bluuebunny/crossref_metadata_2025_split huggingface dataset:
Major Antigenic Proteins of the Agent of Human Granulocytic Ehrlichiosis Are Encoded by Members of a Multigene Family
Authors: Murphy Cheryl I. , Storey James R. , Recchia Joanne , Doros-Richert Linda A. , Gingrich-Baker Cindy , Munroe Kenneth , Bakken Johan S. , Coughlin Richard T. , Beltz Gerald A. 
Published: December 2019

ABSTRACT

Western blot analysis of proteins from a cell culture isolate (USG3) of the human granulocytic ehrlichiosis (HGE) agent has identified a number of immunoreactive proteins, including major antigenic proteins of 43 and 45 kDa. Peptides derived from the 43- and 45-kDa proteins were sequenced, and degenerate PCR primers based on these sequences were used to amplify DNA from USG3. Sequencing of a 550-bp PCR product revealed that it encodes a protein homologous to the MSP-2 proteins of
Anaplasma marginale
. Concurrently, an expression library made from USG3 genomic DNA was screened with granulocytic
Ehrlichia
(GE)-positive immune sera. Analysis of two clones showed that they contain one partial and three full-length highly related genes, suggesting that they are part of a multigene family. Amino acid alignment showed conserved amino- and carboxy-terminal regions which flank a variable region. The conserved regions of these proteins are also homologous to the MSP-2 proteins of
A. marginale
; thus, they were designated GE MSP-2A (45 kDa), MSP-2B (34 kDa), and MSP-2C (38 kDa). The PCR fragment obtained as a result of peptide sequencing was completely contained within the
msp-2A
clone, and all of the sequenced peptides were found in the GE MSP-2 proteins. Recombinant MSP-2B protein and an MSP-2A fusion protein were expressed in
Escherichia coli
and reacted with human sera positive for the HGE agent by immunofluorescence assay. These data suggest that the 43- and 45-kDa proteins of the HGE agent are encoded by members of the GE MSP-2 multigene family.
Only Viable Parasites Are Detected by PCR following Clearance of Rodent Malarial Infections by Drug Treatment or Immune Responses
Authors: Jarra William , Snounou Georges 
Published: December 2019

ABSTRACT

Detection and analysis of pathogens by PCR plays an important role in infectious disease research. The value of these studies would be diminished if nuclear material from dead parasites were found to remain in circulation for extended periods and thus result in positive amplification. This possibility was tested in experimental rodent malaria infections. Blood samples were obtained from infected mice during and following drug or immune clearance of
Plasmodium chabaudi chabaudi
parasitemias. Detection of parasite DNA by a sensitive
Plasmodium
-specific PCR amplification assay was associated with the presence of viable parasites, as detected by subinoculation. No parasite DNA could be detected by PCR 48 h after the injection of killed parasites into mice. Nuclear material from parasites removed by drug or immune responses is rapidly cleared from the circulation and does not contribute significantly to amplification. Thus, results from PCR analysis of malaria-infected blood accurately reflect the presence of live parasites.
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

ABSTRACT

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

ABSTRACTTo 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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACTLeukocytes 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

ABSTRACTMerozoite 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

ABSTRACTPeriodontitis 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

ABSTRACTThe 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

ABSTRACT

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

ABSTRACTMycobacterium 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

ABSTRACT

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

ABSTRACT

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.