bluuebunny/crossref_metadata_2025_split huggingface dataset:
Defects in Type III Secretion Correlate with Internalization of
Pseudomonas aeruginosa
by Epithelial Cells
Authors: Hauser Alan R. , Fleiszig Suzanne , Kang Pil Jung , Mostov Keith , Engel Joanne N. 
Published: December 2019

ABSTRACT

Previous characterization of
Pseudomonas aeruginosa
clinical isolates has demonstrated an inverse correlation between cytotoxicity and internalization by epithelial cells. To further investigate this relationship, we tested PA103, a cytotoxic
P. aeruginosa
strain, and 33 isogenic noncytotoxic transposon mutants for internalization by Madin-Darby canine kidney cells. The majority of the mutants were not internalized, demonstrating that an inverse correlation between cytotoxicity and bacterial uptake by epithelial cells is not absolute. Six of the noncytotoxic mutants, however, demonstrated measurable levels of internalization by standard aminoglycoside exclusion assays even though internalization of wild-type strain PA103 was not detectable. All six had evidence of protein secretion defects involving two proteins, a 40-kDa protein and a 32-kDa protein. These proteins, designated PepB (for
Pseudomonas
exoprotein B) and PepD, respectively, each had characteristics of type III transported proteins. In addition, nucleotide sequencing studies demonstrated that PepB and PepD are homologs of YopB and YopD, respectively, type III secreted proteins of
Yersinia
spp. necessary for the translocation of effector molecules into the cytoplasmic compartment of eukaryotic cells. Thus, while many mutations in PA103 result in loss of cytotoxicity without an appreciable increase in internalization, defects in transport of type III secretion proteins PepB and PepD correlate with both loss of cytotoxicity and gain of internalization. These results are consistent with type III secretion of an inhibitor of internalization that requires PepB and PepD for translocation into the host cell.
Molecular Analysis of Shiga Toxigenic
Escherichia coli
O111:H
−
Proteins Which React with Sera from Patients with Hemolytic-Uremic Syndrome
Authors: Voss Elena , Paton Adrienne W. , Manning Paul A. , Paton James C. 
Published: December 2019

ABSTRACT

Western blot analysis was used to assess the reactivity of convalescent-phase sera from patients who were associated with an outbreak of hemolytic-uremic syndrome (HUS) caused by fermented sausage contaminated with Shiga toxin-producing
Escherichia coli
(STEC). The predominant STEC isolated from HUS patients belonged to serotype O111:H
−
, and reactivity to O111:H
−
whole-cell lysates, treated or untreated with proteinase K, was examined. As expected, all five serum samples demonstrated a marked anti-lipopolysaccharide response, but several protein bands were also immunoreactive, particularly one with an apparent size of 94 kDa. One convalescent-phase serum sample was subsequently used to screen an O111:H
−
cosmid bank and 2 of 900 cosmid clones were found to be positive, both of which contained a similar DNA insert. Western blot analysis of one of these clones identified three major immunoreactive protein bands of approximately 94, 70, and 50 kDa. An immune response to the three proteins was detectable with all five convalescent-phase serum samples but not with healthy human serum. Immunoreactive 94- and 50-kDa species were produced by a deletion derivative of the cosmid containing a 7-kb STEC DNA insert. Sequence analysis of this region indicated that it is part of the locus for enterocyte effacement, including the
eaeA
gene which encodes intimin. The deduced amino acid sequence of the O111:H
−
intimin was 88.6% identical to intimin from O157:H7 STEC, and the most divergent region was the 200 residues at the carboxyl terminus, which were only 75% identical. Such variation may be antigenically significant as serum from a HUS patient infected only with the O111:H
−
STEC reacted with intimin from an enteropathogenic
E. coli
O111 strain, as well as several other
eaeA
-positive STEC isolates, but not with an
eaeA
-positive STEC belonging to serotype O157:H
−
. Sera from two of the other HUS patients also failed to react with intimin from this latter strain. However, intimin from O157:H
−
STEC did react with serum from a patient infected with both O111:H
−
and O157:H
−
STEC.
Sequence Analysis of the
mip
Gene of the Soilborne Pathogen
Legionella longbeachae
Authors: Doyle Robyn M. , Steele Trevor W. , McLennan Alan M. , Parkinson Ian H. , Manning Paul A. , Heuzenroeder Michael W. 
Published: December 2019

ABSTRACT

To understand the basis of pathogenesis by
Legionella longbeachae
serogroup 1, the importance of the Mip protein in this species was examined. Amino-terminal analysis of the purified, cloned
L. longbeachae
serogroup 1 ATCC 33462 Mip protein confirmed that the cloned gene protein was expressed and processed in an
Escherichia coli
background. DNA sequence analysis of plasmid pIMVS27, containing the entire
L. longbeachae
serogroup 1
mip
gene, revealed a high degree of homology to the
mip
gene of
Legionella pneumophila
serogroup 1, 76% homology at the DNA level and 87% identity at the amino acid level. Primer extension analysis determined that the start site of transcription was the same for both species, with some differences observed for the −10 and −35 promoter regions. Primers designed from the
mip
gene sequence obtained for
L. longbeachae
serogroup 1 ATCC 33462 were used to amplify the
mip
genes from
L. longbeachae
serogroup 2 ATCC 33484 and an Australian clinical isolate of
L. longbeachae
serogroup 1 A5H5. The
mip
gene from A5H5 was 100% identical to the type strain sequence. The serogroup 2 strain of
L. longbeachae
differed by 2 base pairs in third-codon positions. Allelic exchange mutagenesis was used to generate an isogenic
mip
mutant in ATCC 33462 and strain A5H5. The ATCC
mip
mutant was unable to infect a strain of
Acanthamoebae
sp. both in liquid and in a potting mix coculture system, while the A5H5
mip
mutant behaved in a manner siilar to that of
L. pneumophila
serogroup 1, i.e., it displayed a reduced capacity to infect and multiply within
Acanthamoebae
. To determine if this mutation resulted in reduced virulence in the guinea pig animal model, the A5H5
mip
mutant and its parent strain were assessed for their abilities to establish an infection after aerosol exposure. Unlike the virulent parent strain, the mutant strain did not kill any animals under two different dose regimes. The data indicate that the Mip protein plays an important role in the intracellular life cycle of
L. longbeachae
serogroup 1 species and is required for full virulence.
Phlebotomus papatasiSaliva Inhibits Protein Phosphatase Activity and Nitric Oxide Production by Murine Macrophages
Authors: Waitumbi John , Warburg Alon 
Published: December 2019

ABSTRACTLeishmaniaparasites, transmitted by phlebotomine sand flies, are obligate intracellular parasites of macrophages. The sand flyPhlebotomus papatasiis the vector ofLeishmania major, a causative agent of cutaneous leishmaniasis in the Old World, and its saliva exacerbates parasite proliferation and lesion growth in experimental cutaneous leishmaniasis. Here we show thatP. papatasisaliva contains a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A of murine macrophages. We further demonstrate thatP. papatasisaliva down regulates expression of the inducible nitric oxide synthase gene and reduces nitric oxide production in murine macrophages. Partial biochemical characterization of the protein phosphatase and nitric oxide inhibitor indicated that it is a small, ethanol-soluble molecule resistant to boiling, proteolysis, and DNase and RNase treatments. We suggest that theP. papatasisalivary protein phosphatase inhibitor interferes with the ability of activated macrophages to transmit signals to the nucleus, thereby preventing up regulation of the induced nitric oxide synthase gene and inhibiting the production of nitric oxide. Since nitric oxide is toxic to intracellular parasites, the salivary protein phosphatase inhibitor may be the mechanism by whichP. papatasisaliva exacerbates cutaneous leishmaniasis.
Antigen-Specific CD8+T Cells Protect against Lethal Toxoplasmosis in Mice Infected withNeospora caninum
Authors: Kasper Lloyd H. , Khan Imtiaz A. 
Published: December 2019

ABSTRACTNeospora caninumis a coccidial protozoan parasite that appears morphologically indistinguishable fromToxoplasma gondiiand that infects a large range of mammals. Both inbred and outbred strains of mice exhibit a high degree of resistance to infection withN. caninum. Three inbred strains of mice (A/J, BALB/c, and C57BL/6) that were infected intraperitoneally withN. caninumwere protected against a lethal challenge fromT. gondii. Vaccine-induced protection wasNeosporadose dependent. A rise in the CD8+T-cell population in mice that had been vaccinated withN. caninumand challenged withT. gondiiwas observed. Adoptive transfer of CD8+T-cell splenocytes fromN. caninum-infected mice was protective against challenge withToxoplasma. The CD8+T cells fromNeospora-infected mice proliferate to bothNeosporaandToxoplasmaantigens in vitro and secrete substantial quantities of gamma interferon when pulsed with the parasite antigen. These observations demonstrate thatN. caninumprotects against lethalT. gondiiinfection by the induction of CD8+T cells that are immunoreactive to both parasites.
Characterization of Leptospiral Outer Membrane Lipoprotein LipL36: Downregulation Associated with Late-Log-Phase Growth and Mammalian Infection
Authors: Haake David A. , Martinich Carleen , Summers Theresa A. , Shang Ellen S. , Pruetz Jay D. , McCoy Adam M. , Mazel Mary K. , Bolin Carole A. 
Published: December 2019

ABSTRACTWe report the cloning of the gene encoding a 36-kDa leptospiral outer membrane lipoprotein, designated LipL36. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe. A Lambda-Zap II library containingEcoRI fragments ofLeptospira kirschneriDNA was screened, and a 2.3-kb DNA fragment which contained the entire structurallipL36gene was identified. Several lines of evidence indicate that LipL36 is lipid modified in a manner similar to that of LipL41, a leptospiral outer membrane lipoprotein we described in a previous study (E. S. Shang, T. A. Summers, and D. A. Haake, Infect. Immun. 64:2322–2330, 1996). The deduced amino acid sequence of LipL36 would constitute a 364-amino-acid polypeptide with a 20-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. LipL36 is solubilized by Triton X-114 extraction ofL. kirschneri; phase separation results in partitioning of LipL36 exclusively into the hydrophobic, detergent phase. LipL36 is intrinsically labeled during incubation ofL. kirschneriin media containing [3H]palmitate. Processing of LipL36 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. After processing, LipL36 is exported to the outer membrane along with LipL41 and lipopolysaccharide. Unlike LipL41, there appears to be differential expression of LipL36. In early-log-phase cultures, LipL36 is one of the most abundantL. kirschneriproteins. However, LipL36 levels drop considerably beginning in mid-log phase. LipL36 expression in vivo was evaluated by examining the humoral immune response to leptospiral antigens in the hamster model of leptospirosis. Hamsters surviving challenge with culture-adapted virulentL. kirschnerigenerate a strong antibody response to LipL36. In contrast, sera from hamsters surviving challenge with host-adaptedL. kirschnerido not recognize LipL36. These findings suggest that LipL36 expression is downregulated during mammalian infection, providing a marker for studying the mechanisms by which pathogenicLeptospiraspecies adapt to the host environment.
Purification and Characterization of a Cytotoxic Exolipid of
Burkholderia pseudomallei
Authors: Häußler S. , Nimtz M. , Domke T. , Wray V. , Steinmetz I. 
Published: December 2019

ABSTRACT

Burkholderia pseudomallei
is the causative agent of melioidosis, an infectious disease, which is increasingly recognized as an important public health problem in various tropical regions. This study describes the identification and characterization of a heat-stable extracellular toxin of
B. pseudomallei
. After cultivation of
B. pseudomallei
in liquid media, the heated cell-free supernatant was concentrated by ultrafiltration. The concentrate exhibited a cytotoxic and hemolytic activity which showed remarkable resistance against alkaline and acidic treatments. For further purification, reversed-phase chromatography using a fast-performance liquid chromatography system was performed. After elution with an acetonitrile gradient, a single cytotoxic and hemolytic peak was detected. Structural characterization of the toxin was performed by a combination of mass spectrometric and nuclear magnetic resonance spectroscopic techniques. A highly purified glycolipid, 2-
O
-α-
l
-rhamnopyranosyl-α-
l
-rhamnopyranosyl-β-hydroxytetradecanoyl-β-hydroxytetradecanoate (Rha-Rha-C
14
-C
14
), with a molecular mass of 762 Da was identified. The purified exolipid showed a time- and dose-dependent cytotoxic effect on phagocytic (HL60) and nonphagocytic (HeLa) cell lines. In addition, a time- and dose-dependent hemolysis of erythrocytes from various species was observed. The toxin structure makes a detergentlike action most probable. Interestingly, the cytotoxic and hemolytic activities of the glycolipid could be neutralized by albumin. Future studies will concentrate on the role of this exolipid as a virulence factor in the pathogenesis of melioidosis.
Expressed Sequence Tag Analysis of the Bradyzoite Stage of
Toxoplasma gondii
: Identification of Developmentally Regulated Genes
Authors: Manger Ian D. , Hehl Adrian , Parmley Steve , Sibley L. David , Marra Marco , Hillier Ladeana , Waterston Robert , Boothroyd John C. 
Published: December 2019

ABSTRACT

Toxoplasma gondii
is a protozoan parasite responsible for widespread infections in humans and animals. Two major asexual forms are produced during the life cycle of this parasite: the rapidly dividing tachyzoite and the more slowly dividing, encysted bradyzoite. To further study the differentiation between these two forms, we have generated a large number of expressed sequence tags (ESTs) from both asexual stages. Previously, we obtained data on ∼7,400 ESTs from tachyzoites (J. Ajioka et al., Genome Res. 8:18–28, 1998). Here, we report the results from analysis of ∼2,500 ESTs from bradyzoites purified from the cysts of infected mice. We also report the results from analysis of 760 ESTs from parasites induced to differentiate from tachyzoites to bradyzoites in vitro. Comparison of the data sets from bradyzoites and tachyzoites reveals many previously uncharacterized sequence clusters which are largely or completely specific to one or other developmental stage. This class includes a bradyzoite-specific form of enolase. Combined with the previously identified bradyzoite-specific form of lactate dehydrogenase, this finding suggests significant differences in flux through the lower end of the glycolytic pathway in this stage. Thus, the generation of this data set provides valuable insights into the metabolism and growth of the parasite in the encysted form and represents a substantial body of information for further study of development in
Toxoplasma.
Local Chemokine Paralysis, a Novel Pathogenic Mechanism forPorphyromonas gingivalis
Authors: Darveau Richard P. , Belton Carol M. , Reife Robert A. , Lamont Richard J. 
Published: December 2019

ABSTRACTPeriodontitis, which is widespread in the adult population, is a persistent bacterial infection associated withPorphyromonas gingivalis. Gingival epithelial cells are among the first cells encountered by bothP. gingivalisand commensal oral bacteria. The chemokine interleukin 8 (IL-8), a potent chemoattractant and activator of polymorphonuclear leukocytes, was secreted by gingival epithelial cells in response to components of the normal oral flora. In contrast,P. gingivaliswas found to strongly inhibit IL-8 accumulation from gingival epithelial cells. Inhibition was associated with a decrease in mRNA for IL-8. Antagonism of IL-8 accumulation did not occur in KB cells, an epithelial cell line that does not support high levels of intracellular invasion byP. gingivalis. Furthermore, a noninvasive mutant ofP. gingivaliswas unable to antagonize IL-8 accumulation. Invasion-dependent destruction of the gingival IL-8 chemokine gradient at sites ofP. gingivaliscolonization (local chemokine paralysis) will severely impair mucosal defense and represents a novel mechanism for bacterial colonization of host tissue.
Adequate Expression of Protective Immunity in the Absence of Granuloma Formation inMycobacterium tuberculosis-Infected Mice with a Disruption in the Intracellular Adhesion Molecule 1 Gene
Authors: Johnson Christine M. , Cooper Andrea M. , Frank Anthony A. , Orme Ian M. 
Published: December 2019

ABSTRACTIt remains unknown whether the expression of cell-mediated protective immunity and the capacity to mount a delayed-type hypersensitivity (DTH) reaction in tuberculosis infection represent two manifestations of a basic response or are dissociable events. In this study, we present data in favor of the latter hypothesis, by showing that tuberculosis infection in the lungs of mice possessing only a truncated form of intracellular adhesion molecule 1 due to gene disruption was still adequately controlled by the expression of protective immunity in the absence of any sustained influx of macrophages and the lack of formation of appreciable granulomas. These animals also had no detectable DTH response to mycobacterial proteins in the footpad assay, indicating that the accumulation of blood-borne macrophages at sites of mycobacterial infection or antigen deposition is not essential to control of the infection. These data support the hypothesis that the DTH component of the cellular response is not protective but contributes by walling off the sites of infection to prevent dissemination and reactivation disease.
Reduced Virulence of Group A Streptococcal Tn
916
Mutants That Do Not Produce Streptolysin S
Authors: Betschel Stephen D. , Borgia Sergio M. , Barg Neil L. , Low Donald E. , De Azavedo Joyce C. S. 
Published: December 2019

ABSTRACT

Streptolysin S (SLS) is a potent cytolytic toxin produced by nearly all group A streptococci (GAS). SLS-deficient Tn
916
insertional mutants were generated from two clinical isolates of GAS, MGAS166s and T18Ps (M serotypes 1 and 18, respectively), by transposon mutagenesis using Tn
916
donor strain
Enterococcus faecalis
CG110. Representative nonhemolytic transconjugants SBNH5 and SB30-2 each harbored a single Tn
916
insertion in identical loci. The insertion in SBNH5 was located in the promoter region of an open reading frame, designated
sagA
, rendering it transcriptionally inactive. Protease, streptolysin O, and DNase activities and the production of M protein remained the same in the nonhemolytic mutants and the wild-type strains, as did the growth rates and exoprotein profiles. Transconjugants were evaluated in an established murine model by injecting the organisms subcutaneously and monitoring the mice for alterations in weight and the development of necrotic lesions. Animals infected with SBNH5, compared to those infected with MGAS166s, gained weight during the first 24 h (+1.15 versus −1.16 g;
P
< 0.05) and had fewer necrotic lesions (0 versus 7;
P
= 0.0007). Animals infected with SB30-2, compared to those infected with T18Ps, also gained weight within the first 24 h (+0.54 versus −0.66 g;
P
< 0.05) and produced fewer necrotic lesions (1 versus 8;
P
= 0.001). Revertants of the mutants in which Tn
916
had been excised regained the hemolytic phenotype and the virulence profile of the wild-type strains. This study demonstrates that SLS-deficient mutants of GAS, belonging to different M serotypes and containing identical Tn
916
mutations, are markedly less virulent than their isogenic parents.
Characteristics of Invasive Candidiasis in Gamma Interferon- and Interleukin-4-Deficient Mice: Role of Macrophages in Host Defense againstCandida albicans
Authors: Káposzta Rita , Tree Peter , Maródi László , Gordon Siamon 
Published: December 2019

ABSTRACTMurine models of invasive candidiasis were used to study the in vivo importance of gamma interferon (IFN-γ) and interleukin-4 (IL-4) in host defense againstCandida albicansand to characterize the tissue inflammatory reactions, with special reference to macrophages (Mφ). Knockout (KO) IFN-γ-deficient (GKO) and IL-4-deficient (IL-4 KO) and C57BL/6 parental mouse strains were challenged intraperitoneally with 108C. albicansblastoconidia. Survival of GKO mice was significantly lower (16.7%) than that of C57BL/6 control (55.5%) and IL-4 KO (61.1%) animals, but was not correlated with the extent of organ colonization. Immunohistological analysis with a panel of myeloid and lymphoid markers revealed multiple renal abscesses, myocarditis, hepatitis, meningoencephalitis, and pneumonia in each strain, with a dominant presence of Mφ. In the absence of IFN-γ,C. albicansinduced striking changes in the phenotype of alveolar Mφ and extensive perivascular lymphoid infiltrates in the lung. Impairment in nitric oxide production by peritoneal Mφ was shown only in GKO mice, and they producedCandida-specific immunoglobulin G (IgG), IgM, IgA, and IgG subclasses in lower titers. Our in vivo studies with KO mice elucidate a critical role for IFN-γ, but not IL-4, in host defense againstC. albicans.
Molecular Characterization of the Fragilysin Pathogenicity Islet of Enterotoxigenic
Bacteroides fragilis
Authors: Moncrief J. Scott , Duncan A. Jane , Wright Rhonda L. , Barroso Lisa A. , Wilkins Tracy D. 
Published: December 2019

ABSTRACT

Enterotoxigenic strains of
Bacteroides fragilis
produce an extracellular metalloprotease toxin (termed fragilysin) which is cytopathic to intestinal epithelial cells and induces fluid secretion and tissue damage in ligated intestinal loops. We report here that the fragilysin gene is contained within a small genetic element termed the fragilysin pathogenicity islet. The pathogenicity islet of
B. fragilis
VPI 13784 was defined as 6,033 bp in length and contained nearly perfect 12-bp direct repeats near its ends. Sequencing across the ends of the pathogenicity islet from two additional enterotoxigenic strains, along with PCR analysis of 20 additional enterotoxigenic strains, revealed that the islet is inserted at a specific site on the
B. fragilis
chromosome. The site of integration in three nontoxigenic strains contained a 17-bp GC-rich sequence which was not present in toxigenic strains and may represent a target sequence for chromosomal integration. In addition to the fragilysin gene, we identified an open reading frame encoding a predicted protein with a size and structural features similar to those of fragilysin. The deduced amino acid sequence was 28.5% identical and 56.3% similar to fragilysin and contained a nearly identical zinc-binding motif and methionine-turn region.
Agents That Inhibit Rho, Rac, and Cdc42 Do Not Block Formation of Actin Pedestals in HeLa Cells Infected with Enteropathogenic
Escherichia coli
Authors: Ben-Ami Goni , Ozeri Vered , Hanski Emanuel , Hofmann Fred , Aktories Klaus , Hahn Klaus M. , Bokoch Gary M. , Rosenshine Ilan 
Published: December 2019

ABSTRACT

Enteropathogenic
Escherichia coli
(EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including
Clostridium difficile
toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.