bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Two
Shigella flexneri
Chromosomal Loci Involved in Intercellular Spreading
Authors: Hong Mei , Gleason Yuki , Wyckoff Elizabeth E. , Payne Shelley M. 
Published: December 2019

ABSTRACT

The ability of
Shigella flexneri
to multiply within colonic epithelial cells and spread to adjacent cells is essential for production of dysentery. Two
S. flexneri
chromosomal loci that are required for these processes were identified by screening a pool of Tn
phoA
insertion mutants. These mutants were able to invade cultured epithelial cells but could not form wild-type plaques. Analysis of the nucleotide sequence indicated that the sites of Tn
phoA
insertion were within two different regions that are almost identical to
Escherichia coli
K-12 chromosomal sequences of unknown functions. One region is located at 70 min on the
E. coli
chromosome, upstream of
murZ
, while the other is at 28 min, downstream of
tonB
. The mutant with the insertion at 70 min was named
vpsC
because it showed an altered pattern of virulence protein secretion. The
vpsC
mutant formed pinpoint-sized plaques, was defective in recovery from infected tissue culture cells, and was sensitive to lysis by the detergent sodium dodecyl sulfate. Recombinant plasmids carrying the
S. flexneri vpsA
, -
B
, and -
C
genes complemented all of the phenotypes of the
vpsC
mutant. A mutation in
vpsA
resulted in the same phenotype as the
vpsC
mutation, suggesting that these two genes are part of a virulence operon in
S. flexneri
. The mutant with the insertion at 28 min was interrupted in the same open reading frame as
S. flexneri ispA
. This
ispA
mutant could not form plaques and was defective in bacterial septation inside tissue culture cells.
hgpB
, a Gene Encoding a Second
Haemophilus influenzae
Hemoglobin- and Hemoglobin-Haptoglobin-Binding Protein
Authors: Ren Zhen , Jin Hongfan , Morton Daniel J. , Stull Terrence L. 
Published: December 2019

ABSTRACT

Haemophilus influenzae
requires heme for growth and can utilize both hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified a hemoglobin- and hemoglobin-haptoglobin-binding protein, HgpA, in
H. influenzae
HI689. Mutation of
hgpA
did not affect binding or utilization of either heme source. The
hgpA
mutant exhibited loss of a 120-kDa protein and increased expression of a 115-kDa protein. These data suggested that at least one other gene product is involved in binding of these heme sources by
H. influenzae
. A 3.2-kbp PCR product derived from HI689 was cloned. The nucleotide sequence indicated a separate, distinct gene with high homology to
hgpA
, which would encode a 115-kDa protein. Primers were designed for directional cloning of the structural gene in the correct reading frame. Sonicates of induced
Escherichia coli
harboring the cloned open reading frame bound both hemoglobin and hemoglobin-haptoglobin. An insertion/deletion mutant of
H. influenzae
at the newly identified locus, designated
hgpB
, was constructed. The 115-kDa protein was not detected in the mutant after affinity purification using biotinylated hemoglobin. An
hgpA hgpB
double-mutant strain exhibited a reduced ability to utilize hemoglobin-haptoglobin, although it was unaltered in the ability to utilize hemoglobin. Affinity isolation of hemoglobin-binding proteins from the double mutant resulted in isolation of an approximately 120-kDa protein. Internal peptide sequencing revealed this protein to be a third distinct protein, highly homologous to HgpA and HgpB. In summary a second hemoglobin- and hemoglobin-haptoglobin-binding protein of
H. influenzae
has been identified and characterized, and the presence of an additional protein of similar function has been revealed.
Comparison of the PspA Sequence fromStreptococcus pneumoniaeEF5668 to the Previously Identified PspA Sequence from Strain Rx1 and Ability of PspA from EF5668 To Elicit Protection against Pneumococci of Different Capsular Types
Authors: McDaniel Larry S. , McDaniel D. Olga , Hollingshead Susan K. , Briles David E. 
Published: December 2019

ABSTRACTPspA (pneumococcal surface protein A) is a serologically varied virulence factor ofStreptococcus pneumoniae. In mice, PspA has been shown to elicit an antibody response that protects against fatal challenge with encapsulatedS. pneumoniae, and the protection-eliciting residues have been mapped to the α-helical N-terminal half of the protein. To date, a published DNA sequence forpspAis available only forS. pneumoniaeRx1, a laboratory strain. PspA/EF5668 (EF5668 indicates the strain of origin of the PspA) is serologically distinct from PspA/Rx1. Sequencing of the gene encoding PspA/EF5668 revealed 71% identity with that of PspA/Rx1. The greatest amount of divergence between the two proteins was seen in their α-helical portions, which are surface exposed and probably under selective pressure to diversify serologically. In spite of the diversity within the α-helical regions of PspAs, we have observed that recombinant PspA (rPspA)/EF5668, like rPspA/Rx1, can elicit cross-protection against pneumococci of different capsular and PspA serological types.
Specificity of Bactericidal Antibody Response to Serogroup B Meningococcal Strains in Brazilian Children after Immunization with an Outer Membrane Vaccine
Authors: Milagres Lucimar G. , Gorla Maria Cecília A. , Sacchi Claudio T. , Rodrigues Mauricio M. 
Published: December 2019

ABSTRACT
Pre- and postvaccination serum samples from 77 children aged 2 to 6 years, who received the Cuban BC vaccine (B:4:P1.15), were analyzed for bactericidal antibodies against a local B:4:P1.15 strain (N44/89). Sera from 16 individuals with bactericidal antibodies against the B:4:P1.15 strain were tested against 23 Brazilian isolates. These include B:4 strains of distinct serosubtypes: P1.15, P1.7,1, P1.3, P1.9, P1.nt, and a B:8,19,23:P1.16 strain. A Cuban B:4:P1.15 strain (Cu385/83) was also included in the study. The specificities of bactericidal antibodies were analyzed by using mutant strains lacking a class 1 protein (PorA protein) or a class 5 protein or both. The results indicated that PorA and class 5 proteins are the main targets recognized by the bactericidal antibodies of vaccinees. Nonetheless, a complex pattern of recognition by bactericidal antibodies was found, and vaccinees were grouped according to antibody specificity. Antibodies from some individuals recognized PorA of serosubtype P1.15. However, antibodies from these individuals could not kill all P1.15 strains tested. Antibodies from a second group recognized both PorA and class 5 proteins, and antibodies from a third group recognized an as yet unidentified target antigen. The results demonstrate the importance of determining the fine epitope specificity of bactericidal antibodies to improve the existing vaccines against B meningococci.
Age-Related Buildup of Humoral Immunity against Epitopes for Rosette Formation and Agglutination in African Areas of Malaria Endemicity
Authors: Barragan Antonio , Kremsner Peter G. , Weiss Walter , Wahlgren Mats , Carlson Johan 
Published: December 2019

ABSTRACTIn this report, we show an age-related buildup of agglutinating activity as well as serum activity against rosette formation in children living in areas of Kenya and Gabon where malaria is endemic. Sera from Kenyans in general exhibited a stronger and wider immune response toward the epitopes, probably reflecting a difference in transmission patterns between the two areas. Thus, our results indicate that repeated malaria attacks in areas of endemicity, and consequently exposure to different isolate-specific antigens, will elicit an antibody-mediated response eventually enabling recognition of the majority of rosetting and agglutinating antigens. The correlation between antirosetting and agglutinating capacity was poor in individual cases, indicating that the rosetting epitopes are only a minor part of the highly diverse surface-exposed antigens (mainly PfEMP1) on the surface of parasitized erythrocytes toward which antibodies may react. These data together with our previous findings that the protection against cerebral malaria correlates with presence of antirosetting antibodies shed new light on our understanding of the gradual acquisition of immunity toward severe complications of malarial infection which children reared in areas of endemicity attain.
Toxoplasma GondiiBradyzoites Form Spontaneously during Sporozoite-Initiated Development
Authors: Jerome M. E. , Radke J. R. , Bohne W. , Roos D. S. , White M. W. 
Published: December 2019

ABSTRACTTachyzoites (VEG strain) that emerge from host cells infected withToxoplasma gondiisporozoites proliferate relatively fast and double their number every 6 h. This rate of growth is intrinsic, as neither the number of host cells invaded nor host cell type appears to influence emergent tachyzoite replication. Fast tachyzoite growth was not persistent, and following ∼20 divisions, the population uniformly shifted to slower growth. Parasites 10 days post-sporozoite infection doubled only once every 15 h and, unlike emergent tachyzoites, they grew at this slower rate over several months of continuous cell culture. The spontaneous change in tachyzoite growth rate preceded the expression of the bradyzoite-specific marker,BAG1. Within 24 h of the growth shift, 2% of the population expressedBAG1, and by 15 days post-sporozoite infection, 50% of the parasites were positive for this marker. SpontaneousBAG1expression was not observed in sporozoites or in tachyzoites during fast growth (through day 6 post-sporozoite inoculation), although these tachyzoites could be induced to expressBAG1earlier by culturing sporozoite-infected cells at pH 8.3. However, alkaline treatment also reduced the replication of emergent tachyzoites to the rate of growth-shifted parasites, supporting a link between reduced parasite growth and bradyzoite differentiation. The shift to slower growth was closely correlated with virulence in mice, as the initially fast-growing emergent tachyzoites were avirulent (100% lethal dose, >104parasites), while a mutant VEG strain (MS-J) that is unable to growth shift caused 100% mortality in mice inoculated with 10 parasites. Parasites recovered from gamma interferon knockout mice inoculated with emergent tachyzoites grew at a slow rate and expressedBAG1, confirming that the replication switch occurs in animals and in the absence of a protective immune response.
Mannan-Specific Immunoglobulin G Antibodies in Normal Human Serum Accelerate Binding of C3 toCandida albicansvia the Alternative Complement Pathway
Authors: Zhang Mason X. , Kozel Thomas R. 
Published: December 2019

ABSTRACTCandida albicansactivates the classical and alternative complement pathways, leading to deposition of opsonic complement fragments on the cell surface. Our previous studies found that antimannan immunoglobulin G (IgG) in normal human serum (NHS) allowsC. albicansto initiate the classical pathway. The purpose of this study was to determine whether antimannan IgG also plays a role in initiation of the alternative pathway. Pooled NHS was rendered free of classical pathway activity by chelation of serum Ca2+with EGTA alone or in combination with immunoaffinity removal of antimannan antibodies. Kinetic analysis revealed a 6-min lag in detection of C3 binding toC. albicansincubated in EGTA-chelated NHS, compared to a 12-min lag in NHS that was both EGTA chelated and mannan absorbed. The 12-min lag was shortened to 6 min by addition of affinity-purified antimannan IgG. The accelerating effect of antimannan IgG on alternative pathway initiation was dose dependent and was reproduced in a complement binding reaction consisting of six purified proteins of the alternative pathway. Both Fab and F(ab′)2fragments of antimannan IgG facilitated alternative pathway initiation in a manner similar to that observed with intact antibody. Immunofluorescence analysis showed that addition of antimannan IgG to EGTA-chelated and mannan-absorbed serum promoted an early deposition of C3 molecules on the yeast cells but had little or no effect on distribution of the cellular sites for C3 activation. Thus, antimannan IgG antibodies play an important regulatory role in interactions between the host complement system andC. albicans.
Characterization of the Hemorrhagic Reaction Caused by
Vibrio vulnificus
Metalloprotease, a Member of the Thermolysin Family
Authors: Miyoshi Shin-ichi , Nakazawa Hiromi , Kawata Koji , Tomochika Ken-ichi , Tobe Kazuo , Shinoda Sumio 
Published: December 2019

ABSTRACT

Vibrio vulnificus
is an opportunistic human pathogen causing wound infections and septicemia, characterized by hemorrhagic and edematous damage to the skin. This human pathogen secretes a metalloprotease (
V. vulnificus
protease [VVP]) as an important virulence determinant. When several bacterial metalloproteases including VVP were injected intradermally into dorsal skin, VVP showed the greatest hemorrhagic activity. The level of the in vivo hemorrhagic activity of the bacterial metalloproteases was significantly correlated with that of the in vitro proteolytic activity for the reconstituted basement membrane gel. Of two major basement membrane components (laminin and type IV collagen), only type IV collagen was easily digested by VVP. Additionally, the immunoglobulin G antibody against type IV collagen, but not against laminin, showed sufficient protection against the hemorrhagic reaction caused by VVP. Capillary vessels are known to be stabilized by binding of the basal surface of vascular endothelial cells to the basement membrane. Therefore, specific degradation of type IV collagen may cause destruction of the basement membrane, breakdown of capillary vessels, and leakage of blood components including erythrocytes.
Localization of a T-Cell Epitope of Superantigen Toxic Shock Syndrome Toxin 1 to Residues 125 to 158
Authors: Hu Wei-Gang , Zhu Xi-Hua , Wu Yu-Zhang , Jia Zheng-Cai 
Published: December 2019

ABSTRACTToxic shock syndrome toxin 1 (TSST-1) is a member of the staphylococcal enterotoxin superantigen family. So far, little is known about T-cell epitopes on superantigens. In this study, we developed an improved method for localizing T-cell epitopes on superantigens that involved synthetic peptides plus costimulation by CD28 or phorbol myristate acetate. Using this method, we localized a T-cell epitope to a 34-residue region, TSST-1 (residues 125 to 158), which possessed only two of four TSST-1-targeted β-chain variable element (Vβ) specificities of T-cell receptors in humans and mice, human Vβ2 and murine Vβ15.
Staphylococcal Enterotoxin B Primes Cytokine Secretion and Lytic Activity in Response to Native Bacterial Antigens
Authors: Mason Kevin M. , Dryden Tricia D. , Bigley Nancy J. , Fink Pamela S. 
Published: December 2019

ABSTRACTSuperantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured withStreptococcus mutansproduced significantly greater amounts of gamma interferon (IFN-γ) and interleukin-12 than did sham-injected controls. The majority of IFN-γ production appeared to be CD8+T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-γ. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to “bystander” fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-γ decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo.
Novel Selection for Isoniazid (INH) Resistance Genes Supports a Role for NAD
+
-Binding Proteins in Mycobacterial INH Resistance
Authors: Chen Ping , Bishai William R. 
Published: December 2019

ABSTRACT

The genetic basis of isoniazid (INH) resistance remains unknown for a significant proportion of clinical isolates. To identify genes which might confer resistance by detoxifying or sequestering INH, we transformed the
Escherichia coli oxyR
mutant, which is relatively sensitive to INH, with a
Mycobacterium tuberculosis
plasmid library and selected for INH-resistant clones. Three genes were identified and called
ceo
for their ability to complement the
Escherichia coli oxyR
mutant.
ceoA
was the previously identified
M. tuberculosis glf
gene, which encodes a 399-amino-acid NAD
+
- and flavin adenine dinucleotide-requiring enzyme responsible for catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The proteins encoded by the
ceoBC
pair were homologous with one another and with the N terminus of the potassium uptake regulatory protein TrkA. Each of the three Ceo proteins contains a motif common to NAD
+
binding pockets. Overexpression of the
M. tuberculosis glf
gene by placing it under the control of the
hsp60
promoter on a multicopy plasmid in
Mycobacterium bovis
BCG produced a strain for which the INH MIC was increased 50% compared to that for the control strains, while similar overexpression of the
ceoBC
pair had no effect on INH susceptibility in BCG. Mycobacterial extracts containing the overexpressed Glf protein did not bind radiolabeled INH directly, suggesting a more complex mechanism than the binding of unmodified INH. Our results support the hypothesis that upregulated mycobacterial proteins such as Glf may contribute to INH resistance in
M. tuberculosis
by binding a modified form of INH or by sequestering a factor such as NAD
+
required for INH activity.
Immunological Characterization of Asp f 2, a Major Allergen fromAspergillus fumigatusAssociated with Allergic Bronchopulmonary Aspergillosis
Authors: Banerjee Banani , Greenberger Paul A. , Fink Jordan N. , Kurup Viswanath P. 
Published: December 2019

ABSTRACTThe 37-kDa recombinant protein Asp f 2, encoding an allergen ofAspergillus fumigatus, was expressed in a prokaryotic expression system and immunologically evaluated for its functional and structural properties. The open reading frame for a 310-amino-acid-long protein was shown to encode a signal peptide of 31 amino acids. A native 37-kDa culture filtrate protein and a 55-kDa mycelial glycoprotein (gp55) exhibited complete N-terminal sequence homology to Asp f 2. A GenBank search for homologous proteins revealed 60 and 44% sequence homologies to the cytosolic protein ASPND1 fromAspergillus nidulansand fibrinogen binding protein fromCandida albicans, respectively. The glycosylation sites and cysteine molecules are conserved in all the three proteins. The extracellular matrix protein laminin showed a dose-dependent interaction with Asp f 2. This protein, expressed as a major cell-associated protein within 24 h of in vitro fungal culture, comprises 20 to 40% of total fungal protein. Furthermore, both native and recombinant Asp f 2 exhibited specific immunoglobulin (IgE) binding with allergic bronchopulmonary aspergillosis (ABPA) and cystic fibrosis-ABPA patients, whereasA. fumigatus-sensitized allergic asthma and normal control subjects failed to show IgE binding with Asp f 2. These results indicate that Asp f 2 is a major allergen ofA. fumigatusexhibiting IgE antibody binding with sera from patients with ABPA. The antigen should be explored further for its potential role in the differential diagnosis ofA. fumigatus-associated allergic diseases.
In Situ Detection of Apoptosis at Sites of Chronic Bacterially Induced Inflammation in Human Gingiva
Authors: Tonetti Maurizio S. , Cortellini Davide , Lang Niklaus P. 
Published: December 2019

ABSTRACTApoptosis is a key phenomenon in the regulation of the life span of terminally differentiated leukocytes. Human gingiva represents an established model to study immune responses to bacterial infection. In this investigation, we used the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) technique to evaluate presence and topographic location of apoptosis-associated DNA damage in human gingival biopsies along with the expression of the p53 and Bcl-2 apoptosis-regulating proteins. Qualitative data analysis showed high densities of cells expressing DNA damage and p53 both within the epithelial attachment to the tooth and in the perivascular infiltrate (infiltrated connective tissue [ICT]) immediately underlying the site of chronic bacterial aggression. Topographic consistency between DNA damage- and p53-positive cells was consistently observed. Quantitative analysis of the ICT showed mean densities of DNA damage- and p53-positive cells of 345 ± 278 and 403 ± 182 cells/mm2, respectively. Numerical consistency was confirmed by multivariate regression analysis: densities of DNA damage-positive cells were significantly predicted by densities of p53-positive cells (P= 0.001,r2= 0.84). In the ICT, cells displaying biotinylated DNA nicks were 3.8% ± 2.7% of total cellularity, while p53- and Bcl-2-positive cells represented 4.4% ± 1.7% and 15.4% ± 6.7% of total cells, respectively. It is suggested that p53 expression associated with DNA damage is a prevalent phenomenon in chronically inflamed human gingiva, and that apoptosis may be a relevant process for the maintenance of local immune homeostasis at sites of chronic bacterial challenge in vivo.
The Major Outer Membrane Protein ofChlamydia psittaciFunctions as a Porin-Like Ion Channel
Authors: Wyllie Susan , Ashley Richard H. , Longbottom David , Herring Alan J. 
Published: December 2019

ABSTRACTThe major outer membrane protein (MOMP) ofChlamydiaspecies shares several biochemical properties with classical porin proteins. Secondary structure analysis by circular dichroism now reveals that MOMP purified fromChlamydia psittacihas a predominantly β-sheet content (62%), which is also typical of bacterial porins. Can MOMP form functional ion channels? To directly test the “porin channel” hypothesis at the molecular level, the MOMP was reconstituted into planar lipid bilayers, where it gave rise to multibarreled channels, probably trimers, which were modified by an anti-MOMP monoclonal antibody. These observations are consistent with the well-characterized homo-oligomeric nature of MOMP previously revealed by biochemical analysis and with the triple-barreled behavior of other porins. MOMP channels were weakly anion selective (PCl/PK∼ 2) and permeable to ATP. They may therefore be a route by whichChlamydiacan take advantage of host nucleoside triphosphates and explain why some anti-MOMP antibodies neutralize infection. These findings have broad implications on the search for an effective chlamydial vaccine to control the significant human and animal diseases caused by these organisms.