bluuebunny/crossref_metadata_2025 huggingface dataset:
Induced Expression of the
Legionella pneumophila
Gene Encoding a 20-Kilodalton Protein during Intracellular Infection
Authors: Kwaik Yousef Abu
Published: December 2019

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

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

Chronic 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

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

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

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

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

We 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

The 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

We 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

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

There 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

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.
Interleukin-12 Is Required for Control of the Growth of Attenuated Aromatic-Compound-Dependent Salmonellae in BALB/c Mice: Role of Gamma Interferon and Macrophage Activation
Authors: Mastroeni Pietro , Harrison J. A. , Robinson J. H. , Clare S. , Khan S. , Maskell D. J. , Dougan G. , Hormaeche C. E.
Published: December 2019

The attenuatedS. typhimuriumSL3261 (aroA) strain causes mild infections in BALB/c mice. We were able to exacerbate the disease by administering anti-interleukin-12 (IL-12) antibodies, resulting in bacterial counts in the spleens and livers of anti-IL-12-treated mice that were 10- to 100-fold higher than the ones normally observed in premortem mice; yet the animals showed only mild signs of illness. Nevertheless, they eventually died of a slow, progressive disease. Mice infected with salmonellae become hypersusceptible to endotoxin. We found that IL-12 neutralization prevented the death of infected mice following subcutaneous injection of lipopolysaccharide. Granulomatous lesions developed in the spleens and livers of control animals, as opposed to a widespread infiltration of mononuclear cells seen in the organs of anti-IL-12-treated mice. In the latter (heavily infected), salmonellae were seen within mononuclear cells, indicating an impairment of the bactericidal or bacteriostatic ability of the phagocytes in the absence of biologically active IL-12. Gamma interferon (IFN-γ) levels were reduced in the sera and tissue homogenates from anti-IL-12-treated mice compared to those in control animals. Furthermore, fluorescence-activated cell sorter analysis on spleen cells showed that IL-12 neutralization impaired the upregulation of I-Ad/I-Edantigens on macrophages from infected mice. Inducible nitric oxide synthase and IFN-γ mRNA production was down-regulated in anti-IL-12-treated mice, which also showed an increased production of IL-10 mRNA and a decrease in nitric oxide synthase activity in the tissues. Administration of recombinant IFN-γ to anti-IL-12-treated mice was able to restore host resistance, granuloma formation, and expression of major histocompatibility complex class II antigens in F4/80+and CD11b+spleen cells.
Contact-Dependent Protein Secretion in
Porphyromonas gingivalis
Authors: Park Yoonsuk , Lamont Richard J.
Published: December 2019

Porphyromonas gingivalis
can induce its uptake by host epithelial cells; however, the nature and role of the
P. gingivalis
molecules involved in this invasion process have yet to be determined. In this study, modulation of secreted
P. gingivalis
proteins following association with gingival epithelial cells was investigated. Western immunoblot analysis showed that contact with epithelial cells or epithelial cell growth media induces
P. gingivalis
33277 to secrete several proteins with molecular masses between 35 and 95 kDa. Secretion of the Arg-gingipain and Lys-gingipain proteases was repressed under these conditions. The contact-induced secreted protein profile was altered in Arg-gingipain-deficient and Lys-gingipain-deficient mutants, indicating a possible role for these proteases in the secretion pathway. The
P. gingivalis
contact-dependent protein secretion pathway differs to some extent from type III protein secretion pathways in enteric pathogens, as a gene homologous to the
invA
family genes was not detected in
P. gingivalis
. The secreted proteins of
P. gingivalis
may play a role in the interactions of the organism with host cells.
Characterization of
Clostridium botulinum
Type B Neurotoxin Associated with Infant Botulism in Japan
Authors: Kozaki Shunji , Kamata Yoichi , Nishiki Tei-ichi , Kakinuma Hiroaki , Maruyama Hiromi , Takahashi Hiroaki , Karasawa Tadahiro , Yamakawa Kiyotaka , Nakamura Shinichi
Published: December 2019

The neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a food-borne botulism-related strain, Okra. The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT. The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT. Binding experiments with rat brain synaptosomes revealed that
125
I-labeled 111/NT bound to a single binding site with a dissociation constant (

K
d

) of 2.5 nM; the value was rather lower than that (0.42 nM) of
125
I-Okra/NT for the high-affinity binding site. In the lipid vesicles reconstituted with ganglioside GT1b,
125
I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the

K
d

values accorded with those of the low- and high-affinity binding sites in synaptosomes. However,
125
I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner. These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity.
Modulation of Enzymatic Activity and Biological Function ofListeria monocytogenesBroad-Range Phospholipase C by Amino Acid Substitutions and by Replacement with theBacillus cereusOrtholog
Authors: Zückert Wolfram R. , Marquis Hélène , Goldfine Howard
Published: December 2019

The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) ofListeria monocytogenesplays a role in the bacterium’s ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs,Clostridium perfringensα-toxin andBacillus cereusPLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBcactive site, while a gene replacement resulted inL. monocytogenessecreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBcexpressed byL. monocytogeneswas highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBcwas significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells.
Surface Structure, Hydrophobicity, Phagocytosis, and Adherence to Matrix Proteins of
Bacillus cereus
Cells with and without the Crystalline Surface Protein Layer
Authors: Kotiranta Anja , Haapasalo Markus , Kari Kirsti , Kerosuo Eero , Olsen Ingar , Sorsa Timo , Meurman Jukka H. , Lounatmaa Kari
Published: December 2019

Nonopsonic phagocytosis of
Bacillus cereus
by human polymorphonuclear leukocytes (PMNs) with particular attention to bacterial surface properties and structure was studied. Two reference strains (ATCC 14579
T
and ATCC 4342) and two clinical isolates (OH599 and OH600) from periodontal and endodontic infections were assessed for adherence to matrix proteins, such as type I collagen, fibronectin, laminin, and fibrinogen. One-day-old cultures of strains OH599 and OH600 were readily ingested by PMNs in the absence of opsonins, while cells from 6-day-old cultures were resistant. Both young and old cultures of the reference strains of
B. cereus
were resistant to PMN ingestion. Preincubation of PMNs with the phagocytosis-resistant strains of
B. cereus
did not affect the phagocytosis of the sensitive strain. Negatively stained cells of OH599 and OH600 studied by electron microscopy had a crystalline protein layer on the cell surface. In thin-sectioned cells of older cultures (3 to 6 days old), the S-layer was observed to peel off from the cells. No S-layer was detected on the reference strains. Extraction of cells with detergent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major 97-kDa protein from the strains OH599 and OH600 but only a weak 97-kDa band from the reference strain ATCC 4342. One-day-old cultures of the clinical strains (hydrophobicity, 5.9 to 6.0%) showed strong binding to type I collagen, laminin, and fibronectin. In contrast, reference strains (hydrophobicity, −1.0 to 4.2%) as well as 6-day-old cultures of clinical strains (hydrophobicity, 19.0 to 53.0%) bound in only low numbers to the proteins. Gold-labelled biotinylated fibronectin was localized on the S-layer on the cell surface as well as on fragments of S-layer peeling off the cells of a 6-day-old culture of
B. cereus
OH599. Lactose, fibronectin, laminin, and antibodies against the S-protein reduced binding to laminin but not to fibronectin. Heating the cells at 84°C totally abolished binding to both proteins. Benzamidine, a noncompetitive serine protease inhibitor, strongly inhibited binding to fibronectin whereas binding to laminin was increased. Overall, the results indicate that changes in the surface structure, evidently involving the S-layer, during growth of the clinical strains of
B. cereus
cause a shift from susceptibility to PMN ingestion and strong binding to matrix and basement membrane proteins. Furthermore, it seems that binding to laminin is mediated by the S-protein while binding to fibronectin is dependent on active protease evidently attached to the S-layer.
Expression of the Virulence Plasmid-Carried Apyrase Gene (
apy
) of Enteroinvasive
Escherichia coli
and
Shigella flexneri
Is under the Control of H-NS and the VirF and VirB Regulatory Cascade
Authors: Berlutti Francesca , Casalino Mariassunta , Zagaglia Carlo , Fradiani Piera Assunta , Visca Paolo , Nicoletti Mauro
Published: December 2019

The transcription of the virulence plasmid (pINV)-carried invasion genes of
Shigella flexneri
and enteroinvasive
Escherichia coli
(EIEC) is induced at 37°C and repressed at 30°C. In this work, we report that the O135: K−:H− EIEC strain HN280 and
S. flexneri
SFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce apyrase (ATP-diphosphohydrolase), the product of the
apy
gene. In addition, the
S. flexneri
strains, but not the EIEC strain, produce a nonspecific phosphatase encoded by the
phoN-Sf
gene. Both
apy
and
phoN-Sf
are pINV-carried loci whose contribution to the pathogenicity of enteroinvasive microorganisms has been hypothesized but not yet established. We found that, like that of virulence genes, the expression of both the
apy
and the
phoN-Sf
genes was temperature regulated. Strain HN280/32 (a pINV-integrated avirulent derivative of HN280 which has a severe reduction of
virB
transcription) expressed the
apy
gene in a temperature-regulated fashion but to a much lower extent than wild-type HN280, while the introduction of the Δ
hns
deletion in HN280 and in HN280/32 induced the wild-type temperature-independent expression of apyrase. These results indicated that a reduction of
virB
transcription, which is known to occur in the pINV-integrated strain HN280/32, accounts for reduced apyrase expression and that the histone-like protein H-NS is involved in this regulatory network. Independent spontaneously generated mutants of HN280 and of SFZM53 which had lost the capacity to bind Congo red dye (Crb
−
) were isolated, and the molecular alterations of pINV were evaluated by PCR analysis. Alterations of pINV characterized by the absence of
virF
or
virB
and by the presence of the intact
apy
locus or intact
apy
and
phoN-Sf
loci were detected among Crb
−
mutants of HN280 and SFZM53, respectively. While all Crb
−
apy
+
mutants of HN280 failed to produce apyrase, Crb
−

apy
+
phoN-Sf

+
mutants of SFZM53 lacked apyrase activity but produced a nonspecific phosphatase, like parental SFZM53. Moreover, the introduction of recombinant plasmids carrying cloned
virF
(pMYSH6504) or
virB
(pBN1) into Crb
−
mutants of HN280 and SFZM53 lacking
virF
or
virB
, respectively, fully restored temperature-dependent apyrase expression to levels resembling those of the parental strains. Taken together, our results demonstrate that, as has already been shown for invasion genes,
apy
is another locus whose expression is controlled by temperature, H-NS, and the VirF and VirB regulatory cascade. In contrast, the temperature-regulated expression of the nonspecific phosphatase does not appear to be under the control of the same regulatory network. These findings led us to speculate that apyrase may play a role in the pathogenicity of enteroinvasive bacteria.
Plasminogen Binding and Activation at the Surface of
Helicobacter pylori
CCUG 17874
Authors: Pantzar Martina , Ljungh Åsa , Wadström Torkel
Published: December 2019

The binding of iodine-labelled plasminogen to
Helicobacter pylori
CCUG 17874 was characterized. Inhibition of the binding was observed after preincubation of
H. pylori
cells with nonradiolabelled plasminogen, lysine, or the lysine analogue ɛ-aminocaproic acid. Fragments of plasminogen, kringles 1 to 3, kringle 4, and mini-plasminogen, were also studied as potential inhibitors. Mini-plasminogen caused total inhibition of the plasminogen binding, while the other fragments caused only partial inhibition. These findings suggest that
H. pylori
binds specifically the fifth kringle structure of the plasminogen molecule. Plasminogen binding to
H. pylori
seems to be independent of culture media and independent of the presence of the cytotoxin-associated CagA antigen. Immunoblot analysis identified two plasminogen binding proteins of 57 and 42 kDa. Scatchard plot analysis revealed one binding mechanism with a

K
d

value of 7 × 10
−7
M. Conversion of
H. pylori
cell-bound plasminogen to plasmin in the presence of a tissue-type plasminogen activator was demonstrated by digestion of the chromogenic substrate S-2251. No activation was noted when plasminogen or tissue-type plasminogen activator was incubated with
H. pylori
cells alone. Formation of
H. pylori
cell surface-bound plasmin may be important to provide a powerful proteolytic mechanism for gastric tissue penetration in type B gastritis and peptic ulcer disease, since plasmin degrades not only fibrin but also extracellular matrix proteins such as various collagens and fibronectin.
Mutational Analysis of Superantigen Activity Responsible for the Induction of Skin Erythema by Streptococcal Pyrogenic Exotoxin C
Authors: Yamaoka Junichi , Nakamura Eijiro , Takeda Yoshifumi , Imamura Sadao , Minato Nagahiro
Published: December 2019

Streptococcal pyrogenic exotoxin C (SPEC), when injected intradermally, induces erythema in unsensitized rabbits. In the present study, we examined whether this erythema induction is due to the T-cell stimulatory activity of SPEC as a superantigen. Analysis by using single-residue mutant SPECs indicated that mutant SPECs Y15I, A16E, and Y17I, in which tyrosine 15, alanine 16, and tyrosine 17 were replaced with isoleucine, glutamic acid, and isoleucine, respectively, exhibited significantly reduced mitogenic activity for Vβ2+human T cells in vitro, and Y15I showed as much as a 1,000-fold reduction. Y15I mutant SPEC, however, retained the ability to bind to major histocompatibility complex class II antigen and to form a homodimer, implying that residue 15 is critically important for the interaction of SPEC with T-cell antigen receptor β chains. When injected intradermally into normal rabbits, wild-type SPEC induced a characteristic erythema after 3 h in a dose-dependent fashion, which was associated with polymorphonuclear and mononuclear cell infiltration. This erythema formation was found to be severely suppressed by systemic pretreatment with cyclosporin A, suggesting the involvement of host T cells. Y15I mutant SPEC exhibited nearly 1,000-fold less erythema induction in vivo than wild-type SPEC. Altogether, the present results strongly suggest that erythema induction in rabbits by SPEC is attributable mostly to its T-cell stimulatory activity as a superantigen.
CD95 Expression in Aged Mice Infected with Tuberculosis
Authors: Roberts A. D. , Orme Ian M.
Published: December 2019

The interaction between CD95 and its ligand is an important homeostatic mechanism that leads to the induction of apoptosis in activated T cells. In view of recent evidence that this pathway might be defective in aged mice, this study investigated CD95 expression on T cells in old mice activated by infection withMycobacterium tuberculosis. The results of the study do not support the hypothesis that CD95 is poorly expressed on CD4 T cells from old mice; instead, it was found that similar numbers of T cells from young and old mice expressed CD95, with the intensity of expression if anything higher on the cells from the old mice. In addition, the study demonstrated that changes in CD44 and CD45RB expression previously observed in young infected mice proceeded in a similar fashion in old animals and, as would be predicted, that CD95hiexpression was primarily associated with CD4 T cells expressing the activated CD44hiCD45RBhiphenotype.
Acquisition of Iron byGardnerella vaginalis
Authors: Jarosik Gregory P. , Land Carol Beth , Duhon Patrice , Chandler Roderick , Mercer Tammy
Published: December 2019

SixGardnerella vaginalisstrains were examined for the ability to utilize various iron-containing compounds as iron sources. In a plate bioassay, all six strains acquired iron from ferrous chloride, ferric chloride, ferrous sulfate, ferric ammonium citrate, ferrous ammonium sulfate, bovine and equine hemin, bovine catalase, and equine, bovine, rabbit, and human hemoglobin. All six strains also acquired iron from human lactoferrin, but not from human transferrin, as determined by a liquid broth growth assay. Siderophore production was detected in eightG. vaginalisstrains by the chrome azurol S universal chemical assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cytoplasmic membrane proteins isolated fromG. vaginalis594 grown under iron-replete and iron-restricted conditions revealed several iron-regulated proteins ranging in molecular mass from 33 to 94 kDa. These results indicate thatG. vaginalismay acquire iron from iron salts and host iron compounds.
Expression of CD14 by Hepatocytes: Upregulation by Cytokines during Endotoxemia
Authors: Liu Shubing , Khemlani Lajwanti S. , Shapiro Richard A. , Johnson Mark L. , Liu Kaihong , Geller David A. , Watkins Simon C. , Goyert Sanna M. , Billiar Timothy R.
Published: December 2019

Studies were undertaken to examine hepatocyte CD14 expression during endotoxemia. Our results show that lipopolysaccharide (LPS) treatment in vivo caused a marked upregulation in CD14 mRNA and protein levels in rat hepatocytes. Detectable increases in mRNA were seen as early as 1.5 h after LPS treatment; these increases peaked at 20-fold by 3 h and returned to baseline levels by 24 h. In situ hybridization localized the CD14 mRNA expression to hepatocytes both in vitro and in vivo. Increases in hepatic CD14 protein levels were detectable by 3 h and peaked at 12 h. Hepatocytes from LPS-treated animals expressed greater amounts of cell-associated CD14 protein, and more of the soluble CD14 was released by hepatocytes from LPS-treated rats in vitro. The increases in hepatocyte CD14 expression during endotoxemia occurred in parallel to increases of CD14 levels in plasma. To provide molecular identification of the hepatocyte CD14, we cloned the rat liver CD14 cDNA. The longest clone consists of a 1,591-bp insert containing a 1,116-bp open reading frame. The deduced amino acid sequence is 372 amino acids long, has 81.8 and 62.8% homology to the amino acid sequences of mouse and human CD14, respectively, and is identical to the rat macrophage CD14. The expressed CD14 protein from this clone was functional, as indicated by NF-κB activation in response to LPS and fluorescein isothiocyanate-LPS binding in CHO cells stably transfected with rat CD14. A nuclear run-on assay showed that CD14 transcription rates were significantly increased in hepatocytes from LPS-treated animals, indicating that the upregulation in CD14 mRNA levels observed in rat hepatocytes after LPS treatment is dependent, in part, on increased transcription. In vitro and in vivo experiments indicated that interleukin-1β and/or tumor necrosis factor α participate in the upregulation of CD14 mRNA levels in hepatocytes. Our data indicate that hepatocytes express CD14 and that hepatocyte CD14 mRNA and protein levels increase rapidly during endotoxemia. Our observations also support the idea that soluble CD14 is an acute-phase protein and that hepatocytes could be a source for soluble CD14 production.
Differential Growth Characteristics and Streptomycin Susceptibility of Virulent and Avirulent
Mycobacterium tuberculosis
Strains in a Novel Fibroblast-Mycobacterium Microcolony Assay
Authors: Byrd Thomas F. , Green Gary M. , Fowlston Sharon E. , Lyons C. Rick
Published: December 2019

The ability to spread from cell to cell may be an important virulence determinant of
Mycobacterium tuberculosis
. An in vitro assay was developed to characterize this ability among four strains of
M. tuberculosis
: the attenuated strain H37Ra, the virulent strains H37Rv and Erdman, and a virulent clinical isolate (Stew). Confluent monolayers of human skin fibroblasts were infected with these strains and overlaid with agar-medium.
M. tuberculosis
infection developed over 21 days as microcolonies originating within the plane of the fibroblasts. Microcolonies of the virulent strains had an elongated appearance and exhibited extensive cording. The cords appeared to invade adjacent cells within the plane of the monolayer. Microcolony diameter of the Erdman strain was significantly larger than that of the other virulent strains, indicating that virulent strains can have distinguishing phenotypes in this assay. In contrast, avirulent H37Ra microcolonies were rounded and noncorded. H37Ra microcolonies were significantly smaller than those of the virulent strains. Microcolony diameter of the virulent strains was not reduced by the extracellularly acting antibiotic streptomycin at concentrations of up to 5.0 μg/ml. In contrast, H37Ra microcolony size was reduced at concentrations as low as 0.5 μg/ml. Growth of all strains was similarly inhibited by 1.0 μg of streptomycin per ml in fibroblast-conditioned tissue culture medium alone. When fibroblasts were infected with the
M. tuberculosis
strains without an agar overlay, with and without streptomycin, numbers of CFU mirrored the changes observed in the microcolony assay. There was a statistically significant decrease in H37Ra CFU compared to virulent strains after treatment with streptomycin. These differences between H37Ra and virulent strains in human fibroblasts suggest that H37Ra may be lacking a virulence determinant involved in cell-to-cell spread of
M. tuberculosis
.