bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induced Expression of the
Legionella pneumophila
Gene Encoding a 20-Kilodalton Protein during Intracellular Infection
Authors: Kwaik Yousef Abu 
Published: December 2019

ABSTRACT

The eukaryotic protein synthesis inhibitor cycloheximid has been used by many investigators to selectively radiolabel intracellular bacteria. Although cycloheximide has no direct effect on bacterial gene expression, there are concerns that long-term inhibition of the host cell protein synthesis may have secondary effects on bacterial gene expression. Therefore, prior to further identification and cloning of the macrophage-induced (MI) genes of
Legionella pneumophila
, the effects of cycloheximide on
L. pneumophila
-infected U937 cells were evaluated by transmission electron microscopy. Inhibition of protein synthesis of the host cell for 6 h had no major effect on the ultrastructure of the host cell, on the formation of rough endoplasmic reticulum-surrounded replicative phagosome, or on initiation of intracellular bacterial replication. In contrast, by 15 h of cycloheximide treatment, there was profound deterioration in the host cell as well as in the phagosome. To examine protein synthesis by
L. pneumophila
during the intracellular infection, U937 macrophage-like cells were infected with
L. pneumophila
, and intracellular bacteria were radiolabeled during a 2-h cycloheximide treatment or following 12 h of cycloheximide treatment. Comparison by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein profile of radiolabeled in vitro-grown
L. pneumophila
to that of intracellularly radiolabeled bacteria showed that 23 proteins were induced in response to the intracellular environment during 2 h of inhibition of host cell protein biosynthesis. Twelve MI proteins of
L. pneumophila
were artifactually induced due to prolonged inhibition of the host cell protein synthesis. The gene encoding a 20-kDa MI protein was cloned by a reverse genetics technique. Sequence analysis showed that the cloned gene encoded a protein that was 80% similar to the enzyme inorganic pyrophosphatase. Studies of promoter fusion to a promoterless
lacZ
gene showed that compared to in vitro-grown bacteria, expression of the pyrophosphatase gene (
ppa
) was induced fourfold throughout the intracellular infection. There was no detectable induction in transcription of the
ppa
promoter during exposure to stress stimuli in vitro. The
ppa
gene of
L. pneumophila
is the first example of a regulated
ppa
gene which is selectively induced during intracellular infection and which may reflect enhanced capabilities of macromolecular biosynthesis by intracellular
L. pneumophila
. The data indicate caution in the long-term use of inhibition of host cell protein synthesis to selectively examine gene expression by intracellular bacteria.
Streptococcal Histone-Like Protein: Primary Structure of
hlpA
and Protein Binding to Lipoteichoic Acid and Epithelial Cells
Authors: Stinson Murray W. , McLaughlin Robert , Choi Suk Ho , Juarez Zaira E. , Barnard John 
Published: December 2019

ABSTRACT

In addition to its role in the nucleoid, the histone-like protein (HlpA) of
Streptococcus pyogenes
is believed to act as a fortuitous virulence factor in delayed sequelae by binding to heparan sulfate-proteoglycans in the extracellular matrix of target organs and acting as a nidus for in situ immune complex formation. To further characterize this protein, the
hlpA
genes were cloned from
S. pyogenes
,
S. gordonii
,
S. mutans
, and
S. sobrinus
, using PCR amplification, and sequenced. The encoded HlpA protein of
S. pyogenes
has 91 amino acids, a predicted molecular mass of 9,647 Da, an isoelectric point of 9.81, and 90% to 95% sequence identity with HlpA of several oral streptococci. The consensus sequence of streptococcal HlpA has 69% identity with the consensus sequence of the histone-like HB protein of
Bacillus
species. Oral viridans group streptococci, growing in chemically defined medium at pH 6.8, released HlpA into the milieu during stationary phase as a result of limited cell lysis. HlpA was not released by these bacteria when grown at pH 6.0 or below.
S. pyogenes
did not release HlpA during growth in vitro; however, analyses of sera from 155 pharyngitis patients revealed a strong correlation (
P
< 0.0017) between the production of antibodies to HlpA and antibodies to streptolysin O, indicating that the histone-like protein is released by group A streptococci growing in vivo. Extracellular HlpA formed soluble complexes with lipoteichoic acid in vitro and bound readily to heparan sulfate on HEp-2 cell surfaces. These results support a potential role for HlpA in the pathogenesis of streptococcus-induced tissue inflammation.
Microbial Pathogenesis in Cystic Fibrosis: Pulmonary Clearance of MucoidPseudomonas aeruginosaand Inflammation in a Mouse Model of Repeated Respiratory Challenge
Authors: Yu H. , Hanes M. , Chrisp C. E. , Boucher J. C. , Deretic V. 
Published: December 2019

ABSTRACTChronic endobronchiolitis compounded by recurringPseudomonas aeruginosainfections is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). In this study, a mouse model of repeated respiratory exposure toP. aeruginosawas established to facilitate investigations of factors contributing toP. aeruginosapersistence and associated inflammatory processes in the lung. While a single exposure toP. aeruginosaaerosols resulted in only mild histopathological changes, repeated exposure caused significant lung pathology in C57BL/6J mice. The peak of histopathological changes and inflammation in C57BL/6J mice was characterized by subacute lymphohistiocytic bronchopneumonia and persistent elevation of tumor necrosis factor alpha and macrophage inflammatory protein 2 in the lung but not in the serum. When isogenic nonmucoid (mucA+) and mucoid (mucA22)P. aeruginosastrains were compared, the mucoid cells were cleared several-fold less efficiently than the parental nonmucoid strain during the initial stages of the aerosol exposure regimen. However, the microscopic pathology findings and proinflammatory cytokine levels were similar in mice exposed to nonmucoid and mucoidP. aeruginosathroughout the infection. We also tested lung histopathology and proinflammatory cytokines in interleukin 10 (IL-10)-deficient transgenic (IL-10T) mice. Significant mortality was seen in IL-10T mice on initial challenge withP. aeruginosa, although no histopathological differences could be observed in the lungs of C57BL/6J and surviving IL-10T mice after a single exposure. However, increased pathology was detected in IL-10T mice relative to C57BL/6J after repeated challenge withP. aeruginosa. This observation supports the proposals that anti-inflammatory cytokines may play a role in suppressingP. aeruginosa-induced tissue damage during chronic infection.
Use of Green Fluorescent Protein To Assess Urease Gene Expression by Uropathogenic
Proteus mirabilis
during Experimental Ascending Urinary Tract Infection
Authors: Zhao Hui , Thompson Richard B. , Lockatell Virginia , Johnson David E. , Mobley Harry L. T. 
Published: December 2019

ABSTRACT

Proteus mirabilis
, a cause of complicated urinary tract infection, expresses urease when exposed to urea. While it is recognized that the positive transcriptional activator UreR induces gene expression, the levels of expression of the enzyme during experimental infection are not known. To investigate in vivo expression of
P. mirabilis
urease, the gene encoding green fluorescent protein (GFP) was used to construct reporter fusions. Translational fusions of urease accessory gene
ureD
, which is preceded by a urea-inducible promoter, were made with
gfp
(modified to express S65T/V68L/S72A [B. P. Cormack et al. Gene 173:33–38, 1996]). Constructs were confirmed by sequencing of the fusion junctions. UreD-GFP fusion protein was induced by urea in both
Escherichia coli
DH5α and
P. mirabilis
HI4320. By using Western blotting with antiserum raised against GFP, expression level was shown to correlate with urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mM urea. Fluorescent
E. coli
and
P. mirabilis
bacteria were observed by fluorescence microscopy following urea induction, and the fluorescence intensity of GFP in cell lysates was measured by spectrophotofluorimetry.
P. mirabilis
HI4320 carrying the UreD-GFP fusion plasmid was transurethrally inoculated into the bladders of CBA mice. One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensity of bacteria in bladder tissue was higher than that in the kidney. Kidneys were primarily infected with single-cell-form fluorescent bacteria, while aggregated bacterial clusters were observed in the bladder. Elongated swarmer cells were only rarely observed. These observations demonstrate that urease is expressed in vivo and that using GFP as a reporter protein is a viable approach to investigate in vivo expression of
P. mirabilis
virulence genes in experimental urinary tract infection.
A New Gene Family (
ariel
) Encodes Asparagine-Rich
Entamoeba histolytica
Antigens, Which Resemble the Amebic Vaccine Candidate Serine-Rich
E. histolytica
Protein
Authors: Mai Zhiming , Samuelson John 
Published: December 2019

ABSTRACT

A family of genes, called
ariel
, are named for and encode asparagine-rich
Entamoeba histolytica
antigens containing 2 to 16 octapeptide repeats. Ariel proteins, which are constitutively expressed by trophozoites, belong to a large antigen family that includes the serine-rich
E. histolytica
protein (SREHP), an amebic vaccine candidate.
Molecular Epidemiology of
Staphylococcus aureus
and
Enterococcus faecalis
in Endophthalmitis
Authors: Booth Mary C. , Hatter Kenneth L. , Miller Darlene , Davis Janet , Kowalski Regis , Parke David W. , Chodosh James , Jett Bradley D. , Callegan Michelle C. , Penland Rebecca , Gilmore Michael S. 
Published: December 2019

ABSTRACT

Genomic DNA fingerprint analysis was performed on 39
Staphylococcus aureus
and 28
Enterococcus faecalis
endophthalmitis isolates collected from multiple clinical centers. Among 21
S. aureus
genomic DNA fingerprint patterns identified, five clonotypes were recovered from multiple unrelated patients and accounted for 58.9% (23 of 39) of the isolates analyzed. Compared with strains having unique genomic DNA fingerprint patterns, the
S. aureus
clonotypes occurring more than once were more likely to result in visual acuities of 20/200 or worse (
P
= 0.036 [χ
2
test]). In contrast to the
S. aureus
isolates, the
E. faecalis
endophthalmitis isolates were a clonally diverse population, enriched for the expression of a known toxin, cytolysin, which is plasmid encoded.
Mannose Induces the Release of Cytopathic Factors from
Acanthamoeba castellanii
Authors: Leher Henry , Silvany Robert , Alizadeh Hassan , Huang Jing , Niederkorn Jerry Y. 
Published: December 2019

ABSTRACT

Acanthamoeba
keratitis is a chronic inflammatory disease of the cornea which is highly resistant to many antimicrobial agents. The pathogenic mechanisms of this disease are poorly understood. However, it is believed that the initial phases in the pathogenesis of
Acanthamoeba
keratitis involve parasite binding and lysis of the corneal epithelium. These processes were examined in vitro, using
Acanthamoeba castellanii
trophozoites. Parasites readily adhered to Chinese hamster corneal epithelial cells in vitro; however, parasite binding was strongly inhibited by mannose but not by lactose. Although mannose prevented trophozoite binding, it did not affect cytolysis of corneal epithelial cells. Moreover, mannose treatment induced trophozoites to release cytolytic factors that lysed corneal epithelial cells in vitro. These factors were uniquely induced by mannose because supernatants collected from either untreated trophozoites or trophozoites treated with other sugars failed to lyse corneal cells. The soluble factors were size fractionated in centrifugal concentrators and found to be ≥100 kDa. Treatment of the supernatants with the serine protease inhibitor phenylmethylsulfonyl fluoride inhibited most, but not all, of the cytopathic activity. These data suggest that the binding of
Acanthamoeba
to mannosylated proteins on the corneal epithelium may exacerbate the pathogenic cascade by initiating the release of cytolytic factors.
Immunogenicity of IpaC-Hybrid Proteins Expressed in theShigella flexneri2a Vaccine Candidate SC602
Authors: Bârzu Simona , Arondel Josette , Guillot Sophie , Sansonetti Philippe J. , Phalipon Armelle 
Published: December 2019

ABSTRACTWe have investigated the capacity of live attenuatedShigella flexneristrains to act as vectors for the induction of local and systemic antibody responses against heterologous epitopes. TheS. flexneriIpaC antigen was selected as a carrier protein into which the C3 neutralizing epitope of the poliovirus VP1 protein was inserted in eight sites distributed along IpaC. The resulting IpaC-C3 hybrid proteins were expressed from recombinant plasmids in theS. flexneri2a vaccine candidate, SC602. Their production was similar to that of wild-type IpaC. All of the hybrid proteins but one were secreted as efficiently as wild-type IpaC. Immunization of mice with each of the recombinant SC602 derivatives reveals that one construct is able to induce serum and local anti-C3 antibodies, showing that at least one permissive site of insertion within IpaC can be defined. Furthermore, mouse-to-mouse variability in the anti-C3 response indicates that the amount of hybrid proteins produced in the host by SC602 should be improved for optimal use ofS. flexnerilive attenuated strains as mucosal vectors for foreign epitopes.
DNA Sequencing and Analysis of the Low-Ca2+-Response Plasmid pCD1 ofYersinia pestisKIM5
Authors: Perry Robert D. , Straley Susan C. , Fetherston Jacqueline D. , Rose Debra J. , Gregor Jason , Blattner Frederick R. 
Published: December 2019

ABSTRACTThe low-Ca2+-response (LCR) plasmid pCD1 of the plague agentYersinia pestisKIM5 was sequenced and analyzed for its genetic structure. pCD1 (70,509 bp) has an IncFIIA-like replicon and a SopABC-like partition region. We have assigned 60 apparently intact open reading frames (ORFs) that are not contained within transposable elements. Of these, 47 are proven or possible members of the LCR, a major virulence property of human-pathogenicYersiniaspp., that had been identified previously in one or more ofY. pestisor the enteropathogenic yersiniaeYersinia enterocoliticaandYersinia pseudotuberculosis. Of these 47 LCR-related ORFs, 35 constitute a continuous LCR cluster. The other LCR-related ORFs are interspersed among three intact insertion sequence (IS) elements (IS100and two new IS elements, IS1616and IS1617) and numerous defective or partial transposable elements. Regional variations in percent GC content and among ORFs encoding effector proteins of the LCR are additional evidence of a complex history for this plasmid. Our analysis suggested the possible addition of a new Syc- and Yop-encoding operon to the LCR-related pCD1 genes and gave no support for the existence of YopL. YadA likely is not expressed, as was the case forY. pestisEV76, and the gene for the lipoprotein YlpA found inY. enterocoliticalikely is a pseudogene inY. pestis. TheyopMgene is longer than previously thought (by a sequence encoding two leucine-rich repeats), the ORF upstream ofypkA-yopJis discussed as a potential Syc gene, and a previously undescribed ORF downstream ofyopEwas identified as being potentially significant. Eight other ORFs not associated with IS elements were identified and deserve future investigation into their functions.
Invasion bySalmonella typhimuriumInduces Increased Expression of the LMP, MECL, and PA28 Proteasome Genes and Changes in the Peptide Repertoire of HLA-B27
Authors: Maksymowych Walter P. , Ikawa Takashi , Yamaguchi Akihiro , Ikeda Makoto , McDonald Darrin , Laouar Leila , Lahesmaa Riitta , Tamura Naoto , Khuong Arranny , Yu David T. Y. , Kane Kevin P. 
Published: December 2019

ABSTRACTWe have analyzed proteasomal adaptation and associated changes in the B27-bound peptide repertoire in response to cellular invasion withSalmonella. The peptide repertoire of HLA-B27 complexes was analyzed by two different methods: (i) high-pressure liquid chromatography (HPLC) profiles of newly synthesized peptides eluted from B27 following metabolic labeling with arginine and (ii) reactivities with two B27 monoclonal antibodies, Ye-2 and B27.M2, sensitive to peptide-induced conformational changes. LMP, MECL, and PA28 expression was analyzed by reverse transcription-PCR (RT-PCR) of mRNA and by Western blot analysis for LMP2. Invasion of HLA-B27-transfected HeLa cells bySalmonella typhimuriuminduced significant changes in the reactivities of HLA-B27 with these two antibodies, which was accompanied by significant quantitative and qualitative changes in the HPLC profile of peptides eluted from HLA-B27. We also observed increases in the RT-PCR values for the LMP2, LMP7, and MECL proteasome subunit genes, as well as the proteasomal activator PA28α and -β genes, and increased expression of the LMP2 protein by Western blotting. Upregulation of LMP2, but not LMP7, gene expression showed a close correlation with the changes in antibody reactivities observed upon bacterial invasion. We observed similar changes in reactivity with the Ye-2 or the B27.M2 antibody of lymphoblastoid cells upon gamma interferon treatment, which significantly correlated with the increased RT-PCR values for the LMP2 gene. This was accompanied by consistent HPLC profile changes for eluted peptides. Thus,Salmonellainvasion leads to serologically recognizable changes in the B27-bound peptide repertoire, which may include peptides of host origin potentially through modulation of proteasome LMP2 subunit expression and, as a consequence, proteasomal activities.
Binding Properties of
Streptococcus gordonii
SspA and SspB (Antigen I/II Family) Polypeptides Expressed on the Cell Surface of
Lactococcus lactis
MG1363
Authors: Holmes Ann R. , Gilbert Christophe , Wells Jeremy M. , Jenkinson Howard F. 
Published: December 2019

ABSTRACT

The oral bacterium
Streptococcus gordonii
expresses two cell wall-associated polypeptides, designated SspA (1,542 amino acid residues) and SspB (1,462 amino acid residues), that have 70% sequence identity. These polypeptides are members of the antigen I/II family of oral streptococcal adhesins and mediate the binding of streptococci to salivary glycoproteins, collagen, and other oral microorganisms such as
Actinomyces naeslundii
. To determine if SspA and SspB have differential binding properties, the coding sequences of the
sspA
and
sspB
genes were cloned into expression plasmid vector pTREX1-usp45LS to generate pTREX1-sspA and pTREX1-sspB, respectively, and the Ssp polypeptides were displayed on the cell surface of
Lactococcus lactis
MG1363. Lactococcal cells expressing similar levels of surface SspA or SspB polypeptide were then compared for their abilities to adhere to a range of antigen I/II polypeptide substrates. More than twice as many
L. lactis
cells expressing SspA bound to immobilized salivary agglutinin glycoprotein (SAG) as did
L. lactis
cells expressing SspB. In contrast, lactococci expressing SspB adhered twice as well as lactococci producing SspA to collagen type I and to
Candida albicans
. The binding of
A. naeslundii
to lactococci was only weakly enhanced by surface expression of Ssp polypeptides.
L. lactis
(pTREX1-sspB) cells bound in greater numbers to SAG than did
Enterococcus faecalis
JH2-2 cells expressing SspB from pAM401EB-5. The results suggest that SspA and SspB have markedly different binding affinities for their oral substrates and thus may function to promote site diversity in colonization by
S. gordonii
.
Differential Normalization of Mucosal Interleukin-8 and Interleukin-6 Activity afterHelicobacter pyloriEradication
Authors: Ando Takafumi , Kusugami Kazuo , Ohsuga Masahiro , Ina Kenji , Shinoda Masataka , Konagaya Toshihiro , Sakai Tetsu , Imada Akira , Kasuga Naomi , Nada Toshi , Ichiyama Satoshi , Blaser Martin J. 
Published: December 2019

ABSTRACTThere is differential resolution of mucosal infiltration with neutrophils and mononuclear cells following successfulHelicobacter pylorieradication. We investigated the effects ofH. pylorieradication on mucosal interleukin-8 (IL-8) and IL-6 activity in relation to the resolution ofH. pylori-associated gastritis. Eighty-one duodenal ulcer patients withH. pyloriinfection received dual- or triple-treatment eradication therapy, and mucosal biopsy specimens obtained at the initial and follow-up endoscopic examinations were cultured in vitro for 24 h. The levels of IL-8 and IL-6 were measured by enzyme-linked immunosorbent assays. In the 42 patients in whomH. pylorieradication failed, there was little change in the numbers of neutrophils and mononuclear cells infiltrating the mucosa and in IL-8 and IL-6 activity. In the 39 patients in whomH. pyloriwas eradicated, there was normalization both in the numbers of infiltrating neutrophils and in mucosal IL-8 activity, which was evident within 1 month following therapy. In contrast, there was a gradual resolution of mononuclear cell infiltration over a 6-month period, accompanied by a gradual normalization in IL-6 levels. Addition ofH. pylorito cultures of mucosal tissues induced a significant increase in IL-8 activity in both uninfected control subjects and patients from whomH. pyloriwas eradicated. However, this introduction yielded a significant increase in IL-6 activity only in the latter group. This study indicates a dichotomy in the changes of mucosal IL-8 and IL-6 activity afterH. pylorieradication. The rapid normalization of IL-8 afterH. pylorieradication and the ability ofH. pyloricells to stimulate IL-8 in control tissues indicate that IL-8 induction is a part of the innate (nonimmune) responses to this organism. In contrast, the results of experiments analyzing IL-6 activity in cultured mucosal tissues suggest that the gradual resolution of mucosal IL-6 activity and mononuclear infiltration after successful eradication observed in vivo may reflect gradually diminishing residual immune responses againstH. pylori.
Identification of the Cilium Binding Epitope of the
Mycoplasma hyopneumoniae
P97 Adhesin
Authors: Hsu Tsungda , Minion F. Chris 
Published: December 2019

ABSTRACT

Mycoplasma hyopneumoniae
colonizes the swine respiratory tract at the level of ciliated cells by attaching specifically to the cilium membrane. This interaction involves an adhesin called P97; the cilium binding activity of this protein was localized to the carboxy terminus, which included two repeat regions, R1 and R2 (T. Hsu, S. Artiushin, and F. C. Minion, J. Bacteriol. 179:1317–1323, 1997). To further delineate the molecular mechanisms of
M. hyopneumoniae
interactions with ciliated epithelium, we used a bank of transposon inserts in the cloned P97 gene to identify the site for cilium binding by testing the truncated gene products in an in vitro microtiter plate adherence assay. These studies showed that the cilium binding site was located in the AAKPV(E) repeat sequence of P97, referred to as the R1 repeat. For functional binding, at least seven AAKPV(E) repeats were required. The adherence-blocking monoclonal antibody F1B6 also recognized this region but required fewer AAKPV(E) repeats for recognition. We then constructed R1 region-
lacZ
gene fusions and used the resulting R1 repeat–β-galactosidase fusion proteins in an in vitro assay to confirm the role of R1 in cilium binding. A comparison of the R1 regions of
M. hyopneumoniae
strains displaying variation in cilium adherence failed to identify changes that could account for the differences in adherence shown by the strains. Thus, we concluded that other proteins, in addition to P97, must be involved in cilium adherence, possibly in combination with P97.