scholarly journals Edwardsiella ictaluri Encodes an Acid-Activated Urease That Is Required for Intracellular Replication in Channel Catfish (Ictalurus punctatus) Macrophages

2009 ◽  
Vol 75 (21) ◽  
pp. 6712-6720 ◽  
Author(s):  
Natha J. Booth ◽  
Judith B. Beekman ◽  
Ronald L. Thune
Keyword(s):  
Ictalurus Punctatus ◽  
Urease Activity ◽  
Genomic Analysis ◽  
Acid Tolerance ◽  
Head Kidney ◽  
Edwardsiella Ictaluri ◽  
Wild Type ◽  
Intracellular Replication ◽  
Two Dimensional Gel Electrophoresis

ABSTRACT Genomic analysis indicated that Edwardsiella ictaluri encodes a putative urease pathogenicity island containing the products of nine open reading frames, including urea and ammonium transporters. In vitro studies with wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significantly tolerant of acid conditions (pH 3.0) but that urease activity is not required for acid tolerance. Growth studies demonstrated that E. ictaluri is unable to grow at pH 5 in the absence of urea but is able to elevate the environmental pH from pH 5 to pH 7 and grow when exogenous urea is available. Substantial production of ammonia was observed for wild-type E. ictaluri in vitro in the presence of urea at low pH, and optimal activity occurred at pH 2 to 3. No ammonia production was detected for the urease mutant. Proteomic analysis with two-dimensional gel electrophoresis indicated that urease proteins are expressed at both pH 5 and pH 7, although urease activity is detectable only at pH 5. Urease was not required for initial invasion of catfish but was required for subsequent proliferation and virulence. Urease was not required for initial uptake or survival in head kidney-derived macrophages but was required for intracellular replication. Intracellular replication of wild-type E. ictaluri was significantly enhanced when urea was present, indicating that urease plays an important role in intracellular survival and replication, possibly through neutralization of the acidic environment of the phagosome.

scholarly journals Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro

2020 ◽  
Vol 58 (1) ◽  
pp. 21-33
Author(s):  
Pingping Shen ◽  
Johnny Dang ◽  
Zerui Wang ◽  
Weiguanliu Zhang ◽  
Jue Yuan ◽  
...  
Keyword(s):  
Protein Misfolding ◽  
Neuroblastoma Cells ◽  
Cellular Prion Protein ◽  
Wild Type ◽  
Gpi Anchor ◽  
Two Dimensional Gel Electrophoresis ◽  
Molecular Weights ◽  
Prion Propagation

AbstractAlteration in cellular prion protein (PrPC) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrPSc). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrPSc and cellular trafficking of PrPQ227X remain to be determined. Here, we show that PrPSc in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrPWt), suggesting pathological PrPQ227X is incapable of recruiting PrPWt in vivo. This mutant anchorless protein, however, is able to recruit PrPWt from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrPSc-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrPQ227X or PrPWt were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrPWt is recovered in the cell lysate fraction, while most of the mutant PrPQ227X is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrPQ227X spots clustered at molecular weights of 22–25 kDa with an isoelectric point (pI) of 3.5–5.5, whereas protein spots from the medium are at 18–26 kDa with a pI of 7–10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.

scholarly journals Streptococcus thermophiles DMST-H2 Promotes Recovery in Mice with Antibiotic-Associated Diarrhea

2020 ◽  
Vol 8 (11) ◽  
pp. 1650
Author(s):  
Jin-Shuang Hu ◽  
Yan-Yan Huang ◽  
Jia-Hua Kuang ◽  
Jia-Jia Yu ◽  
Qin-Yu Zhou ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Intestinal Flora ◽  
Intestinal Barrier ◽  
Genomic Analysis ◽  
Acid Tolerance ◽  
Interferon Γ ◽  
Common Side Effect ◽  
Gastrointestinal Conditions ◽  
Antibiotic Associated Diarrhea

Antibiotic-associated diarrhea (AAD) is the most common side effect of antibiotics and is routinely treated with probiotics in clinical. Streptococcus thermophiles, extensively utilized for producing dairy foods, has recently been regarded as a new promising probiotic candidate. In this study, the efficacy of Streptococcus thermophiles DMST-H2 (DMST-H2) for AAD treatment in mice was investigated. DMST-H2 was isolated from Chinese traditional yogurt, proved to be non-toxic, and presented tolerance against simulated gastrointestinal conditions in vitro. Additionally, genomic analysis revealed that it possessed genes related to acid tolerance, bile salt tolerance, adhesion, oxidative stress and bacteriocin production. The animal experiment results showed that both DMST-H2 treatment and natural recovery could reduce fecal water content. Compared with spontaneous recovery, DMST-H2 accelerated the recovery of the enlarged caecum and intestinal barrier injury from AAD, and further decreased endotoxin (ET), D-lactate (D-LA) and diamine oxidase (DAO) content in serum. Moreover, pro-inflammatory cytokines (TNF-α) were reduced, while interferon-γ (IFN-γ) and anti-inflammatory cytokines (IL-10) increased after treating with DMST-H2. Furthermore, DMST-H2 better restored the structure of intestinal flora. At the phylum level, Firmicutes increased and Proteobacteria decreased. These findings indicate that DMST-H2 could promote recovery in mice with antibiotic-associated diarrhea.

scholarly journals Novel Salmonella enterica Serovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication

2001 ◽  
Vol 69 (12) ◽  
pp. 7413-7418 ◽  
Author(s):  
Tahar van der Straaten ◽  
Angela van Diepen ◽  
Kitty Kwappenberg ◽  
Sjaak van Voorden ◽  
Kees Franken ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Function ◽  
Salmonella Enterica ◽  
Host Cells ◽  
Wild Type ◽  
Intracellular Replication ◽  
Oxygen Intermediates ◽  
Serovar Typhimurium

ABSTRACT Upon contact with host cells, the intracellular pathogenSalmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribedSalmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonellachromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 104 to 107bacteria in C3H/HeN and 101 to 104 bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.

scholarly journals Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Yoon-Suk Kang ◽  
James E. Kirby
Keyword(s):  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Life Cycles ◽  
Wild Type ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv ◽  
Reporter Systems ◽  
Brucella Neotomae

ABSTRACT We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila. Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.

In vitro and in vivo interaction of macrophages from vaccinated and non-vaccinated channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri

2009 ◽  
Vol 26 (3) ◽  
pp. 543-552 ◽  
Author(s):  
Riccardo Russo ◽  
Craig A. Shoemaker ◽  
Victor S. Panangala ◽  
Phillip H. Klesius
Keyword(s):  
Ictalurus Punctatus ◽  
Channel Catfish ◽  
Edwardsiella Ictaluri

scholarly journals The Human Cytomegalovirus UL44 Protein Is a Substrate for the UL97 Protein Kinase

2003 ◽  
Vol 77 (14) ◽  
pp. 7720-7727 ◽  
Author(s):  
Paula M. Krosky ◽  
Moon-Chang Baek ◽  
Wan Jin Jahng ◽  
Imma Barrera ◽  
Robert J. Harvey ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Human Cytomegalovirus ◽  
Nucleoside Analogs ◽  
Wild Type Virus ◽  
Null Mutant ◽  
Natural Substrate ◽  
Wild Type ◽  
Two Dimensional Gel Electrophoresis ◽  
Infected Cells

ABSTRACT The human cytomegalovirus UL97 protein is an unusual protein kinase that is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. However, no natural substrate of UL97 in infected cells has been identified. We report here that recombinant UL44 protein became radiolabeled when incubated with recombinant UL97 and [32P]ATP and that both proteins could be coimmunoprecipitated by an antibody that recognizes either protein. Subsequent studies showed that highly purified, recombinant UL97 phosphorylated purified, recombinant UL44. This phosphorylation occurred on serine and threonine residues and was sensitive to inhibition by maribavir and to a mutation that inactivates UL97 catalytic activity. Two-dimensional gel electrophoresis revealed the absence of specific phosphorylated forms of UL44 in immunoprecipitates from lysates of cells infected with a UL97 null mutant virus or with wild-type virus in the presence of maribavir. The results indicate that UL97 is sufficient to phosphorylate UL44 in vitro and is necessary for the normal phosphorylation of UL44 in infected cells. This strongly suggests that UL44 is a natural substrate of UL97.

scholarly journals Brucella abortus VirB12 Is Expressed during Infection but Is Not an Essential Component of the Type IV Secretion System

2005 ◽  
Vol 73 (9) ◽  
pp. 6048-6054 ◽  
Author(s):  
Yao-Hui Sun ◽  
Hortensia G. Rolán ◽  
Andreas B. den Hartigh ◽  
David Sondervan ◽  
Renée M. Tsolis
Keyword(s):  
Brucella Abortus ◽  
Secretion System ◽  
Splenic Tissue ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Deletion Mutants ◽  
Wild Type ◽  
Intracellular Replication ◽  
Type Iv

ABSTRACT The Brucella abortus virB operon, consisting of 11 genes, virB1 to virB11, and two putative genes, orf12 (virB12) and orf13, encodes a type IV secretion system (T4SS) that is required for intracellular replication and persistent infection in the mouse model. This study was undertaken to determine whether orf12 (virB12) encodes an essential part of the T4SS apparatus. The virB12 gene was found to encode a 17-kDa protein, which was detected in vitro in B. abortus grown to stationary phase. Mice infected with B. abortus 2308 produced an antibody response to the protein encoded by virB12, showing that this gene is expressed during infection. Expression of virB12 was not required for survival in J774 macrophages. VirB12 was also dispensable for the persistence of B. abortus, B. melitensis, and B. suis in mice up to 4 weeks after infection, since deletion mutants lacking virB12 were recovered from splenic tissue at wild-type levels. These results show that VirB12 is not essential for the persistence of the human-pathogenic Brucella spp. in the mouse and macrophage models of infection.

Effects of light- and altered-cercosporin phenotypes on gene expression in Cercospora kikuchii

1993 ◽  
Vol 39 (1) ◽  
pp. 118-124 ◽  
Author(s):  
J. A. Rollins ◽  
M. Ehrenshaft ◽  
R. G. Upchurch
Keyword(s):  
In Vitro Translation ◽  
Complete Medium ◽  
Wild Type ◽  
In Planta ◽  
Logarithmic Growth Phase ◽  
Two Dimensional Gel Electrophoresis ◽  
Cercospora Kikuchii ◽  
Potato Dextrose Broth ◽  
Type Isolate

Cercospora kikuchii is a fungal pathogen of soybean that produces a photosensitizing and phytotoxic polyketide, cercosporin, in culture and in planta. We have studied the influence of growth stage, light, and growth medium on cercosporin accumulation in a wild-type isolate and three mutant strains with altered toxin phenotypes. After an initial logarithmic growth phase, the wild-type isolate accumulated high levels of cercosporin on either complete medium or potato dextrose broth, but only when cultured in the light. Dark-grown cultures of the wild-type and light-grown cultures of two uv-induced mutant derivatives accumulated 100-fold lower cercosporin levels. A third mutant strain accumulated wild-type cercosporin levels, but only when cultured in the light in potato dextrose broth. Two-dimensional gel electrophoresis of both extracted proteins and in vitro translation products from wild-type cultures revealed the presence of polypeptides and poly A + RNAs whose accumulation was positively regulated by light. Comparison of translated polypeptide patterns from wild-type and mutant cultures also demonstrated differential accumulation of translatable poly A + RNAs in cercosporin-producing and nonproducing cultures.Key words: nonspecific toxin, photo induction, in vitro translation.

scholarly journals Pathogenicity in isolates of Salmonella enterica serotype Enteritidis PT4 which differ in RpoS expression: effects of growth phase and low temperature

1998 ◽  
Vol 121 (2) ◽  
pp. 295-301 ◽  
Author(s):  
T. J. HUMPHREY ◽  
A. WILLIAMS ◽  
K. McALPINE ◽  
F. JØRGENSEN ◽  
C. O'BYRNE
Keyword(s):  
Growth Phase ◽  
Salmonella Enterica ◽  
Acid Tolerance ◽  
Wild Type ◽  
Potential Marker ◽  
Sensitive Isolate ◽  
Acid Tolerant ◽  
Positive Isolate ◽  
Hostile Environments

Experiments with 2 wild type isolates of Salmonella enterica serotype Enteritidis PT4, which differed in RpoS expression, tolerance to certain hostile environments and pathogenicity, found that changes in in vitro acid, heat, or peroxide tolerance had no effect on the ability of the isolates to multiply in the spleens of C57/BL7/J mice infected orally. Thus, with the pathogenic RpoS-positive isolate, the infectivity of log phase chilled cells, which are profoundly acid-sensitive, was the same as that of non-chilled stationary phase cells which are acid-tolerant. Similarity the infectivity of the RpoS-negative, sensitive isolate, was not enhanced by increases in any tolerance. The ability to survive on surfaces, like infectivity, was also largely unaffected by either growth phase or cold exposure. These two attributes may thus be related and, given that the pathogenic PT4 isolate is capable of prolonged survival and the non-pathogenic isolate survives poorly, survival could serve as a potential marker of pathogenicity. Although the pathogenicity of the two isolates was very different, they showed an almost identical increase in acid tolerance following culture at pH 4·0 for up to 60 min.

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