal antibodies. HA−cultures lacked the 53-kDa proteins and synthesized truncated 27- to 30-kDa forms of the 48- to 50-kDa proteins. Inoculation of cyclosporin A (CsA) into infected joints significantly decreased the frequency of acute synovitis (P= 0.001). Moreover, repeated intra-articular inoculation of CsA (three doses of 1 mg at 2-day intervals) significantly reduced the local antibody response toM. synoviaein the joints treated with CsA.
Chlamydia trachomatis
IncA Is Localized to the Inclusion Membrane and Is Recognized by Antisera from Infected Humans and Primates
Authors: Bannantine John P. , Stamm Walter E. , Suchland Robert J. , Rockey Daniel D.
Published: December 2019

Chlamydia psittaci
produces a collection of proteins, termed IncA, IncB, and IncC, that are localized to the chlamydial inclusion membrane. In this report we demonstrate that IncA is also produced by
Chlamydia trachomatis. C. trachomatis
IncA is structurally similar to
C. psittaci
IncA and is also localized to the inclusion membrane. Immunoblot analysis demonstrated that sera from
C. trachomatis
-infected patients and from experimentally infected monkeys both recognized
C. trachomatis
IncA.
Population Structure of the Relapsing Fever Spirochete
Borrelia hermsii
as Indicated by Polymorphism of Two Multigene Families That Encode Immunogenic Outer Surface Lipoproteins
Authors: Hinnebusch B. Joseph , Barbour Alan G. , Restrepo Blanca I. , Schwan Tom G.
Published: December 2019

The tick-borne relapsing fever spirochete
Borrelia hermsii
evades the mammalian immune system by periodically switching expression among members of two multigene families that encode immunogenic, antigenically distinct outer surface proteins. The type strain,
B. hermsii
HS1, has at least 40 complete genes and pseudogenes that participate in this multiphasic antigenic variation. Originally termed
vmp
(for variable major protein) genes, they have been reclassified as
vsp
(for variable small protein) and
vlp
(for variable large protein) genes, based on size and amino acid sequence similarities. To date, antigenic variation in
B. hermsii
has been studied only in the type strain, HS1. Nucleotide sequence comparisons of 23
B. hermsii
HS1 genes revealed five distinct groups, the
vsp
gene family and four subfamilies of
vlp
genes. We used PCR with family- and subfamily-specific primers, followed by restriction fragment length polymorphism analysis, to compare the
vsp
and
vlp
repertoires of HS1 and seven other
B. hermsii
isolates from Washington, Idaho, and California. This analysis, together with pulsed-field gel electrophoresis genome profiles, revealed that the eight isolates formed three distinct groups, which likely represent clonal lineages. Members of the three groups coexisted in the same geographic area, but they could also be isolated across large geographical distances. This population structure may result from immune selection by the host, as has been proposed for other pathogens with polymorphic antigens.
Interleukin-4-Dependent Immunoglobulin G1 Isotype Switch in the Presence of a Polarized Antigen-Specific Th1-Cell Response to the Trypanosome Variant Surface Glycoprotein
Authors: Schopf Lisa R. , Filutowicz Hanna , Bi Xiao-Juan , Mansfield John M.
Published: December 2019

This study examines B-cell immunoglobulin (Ig) class-switching events in the context of parasite antigen-specific Th-cell responses in experimental African trypanosomiasis. Inbred mice were infected withTrypanosoma brucei rhodesiense, and the coordinate stimulation of Th-cell cytokine responses and B-cell responses to the trypanosome variant surface glycoprotein (VSG) was measured. The cytokines produced by T cells in response to VSG, at both the transcript and protein levels, were gamma interferon and interleukin-2 (IL-2) but not IL-4 or IL-5. Isotype profiles of antibodies specific for VSG showed that IgG1, IgG2a, and IgG3 switch responses predominated; no VSG-specific IgE responses were detected. To determine whether cryptic IL-4 responses played a role in promoting the unexpected IgG1 switch response, IL-4 knockout mice were infected; the cytokine responses and Ig isotype profiles of IL-4 knockout mice were identical to those of the wild-type control mice except for dramatically reduced IgG1 levels in response to VSG. Thus, these results revealed an IL-4-dependent component of the VSG-driven B-cell Cμ-to-Cγ1 switch. We speculate that an IL-4 response is mediated primarily by cells other than T lymphocytes since IL-4-secreting but parasite antigen-unresponsive, “background” cells were detected in all infected mice and since infectednudemice also displayed a detectable IgG1 switch response. Overall, our results suggest that B-cell clonal stimulation, maturation, and Ig class switching in African trypanosomiasis may be partially regulated by unusual mechanisms that do not include antigen-specific Th1 or Th2 cells.
Mucosal Immunogenicity of a Holotoxin-Like Molecule Containing the Serine-RichEntamoeba histolyticaProtein (SREHP) Fused to the A2Domain of Cholera Toxin
Authors: Sultan Faisal , Jin Ling-ling , Jobling Michael G. , Holmes Randall K. , Stanley Samuel L.
Published: December 2019

One strategy for the induction of mucosal immune responses by oral immunization is to administer the antigen in conjunction with cholera toxin. Cholera toxin consists of one A polypeptide (CTA) which is noncovalently linked to five B subunits (CTB) via the A2portion of the A subunit (CTA2). Coupling of antigens to the nontoxic B subunit of cholera toxin may improve the immunogenicity of antigens by targeting them to GM1 ganglioside on M cells and intestinal epithelial cells. Here, we describe the construction of a translational fusion protein containing the serine-richEntamoeba histolyticaprotein (SREHP), a protective amebic antigen, fused to a maltose binding protein (MBP) and to CTA2. When coexpressed inEscherichia coliwith the CTB gene, these proteins assembled into a holotoxin-like chimera containing MBP-SREHP-CTA2and CTB. This holotoxin-like chimera (SREHP-H) inhibited the binding of cholera toxin to GM1 ganglioside. Oral vaccination of mice with SREHP-H induced mucosal immunoglobulin A (IgA) and serum IgG antiamebic antibodies and low levels of mucosal anti-CTB antibodies. Our studies confirm that the genetic coupling of antigens to CTA2and their coexpression inE. colican produce holotoxin-like molecules that are mucosally immunogenic without the requirement for supplemental cholera toxin, and they establish the SREHP-H protein as a candidate for evaluation as a vaccine to prevent amebiasis.