bluuebunny/crossref_metadata_2025 huggingface dataset:
The Globoseries Glycosphingolipid Sialosyl Galactosyl Globoside Is Found in Urinary Tract Tissues and Is a Preferred Binding Receptor In Vitro for Uropathogenic
Escherichia coli
Expressing
pap
-Encoded Adhesins
Authors: Stapleton A. E. , Stroud M. R. , Hakomori S. I. , Stamm W. E.
Published: December 2019

Women with a history of recurrent
Escherichia coli
urinary tract infections (UTIs) are significantly more likely to be nonsecretors of blood group antigens than are women without such a history, and vaginal epithelial cells (VEC) from women who are nonsecretors show enhanced adherence of uropathogenic
E. coli
isolates compared with cells from secretors. We previously extracted glycosphingolipids (GSLs) from native VEC and determined that nonsecretors (but not secretors) selectively express two extended globoseries GSLs, sialosyl galactosyl globoside (SGG) and disialosyl galactosyl globoside (DSGG), which specifically bound uropathogenic
E. coli
R45 expressing a P adhesin. In this study, we demonstrated, by purifying the compounds from this source, that SGG and DSGG are expressed in human kidney tissue. We also demonstrated that SGG and DSGG isolated from human kidneys bind uropathogenic
E. coli
isolates expressing each of the three classes of
pap
-encoded adhesins, including cloned isolates expressing PapG from J96, PrsG from J96, and PapG from IA2, and the wild-type isolates IA2 and R45. We metabolically
35
S labeled these five
E. coli
isolates and measured their relative binding affinities to serial dilutions of SGG and DSGG as well as to globotriaosylceramide (Gb
3
) and globotetraosylceramide (Gb
4
), two other globoseries GSLs present in urogenital tissues. Each of the five
E. coli
isolates bound to SGG with the highest apparent avidity compared with their binding to DSGG, Gb
3
, and Gb
4
, and each isolate had a unique pattern of GSL binding affinity. These studies further suggest that SGG likely plays an important role in the pathogenesis of UTI and that its presence may account for the increased binding of
E. coli
to uroepithelial cells from nonsecretors and for the increased susceptibility of nonsecretors to recurrent UTI.
Infection of the Laboratory Mouse with the Intracellular PathogenEhrlichia chaffeensis
Authors: Winslow Gary M. , Yager Eric , Shilo Konstantin , Collins Doris N. , Chu Frederick K.
Published: December 2019

To determine the basis of susceptibility and resistance to human monocytic ehrlichiosis (HME), immunocompetent and immunocompromised mice were infected withEhrlichia chaffeensisand bacterial loads were measured by PCR and by immunohistochemistry. Immunocompetent (C.B-17 and C57BL/6) mice cleared the bacteria within 10 days, but immunocompromised SCID and SCID/BEIGE mice developed persistent infection in the spleen, liver, peritoneal cavity, brain, lung, and bone marrow and became moribund within 24 days. Both immunocompromised strains lack T and B lymphocytes, but the SCID/BEIGE strain is also deficient in natural killer (NK) cell function. During advanced stages of disease, the infections were associated with wasting, splenomegaly, lymphadenopathy, liver granulomas and necroses, intravascular coagulation, and granulomatous inflammation. Histochemical and immunohistochemical localization studies confirmed the presence of bacteria in tissues, and viable bacteria were cultured from infected animals. The data reveal that T and/or B cells play an essential role during resistance of immunocompetent mice to infection withE. chaffeensisand demonstrate the utility of immunocompromised mice as an experimental model for the study of HME.
Inactivation of DsbA, but Not DsbC and DsbD, Affects the Intracellular Survival and Virulence of
Shigella flexneri
Authors: Yu Jun
Published: December 2019

In this study, three mutants,
dsbA
::
kan
,
dsbC-kan
, and
dsbD-kan
, of
Shigella flexneri
serotype 5 were constructed and characterized to investigate the role of the periplasmic thiol:disulfide oxidoreductases in pathogenicity. In gentamicin protection assays and the Serény test, the
dsbA
mutant showed reduced virulence while the
dsbC
and
dsbD
mutants were similar to the wild type. That inactivation of
dsbA
was responsible for the reduced virulence was verified by complementation with the cloned wild-type gene in in vitro and in vivo assays. Despite the changed virulence behavior, the
dsbA
mutant could penetrate HeLa cells 15 min postinfection, consistent with the fact that it actively secretes Ipa proteins upon Congo red induction. Furthermore, the
dsbA
mutant was able to produce actin comets and protrusions, indicating its capacity for intra- and intercellular spread. However, a kinetic analysis of intracellular growth showed that the
dsbA
mutant barely grew in HeLa cells during a 4-h infection whereas the wild type had a doubling time of 41 min. Electron microscopy analysis revealed that
dsbA
mutant bacteria were trapped in protrusion-derived vacuoles surrounded by double membranes, resembling an
icsB
mutant reported previously. Moreover, the trapped bacteria appeared to be lysed simultaneously with the double membranes, resulting in characteristic empty vacuoles in the host cell cytosol. Thus, the attenuation mechanism for
dsbA
mutant appears to be more complicated than was previously suggested.
Effective and Long-Lasting Immunity against the ParasiteLeishmania majorin CD8-Deficient Mice
Authors: Huber Magdalena , Timms Emma , Mak Tak W. , Röllinghoff Martin , Lohoff Michael
Published: December 2019

The results of earlier investigations that tested whether CD8+T cells are required in the defense againstLeishmania majorhave been inconsistent. We used CD8-deficient mice to directly address this issue. After primary infection withL. major, CD8-deficient mice controlled the infection for over 1 year and mounted strong T helper 1 cell responses. Thus, CD8+T cells are not required for the long-term control of a primary infection withL. major.
Modulation of Endotoxin- and Enterotoxin-Induced Cytokine Release by In Vivo Treatment with β-(1,6)-Branched β-(1,3)-Glucan
Authors: Soltys Jindrich , Quinn Mark T.
Published: December 2019

Leukocytes activated by endotoxin or enterotoxins release proinflammatory cytokines, thereby contributing to the cascade of events leading to septic shock. In the present studies, we analyzed the effects of in vivo administration of a soluble immunomodulator, β-(1,6)-branched β-(1,3)-glucan (soluble β-glucan), on toxin-stimulated cytokine production in monocytes and lymphocytes isolated from treated mice. In vitro stimulation of lymphocytes isolated from soluble β-glucan-treated mice with lipopolysaccharide (LPS) resulted in enhanced production of interleukin-6 (IL-6) and suppressed production of tumor necrosis factor alpha (TNF-α), while stimulation of these cells with staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin 1 (TSST-1) resulted in enhanced production of gamma interferon (IFN-γ) and suppressed production of IL-2 and TNF-α compared to that in cells isolated from untreated mice. In vitro stimulation of monocytes isolated from soluble β-glucan-treated mice with LPS also resulted in suppressed TNF-α production, while stimulation of these cells with SEB or TSST-1 resulted in suppressed IL-6 and TNF-α production compared to that in cells isolated from untreated mice. Thus, the overall cytokine pattern of leukocytes from soluble β-glucan-treated mice reflects suppressed production of proinflammatory cytokines, especially TNF-α. Taken together, our results suggest that treatment with soluble β-glucan can modulate the induction cytokines during sepsis, resulting in an overall decrease in host mortality.
Partial Protection againstPlasmodium vivaxBlood-Stage Infection inSaimiriMonkeys by Immunization with a Recombinant C-Terminal Fragment of Merozoite Surface Protein 1 in Block Copolymer Adjuvant
Authors: Yang Chunfu , Collins William E. , Sullivan JoAnn S. , Kaslow David C. , Xiao Lihua , Lal Altaf A.
Published: December 2019

Merozoite surface protein 1 is a candidate for blood-stage vaccines against malaria parasites. We report here an immunization study ofSaimirimonkeys with a yeast-expressed recombinant protein containing the C terminus ofPlasmodium vivaxmerozoite surface protein 1 and two T-helper epitopes of tetanus toxin (yP2P30Pv20019), formulated in aluminum hydroxide (alum) and block copolymer P1005. Monkeys immunized three times with yP2P30Pv20019in block copolymer P1005 had significantly higher prechallenge titers of immunoglobulin G (IgG) antibodies against the immunogen and asexual blood-stage parasites than those immunized with yP2P30Pv20019in alum, antigen alone, or phosphate-buffered saline (PBS) (P< 0.05). Their peripheral blood mononuclear cell proliferative responses to immunogen stimulation 4 weeks after the second immunization were also significantly higher than those from the PBS control group (P< 0.05). Upon challenge with 100,000 asexual blood-stage parasites 5 weeks after the last immunization, monkeys immunized with yP2P30Pv20019in block copolymer P1005 had prepatent periods longer than those for the control alone group (P> 0.05). Three of the five animals in this group also had low parasitemia (peak parasitemia, ≤20 parasites/μl of blood). Partially protected monkeys had significantly higher levels of prechallenge antibodies against the immunogen than those unprotected (P< 0.05). There was also a positive correlation between the prepatent period and titers of IgG antibodies against the immunogen and asexual blood-stage parasites and a negative correlation between accumulated parasitemia and titers of IgG antibodies against the immunogen (P< 0.05). These results indicate that when combined with block copolymer and potent T-helper epitopes, the yeast-expressed P2P30Pv20019recombinant protein may offer some protection against malaria.
Interleukin-6 (IL-6) and IL-8 Are Induced in Human Oral Epithelial Cells in Response to Exposure to PeriodontopathicEikenella corrodens
Authors: Yumoto Hiromichi , Nakae Hideaki , Fujinaka Keiko , Ebisu Shigeyuki , Matsuo Takashi
Published: December 2019

Periodontitis is the inflammatory response in periodontal tissues elicited by bacterial colonization in periodontal pockets. In this response, pocket epithelial cells are the first cells to come into contact with bacteria. To elucidate this mechanism, we determined the adherence of the periodontopathic bacteriumEikenella corrodens1073, which has a GalNAc-sensitive lectin-like adhesin (EcLS), to a human oral epithelial carcinoma cell line (KB) and the induction of proinflammatory cytokine production in the cells following exposure to this bacterium in vitro. In the adherence assay, EcLS played a role as the adhesin of this bacterium in adherence to KB cells. In a reverse transcriptase PCR, significant interleukin-8 (IL-8) and IL-6 mRNA levels were induced in response to exposure to this bacterium. In an enzyme-linked immunosorbent assay after an 8-h bacterial exposure, the IL-8 and IL-6 protein levels were 13.5- and 8.3-fold higher than those in the nonexposed controls, respectively. These protein responses were time dependent. Interestingly, whenE. corrodenswas separated from KB cells by cell culture inserts, a slight stimulation of the IL-6 and IL-8 mRNA and secreted protein levels was seen. These results imply that the direct contact ofE. corrodens1073 with oral epithelial cells is not necessarily required for the stimulation of IL-6 and IL-8 secretion. We suggest thatE. corrodensinduces the epithelial cells to secrete proinflammatory cytokines which serve as an early signaling system to host immune and inflammatory cells in underlying connective tissues.
Detection of LiveTrypanosoma cruziin Tissues of Infected Mice by Using Histochemical Stain for β-Galactosidase
Authors: Buckner Frederick S. , Wilson Aaron J. , Van Voorhis Wesley C.
Published: December 2019

The pathogenesis of tissue damage in chronicTrypanosoma cruziinfection has been a subject of long-standing debate. Conventional staining methods reveal a paucity of parasites in tissues from chronically infected individuals, which has led to the theory that the pathologic findings may be primarily autoimmune in origin. Immunostaining forT. cruziantigens or in situ PCR methods show evidence for parasite components in chronic tissues; however, these methods do not address whether the stained material represents parasite debris or live organisms. An improved method for detecting intactT. cruziin tissues was developed by making a genetically engineered strain that expressesEscherichia coliβ-galactosidase. The expression of this enzyme allows the detection ofT. cruziin tissues by using the histochemical stain 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal). The technique was used to monitor tissue parasitism and its relation to pathologic findings in the mouse model of Chagas’ disease. Parasites were easily visible as bright blue structures in skeletal muscle, heart, bladder, peripheral nerve, liver, spleen, adrenal gland, brain, and adipose tissue in acutely infected mice. The number of viable parasites diminished >100-fold when tissues from 3-week-infected mice were compared with those from 10-month-infected mice. However, even at the lower level, parasites were clearly recognizable in sections of skeletal muscle and bladder at the 10-month time point. Inflammation remained robust in skeletal muscle, bladder, and sciatic nerve despite the near disappearance of parasites, suggesting three possibilities: exuberant host reactions to the few remaining parasites, autoimmune inflammation, or reactions to retained parasite antigens in the tissues.
Identification and Functional Characterization of the
Neisseria gonorrhoeae lbpB
Gene Product
Authors: Biswas Gour D. , Anderson James E. , Chen Ching-Ju , Cornelissen Cynthia Nau , Sparling P. Frederick
Published: December 2019

We cloned
lbpB
, encoding a predicted 80-kDa lipoprotein, upstream of
lbpA
. A nonpolar mutant (LbpB
−
LbpA
+
) had normal lactoferrin (LF) binding and grew normally with LF as an iron source, whereas LbpB
−
LbpA
−
and LbpB
+
LbpA
−
strains had reduced binding of LF and did not grow with LF as an iron source. LbpB bound LF directly in an affinity purification, suggesting that LbpB might play a still-uncharacterized role in the LF iron utilization.
Mycobacterium tuberculosisCatalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro
Authors: Manca Claudia , Paul Simon , Barry Clifton E. , Freedman Victoria H. , Kaplan Gilla
Published: December 2019

Mycobacterium tuberculosishas a relatively high resistance to killing by hydrogen peroxide and organic peroxides. Resistance may be mediated by mycobacterial catalase-peroxidase (KatG) and possibly by alkyl hydroperoxide reductase (AhpC). To determine the interrelationship between sensitivity to H2O2, catalase and peroxidase activities, and bacillary growth rates measured both intracellularly in human monocytes and in culture medium, we examined one laboratory strain, two clinical isolates, and three recombinant strains ofM. tuberculosiswith differing levels of KatG and AhpC. Five of the mycobacterial strains had intracellular doubling times of 27 to 32 h, while one KatG-deficient clinical isolate (ATCC 35825) doubled in ∼76 h. Killing of mycobacteria by exogenously added H2O2was more pronounced for intracellular bacilli than for those bacilli derived from disrupted monocytes. Strains with no detectable KatG expression or catalase activity were relatively sensitive to killing (43 to 67% killing) by exogenous H2O2. However, once even minimal catalase activity was present, mycobacterial catalase activity over a 10-fold range (0.56 to 6.2 U/mg) was associated with survival of 85% of the bacilli. Peroxidase activity levels correlated significantly with resistance of the mycobacterial strains to H2O2-mediated killing. An endogenous oxidative burst induction by 4β-phorbol 12β-myristate 13α-acetate treatment of infected monocytes reduced the viability of the KatG null strain (H37Rv Inhr) but not the KatG-overexpressing strain [H37Rv(pMH59)]. These results suggest that mycobacterial resistance to oxidative metabolites (including H2O2and other peroxides) may be an important mechanism of bacillary survival within the host phagocyte.
Transposition of the Endogenous Insertion Sequence Element IS
1126
Modulates Gingipain Expression in
Porphyromonas gingivalis
Authors: Simpson Waltena , Wang Chin-Yen , Mikolajczyk-Pawlinska Jowita , Potempa Jan , Travis James , Bond Vincent C. , Genco Caroline Attardo
Published: December 2019

We have previously reported on a Tn
4351
-generated mutant of
Porphyromonas gingivalis
(MSM-3) which expresses enhanced arginine-specific proteinase activity and does not utilize hemin or hemoglobin for growth (C. A. Genco et al., Infect. Immun. 63:2459–2466, 1995). In the process of characterizing the genetic lesion in
P. gingivalis
MSM-3, we have determined that the endogenous
P. gingivalis
insertion sequence element IS
1126
is capable of transposition within
P. gingivalis
. We have also determined that IS
1126
transposition modulates the transcription of the genes encoding the lysine-specific proteinase, gingipain K (
kgp
) and the arginine-specific proteinase, gingipain R2 (
rgpB
). Sequence analysis of
P. gingivalis
MSM-3 revealed that Tn
4351
had inserted 60 bp upstream of the
P. gingivalis
endogenous IS element IS
1126
. Furthermore,
P. gingivalis
MSM-3 exhibited two additional copies of IS
1126
compared to the parental strain A7436. Examination of the first additional IS
1126
element, IS
1126
1
, indicated that it has inserted into the putative promoter region of the
P. gingivalis kgp
gene. Analysis of total RNA extracted from
P. gingivalis
MSM-3 demonstrated no detectable
kgp
transcript; likewise,
P. gingivalis
MSM-3 was devoid of lysine-specific proteinase activity. The increased arginine-specific proteinase activity exhibited by
P. gingivalis
MSM-3 was demonstrated to correlate with an increase in the
rgpA
and
rgpB
transcripts. The second additional IS
1126
element, IS
1126
2
, was found to have inserted upstream of a newly identified gene,
hmuR
, which exhibits homology to a number of TonB-dependent genes involved in hemin and iron acquisition. Analysis of total RNA from
P. gingivalis
MSM-3 demonstrated that
hmuR
is transcribed, indicating that the insertion of IS
1126
had not produced a polar effect on
hmuR
transcription. The hemin-hemoglobin defect in
P. gingivalis
MSM-3 is proposed to result from the inactivation of Kgp, which has recently been demonstrated to function in hemoglobin binding. Taken together, the results presented here demonstrate that the introduction of Tn
4351
into the
P. gingivalis
chromosome has resulted in two previously undocumented phenomena in
P. gingivalis
: (i) the transposition of the endogenous insertion sequence element IS
1126
and (ii) the modulation of gingipain transcription and translation as a result of IS
1126
transposition.
Local and Systemic Neutralizing Antibody Responses Induced by Intranasal Immunization with the Nontoxic Binding Domain of Toxin A from
Clostridium difficile
Authors: Ward Stephen J. , Douce Gill , Dougan Gordon , Wren Brendan W.
Published: December 2019

Fourteen of the 38 C-terminal repeats from
Clostridium difficile
toxin A (14CDTA) were cloned and expressed either with an N-terminal polyhistidine tag (14CDTA-HIS) or fused to the nontoxic binding domain from tetanus toxin (14CDTA-TETC). The recombinant proteins were successfully purified by bovine thyroglobulin affinity chromatography. Both
C. difficile
toxin A fusion proteins bound to known toxin A ligands present on the surface of rabbit erythrocytes. Intranasal immunization of BALB/c mice with three separate 10-μg doses of 14CDTA-HIS or -TETC generated significant levels of anti-toxin A serum antibodies compared to control animals. The coadministration of the mucosal adjuvant heat labile toxin (LT) from
Escherichia coli
(1 μg) significantly increased the anti-toxin A response in the serum and at the mucosal surface. Importantly, the local and systemic antibodies generated neutralized toxin A cytotoxicity. Impressive systemic and mucosal anti-toxin A responses were also seen following coadministration of 14CDTA-TETC with LTR72, an LT derivative with reduced toxicity which shows potential as a mucosal adjuvant for humans.
In Vivo Distribution ofHelicobacter felisin the Gastric Mucus of the Mouse: Experimental Method and Results
Authors: Schreiber Sören , Stüben Manuela , Josenhans Christine , Scheid Peter , Suerbaum Sebastian
Published: December 2019

We describe a method that permits the collection of very small samples (2 nl) from precisely defined positions within the gastric mucus of anesthetized mice. This method was used to study the in vivo local distribution of bacteria within the mucus ofHelicobacter felis-infected mice. A total of 200 samples from 40 mice were analyzed. Each sample was microscopically analyzed, within less than 1 min, as a native preparation. To avoid changes in bacterial location within the mucus after collection and to improve the counting accuracy, bacterial motility was blocked by adjusting the pH inside the collecting pipette to 4.5. The mucus in a collected sample was subdivided into three layers, an epithelial layer (the first 25 μm of mucus from the tissue-mucus interface), a luminal layer (the last 25 μm to the mucus-lumen interface), and the remaining central mucus layer. The volume of the analyzed segments in the sample was between 4 and 9 pl. The concentration of bacteria inside the epithelial mucus layer was 3,400 per nl, but it was only 50 per nl inside the central mucus layer. The mean distance ofH. felisto the epithelial surface was 16 μm. A total of 75% of allH. felisbacteria resided in the mucus zone between 5 and 20 μm from the tissue surface, with no bacteria closer than 5 μm to the epithelial surface. This method permits the study of factors determining the density of colonization and distribution of bacteria along chemical gradients with a high precision.
Immune Responses Induced by Gene Gun or Intramuscular Injection of DNA Vaccines That Express Immunogenic Regions of the Serine Repeat Antigen fromPlasmodium falciparum
Authors: Belperron Alexia A. , Feltquate David , Fox Barbara A. , Horii Toshihiro , Bzik David J.
Published: December 2019

The liver- and blood-stage-expressed serine repeat antigen (SERA) ofPlasmodium falciparumis a candidate protein for a human malaria vaccine. We compared the immune responses induced in mice immunized with SERA-expressing plasmid DNA vaccines delivered by intramuscular (i.m.) injection or delivered intradermally by Gene Gun immunization. Mice were immunized with a pcdna3 plasmid encoding the entire 47-kDa domain of SERA (amino acids 17 to 382) or the N-terminal domain (amino acids 17 to 110) of SERA. Minimal antibody responses were detected following DNA vaccination with the N-terminal domain of SERA, suggesting that the N-terminal domain alone is not highly immunogenic by this route of vaccine delivery. Immunization of mice by Gene Gun delivery of the 47-kDa domain of SERA elicited a significantly higher serum antibody titer to the antigen than immunization of mice by i.m. injection with the same plasmid did. The predominant isotype subclass of the antibodies elicited to the SERA protein following i.m. and Gene Gun immunizations with SERA plasmid DNA was immunoglobulin G1. Coimmunization of mice with SERA plasmid DNA and a plasmid expressing the hepatitis B surface antigen (pCMV-s) by the i.m. route resulted in higher anti-SERA titers than those generated in mice immunized with the SERA DNA plasmid alone. Vaccination with DNA may provide a viable alternative or may be used in conjunction with protein-based subunit vaccines to maximize the efficacy of a human malaria vaccine that includes immunogenic regions of the SERA protein.
Induction of Cytokine Synthesis by Flagella from Gram-Negative Bacteria May Be Dependent on the Activation or Differentiation State of Human Monocytes
Authors: Ciacci-Woolwine Federica , McDermott Patrick F. , Mizel Steven B.
Published: December 2019

We have previously demonstrated that salmonellae, but notEscherichia coliorYersinia enterocolitica, stimulates tumor necrosis factor alpha (TNFα) production in the human promonocytic cell line U38. Subsequent analysis revealed that the TNFα-inducing activity of salmonellae was associated with flagellin, a major component of flagella from gram-negative bacteria. In the present study, we have explored the basis for the apparent specificity of action ofSalmonellaflagella on TNFα expression in U38 cells and have extended this analysis to normal human peripheral blood mononuclear cells (PBMC). Flagella from the enteropathogenicE. colistrain E2348/69,Y. enterocoliticaJB580, andPseudomonas aeruginosaPAO1, which did not induce significant levels of TNFα production in U38 cells, were as potent asSalmonellaflagella in terms of TNFα and interleukin 1β activation in PBMC. However, TNFα production in U38 cells was greatly enhanced when these cells were stimulated with flagella fromE. coli,Y. enterocolitica, andP. aeruginosain the presence of a costimulant, phorbol 13-myristate acetate. These findings are consistent with the hypothesis that the activation or differentiation state of a monocyte may have a substantial effect on the cell’s responsiveness to flagellum stimulation of cytokine synthesis. Furthermore, these results indicate that cytokine induction in monocytes may be a general property of flagella from gram-negative bacteria.
P2Z-Independent and P2Z Receptor-Mediated Macrophage Killing byPseudomonas aeruginosaIsolated from Cystic Fibrosis Patients
Authors: Zaborina Olga , Misra Namita , Kostal Jan , Kamath Shilpa , Kapatral Vinayak , El-Idrissi M. El-Azami , Prabhakar B. S. , Chakrabarty A. M.
Published: December 2019

We demonstrate that a mucoid, alginate-producing strain ofPseudomonas aeruginosaisolated from the lungs of a cystic fibrosis (CF) patient secretes multiple enzymes with nucleoside diphosphate kinase (Ndk), ATPase, adenylate kinase, 5′-nucleotidase, and ATP-modifying enzymatic activities. The secretion is triggered at high cell density and in complex media but is greatly reduced when the mucoid cells are grown in mineral salts media or in presence of 5.0 mM Ca2+or Mg2+. Interestingly, the secretion is triggered primarily in the mucoid CF isolate of strain 8821M (or in strain FRD1) but not in a nonmucoid laboratory strain, PAO1. The purified secreted Ndk shows 100% match in its N-terminal amino acid sequence with that of purified intracellular Ndk and demonstrates similar enzymatic properties. The N-terminal sequence of the purified ATPase isolated from anndkknockout mutant shows its identity with that of the heat shock chaperonin Hsp60. During fractionation, the flowthrough fraction from a Mono Q column demonstrates the presence of 5′-nucleotidase, adenylate kinase, and a putative ATP reductase activity. These fractions demonstrate high cytotoxic activities for murine peritoneal primary macrophages which can be further stimulated in the presence of ATP or inhibited by pretreatment of macrophages with oxidized ATP (oATP). The cytotoxicity associated with ATP-induced stimulation is believed to be due to activation of macrophage surface-associated P2Z (P2X7) receptors, which are one of the purinergic receptors responsible for pore formation on macrophage membrane. Blocking of these receptors by pretreatment with oATP blocks ATP-induced macrophage cell death. Thus mucoidP. aeruginosacells elaborate enzymes that modulate the external ATP levels of macrophages, thereby modulating macrophage cell death through P2Z receptor activation. Evidence for the presence of secreted cytotoxic agents that act independently of P2Z receptor activation is also presented.
Tyrosine Phosphatase SHP-1 Is Involved in CD66-Mediated Phagocytosis of Opa52-ExpressingNeisseria gonorrhoeae
Authors: Hauck Christof R. , Gulbins Erich , Lang Florian , Meyer Thomas F.
Published: December 2019

Opa proteins ofNeisseria gonorrhoeaebind to CD66 receptors on human phagocytes, thereby inducing efficient uptake of the bacteria in the absence of opsonins. The interaction of Opa proteins and CD66 receptors leads to activation of Src family tyrosine kinases, a process that is of critical importance for the efficient, CD66-mediated internalization. Here we show that during Opa-mediated stimulation of CD66 the activity of the host cell tyrosine phosphatase SHP-1 is strongly downregulated, concomitant with increases in the tyrosine phosphorylation of several cellular proteins. Since the SHP-1 tyrosine phosphorylation level itself is influenced by Opa-induced events, this phosphatase comprises an important regulatory checkpoint of the pathogen-triggered signaling cascade in human phagocytes.
Rational Live Oral Carrier Vaccine Design by Mutating Virulence-Associated Genes of
Yersinia enterocolitica
Authors: Igwe Emeka I. , Rüssmann Holger , Roggenkamp Andreas , Noll Annette , Autenrieth Ingo B. , Heesemann J.
Published: December 2019

Three different
Yersinia enterocolitica
serotype O8 strains harboring mutations in virulence-associated genes coding for
Yersinia
adhesin A (YadA), Mn-cofactored superoxide dismutase (SodA), and high-molecular-weight protein 1 were analyzed for their ability to colonize and persist in tissues after orogastric immunization of C57BL/6 mice. We demonstrated that all three
Yersinia
mutant strains were markedly impaired in their ability to disseminate into the spleens and livers of immunized mice but were able to colonize the Peyer’s patches for at least 12 days, resulting in the induction of significant antibody titers against
Yersinia
outer proteins (Yops) and in the priming of
Yersinia
antigen-specific CD4
+
Th1 cells isolated from spleens. The high level of attenuation did not diminish the immunogenic properties of the mutant strains. In fact, mice immunized with a single oral dose of any of the mutant strains were protected against a lethal oral-challenge infection with wild-type
Y. enterocolitica
. Moreover, adoptive transfer of
Yersinia
-specific antibodies from sera of mice immunized with the mutant WAP-314
sodA
revealed that this protection could be mediated by
Yersinia
-specific immunoglobulins.
Differentiation of Monocytes to Macrophages Primes Cells for Lipopolysaccharide Stimulation via Accumulation of Cytoplasmic Nuclear Factor κB
Authors: Takashiba Shogo , Van Dyke Thomas E. , Amar Salomon , Murayama Yoji , Soskolne Aubrey W. , Shapira Lior
Published: December 2019

During infection, circulating blood monocytes migrate from the vasculature to the extravascular compartments where they mature into tissue macrophages. The maturation process prepares the cell to actively participate in the inflammatory and the immune responses, and many transcription factors have been found to be involved. Here we report on a novel role for nuclear factor κB (NF-κB) in this process. Its accumulation in the cytoplasm of differentiated macrophages is responsible for the enhanced ability of the cell to respond to lipopolysaccharide (LPS) stimulation, as determined by tumor necrosis factor alpha (TNF-α) secretion. Differentiation of the human monocytic cell line THP-1 into macrophage-like cells was induced by exposure of the cells to phorbol myristate acetate. DNA-bindable NF-κB was not detected in the cytoplasm of undifferentiated THP-1 cells but accumulated in the cytoplasm of the cells following differentiation. No TNF-α was detected in the media of resting differentiated and nondifferentiated THP-1 cells. Stimulation with LPS of differentiated cells induced the production of higher levels of TNF-α than stimulation of nondifferentiated cells. This hyperresponsiveness to LPS was found in the mRNA and secreted TNF-α levels. Furthermore, stimulation with LPS induced the translocation of NF-κB from the cytoplasm into the nucleus. This translocation process was more rapid in the differentiated cells than in the nondifferentiated cells, and the resultant accumulated levels of NF-κB in the nucleus were higher. The DNA-bindable NF-κB was identified as a heterodimer of p65 and p50. The results suggest that NF-κB accumulation in the cytoplasm during maturation of monocytes to macrophages primes the cells for enhanced responsiveness to LPS and results in the rapid secretion of inflammatory mediators, such as TNF-α, by mature macrophages following LPS challenge.
Endothelial Adhesion Molecule Expression and Its Inhibition by Recombinant Bactericidal/Permeability-Increasing Protein Are Influenced by the Capsulation and Lipooligosaccharide Structure ofNeisseria meningitidis
Authors: Dixon Garth L. J. , Heyderman Robert S. , Kotovicz Karolena , Jack Dominic L. , Andersen Svein R. , Vogel Ulrich , Frosch Matthias , Klein Nigel
Published: December 2019

Vascular endothelial injury is responsible for many of the clinical manifestations of severe meningococcal disease. Binding and migration of activated host inflammatory cells is a central process in vascular damage. The expression and function of adhesion molecules regulate interactions between leukocytes and endothelial cells. Little is known about how meningococci directly influence these receptors. In this study we have explored the effect ofNeisseria meningitidison endothelial adhesion molecule expression and found this organism to be a potent inducer of the adhesion molecules CD62E, ICAM-1, and VCAM-1. Exposure of endothelium to a serogroup B strain ofNeisseria meningitidis, B1940, and a range of isogenic mutants revealed that lipooligosaccharide (LOS) structure and capsulation influence the expression of adhesion molecules. Following only a brief exposure (15 min) to the bacteria, there were large differences in the capacity of the different mutants to induce vascular cell adhesion molecules, with the unencapsulated and truncated LOS strains being most potent (P< 0.05). Furthermore, the pattern of cell adhesion molecule expression was different with purified endotoxin from that with intact bacteria. Meningococci were more potent stimuli of CD62E expression than was endotoxin, whereas endotoxin was at least as effective as meningococci in inducing ICAM-1 and VCAM-1. The effect of bactericidal/permeability increasing protein (rBPI21), an antibacterial molecule with antiendotoxin properties, was also dependent on LOS structure. The strains which possessed a truncated or nonsialylated LOS, whether capsulated or not, were more sensitive to the inhibitory effects of rBPI21. These findings could have important implications for the use of antiendotoxin therapy in meningococcal disease.
Monoclonal Antibody to Shiga Toxin 2 Which Blocks Receptor Binding and Neutralizes Cytotoxicity
Authors: Nakao Hiroshi , Kiyokawa Nobutaka , Fujimoto Junichiro , Yamasaki Shinji , Takeda Tae
Published: December 2019

A monoclonal antibody (MAb) was raised against Shiga toxin 2 (Stx2) of
Escherichia coli
O157:H7. MAb VTm1.1 belonged to the immunoglobulin G1 subclass and had a κ light chain, and it could neutralize the cytotoxic activity of Stx2 and variants derived from patient strains but not that of variants derived from animals. MAb VTm1.1 was shown to bind to the B subunit of these neutralized Stx2s by Western blotting. Comparison of B-subunit amino acid sequences and reactivities to these Stxs suggested six amino acids (Ser30, Ser53, Glu56, Gln65, Asn68, and Asp69) that were candidates for the MAb VTm1.1 epitope. Consequently, five Stx2 mutants (S30N, S53N, E56H, Q65K, and N68Ter) were prepared by site-directed mutagenesis to determine which residue is essential for the epitope. All of these mutants showed cytotoxicity almost equal to that of the wild-type Stx2. Of the five Stx2 mutants, only E56H could not be neutralized by MAb VTm1.1. Western blot analysis also showed that MAb VTm1.1 could not bind to the E56H B subunit. These results indicated that Glu56 is an important residue recognized by MAb VTm1.1. Immunofluorescence analysis further indicated that MAb VTm1.1 inhibits the binding of Stx2 to its receptors. MAb VTm1.1 could be a useful therapeutic agent for Shiga toxin-producing
E. coli
infection.
Emergence of
Anaplasma marginale
Antigenic Variants during Persistent Rickettsemia
Authors: French Dorothy M. , Brown Wendy C. , Palmer Guy H.
Published: December 2019

Anaplasma marginale
is an ehrlichial pathogen of cattle, in the order
Rickettsiales
, that establishes persistent cyclic rickettsemia in the infected host. Within each rickettsemic cycle,
A. marginale
expressing antigenically variant major surface protein 2 (MSP2) emerge. By cloning 17 full-length
msp2
transcripts expressed during cyclic rickettsemia, we determined that emergent variants have a single, central hypervariable region encoding variant B-cell epitopes. The N- and C-terminal regions are highly conserved among the expressed
A. marginale
variants, and similar sequences define the MSP2 homologues in the agent of human granulocytic ehrlichiosis (HGE). This is in contrast to the MSP2 homologues in ehrlichial genogroup I pathogens,
Ehrlichia chaffeensis
,
Ehrlichia canis
, and
Cowdria ruminantium
, that have multiple hypervariable regions. By defining the variable and conserved regions, we were able to show that the single hypervariable region of
A. marginale
MSP2 encodes epitopes that are immunogenic and induce variant-specific antibody responses during persistent infection. These findings demonstrate that the MSP2 structural variants that emerge during each cycle of persistent rickettsemia are true antigenic variants, consistent with MSP2 antigenic variation as a mechanism of
A. marginale
persistence.
Targeted Salivary Gland Immunization with Plasmid DNA Elicits Specific Salivary Immunoglobulin A and G Antibodies and Serum Immunoglobulin G Antibodies in Mice
Authors: Kawabata Shigetada , Terao Yutaka , Fujiwara Taku , Nakagawa Ichiro , Hamada Shigeyuki
Published: December 2019

For the development of vaccines against oral and pharyngeal pathogens invading the mucosal epithelia, both secretory and serum immunoglobulin A (IgA) and IgG antibodies and cytotoxic T lymphocytes (CTL) have been induced. We used a novel approach, targeted salivary gland (TSG) immunization, using plasmid pcDNA3/fimA, coding forPorphyromonas gingivalisfimbriae. Expression of subunit protein, fimbrillin, was observed in eukaryotic cells growing in vitro following transfection with pcDNA3/fimA. In this study, we obtained good humoral and cell-mediated immune responses in BALB/c mice by TSG administration using the above-mentioned DNA vaccine. The production of fimbria-specific IgA and IgG antibodies in saliva and serum IgG antibody was significantly stimulated by TSG immunization. Injection of DNA vaccine into salivary gland elicited high-level production of antigen-specific IgG antibody, similar to that induced following intramuscular immunization. The major IgG subclass that recognized fimbriae was IgG2a in serum from pcDNA3/fimA-immunized mice. Reverse transcription-PCR analysis of mononuclear cells from salivary glands showed that levels of Th2 cytokine-specific mRNA were higher in the immunized group than in the nonimmunized group. In addition, TSG DNA immunization resulted in the generation of antigen-specific CTL in spleen. These results indicate that TSG immunization with plasmid DNA may represent a genetic immunization strategy against infection by oral and pharyngeal pathogens that may invade local, mucosal surfaces.
A DNA Sequence Corresponding to the Gene Encoding Cysteine Proteinase 5 in
Entamoeba histolytica
Is Present and Positionally Conserved but Highly Degenerated in
Entamoeba dispar
Authors: Willhoeft Ute , Hamann Lutz , Tannich Egbert
Published: December 2019

Cysteine proteinases of
Entamoeba histolytica
are considered to be one of the most important classes of molecules responsible for the parasite's ability to destroy human tissues. Interestingly, one particular cysteine proteinase, located on the surface of
E. histolytica
trophozoites and designated cysteine proteinase 5 (CP5), is not expressed in the closely related but nonpathogenic species
Entamoeba dispar
. By comparing the
E. histolytica
and
E. dispar
genomic loci containing the gene for CP5 (
cp5
), it was found that the position of
cp5
within the genomic context is conserved between the two organisms, but that the gene is highly degenerated in
E. dispar
, as it contains numerous nucleotide exchanges, insertions, and deletions, resulting in multiple stop codons within the
cp5
reading frame. An alignment of all available orthologous
E. histolytica
and
E. dispar
DNA sequences suggested that
cp5
started to degenerate in
E. dispar
coincidently when the two organisms began to diverge from a common ancestor.
Importance of B cells, but Not Specific Antibodies, in Primary and Secondary Protective Immunity to the Intracellular BacteriumFrancisella tularensisLive Vaccine Strain
Authors: Elkins Karen L. , Bosio Catharine M. , Rhinehart-Jones Tonya R.
Published: December 2019

Although there appears to be little if any role for specific antibodies in protection against intracellular bacteria, such as the model pathogenF. tularensislive vaccine strain (LVS), the role of B cells themselves in primary and secondary infection with such bacteria has not been examined directly. We show here that mice deficient in mature B cells and antibodies (B-cell knockout mice) are marginally compromised in controlling primary sublethal infection but are 100-fold less well protected against secondary lethal challenge than are their normal counterparts. This defect in optimal specific protective immunity was readily reconstituted by the transfer of primed, and to a lesser degree, unprimed B cells, but not by the transfer of specific antibodies. The results indicate a previously unappreciated role for B cells in secondary immunity to intracellular pathogens through a function other than antibody production.
Laccase ProtectsCryptococcus neoformansfrom Antifungal Activity of Alveolar Macrophages
Authors: Liu Lide , Tewari Ram P. , Williamson Peter R.
Published: December 2019

While laccase ofCryptococcus neoformansis implicated in the virulence of the organism, our recent studies showing absence of melanin in the infected mouse brain has led us to a search for alternative roles for laccase in cryptococcosis. We investigated the role of laccase in protection ofC. neoformansagainst murine alveolar macrophage (AM)-mediated antifungal activity by using a pair of congenic laccase-positive (2E-TUC) and laccase-deficient (2E-TU) strains. The laccase-positive cells with laccase derepression were more resistant to the antifungal activity of AM than a laccase-deficient strain ([28.9 ± 1.2]% versus [40.2 ± 2.6]% killing). Addition ofl-dopa toCryptococcusto produce melanin in a laccase-positive strain resulted in a slight increase in protection ofC. neoformansfrom the antifungal activity of macrophages ([25.4 ± 3.4]% versus [28.9 ± 1.2]% killing). Recombinant cryptococcal laccase exhibited iron oxidase activity in converting Fe(II) to Fe(III). Moreover, recombinant laccase inhibited killing ofC. neoformansby hydroxyl radicals catalyzed by iron in a cell-free system. Addition of the hydroxyl radical scavenger mannitol or dimethyl sulfoxide to AMs prior to the introduction of cryptococcal cells decreased killing of both strains and reduced the difference in susceptibility between the laccase-positive and laccase-deficient strains. Furthermore, laccase-mediated protection from AM killing was inhibited by the addition of Fe(II), presumably by overcoming the effects of the iron oxidase activity of cryptococcal laccase. These results suggest that the iron oxidase activity of laccase may protectC. neoformansfrom macrophages by oxidation of phagosomal iron to Fe(III) with a resultant decrease in hydroxyl radical formation.
Effect of
mutS
and
recD
Mutations on
Salmonella
Virulence
Authors: Zahrt Thomas C. , Buchmeier Nancy , Maloy Stanley
Published: December 2019

Hybrid derivatives of closely related bacteria may be used to dissect strain-specific functions that contribute to virulence within a host. However, mismatches between DNA sequences are a potent barrier to recombination. Recipients with
mutS
and
recD
mutations overcome this barrier, allowing construction of genetic hybrids. To determine whether
Salmonella
hybrids constructed in a
mutS recD
host can be used to study virulence, we assayed the effect of
mutS
and
recD
mutations on the virulence of
Salmonella typhimurium
14028s in mice. Mutants defective in either
mutS
or
recD
do not affect the time course or the 50% lethal dose (LD
50
) of the infection. In contrast, the inactivation of both
mutS
and
recD
results in a synthetic phenotype which substantially increases the time required to cause a lethal infection without changing the LD
50
. This phenotype results from an inability of
mutS recD
double mutants to rapidly adapt to purine-limiting conditions present within macrophages. Although the disease progression is slower,
S. typhimurium mutS recD
mutants retain the ability to cause lethal infections, and, thus, hybrids constructed in
mutS recD
hosts may permit the analysis of virulence factors in a surrogate animal model.
Homotypic and Heterotypic Antibody Responses to a 56-Kilodalton Protein of
Orientia tsutsugamushi
Authors: Choi Myung-Sik , Seong Seung-Yong , Kang Jae-Seung , Kim Yun-Won , Huh Myung-Suk , Kim Ik-Sang
Published: December 2019

We analyzed homotypic and heterotypic antibody responses to a type-specific antigen (Tsa), a 56-kDa protein of
Orientia tsutsugamushi
, by using sera from mice immunized with strains Gilliam, Karp, Kato, and Boryong. We generated a series of deletion constructs of the
tsa
gene and expressed them as MalE fusion proteins. Variable domain I (VD I) showed strong responses to homotypic antibodies. Antigenic domain II (AD II) from Boryong and Karp showed cross-reactivities to each other. VD III showed no responses to any of the antibodies. Sera from Kato-immunized mice showed only homotypic responses to AD III. On the other hand, sera of the mice immunized with Gilliam, Karp, or Boryong showed homotypic as well as heterotypic responses to this region. VD IV showed the strongest heterotypic antibody responses among the fragments tested. These data suggest that VD I is important in homotypic antibody responses and that AD II, AD III, and VD IV are important in heterotypic antibody responses of the mice to Tsa.
P48 Major Surface Antigen of
Mycoplasma agalactiae
Is Homologous to a
malp
Product of
Mycoplasma fermentans
and Belongs to a Selected Family of Bacterial Lipoproteins
Authors: Rosati Sergio , Pozzi Sarah , Robino Patrizia , Montinaro Barbara , Conti Amedeo , Fadda Manlio , Pittau Marco
Published: December 2019

A major surface antigenic lipoprotein of
Mycoplasma agalactiae
, promptly recognized by the host's immune system, was characterized. The mature product, P48, showed significant similarity and shared conserved amino acid motifs with lipoproteins or predicted lipoproteins from
Mycoplasma fermentans
,
Mycoplasma hyorhinis
, relapsing fever
Borrelia
spp.,
Bacillus subtilis
, and
Treponema pallidum
.