bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell Vacuolation Caused by
Vibrio cholerae
Hemolysin
Authors: Figueroa-Arredondo Paula , Heuser John E. , Akopyants Natalia S. , Morisaki J. Hiroshi , Giono-Cerezo Silvia , Enrı́quez-Rincón Fernando , Berg Douglas E.
Published: July 2002

Non-O1 strains of
Vibrio cholerae
implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood. Here we report that at least one-third of diarrhea-associated nonepidemic
V. cholerae
strains from Mexico cause vacuolation of cultured Vero cells. Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin of
Aeromonas
; it involved primarily the endoplasmic reticulum at early times (∼1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (∼16 h). In contrast to vacuolation caused by
Helicobacter pylori
VacA protein, that induced by
V. cholerae
was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases. It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization. The gene for
V. cholerae
vacuolating activity was cloned and was found to correspond to
hlyA
, the structural gene for hemolysin. HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis. Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin. We propose that this vacuolation, in itself, contributes to the virulence of
V. cholerae
strains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses.
Expression of
Chlamydia pneumoniae
Polymorphic Membrane Protein Family Genes
Authors: Grimwood Jane , Olinger Lynn , Stephens Richard S.
Published: July 2002

The genome of the obligate intracellular bacterium
Chlamydia pneumoniae
CWL029 encodes a family of 21 proteins with predicted outer membrane localization. These polymorphic membrane proteins (Pmps) are heterogeneous in both amino acid sequence and predicted size but are unified by the conserved amino acid motifs GGAI and FXXN repeated in the N-terminal half of each protein. Reverse transcriptase PCR analysis showed that all
pmp
genes are transcribed. To determine whether all proteins are expressed, specific antisera were generated by immunization with mutually exclusive synthetic peptides representing each of the 21 predicted Pmps. Each antiserum reacted with, and was typically immunospecific for, the corresponding peptide immunogen by enzyme-linked immunosorbent assay. Western blot analyses of purified elementary bodies showed that 11 of the 21 Pmps were detectable. Attempts to demonstrate by Sarykosyl fractionation that the Pmps were localized to the outer membrane revealed that several of the Pmps were unstable and readily degraded. Analyses of additional
C. pneumoniae
strains showed that although some Pmps are conserved, others vary between strains, in both molecular weight and level of expression.
Involvement of CD14 and Toll-Like Receptors in Activation of Human Monocytes byAspergillus fumigatusHyphae
Authors: Wang J. E. , Warris A. , Ellingsen E. A. , Jørgensen P. F. , Flo T. H. , Espevik T. , Solberg R. , Verweij P. E. , Aasen A. O.
Published: July 2002

Invasive fungal infections represent an increasing problem associated with high mortality. The present study was undertaken to identify leukocyte subsets that are activated by hyphal fragments in a whole-human-blood model, as well as to examine the involvement of CD14 and Toll-like receptors (TLRs) in activation of monocytes by hyphae. Incubation of whole human blood with hyphal fragments fromAspergillus fumigatusandScedosporium prolificansfor 6 h caused induction of mRNAs for tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 in T cells, B cells, and monocytes, but not in granulocytes, as analyzed by reverse transcription-PCR with mRNA isolated from very pure populations of these leukocyte subsets. In primary adherent human monocytes, induction of TNF-α by hyphal fragments was dependent on plasma. Heat treatment of plasma at 56°C for 30 min strongly reduced the ability of plasma to prime for activation. Pretreatment of human monocytes with different concentrations (1, 3, and 10 μg/ml) of monoclonal antibody (MAb) HTA125 (anti-TLR4) or MAb 18D11 (anti-CD14) for 30 min inhibited the release of TNF-α induced by hyphal fragments in a dose-dependent manner. Maximal inhibitions of 35 and 70% were obtained with 10 μg of HTA125 and 18D11 per ml, respectively. In contrast, pretreatment with MAb TL2.1 (anti-TLR2) did not affect signaling induced by hyphae. Pretreatment with the lipid A antagonist B975 blocked lipopolysaccharide signaling but did not inhibit TNF-α production induced by hyphal fragments. Our results suggest that T cells, B cells, and monocytes are involved in the innate immune response to invasive fungal pathogens and that serum components are relevant for activation of monocytes by hyphae. CD14 and TLR4 may be involved in signaling ofAspergillushyphae in monocytes, but further studies to elucidate this issue are warranted.
Mu-Like Prophage in Serogroup B
Neisseria meningitidis
Coding for Surface-Exposed Antigens
Authors: Masignani Vega , Giuliani Marzia Monica , Tettelin Hervé , Comanducci Maurizio , Rappuoli Rino , Scarlato Vincenzo
Published: July 2002

Sequence analysis of the genome of
Neisseria meningititdis
serogroup B revealed the presence of an ∼35-kb region inserted within a putative gene coding for an ABC-type transporter. The region contains 46 open reading frames, 29 of which are colinear and homologous to the genes of
Escherichia coli
Mu phage. Two prophages with similar organizations were also found in serogroup A meningococcus, and one was found in
Haemophilus influenzae
. Early and late phage functions are well preserved in this family of Mu-like prophages. Several regions of atypical nucleotide content were identified. These likely represent genes acquired by horizontal transfer. Three of the acquired genes are shown to code for surface-associated antigens, and the encoded proteins are able to induce bactericidal antibodies.
Antimycobacterial Agent Based on mRNA Encoding Human β-Defensin 2 Enables Primary Macrophages To Restrict Growth ofMycobacterium tuberculosis
Authors: Kisich Kevin O. , Heifets Leonid , Higgins Michael , Diamond Gill
Published: July 2002

Human macrophages are hosts forMycobacterium tuberculosis, the causative agent of tuberculosis, which killed approximately 1.87 million people in 1997. Human alveolar macrophages do not express α- or β-defensins, broad-spectrum antimicrobial peptides which are expressed in macrophages from other species more resistant to infection withM. tuberculosis. It has been previously reported thatM. tuberculosisis susceptible to killing by defensins, which may explain the difference in resistance. Defensin peptides have been suggested as a possible therapeutic strategy for a variety of infectious diseases, but development has been hampered by difficulties in their large-scale production. Here we report the cellular synthesis of human β-defensin 2 via highly efficient mRNA transfection of human macrophages. This enabled mycobactericidal and mycobacteristatic activity by the macrophages. Although human macrophages are difficult to transfect with plasmid vectors, these studies illustrate that primary macrophages are permissive for mRNA transfection, which enabled expression of a potentially therapeutic protein.
Clumping Factor A Mediates Binding of
Staphylococcus aureus
to Human Platelets
Authors: Siboo Ian R. , Cheung Ambrose L. , Bayer Arnold S. , Sullam Paul M.
Published: July 2002

The direct binding of bacteria to platelets may be an important virulence mechanism in the pathogenesis of infective endocarditis. We have previously described
Staphylococcus aureus
strain PS12, a Tn
551
-derived mutant of strain ISP479, with reduced ability to bind human platelets in vitro. When tested in an animal model of endocarditis, the PS12 strain was less virulent than its parental strain, as measured by bacterial densities in endocardial vegetations and incidence of systemic embolization. We have now characterized the gene disrupted in PS12 and its function in platelet binding. DNA sequencing, Southern blotting, and PCR analysis indicate that PS12 contained two Tn
551
insertions within the clumping factor A (ClfA) locus (
clfA
). The first copy was upstream from the
clfA
start codon and appeared to have no effect on ClfA production. The second insertion was within the region encoding the serine aspartate repeat of ClfA and resulted in the production of a truncated ClfA protein that was secreted from the cell. A purified, recombinant form of the ClfA A region, encompassing amino acids 40 through 559, significantly reduced the binding of ISP479C to human platelets by 44% (
P
= 0.0001). Immunoprecipitation of recombinant ClfA that had been incubated with solubilized platelet membranes coprecipitated a 118-kDa platelet membrane protein. This protein does not appear to be glycoprotein IIb. These results indicate that platelet binding by
S. aureus
is mediated in part by the direct binding of ClfA to a novel 118-kDa platelet membrane receptor.
Receptor for Fc on the Surfaces of Schistosomes
Authors: Loukas Alex , Jones Malcolm K. , King Lynette T. , Brindley Paul J. , McManus Donald P.
Published: July 2002

Schistosoma mansonimasks its surface with adsorbed host proteins including erythrocyte antigens, immunoglobulins, major histocompatibility complex class I, and β2-microglobulin (β2m), presumably as a means of avoiding host immune responses. How this is accomplished has not been explained. To identify surface receptors for host proteins, we biotinylated the tegument of liveS. mansoniadults and mechanically transformed schistosomula and then removed the parasite surface with detergent. Incubation of biotinylated schistosome surface extracts with human immunoglobulin G (IgG) Fc-Sepharose resulted in purification of a 97-kDa protein that was subsequently identified as paramyosin (Pmy), using antiserum specific for recombinant Pmy. Fc also bound recombinantS. mansoniPmy and nativeS. japonicumPmy. Antiserum to Pmy decreased the binding of Pmy to Fc-Sepharose, and no proteins bound after removal of Pmy from extracts. Fluoresceinated human Fc bound to the surface, vestigial penetration glands, and nascent oral cavity of mechanically transformed schistosomula, and rabbit anti-Pmy Fab fragments ablated the binding of Fc to the schistosome surface. Pmy coprecipitated with host IgG from parasite surface extracts, indicating that complexes formed on the parasite surface as well as in vitro. Binding of Pmy to Fc was not inhibited by soluble protein A, suggesting that Pmy does not bind to the region between the CH2 and CH3 domains used by many other Fc-binding proteins. β2m did not bind to the schistosome Fc receptor (Pmy), a finding that contradicts reports from earlier workers but did bind to a heteromultimer of labeled schistosomula surface proteins. This is the first report of the molecular identity of a schistosome Fc receptor; moreover it demonstrates an additional aspect of the unusual and multifunctional properties of Pmy from schistosomes and other parasitic flatworms.
Antibodies to Variant Antigens on the Surfaces of Infected Erythrocytes Are Associated with Protection from Malaria in Ghanaian Children
Authors: Dodoo Daniel , Staalsoe Trine , Giha Haider , Kurtzhals Jørgen A. L. , Akanmori Bartholomew D. , Koram Kojo , Dunyo Samuel , Nkrumah Francis K. , Hviid Lars , Theander Thor G.
Published: July 2002

Plasmodium falciparum
erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of infected erythrocytes. Each parasite genome contains about 40 PfEMP1 genes, but only 1 PfEMP1 gene is expressed at a given time. PfEMP1 serves as a parasite-sequestering ligand to endothelial cells and enables the parasites to avoid splenic passage. PfEMP1 antibodies may protect from disease by inhibiting sequestration, thus facilitating the destruction of infected erythrocytes in the spleen. In this study, we have measured antibodies in Ghanaian children to a conserved region of PfEMP1 by enzyme-linked immunosorbent assay and antibodies to variant molecules on erythrocytes infected with field isolates of
P. falciparum
by flow cytometry. Based on close clinical monitoring, the children were grouped into those who did (susceptible) and those who did not (protected) have malaria during the season. The prevalences of antibodies to both the conserved PfEMP1 peptide and the variant epitopes were greater than 50%, and the levels of immunoglobulin G (IgG) correlated with age. The levels of antibodies to both the conserved peptide and the variant epitopes were higher in protected than in susceptible children. After correcting for the effect of age, the levels of IgG to variant antigens on a Sudanese and a Ghanaian parasite isolate remained significantly higher in protected than in susceptible children. Thus, the levels of IgG to variant antigens expressed on the surface of infected erythrocytes correlated with protection from clinical malaria. In contrast, the levels of IgG to a peptide derived from a conserved part of PfEMP1 did not correlate with protection from malaria.
Superoxide Dismutase-Deficient Mutants of
Helicobacter pylori
Are Hypersensitive to Oxidative Stress and Defective in Host Colonization
Authors: Seyler Richard W. , Olson Jonathan W. , Maier Robert J.
Published: July 2002

Superoxide dismutase (SOD) is a nearly ubiquitous enzyme among organisms that are exposed to oxic environments. The single SOD of
Helicobacter pylori
, encoded by the
sodB
gene, has been suspected to be a virulence factor for this pathogenic microaerophile, but mutations in this gene have not been reported previously. We have isolated mutants with interruptions in the
sodB
gene and have characterized them with respect to their response to oxidative stress and ability to colonize the mouse stomach. The
sodB
mutants are devoid of SOD activity, based on activity staining in nondenaturing gels and quantitative assays of cell extracts. Though wild-type
H. pylori
is microaerophilic, the mutants are even more sensitive to O
2
for both growth and viability. While the wild-type strain is routinely grown at 12% O
2
, growth of the mutant strains is severely inhibited at above 5 to 6% O
2
. The effect of O
2
on viability was determined by subjecting nongrowing cells to atmospheric levels of O
2
and plating for survivors at 2-h time intervals. Wild-type cell viability dropped by about 1 order of magnitude after 6 h, while viability of the
sodB
mutant decreased by more than 6 orders of magnitude at the same time point. The mutants are also more sensitive to H
2
O
2
, and this sensitivity is exacerbated by increased O
2
concentrations. Since oxidative stress has been correlated with DNA damage, the frequency of spontaneous mutation to rifampin resistance was studied. The frequency of mutagenesis of an
sodB
mutant strain is about 15-fold greater than that of the wild-type strain. In the mouse colonization model, only 1 out of 23 mice inoculated with an SOD-deficient mutant of a mouse-adapted strain became
H. pylori
positive, while 15 out of 17 mice inoculated with the wild-type strain were shown to harbor the organism. Therefore, SOD is a virulence factor which affects the ability of this organism to colonize the mouse stomach and is important for the growth and survival of
H. pylori
under conditions of oxidative stress.
Growth Phase-Dependent Invasion of
Pseudomonas aeruginosa
and Its Survival within HeLa Cells
Authors: Ha Unhwan , Jin Shouguang
Published: July 2002

Clinical isolates of
Pseudomonas aeruginosa
are classified into invasive and noninvasive (cytolytic) strains. In a noninvasive PA103 background, ExoS and ExoT have recently been shown to function as anti-internalization factors. However, these two factors seemed not to have such a function in an invasive strain PAK background. In this study, using HeLa tissue culture cells, we observed that the internalization of invasive strain PAK is dependent on its growth phases, with the stationary-phase cells internalized about 100-fold more efficiently than the exponential-phase cells. This growth phase-dependent internalization was not observed in the noninvasive PA103 strain. Further analysis of various mutant derivatives of the invasive PAK and the noninvasive PA103 strains demonstrated that ExoS or ExoT that is injected into host cells by a type III secretion machinery functions as an anti-internalization factor in both types of strains. In correlation with the growth phase-dependent internalization, the invasive strain PAK translocates much higher amount of ExoS and ExoT into HeLa cells when it is in an exponential-growth phase than when it is in a stationary-growth phase, whereas the translocation of ExoT by the noninvasive strain PA103 is consistently high regardless of the growth phases, suggesting a difference in the regulatory mechanism of type III secretion between the two types of strains. Consistent with the invasive phenotype of the parent strain, an internalized PAK derivative survived well within the HeLa cells, whereas the viability of internalized PA103 derivative was dramatically decreased and completely cleared within 48 h. These results indicate that the invasive strains of
P. aeruginosa
have evolved the mechanism of intracellular survival, whereas the noninvasive
P. aeruginosa
strains have lost or not acquired the ability to survive within the epithelial cells.
Novel Type of Fimbriae Encoded by the Large Plasmid of Sorbitol-Fermenting Enterohemorrhagic
Escherichia coli
O157:H
−
Authors: Brunder Werner , Khan A. Salam , Hacker Jörg , Karch Helge
Published: July 2002

Sorbitol-fermenting (SF) enterohemorrhagic
Escherichia coli
(EHEC) O157:H
−
have emerged as important causes of diarrheal diseases and the hemolytic-uremic syndrome in Germany. In this study, we characterized a 32-kb fragment of the plasmid of SF EHEC O157:H
−
, pSFO157, which differs markedly from plasmid pO157 of classical non-sorbitol-fermenting EHEC O157:H7. We found a cluster of six genes, termed
sfpA
,
sfpH
,
sfpC
,
sfpD
,
sfpJ,
and
sfpG,
which mediate mannose-resistant hemagglutination and the expression of fimbriae.
sfp
genes are similar to the
pap
genes, encoding P-fimbriae of uropathogenic
E. coli
, but the
sfp
cluster lacks homologues of genes encoding subunits of a tip fibrillum as well as regulatory genes. The major pilin, SfpA, despite its similarity to PapA, does not cluster together with known PapA alleles in a phylogenetic tree but is structurally related to the PmpA pilin of
Proteus mirabilis
. The putative adhesin gene
sfpG
, responsible for the hemagglutination phenotype, shows significant homology neither to
papG
nor to other known sequences. Sfp fimbriae are 3 to 5 nm in diameter, in contrast to P-fimbriae, which are 7 nm in diameter. PCR analyses showed that the
sfp
gene cluster is a characteristic of SF EHEC O157:H
−
strains and is not present in other EHEC isolates, diarrheagenic
E. coli,
or other
Enterobacteriaceae
. The
sfp
gene cluster is flanked by two blocks of insertion sequences and an origin of plasmid replication, indicating that horizontal gene transfer may have contributed to the presence of Sfp fimbriae in SF EHEC O157:H
−
.
Induction of Cell-Mediated Immunity againstMycobacterium tuberculosisUsing DNA Vaccines Encoding Cytotoxic and Helper T-Cell Epitopes of the 38-Kilodalton Protein
Authors: Fonseca Dora P. A. J. , Benaissa-Trouw Barry , van Engelen Marloes , Kraaijeveld Cees A. , Snippe Harm , Verheul André F. M.
Published: July 2002

Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed DNA vaccines encoding cytotoxic T lymphocytes (CTL) and T helper cell (Th) epitopes of the 38-kDa lipoglycoprotein ofMycobacterium tuberculosisand analyzed and compared their immunogenicities with that of pXJ38, a DNA vaccine encoding the entire 38-kDa protein (X. Zhu, N. Venkataprasad, H. S. Thangaraj, M. Hill, M. Singh, J. Ivanyi, and H. M. Vordermeier, J. Immunol. 158:5921–5926, 1997). Plasmid DNAs encoding a CTL epitope, P3 (pP3), a Th epitope (vTh), or both the Th and the P3 epitopes (pThP3) were prepared and tested in C57BL6/J (H-2b) mice. Our results confirmed that DNA immunization with pXJ38 induces strong CD8+CTL and Th1 responses (high gamma interferon [IFN-γ], low interleukin-4 [IL-4]). Coadministration of plasmid DNAs encoding a Th epitope with those encoding a CTL epitope (vTh+pP3) elicited both antigen-specific CD8+CTL and Th1 responses. High levels of IFN-γ were secreted by spleen cells from all plasmid DNA-vaccinated mice after in vitro stimulation with the recombinant 38-kDa protein. Small or undetectable amounts of IL-4 were observed, which indicates the induction of a Th1-like response. Multiple-epitope vaccination by vTh+pP3 or pThP3 resulted in a broader Th1 response to peptide or epitopes than the single-epitope plasmid DNAs. Antigen-specific immunoglobulin G2a was only detected in sera from mice immunized with the plasmid pXJ38, and not in mice immunized with the epitope-based DNA vaccines. Thus, the absence of an antibody response after immunization with epitope plasmid DNAs and their ability to trigger only a specific cellular immune response may prove to be important advantages for a vaccine against tuberculosis.
Reciprocal, Temporal Expression of SpeA and SpeB by Invasive M1T1 Group A Streptococcal Isolates In Vivo
Authors: Kazmi Shahana U. , Kansal Rita , Aziz Ramy K. , Hooshdaran Massoumeh , Norrby-Teglund Anna , Low D. E. , Halim Abdel-Baset , Kotb Malak
Published: July 2002

The streptococcal pyrogenic exotoxins (Spes) play a central role in the pathogenesis of invasive group A streptococcal (GAS) infections. The majority of recent invasive GAS infections have been caused by an M1T1 strain that harbors the genes for several streptococcal superantigens, including
speA
,
speB
,
speF
,
speG
, and
smeZ
. However, considerable variation in the expression of Spe proteins among clonal M1 isolates has been found, and many of the
speA
-positive M1 strains do not produce detectable amounts of SpeA in vitro. This study was designed to test the hypothesis that
speA
gene expression can be induced in vivo. A mouse infection chamber model that allows sequential sampling of GAS isolates at various time points postinfection was developed and used to monitor the kinetics of Spe production in vivo. Micropore Teflon diffusion chambers were implanted subcutaneously in BALB/c mice, and after 3 weeks the pores became sealed with connective tissue and sterile fluid containing a white blood cell infiltrate accumulated inside the infection chambers. Representative clonal M1T1 isolates expressing no detectable SpeA were inoculated into the implanted chambers, and the expression of SpeA in the aspirated aliquots of the chamber fluid was analyzed on successive days postinfection. Expression of SpeA was detected in the chamber fluid as early as days 3 to 5 postinfection in most animals, with a significant increase in expression by day 7 in all infected mice. Isolates recovered from the chamber and grown in vitro continued to produce SpeA even after 21 passages in vitro, suggesting stable switch on of the
speA
gene. A temporal relation between the upregulation of SpeA expression and the downregulation of SpeB expression was observed in vivo. These data suggest that in vivo host and/or environmental signals induced
speA
gene expression and suppressed
speB
gene expression. This underscores the role of the host-pathogen interaction in regulating the expression of streptococcal virulence factors in vivo. The model described here should facilitate such studies.
Predominance of Serotype-Specific Mucosal Antibody Response in
Shigella flexneri
-Infected Humans Living in an Area of Endemicity
Authors: Rasolofo-Razanamparany Voahangy , Cassel-Beraud Anne-Marie , Roux Jean , Sansonetti Philippe J. , Phalipon Armelle
Published: July 2002

The mucosal humoral immune response elicited following
Shigella flexneri
infection in patients living in Antananarivo districts (Madagascar Island) was evaluated by measuring the gut-derived, circulating immunoglobulin A (IgA) antibody-secreting cells (ASC) specific for the major bacterial antigen lipopolysaccharide (LPS). Fifty, 34, 11, and 5% of the
S. flexneri
-positive patients were infected with serotypes 2a, 1a, 4a, and 3a, respectively. The total number of IgA ASC in infected patients increased significantly, compared to the number in healthy controls, early after the onset of disease. The number of anti-homologous LPS IgA ASC varied among individuals and peaked between days 5 and 10 after the onset of the disease. In the
S. flexneri
1a- and 2a-infected patients, the level of IgA ASC cross-reactivity to heterologous
S. flexneri
serotypes was weak. These data indicate that
S. flexneri
2a and 1a are the predominant strains responsible for shigellosis in this area of endemicity and that the anti-LPS antibody response following natural infection is mainly directed against serotype-specific determinants.
Roles of Fructosyltransferase and Levanase-Sucrase of
Actinomyces naeslundii
in Fructan and Sucrose Metabolism
Authors: Bergeron Lori J. , Burne Robert A.
Published: July 2002

The ability of
Actinomyces naeslundii
to convert sucrose to extracellular homopolymers of fructose and to catabolize these types of polymers is suspected to be a virulence trait that contributes to the initiation and progression of dental caries and periodontal diseases. Previously, we reported on the isolation and characterization of the gene,
ftf
, encoding the fructosyltransferase (FTF) of
A. naeslundii
WVU45. Allelic exchange mutagenesis was used to inactivate
ftf
, revealing that FTF-deficient stains were completely devoid of the capacity to produce levan-type (β2,6-linked) polysaccharides. A polyclonal antibody was raised to a histidine-tagged, purified
A. naeslundii
FTF, and the antibody was used to localize the enzyme in the supernatant fluid. A sensitive technique was developed to detect levan formation by proteins that had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the method was used to confirm that the levan-synthesizing activity of
A. naeslundii
existed predominantly in a cell-free form, that a small amount of the activity was cell associated, and that the
ftf
mutant was unable to produce levans. By using the nucleotide sequence of the levanase gene of a genospecies 2
A. naeslundii
, formerly
Actinomyces viscosus
, a portion of a homologue of this gene (
levJ
) was amplified by PCR and inserted into a suicide vector, and the resulting construct was used to inactivate the
levJ
gene in the genospecies 1 strain WVU45. A variety of physiologic and biochemical studies were performed on the wild-type and LevJ-deficient strains to demonstrate that (i) this enzyme was the dominant levanase and sucrase of
A. naeslundii
; (ii) that LevJ was inducible by growth in sucrose; (iii) that the LevJ activity was found predominantly (>90%) in a cell-associated form; and (iv) that there was a second, fructose-inducible fructan hydrolase activity produced by these strains. The data provide the first detailed molecular analysis of fructan production and catabolism in this abundant and important oral bacterium.
Interleukin-2 and Loss of Immunity in ExperimentalMycobacterium aviumInfection
Authors: Mannering Stuart I. , Cheers Christina
Published: July 2002

Experimental infection of mice with a virulent strain ofMycobacterium aviumleads to a slowly progressive disease, which we have previously shown culminates in loss of gamma interferon (IFN-γ) production by T lymphocytes and death of the animals approximately 40 weeks after infection. Here we investigated the changes in T-cell activation, the production of interleukin-2 (IL-2), and the response to IL-2 throughoutM. aviuminfection as a possible explanation for this loss. We found that there is a steady increase in the percentage of T cells expressing activation markers right to the end of infection. However, in vivo T-cell proliferation, measured as a percentage of CD4+and CD8+cells incorporating 5-bromo-2′-deoxyuridine, initially increased but then remained constant. In the final stages of infection there was a decline in proliferation of activated (CD62L−) T cells. Since IL-2 is a major driver of T-cell proliferation, we asked whether this was due to loss of IL-2 responsiveness or production. However, CD25 (IL-2Rα) continued to be highly expressed in the terminal stages of infection, and although IL-2 production declined, addition of recombinant IL-2 to cultures could not rescue the final loss of IFN-γ production.
Genomic Approach for Analysis of Surface Proteins inChlamydia pneumoniae
Authors: Montigiani Silvia , Falugi Fabiana , Scarselli Maria , Finco Oretta , Petracca Roberto , Galli Giuliano , Mariani Massimo , Manetti Roberto , Agnusdei Mauro , Cevenini Roberto , Donati Manuela , Nogarotto Renzo , Norais Nathalie , Garaguso Ignazio , Nuti Sandra , Saletti Giulietta , Rosa Domenico , Ratti Giulio , Grandi Guido
Published: July 2002

Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working withC. pneumoniaeand in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization ofC. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization inC. pneumoniae.
Structure ofCampylobacter jejuniLipopolysaccharides Determines Antiganglioside Specificity and Clinical Features of Guillain-Barré and Miller Fisher Patients
Authors: Ang C. W. , Laman J. D. , Willison H. J. , Wagner E. R. , Endtz H. P. , De Klerk M. A. , Tio-Gillen A. P. , Van den Braak N. , Jacobs B. C. , Doorn P. A. Van
Published: July 2002

Ganglioside mimicry in the lipopolysaccharide (LPS) fraction ofCampylobacter jejuniisolated from Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) patients was compared with isolates from patients with an uncomplicated enteritis. The antibody response toC. jejuniLPS and gangliosides in neuropathy patients and controls was compared as well. LPS from GBS and MFS-associated isolates more frequently contained ganglioside-like epitopes compared to control isolates. Almost all neuropathy patients showed a strong antibody response against LPS and multiple gangliosides in contrast to enteritis patients. Isolates from GBS patients more frequently had a GM1-like epitope than isolates from MFS patients. GQ1b-like epitopes were present in all MFS-associated isolates and was associated with anti-GQ1b antibody reactivity and the presence of oculomotor symptoms. These results demonstrate that the expression of ganglioside mimics is a risk factor for the development of post-Campylobacterneuropathy. This study provides additional evidence for the hypothesis that the LPS fraction determines the antiganglioside specificity and clinical features in post-Campylobacterneuropathy patients.
Osmolarity and pH Growth Conditions Regulate
fim
Gene Transcription and Type 1 Pilus Expression in Uropathogenic
Escherichia coli
Authors: Schwan William R. , Lee Jeffrey L. , Lenard Farrah A. , Matthews Brian T. , Beck Michael T.
Published: July 2002

A comparative study was performed to determine the effects of pH, osmolarity, and human urine on the transcription of several
fim
genes, as well as the overall expression of type 1 pili. Several
fim-lacZYA
fusions were constructed on single-copy plasmids to test a range of pHs and a range of osmolarities. Growth in acidic medium slightly reduced expression from all of the
fim
promoters (
fimA
,
fimB
, and
fimE
). Increased osmolarity in neutral-pH medium repressed
fimA
and
fimB
transcription by approximately 50% when 400 mM NaCl was used and nearly threefold when 800 mM NaCl was used, whereas
fimE
transcription rose slightly as the osmolarity increased. This effect was more pronounced in high-osmolarity acidic media;
fimB
and
fimA
expression decreased fivefold in growth media containing 800 mM NaCl compared to expression in growth media without added NaCl. Moreover,
fimE
expression doubled under the same high-osmolarity conditions compared to expression in a low-osmolarity acidic environment. When a
fimB-lacZ
or
fimE-lacZ
fusion was inserted into the chromosome of strain AAEC189,
fimE
expression changed slightly as the osmolarity increased, but
fimB
expression decreased by 50% in a low-pH high-osmolarity environment. When strain AAEC189 with either a plasmid-borne
fimB-lacZ
fusion or a plasmid-borne
fimE-lacZ
fusion was grown in human urine, similar changes in the levels of
fimB
and
fimE
expression were observed. Limiting-dilution reverse transcription-PCR confirmed that these changes in
fim
expression occurred in clinical isolates of uropathogenic
Escherichia coli
grown in media with different pHs and different osmolarities. Furthermore, the invertible switch region in uropathogenic strain NU149 shifted from favoring the phase-on position in a neutral-pH low-osmolarity environment to favoring the phase-off position in a low-pH high-osmolarity environment. Results obtained with an
ompR
mutant strain demonstrated that
fimB
expression was derepressed and that OmpR may neutralize repression by an acid response regulator of
fimE
expression in a low-pH environment. In addition, H-NS was verified to be important in regulation of
fimB
, but it had only a slight effect on
fimE
under the specific pH and osmotic growth conditions tested. Enzyme immunoassays with anti-type 1 pilus antibody and hemagglutination assays showed that fewer type 1 pili were detected with cells in a low-pH high-osmolarity environment. Together, these observations demonstrate that a combination of low pH and high osmolarity regulates the transcription of
fim
genes, which favors a shift in the invertible element to the phase-off orientation and a loss of type 1 pilus expression. Taken together, our data suggest that the environmental cues that we tested may regulate expression of type 1 pili in specific in vivo niches, such as murine kidneys and possibly human kidneys.
Lymphocyte Recruitment and Protective Efficacy against Pulmonary Mycobacterial Infection Are Independent of the Route of PriorMycobacterium bovisBCG Immunization
Authors: Palendira Umaimainthan , Bean Andrew G. D. , Feng Carl G. , Britton Warwick J.
Published: July 2002

Mycobacterium tuberculosisinfects humans through the lung, and immunity to this chronic infection is mediated primarily by CD4+T lymphocytes. Recently we have demonstrated that the recruitment of lymphocytes to the lung during primary aerosolM. tuberculosisinfection in mice occurs predominantly through the interaction of α4β1integrin on CD4+T cells and vascular cell adhesion molecule-1 on the pulmonary endothelium. To investigate the effect of route of immunization withMycobacterium bovisBCG on the pattern of T-cell recruitment to the lung, we have analyzed the differences in expression of integrins on activated memory CD4+T cells infiltrating the lung following primary BCG immunization by aerosol, intravenous, and subcutaneous routes and after subsequent aerosol challenge withM. tuberculosis. There were marked differences in the patterns of recruitment of activated CD4+T cells to the lung following primary immunization by the three routes. Expansion of CD44hiCD62LlowCD4+T cells in the lung occurred following aerosol and intravenous BCG immunizations, and the lymphocyte recruitment was proportional to the pulmonary bacterial load. The majority of infiltrating CD4+T cells expressed α4β1integrin. On subsequent exposure to aerosol BCG rapid expansion of gamma interferon-secreting α4β1+CD4+T cells occurred to the same extent in all immunized mice, regardless of the route of immunization. Similar expansion of α4β1+CD4+memory T cells occurred followingM. tuberculosischallenge. The three routes of BCG immunization resulted in the same level of protection against aerosolM. tuberculosisor BCG challenge in both the lungs and spleen. Therefore, recruitment of effector T lymphocytes and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior BCG immunization.
Role of Various Enterotoxins in
Aeromonas hydrophila-
Induced Gastroenteritis: Generation of Enterotoxin Gene-Deficient Mutants and Evaluation of Their Enterotoxic Activity
Authors: Sha Jian , Kozlova E. V. , Chopra A. K.
Published: July 2002

Three enterotoxins from the
Aeromonas hydrophila
diarrheal isolate SSU have been molecularly characterized in our laboratory. One of these enterotoxins is cytotoxic in nature, whereas the other two are cytotonic enterotoxins, one of them heat labile and the other heat stable. Earlier, by developing an isogenic mutant, we demonstrated the role of a cytotoxic enterotoxin in causing systemic infection in mice. In the present study, we evaluated the role of these three enterotoxins in evoking diarrhea in a murine model by developing various combinations of enterotoxin gene-deficient mutants by marker-exchange mutagenesis. A total of six isogenic mutants were prepared in a cytotoxic enterotoxin gene (
act
)-positive or -negative background strain of
A. hydrophila
. We developed two single knockouts with truncation in either the heat-labile (
alt
) or the heat-stable (
ast
) cytotonic enterotoxin gene; three double knockouts with truncations of genes encoding (i)
alt
and
ast
, (ii)
act
and
alt
, and (iii)
act
and
ast
genes; and a triple-knockout mutant with truncation in all three genes,
act
,
alt
, and
ast
. The identity of these isogenic mutants developed by double-crossover homologous recombination was confirmed by Southern blot analysis. Northern and Western blot analyses revealed that the expression of different enterotoxin genes in the mutants was correspondingly abrogated. We tested the biological activity of these mutants in a diet-restricted and antibiotic-treated mouse model with a ligated ileal loop assay. Our data indicated that all of these mutants had significantly reduced capacity to evoke fluid secretion compared to that of wild-type
A. hydrophila
; the triple-knockout mutant failed to induce any detectable level of fluid secretion. The biological activity of selected
A. hydrophila
mutants was restored after complementation. Taken together, we have established a role for three enterotoxins in
A. hydrophila
-induced gastroenteritis in a mouse model with the greatest contribution from the cytotoxic enterotoxin Act, followed by the Alt and Ast cytotonic enterotoxins.
Preexisting Inflammation Due toMycobacterium bovisBCG Infection Differentially Modulates T-Cell Priming against a Replicating or Nonreplicating Immunogen
Authors: Dudani Renu , Chapdelaine Yvan , van Faassen Henk , Smith Dean K. , Shen Hao , Krishnan Lakshmi , Sad Subash
Published: July 2002

Induction of T-cell memory by vaccination ensures long-term protection against pathogens. We determined whether on-going inflammatory responses during vaccination influenced T-cell priming. A preexposure of mice toMycobacterium bovisBCG impaired their subsequent ability to prime T cells againstListeria monocytogenes. This was characterized by a decrease inL. monocytogenes-specific gamma interferon (IFN-γ)-secreting CD4+and CD8+T cells. The intensity of T-cell priming towardsL. monocytogenesdepended on the extent ofL. monocytogenesexpansion, and a cessation of this expansion caused byM. bovisBCG-induced inflammation resulted in impairment in T-cell priming. A challenge ofM. bovisBCG-infected mice with a higherL. monocytogenesdose increasedL. monocytogenessurvival and restored T-cell priming towardsL. monocytogenes. Impairment in T-cell priming towardsL. monocytogenesdue toM. bovisBCG-induced inflammation resulted in a compromised protective efficacy in the long term after mice were rechallenged withL. monocytogenes. Preexisting inflammation selectively impaired T-cell priming for replicating immunogens as CD8+T-cell response to ovalbumin administered as an inert antigen (ovalbumin-archaeosomes) was enhanced byM. bovisBCG preimmunization, whereas priming towards ovalbumin administered as a live immunogen (L. monocytogenes-ovalbumin) was impaired. Thus, depending on the nature of the immunogen, the presence of prior inflammatory responses may either impede or boost vaccine efficacy.
Evidence that the Enterococcal Polysaccharide Antigen Gene (
epa
) Cluster Is Widespread in
Enterococcus faecalis
and Influences Resistance to Phagocytic Killing of
E. faecalis
Authors: Teng Fang , Jacques-Palaz Karen D. , Weinstock George M. , Murray Barbara E.
Published: July 2002

In previous studies, we cloned a cluster of genes involved in polysaccharide biosynthesis (
epa
) from
Enterococcus faecalis
strain OG1RF and showed that this gene cluster mediated synthesis of a polysaccharide in
Escherichia coli
. Disruption of two open reading frames in the
epa
gene cluster of OG1RF generated two mutants, TX5179 and TX5180, which were attenuated in a mouse peritonitis model. In the current study, Western blotting was performed with serum from a patient with
E. faecalis
endocarditis and polysaccharide extracts from OG1RF and the mutants TX5179 and TX5180. OG1RF showed a smear in the high-molecular-weight region and discrete bands in the low-molecular-weight region, which were missing from the mutants; periodate treatment and carbohydrate staining confirmed the polysaccharide nature of this material. In a neutrophil killing assay using OG1RF-absorbed normal human serum, the mutants TX5179 and TX5180, respectively, were 50 and 2.4 times more susceptible to killing than wild-type OG1RF (
P
≤ 0.01). With a fluorescence phagocytosis assay, 2.5 to 3 times more of the mutants were taken up by neutrophils than OG1RF (
P
≤ 0.001). Finally, with restriction digestion and hybridization under high-stringency conditions, the
epa
gene cluster of OG1RF (which is also present in the sequenced
E. faecalis
strain V583) was detected in 12 of 12 other clonally distinct
E. faecalis
strains tested: a similar polysaccharide pattern was detected for the 12 strains on Western blots using an
E. faecalis
endocarditis patient serum, and sera from four other patients with
E. faecalis
endocarditis all reacted with polysaccharide extracts of OG1RF. These results indicate that the
epa
gene cluster is widespread among
E. faecalis
and confers some protection against human host defenses.
Haemophilus ducreyi
Requires the
flp
Gene Cluster for Microcolony Formation In Vitro
Authors: Nika Joseph R. , Latimer Jo L. , Ward Christine K. , Blick Robert J. , Wagner Nikki J. , Cope Leslie D. , Mahairas Gregory G. , Munson Robert S. , Hansen Eric J.
Published: July 2002

Haemophilus ducreyi
, the etiologic agent of chancroid, has been shown to form microcolonies when cultured in the presence of human foreskin fibroblasts. We identified a 15-gene cluster in
H. ducreyi
that encoded predicted protein products with significant homology to those encoded by the
tad
(for tight adhesion) locus in
Actinobacillus actinomycetemcomitans
that is involved in the production of fimbriae by this periodontal pathogen. The first three open reading frames in this
H. ducreyi
gene cluster encoded predicted proteins with a high degree of identity to the Flp (fimbria-like protein) encoded by the first open reading frame of the
tad
locus; this 15-gene cluster in
H. ducreyi
was designated
flp
. RT-PCR analysis indicated that the
H. ducreyi flp
gene cluster was likely to be a polycistronic operon. Mutations within the
flp
gene cluster resulted in an inability to form microcolonies in the presence of human foreskin fibroblasts. In addition, the same mutants were defective in the ability to attach to both plastic and human foreskin fibroblasts in vitro. An
H. ducreyi
mutant with an inactivated
tadA
gene exhibited a small decrease in virulence in the temperature-dependent rabbit model for experimental chancroid, whereas another
H. ducreyi
mutant with inactivated
flp-1
and
flp-2
genes was as virulent as the wild-type parent strain. These results indicate that the
flp
gene cluster is essential for microcolony formation by
H. ducreyi
, whereas this phenotypic trait is not linked to the virulence potential of the pathogen, at least in this animal model of infection.
Expression of the Biofilm-Associated Protein Interferes with Host Protein Receptors of
Staphylococcus aureus
and Alters the Infective Process
Authors: Cucarella Carme , Tormo M. Ángeles , Knecht Erwin , Amorena Beatriz , Lasa Íñigo , Foster Timothy J. , Penadés José R.
Published: July 2002

The adherence of
Staphylococcus aureus
to soluble proteins and extracellular-matrix components of the host is one of the key steps in the pathogenesis of staphylococcal infections.
S. aureus
presents a family of adhesins called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that specifically recognize host matrix components. We examined the influence of biofilm-associated protein (Bap) expression on
S. aureus
adherence to host proteins, epithelial cell cultures, and mammary gland sections and on colonization of the mammary gland in an in vivo infection model. Bap-positive strain V329 showed lower adherence to immobilized fibrinogen and fibronectin than isogenic Bap-deficient strain m556. Bacterial adherence to histological sections of mammary gland and bacterial internalization into 293 cells were significantly lower in the Bap-positive strains. In addition, the Bap-negative strain showed significantly higher colonization in vivo of sheep mammary glands than the Bap-positive strain. Taken together, these results strongly suggest that the expression of the Bap protein interferes with functional properties of the MSCRAMM proteins, preventing initial bacterial attachment to host tissues and cellular internalization.
Sulfolobicins, Specific Proteinaceous Toxins Produced by Strains of the Extremely Thermophilic Archaeal Genus
Sulfolobus
Authors: Prangishvili David , Holz Ingelore , Stieger Evelyn , Nickell Stephan , Kristjansson Jakob K. , Zillig Wolfram
Published: July 2002

Several novel strains of “
Sulfolobus islandicus
” produced proteinaceous toxins, termed sulfolobicins, which killed cells of other strains of the same species, as well as of
Sulfolobus solfataricus
P1 and
Sulfolobus shibatae
B12, but not of the producer strains and of
Sulfolobus acidocaldarius
DSM639. The sulfolobicin purified from the strain HEN2/2 had a molecular mass of about 20 kDa. It was found to be associated with the producer cells as well as with cell-derived S-layer-coated spherical membrane vesicles 90 to 180 nm in diameter and was not released from the cells in soluble form.