bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular Trafficking ofBrucella abortusin J774 Macrophages
Authors: Arenas Graciela N. , Staskevich Ana Sandra , Aballay Alejandro , Mayorga Luis S.
Published: July 2002

Brucella abortusis a facultative intracellular bacterium capable of surviving inside professional and nonprofessional phagocytes. The microorganism remains in membrane-bound compartments that in several cell types resemble modified endoplasmic reticulum structures. To monitor the intracellular transport ofB. abortusin macrophages, the kinetics of fusion of phagosomes with preformed lysosomes labeled with colloidal gold particles was observed by electron microscopy. The results indicated that phagosomes containing liveB. abortuswere reluctant to fuse with lysosomes. Furthermore, newly endocytosed material was not incorporated into these phagosomes. These observations indicate that the bacteria strongly affect the normal maturation process of macrophage phagosomes. However, after overnight incubation, a significant percentage of the microorganisms were found in large phagosomes containing gold particles, resembling phagolysosomes. Most of theBrucellabacteria present in phagolysosomes were not morphologically altered, suggesting that they can also resist the harsh conditions prevalent in this compartment. About 50% colocalization ofB. abortuswith LysoSensor, a weak base that accumulates in acidic compartments, was observed, indicating that theB. abortusbacteria do not prevent phagosome acidification. In contrast to what has been described for HeLa cells, only a minor percentage of the microorganisms were found in compartments labeled with monodansylcadaverine, a marker for autophagosomes, and with DiOC6 (3,3′-dihexyloxacarbocyanine iodide), a marker for the endoplasmic reticulum. These results indicate thatB. abortusbacteria alter phagosome maturation in macrophages. However, acidification does occur in these phagosomes, and some of them can eventually mature to phagolysosomes.
Contribution of Clumping Factor B to Pathogenesis of Experimental Endocarditis due to
Staphylococcus aureus
Authors: Entenza J. M. , Foster T. J. , Ni Eidhin D. , Vaudaux P. , Francioli P. , Moreillon P.
Published: July 2002

Staphylococcus aureus
Newman with an insertion mutation in
clfB
, the gene encoding clumping factor B, only marginally decreased infection rate (
P
> 0.05) in rats with experimental endocarditis. In contrast,
clfB
complementation on a multicopy plasmid significantly increased infectivity (
P
< 0.05) over the deleted mutants. Although
clfB
could affect endovascular infection, its importance in experimental endocarditis was limited.
Severe Schistosomiasis in the Absence of Interleukin-4 (IL-4) Is IL-12 Independent
Authors: Patton Elisabeth A. , Brunet Laura Rosa , La Flamme Anne C. , Pedras-Vasconcelos João , Kopf Manfred , Pearce Edward J.
Published: July 2002

An interleukin-4 (IL-4)-dependent Th2 response allows wild-type mice to survive infection with the parasiteSchistosoma mansoni. In the absence of IL-4, infected mice mount a Th1-like proinflammatory response, develop severe disease, and succumb. Neither the Th1 response nor morbidity is IL-12 dependent in this system.
Intracellular Induction of the
Bartonella henselae virB
Operon by Human Endothelial Cells
Authors: Schmiederer Michael , Arcenas Rodney , Widen Raymond , Valkov Nikola , Anderson Burt
Published: July 2002

One of the more recently identified bacterial exportation systems is the type IV secretion mechanism, which is characterized by a multiprotein complex that spans the inner and outer bacterial membranes and contains a pilin component. The most thoroughly studied type IV secretion system is encoded by the
virB
operon of
Agrobacterium tumefaciens
. In
Bartonella henselae,
8 of the 10
virB
operon genes share extensive homology and arrangement with the
virB
operon of
A. tumefaciens
. Sequencing of the region upstream of the
B. henselae virB2
gene revealed a region with sequence homology to the
vir
box of
A. tumefaciens.
This possible promoter region was cloned upstream of the green fluorescent protein reporter gene in the promoterless vector pANT3 and used to transform
B. henselae.
Minimal reporter gene expression was seen in the transformed bacteria cultivated in the absence of host cells, but expression was strongly induced in intracellular bacteria cultivated with human microvascular endothelial cells. Deletion of an 87-bp fragment, which contained the putative
vir
box from the 5′ end of the promoter region, diminished intracellular induction of the reporter gene. Host cell induction of the 17-kDa antigen gene, which replaces
virB5
in
B. henselae
, was also demonstrated at the protein level using specific antiserum. Thus, expression of the
virB
genes of
B. henselae
is induced in bacteria, which have invaded host cells, through a mechanism that may be similar to the environment-sensing mechanism found in the
virB
operon of
A. tumefaciens
.
Involvement of Fungal Cell Wall Components in Adhesion ofSporothrix schenckiito Human Fibronectin
Authors: Lima Osana C. , Figueiredo Camila C. , Previato José O. , Mendonça-Previato Lucia , Morandi Verônica , Lopes Bezerra Leila M.
Published: July 2002

Systemic sporotrichosis is an emerging infection potentially fatal for immunocompromised patients. Adhesion to extracellular matrix proteins is thought to play a crucial role in invasive fungal diseases. Here we report studies of the adhesion ofSporothrix schenckiito the extracellular protein fibronectin (Fn). Both yeast cells and conidia ofS. schenckiiwere able to adhere to Fn as detected by enzyme-linked immunosorbent binding assays. Adhesion of yeast cells to Fn is dose dependent and saturable.S. schenckiiadheres equally well to 40-kDa and 120-kDa Fn proteolytic fragments. While adhesion to Fn was increased by Ca2+, inhibition assays demonstrated that it was not RGD dependent. A carbohydrate-containing cell wall neutral fraction blocked up to 30% of the observed adherence for the yeast cells. The biochemical nature of this fraction suggests the participation of cell surface glycoconjugates in binding by their carbohydrate or peptide moieties. These results provide new data concerningS. schenckiiadhesion mechanisms, which could be important in host-fungus interactions and the establishment of sporotrichosis.
Identification of Independent
Streptococcus gordonii
SspA and SspB Functions in Coaggregation with
Actinomyces naeslundii
Authors: Egland Paul G. , Dû Laurence D. , Kolenbrander Paul E.
Published: July 2002

The initial stages of dental plaque formation involve the adherence of early colonizing organisms such as
Streptococcus gordonii
and
Actinomyces naeslundii
to the saliva-coated tooth surface and to each other. The
S. gordonii
surface proteins SspA and SspB are known to play a role in adherence to salivary proteins and mediate coaggregation with other bacteria. Coaggregation is the adhesin receptor-mediated interaction between genetically distinct cell types and appears to be ubiquitous among oral isolates. To define the function of SspA and SspB separately on the surface of their natural host, we constructed and analyzed the coaggregation properties of an isogenic
sspB
mutant of
S. gordonii
DL1, an
sspAB
double mutant, and a previously described
sspA
mutant.
A. naeslundii
strains have been previously classified into six coaggregation groups based on the nature of their coaggregations with
S. gordonii
DL1 and other oral streptococci. Coaggregation assays with the
sspA
and
sspB
mutants showed that SspA and SspB are the streptococcal proteins primarily responsible for defining these coaggregation groups and, thus, are highly significant in the establishment of early dental plaque. SspA exhibited two coaggregation-specific functions. It participated in lactose-inhibitable and -noninhibitable interactions, while SspB mediated only lactose-noninhibitable coaggregations. Accordingly, the
sspAB
double mutant lacked these functions and allowed us to detect a third coaggregation interaction with one of these organisms. These proteins may play an important role in development of
S. gordonii-A. naeslundii
communities in early dental plaque. Understanding these adhesin proteins will aid investigations of complex microbial communities that characterize periodontal diseases.
Antibodies against Listerial Protein 60 Act as an Opsonin for Phagocytosis ofListeria monocytogenesby Human Dendritic Cells
Authors: Kolb-Mäurer Annette , Pilgrim Sabine , Kämpgen Eckhart , McLellan Alexander D. , Bröcker Eva-Bettina , Goebel Werner , Gentschev Ivaylo
Published: July 2002

Human-monocyte-derived dendritic cells (MoDC) are very efficient in the uptake ofListeria monocytogenes, a gram-positive bacterium which is an important pathogen in humans and animals causing systemic infections with symptoms such as septicemia and meningitis. In this work, we analyzed the influence of blood plasma on the internalization ofL. monocytogenesinto human MoDC and compared the uptake ofL. monocytogeneswith that ofSalmonella entericaserovar Typhimurium andYersinia enterocolitica. While human plasma did not significantly influence the uptake of serovar Typhimurium andY. enterocoliticaby human MoDC, the efficiency of the uptake ofL. monocytogenesby these phagocytes was strongly enhanced by human plasma. In plasma-free medium the internalization ofL. monocytogeneswas very low, whereas the addition of pooled human immunoglobulins resulted in the internalization of these bacteria to a degree comparable to the highly efficient uptake observed with human plasma. All human plasma tested contained antibodies against the 60-kDa extracellular protein ofL. monocytogenes(p60), and anti-p60 antibodies were also found in the commercially available pooled immunoglobulins. Strikingly, in contrast toL. monocytogeneswild type, aniapdeletion mutant (totally deficient in p60) showed only a minor difference in the uptake by human MoDC in the presence or the absence of human plasma. These results support the assumption that antibodies against the listerial p60 protein may play an important role in Fc-receptor-mediated uptake ofL. monocytogenesby human MoDC via opsonization of the bacteria. This process may have a major impact in preventing systemic infection inL. monocytogenesin immunocompetent humans.
Characterization of the
groESL
Operon in
Listeria monocytogenes
: Utilization of Two Reporter Systems (
gfp
and
hly
) for Evaluating In Vivo Expression
Authors: Gahan Cormac G. M. , O'Mahony James , Hill Colin
Published: July 2002

The ability of intracellular pathogens to sense and adapt to the hostile environment of the host is an important factor governing virulence. We have sequenced the operon encoding the major heat shock proteins GroES and GroEL in the gram-positive food-borne pathogen
Listeria monocytogenes
. The operon has a conserved orientation in the order
groES groEL
. Upstream of
groES
and in the opposite orientation is a gene encoding a homologue of the
Bacillus subtilis
protein YdiL, while downstream of
groEL
is a gene encoding a putative bile hydrolase. We used both reverse transcriptase-PCR (RT-PCR) and transcriptional fusions to the UV-optimized
Aequorea victoria
green fluorescent protein (GFP
UV
) to analyze expression of
groESL
under various environmental stress conditions, including heat shock, ethanol stress, and acid shock, and during infection of J774 mouse macrophage cells. Strains harboring GFP
UV
transcriptional fusions to the promoter region of
groESL
demonstrated a significant increase in fluorescence following heat shock that was detected by both fluorimetry and fluorescence microscopy. Using both RT-PCR and GFP technology we detected expression of
groESL
following internalization by J774 cells. Increased intracellular expression of
dnaK
was also determined using RT-PCR. We have recently described a system which utilizes
L. monocytogenes
hemolysin as an in vivo reporter of gene expression within the host cell phagosome (C. G. M. Gahan and C. Hill, Mol. Microbiol. 36:498–507, 2000). In this study a strain was constructed in which hemolysin expression was placed under the control of the
groESL
promoter. In this strain hemolysin expression during infection also confirms transcription from the
groESL
promoter during J774 and murine infection, albeit at lower levels than the known virulence factor
plcA
.
Porphyromonas gingivalisGingipain-R Enhances Interleukin-8 but Decreases Gamma Interferon-Inducible Protein 10 Production by Human Gingival Fibroblasts in Response to T-Cell Contact
Authors: Oido-Mori Mari , Rezzonico Roger , Wang Pao-Li , Kowashi Yusuke , Dayer Jean-Michel , Baehni Pierre C. , Chizzolini Carlo
Published: July 2002

Proteases produced byPorphyromonas gingivalis, an oral pathogen, are considered important virulence factors and may affect the responses of cells equipped with proteinase-activated receptors. The aim of this study was to investigate the effect of the arginine-specific cysteine protease gingipain-R produced byP. gingivalison chemokine production by human gingival fibroblasts (HGF) and the effect of gingipain-R treatment on the subsequent contact-dependent activation of HGF by T cells. HGF incubated in the presence of purified 47-kDa gingipain-R showed increased levels of interleukin-8 (IL-8) mRNA. Cyclooxygenase-2 (COX-2) mRNA was also induced. Further exposure of HGF to activated T cells resulted in the dose- and time-dependent enhancement of IL-8 transcription and release. T-cell membrane-bound tumor necrosis factor (TNF) was the ligand inducing IL-8 production by HGF, since TNF neutralization abrogated HGF responses to T-cell contact. The enhanced IL-8 release was due, at least in part, to prostaglandin-E2production, which was mostly blocked by indomethacin. Gingipain-R proteolytic activity was required since heat inactivation, specific synthetic protease inhibitors, and the natural substrate competitor histatin 5 abrogated its effects. The enhanced production of IL-8 in response to T-cell contact was specific since monocyte chemotactic protein-1 (MCP-1) production was unaffected while interferon-gamma-inducible protein-10 (IP-10) was inhibited. The sum of these activities may result in the recruitment of differential cell types to sites of inflammation since IL-8 preferentially recruits neutrophils and IP-10 attracts activated T cells and may be relevant to the pathogenesis of periodontitis.
Characterization and Protective Potential of the Immune Response toTaenia soliumParamyosin in a Murine Model of Cysticercosis
Authors: Vázquez-Talavera José , Solı́s Carlos F. , Terrazas Luis I. , Laclette Juan P.
Published: July 2002

Paramyosin has been proposed as a vaccine candidate in schistosomiasis and filariasis. However, limited information is available about its protective potential against cysticercosis and the immune response it induces. Immunization of mice with recombinant full-length paramyosin ofTaenia solium(TPmy) results in about a 52% reduction in parasite burden after a subsequent challenge by intraperitoneal inoculation ofTaenia crassicepscysticerci. Immunization assays using recombinant fragments of TPmy, corresponding approximately to thirds on the amino, central, or carboxyl regions, suggest that protective epitopes are located mostly in the amino-end third. Proliferation assays using T cells obtained from mice immunized with the full-length recombinant TPmy also showed a preferential response to the amino-terminal fragment. In contrast, antibodies in the sera from these mice predominantly recognize epitopes located in the carboxyl-terminal fragment, being the immunoglobulin G1 subclass, the predominant antibody isotype. Characterization of the cellular immune response induced against the protective amino-terminal fragment reveals production of gamma interferon and interleukin-2, but not interleukin-4, suggesting a Th1-like profile.
Type 3 Fimbrial Shaft (MrkA) of
Klebsiella pneumoniae
, but Not the Fimbrial Adhesin (MrkD), Facilitates Biofilm Formation
Authors: Langstraat Jennifer , Bohse Megan , Clegg Steven
Published: July 2002

Isolates of
Klebsiella pneumoniae
are responsible for opportunistic infections, particularly of the urinary tract and respiratory tract, in humans. These bacteria express type 3 fimbriae that have been implicated in binding to eucaryotic cells and matrix proteins. The type 3 fimbriae mediate binding to target tissue using the MrkD adhesin that is associated with the fimbrial shaft comprised of the MrkA protein. The formation of biofilms in vitro by strains of
K. pneumoniae
was shown to be affected by the production of fimbriae on the bacterial surface. However, a functional MrkD adhesin was not necessary for efficient biofilm formation. Nonfimbriate strains were impaired in their ability to form biofilms. Using isogenic fimbriate and nonfimbriate strains of
K. pneumoniae
expressing green fluorescent protein it was possible to demonstrate that the presence of type 3 fimbriae facilitated the formation of dense biofilms in a continuous-flowthrough chamber. Transformation of nonfimbriate mutants with a plasmid possessing an intact
mrk
gene cluster restored the fimbrial phenotype and the rapid ability to form biofilms.
Dietary Glycine Prevents Peptidoglycan Polysaccharide-Induced Reactive Arthritis in the Rat: Role for Glycine-Gated Chloride Channel
Authors: Li Xiangli , Bradford Blair U. , Wheeler Michael D. , Stimpson Stephen A. , Pink Heather M. , Brodie Thomas A. , Schwab John H. , Thurman Ronald G.
Published: July 2002

Peptidoglycan polysaccharide (PG-PS) is a primary structural component of bacterial cell walls and causes rheumatoid-like arthritis in rats. Recently, glycine has been shown to be a potential immunomodulator; therefore, the purpose of this study was to determine if glycine would be protective in a PG-PS model of arthritis in vivo. In rats injected with PG-PS intra-articularly, ankle swelling increased 21% in 24 to 48 h and recovered in about 2 weeks. Three days prior to reactivation with PG-PS given intravenously (i.v.), rats were divided into two groups and fed a glycine-containing or nitrogen-balanced control diet. After i.v. PG-PS treatment joint swelling increased 2.1 ± 0.3 mm in controls but only 1.0 ± 0.2 mm in rats fed glycine. Infiltration of inflammatory cells, edema, and synovial hyperplasia in the joint were significantly attenuated by dietary glycine. Tumor necrosis factor alpha (TNF-α) mRNA was detected in ankle homogenates from rats fed the control diet but not in ankles from rats fed glycine. Moreover, intracellular calcium was increased significantly in splenic macrophages treated with PG-PS; however, glycine blunted the increase about 50%. The inhibitory effect of glycine was reversed by low concentrations of strychnine or chloride-free buffer, and it increased radiolabeled chloride influx nearly fourfold, an effect also inhibited by strychnine. In isolated splenic macrophages, glycine blunted translocation of the p65 subunit of NF-κB into the nucleus, superoxide generation, and TNF-α production caused by PG-PS. Further, mRNA for the beta subunit of the glycine receptor was detected in splenic macrophages. This work supports the hypothesis that glycine prevents reactive arthritis by blunting cytokine release from macrophages by increasing chloride influx via a glycine-gated chloride channel.
Muramic Acid Is Not Generally Present in the Human Spleen as Determined by Gas Chromatography-Tandem Mass Spectrometry
Authors: Kozar Michael P. , Laman Jon D. , Fox Alvin
Published: July 2002

It has been hypothesized that bacterial debris may accumulate in tissues of the reticuloendothelial system (RES) serving as an inflammatory stimulus for human disease. In support of this hypothesis, muramic acid (Mur), a component of bacterial peptidoglycan (PG), has previously been reported to be present in culture-negative human spleen. High-performance liquid chromatography (HPLC) was employed in these analyses, and a peak was detected at the retention time of Mur. However, HPLC is best used as a screening technique, and it is vital that these tentative observations be reexamined by the state-of-the-art approach (gas chromatography-tandem mass spectrometry [GC-MS2]). Indeed, in the present work using GC-MS2, Mur was not detected in six out of seven human spleens previously examined by HPLC. However, Mur was categorically detected at minute concentrations, 50 ppb, in one spleen. In conclusion, since Mur is not generally found in culture-negative human spleen, in future studies, these tissues can serve as negative controls. The study of Mur levels in inflammation (e.g., reactive arthritis) could prove important in testing the hypothesis that bacterial debris persisting in tissues could serve as a depot inciting diseases of unknown etiology.
Endogenous Pro- and Anti-Inflammatory Cytokines Differentially Regulate an In Vivo Humoral Response toStreptococcus pneumoniae
Authors: Khan Abdul Q. , Shen Yi , Wu Zheng-Qi , Wynn Thomas A. , Snapper Clifford M.
Published: July 2002

Proinflammatory cytokines play a critical role in innate host defense against extracellular bacteria. However, little is known regarding the effects of these cytokines on the adaptive humoral response. Mice injected with a neutralizing anti-tumor necrosis factor alpha (TNF-α) monoclonal antibody (MAb) at the time of primary immunization with intactStreptococcus pneumoniae(strain R36A) showed a substantial reduction in both the primary immunoglobulin G (IgG) response specific for the cell wall protein, pneumococcal surface protein A (PspA), as well as in the development of PspA-specific memory. In contrast, anti-TNF-α MAb injected only at the time of secondary immunization with R36A failed to alter the boosted anti-PspA response. TNF-α was required only within the first 48 to 72 h after primary immunization with R36A and was induced both by non-B and non-T cells and by lymphoid cells, within 2 to 6 h after immunization, with levels returning to normal by 24 h. Thus, the early innate release of TNF-α was critical for optimal stimulation of the subsequent adaptive humoral response to R36A. Additional proinflammatory (interleukin 1 [IL-1], IL-6, IL-12, and gamma interferon [IFN-γ]) as well as anti-inflammatory (IL-4 and IL-10) cytokines were also transiently induced. Mice genetically deficient in IL-6, IFN-γ, or IL-12 also showed a reduced IgG anti-PspA response of all IgG isotypes. In contrast, IL-4−/−and IL-10−/−mice immunized with R36A showed a significant elevation in the IgG anti-PspA response, except that there was decreased IgG1 in IL-4−/−mice. In this regard, a marked enhancement in the induction of proinflammatory cytokines was observed in the absence of IL-10, relative to controls. Ig isotype titers specific for the phosphorycholine determinant of C-polysaccharide were similarly regulated, but to a much more modest degree. These data suggest that proinflammatory and anti-inflammatory cytokines differentially regulate an in vivo protein- and polysaccharide-specific Ig response to an extracellular bacteria.
Type III Group B Streptococcal Polysaccharide Induces Antibodies That Cross-React withStreptococcus pneumoniaeType 14
Authors: Guttormsen Hilde-Kari , Baker Carol J. , Nahm Moon H. , Paoletti Lawrence C. , Zughaier Susu M. , Edwards Morven S. , Kasper Dennis L.
Published: July 2002

Covalent linkage of a bacterial polysaccharide to a protein greatly enhances the carbohydrate's immunogenicity and its binding to solid surfaces in immunoassays. These findings have spurred the development of glycoconjugate vaccines to prevent serious bacterial infections as well as the use of glycoconjugates as coating antigens in bioassays. We evaluated sera from women immunized with unconjugated group B streptococcal (GBS) type III (GBS III) polysaccharide (IIIPS) or with IIIPS covalently linked to tetanus toxoid to assess specificity, sensitivity, and parallelism in dilution curves in two GBS III enzyme-linked immunosorbent assays (ELISAs). One assay used IIIPS mixed with methylated human serum albumin (IIIPS + mHSA) as the coating antigen, and the other used IIIPS covalently linked to HSA (III-HSA). Each coating antigen was associated with a highly specific GBS III bioassay. The sensitivity was higher in the III-HSA ELISA, in which conjugated IIIPS is bound to the plates. Parallelism in titration curves was observed in the III-HSA but not in the IIIPS + mHSA ELISA. The excellent correlation between the concentrations of GBS IIIPS-specific immunoglobulin G (IgG) and the opsonophagocytic activity of these antibodies indicated that the III-HSA assay can predict functionality of vaccine-induced IgG against GBS III disease. The structure of the repeating unit of the capsular polysaccharide of GBS III differs from that ofStreptococcus pneumoniaetype 14 (Pn14 PS) only by the presence on GBS III of a sialic acid residue at the end of the side chain. The majority of healthy adults responding to GBS III vaccines with a fourfold or greater increase in GBS III-specific IgG antibodies developed antibodies cross-reacting with Pn14 PS (i.e., desialylated GBS IIIPS). The proportion of GBS vaccine responders who developed IgG to the desialylated IIIPS did not depend on whether IIIPS was given in the unconjugated or conjugated form. When present, these vaccine-induced cross-reacting antibodies conferred in vitro antibody-mediated opsonophagocytosis and killing of both GBS III and Pn14, two pathogens that cause invasive disease in young infants.
Mucosal Tolerance to a Bacterial Superantigen Indicates a Novel Pathway To Prevent Toxic Shock
Authors: Collins L. Vincent , Eriksson Kristina , Ulrich Robert G. , Tarkowski Andrej
Published: July 2002

Enterotoxins with superantigenic properties secreted during systemicStaphylococcus aureusinfection are responsible for toxic shock. We show that intranasal administration of staphylococcal enterotoxin A (SEA), but not a recombinant SEA lacking superantigenic activity, protected mice against lethal systemic SEA challenge. Protection was superantigen specific since intranasal exposure to SEA would not protect against death caused by subsequent toxic shock syndrome toxin 1 systemic challenge. Protection was neither due to selective depletion of SEA-specific T-cell receptor Vβ families nor due to production of neutralizing anti-SEA antibodies. Importantly, the production of interleukin 10 (IL-10) induced by “tolerization” (that is, by the induction of immunological tolerance) contributed to the observed protection against lethal superantigen-triggered disease. In support of this notion we found that (i) significantly increased levels of IL-10 in sera of “tolerized” animals (that is, animals rendered tolerant) and (ii) IL-10−/−mice could not be tolerized by mucosal SEA administration. Altogether, this is the first study to show that mucosal tolerance to a superantigen is readily triggered by means of immunodeviation.
Vaccination of Pregnant Dams with Intimin
O157
Protects Suckling Piglets from
Escherichia coli
O157:H7 Infection
Authors: Dean-Nystrom Evelyn A. , Gansheroff Lisa J. , Mills Melody , Moon Harley W. , O'Brien Alison D.
Published: July 2002

Cattle are important reservoirs of enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 that cause disease in humans. Both dairy and beef cattle are asymptomatically and sporadically infected with EHEC. Our long-term goal is to develop an effective vaccine to prevent cattle from becoming infected and transmitting EHEC O157:H7 to humans. We used passive immunization of neonatal piglets (as a surrogate model) to determine if antibodies against EHEC O157 adhesin (intimin
O157
) inhibit EHEC colonization. Pregnant swine (dams) with serum anti-intimin titers of ≤100 were vaccinated twice with purified intimin
O157
or sham-vaccinated with sterile buffer. Intimin
O157
-specific antibody titers in colostrum and serum of dams were increased after parenteral vaccination with intimin
O157
. Neonatal piglets were allowed to suckle vaccinated or sham-vaccinated dams for up to 8 h before they were inoculated with 10
6
CFU of a Shiga toxin-negative (for humane reasons) strain of EHEC O157:H7. Piglets were necropsied at 2 to 10 days after inoculation, and intestinal samples were collected for determination of bacteriological counts and histopathological analysis. Piglets that ingested colostrum containing intimin
O157
-specific antibodies from vaccinated dams, but not those nursing sham-vaccinated dams, were protected from EHEC O157:H7 colonization and intestinal damage. These results establish intimin
O157
as a viable candidate for an EHEC O157:H7 antitransmission vaccine.
Vaccination with Plasmid DNA Encoding TSA/LmSTI1 Leishmanial Fusion Proteins Confers Protection againstLeishmania majorInfection in Susceptible BALB/c Mice
Authors: Campos-Neto A. , Webb J. R. , Greeson K. , Coler R. N. , Skeiky Y. A. W. , Reed S. G.
Published: July 2002

We have recently shown that a cocktail containing two leishmanial recombinant antigens (LmSTI1 and TSA) and interleukin-12 (IL-12) as an adjuvant induces solid protection in both a murine and a nonhuman primate model of cutaneous leishmaniasis. However, because IL-12 is difficult to prepare, is expensive, and does not have the stability required for a vaccine product, we have investigated the possibility of using DNA as an alternative means of inducing protective immunity. Here, we present evidence that the antigens TSA and LmSTI1 delivered in a plasmid DNA format either as single genes or in a tandem digene construct induce equally solid protection againstLeishmania majorinfection in susceptible BALB/c mice. Immunization of mice with either TSA DNA or LmSTI1 DNA induced specific CD4+-T-cell responses of the Th1 phenotype without a requirement for specific adjuvant. CD8 responses, as measured by cytotoxic-T-lymphocyte activity, were generated after immunization with TSA DNA but not LmSTI1 DNA. Interestingly, vaccination of mice with TSA DNA consistently induced protection to a much greater extent than LmSTI1 DNA, thus supporting the notion that CD8 responses might be an important accessory arm of the immune response for acquired resistance against leishmaniasis. Moreover, the protection induced by DNA immunization was specific for infection withLeishmania, i.e., the immunization had no effect on the course of infection of the mice challenged with an unrelated intracellular pathogen such asMycobacterium tuberculosis. Conversely, immunization of BALB/c mice with a plasmid DNA that is protective against challenge withM. tuberculosishad no effect on the course of infection of these mice withL. major. Together, these results indicate that the protection observed with the leishmanial DNA is mediated by acquired specific immune response rather than by the activation of nonspecific innate immune mechanisms. In addition, a plasmid DNA containing a fusion construct of the two genes was also tested. Similarly to the plasmids encoding individual proteins, the fusion construct induced both specific immune responses to the individual antigens and protection against challenge withL. major. These results confirm previous observations about the possibility of DNA immunization against leishmaniasis and lend support to the idea of using a single polygenic plasmid DNA construct to achieve polyspecific immune responses to several distinct parasite antigens.
Recruitment of Mammalian Cell Fibronectin to the Surface of
Chlamydia trachomatis
Authors: Kleba Betsy J. , Banta Erin , Lindquist Erika A. , Stephens Richard S.
Published: July 2002

Pathogenic bacteria exploit the presence of various host cell molecules in order to colonize new tissues. Fibronectin is involved in a wide range of cell functions in vivo, and staphylococci, streptococci, and gonococci have evolved mechanisms to utilize this glycoprotein to mediate host cell binding. We show that elementary bodies (EB) from two biovars of
Chlamydia trachomatis
recruit fibronectin to their surfaces upon lysis of the host cell. We also demonstrate that a heparan sulfate lyase-sensitive molecule on chlamydial EB is responsible for binding at least a portion of this fibronectin.
An IS
257
-Derived Hybrid Promoter Directs Transcription of a
tetA
(K) Tetracycline Resistance Gene in the
Staphylococcus aureus
Chromosomal
mec
Region
Authors: Simpson Alice E. , Skurray Ronald A. , Firth Neville
Published: July 2002

Transcription of the
tetA
(K) tetracycline resistance determinant encoded by an IS
257
-flanked cointegrated copy of a pT181-like plasmid, located within the chromosomal
mec
region of a methicillin-resistant
Staphylococcus aureus
isolate, has been investigated. The results demonstrated that transcription of
tetA
(K) in this strain is directed by both an IS
257
-derived hybrid promoter, which is stronger than the native
tetA
(K) promoter in the autonomous form of pT181, and a complete outwardly directed promoter identified within one end of IS
257
. Despite lower gene dosage, the chromosomal configuration was shown to afford a higher level of resistance than that mediated by pT181 in an autonomous multicopy state. Furthermore, competition studies revealed that a strain carrying the chromosomal
tetA
(K) determinant exhibited a higher level of fitness in the presence of tetracycline but not in its absence. This finding suggests that tetracycline has been a selective factor in the emergence of strains carrying a cointegrated pT181-like plasmid in their chromosomes. The results highlight the potential of IS
257
to influence the expression of neighboring genes, a property likely to enhance its capacity to mediate advantageous genetic rearrangements.
Characterization of the Ends and Target Sites of the Novel Conjugative Transposon Tn
5397
from
Clostridium difficile
: Excision and Circularization Is Mediated by the Large Resolvase, TndX
Authors: Wang Hongmei , Roberts Adam P. , Lyras Dena , Rood Julian I. , Wilks Mark , Mullany Peter
Published: July 2002

Tn
5397
is a conjugative transposon that was originally isolated from
Clostridium difficile
. Previous analysis had shown that the central region of Tn
5397
was closely related to the conjugative transposon Tn
916
. However, in this work we obtained the DNA sequence of the ends of Tn
5397
and showed that they are completely different to those of Tn
916
. Tn
5397
did not contain the
int
and
xis
genes, which are required for the excision and integration of Tn
916
. Instead, the right end of Tn
5397
contained a gene,
tndX
, that appears to encode a member of the large resolvase family of site-specific recombinases. TndX is closely related to the TnpX resolvase from the mobilizable but nonconjugative chloramphenicol resistance transposons, Tn
4451
from
Clostridium perfringens
and Tn
4453
from
C. difficile
. Like the latter elements, inserted copies of Tn
5397
were flanked by a direct repeat of a GA dinucleotide. The Tn
5397
target sites were also shown to contain a central GA dinucleotide. Excision of the element in
C. difficile
completely regenerated the original target sequence. A circular form of the transposon, in which the left and right ends of the element were separated by a GA dinucleotide, was detected by PCR in both
Bacillus subtilis
and
C. difficile
. A Tn
5397
mutant in which part of
tndX
was deleted was constructed in
B. subtilis
. This mutant was nonconjugative and did not produce the circular form of Tn
5397
, indicating that the TndX resolvase has an essential role in the excision and transposition of Tn
5397
and is thus the first example of a member of the large resolvase family of recombinases being involved in conjugative transposon mobility. Finally, we showed that introduction of Tn
916
into a strain containing Tn
5397
induced the loss of the latter element in 95.6% of recipients.
Halocin S8: a 36-Amino-Acid Microhalocin from the Haloarchaeal Strain S8a
Authors: Price Lance B. , Shand Richard F.
Published: July 2002

Halocin S8 is a hydrophobic microhalocin of 36 amino acids (3,580 Da) and is the first microhalocin to be described. This peptide antibiotic is unique since it is processed from inside a much larger, 33,962-Da pro-protein. Halocin S8 is quite robust, as it can be desalted, boiled, subjected to organic solvents, and stored at 4°C for extended periods without losing activity. The complete amino acid sequence of halocin S8 was obtained first by Edman degradation of the purified protein and verified from the
halS8
gene: H
2
N-S-D-C-N-I-N-S-N-T-A-A-D-V-I-L-C-F-N-Q-V-G-S-C-A-L-C-S-P-T-L-V-G-G-P-V-P-COOH. The
halS8
gene is encoded on an ∼200-kbp megaplasmid and contains a 933-bp open reading frame, of which 108 bp are occupied by halocin S8. Both the
halS8
promoter and the “leaderless”
halS8
transcript are typically haloarchaeal. Northern blot analysis revealed three
halS8
transcripts: two abundant and one minor. Inspection of the 3′ end of the gene showed only a single, weak termination site (5′-TTTAT-3′), suggesting that some processing of the larger transcripts may be involved. Expression of the
halS8
gene is growth stage dependent: basal
halS8
transcript levels are present in low concentrations during exponential growth but increase ninefold during the transition to stationary phase. Initially, halocin activity parallels
halS8
transcript levels very closely. However, when halocin activity plateaus, transcripts remain abundant, suggesting inhibition of translation at this point. Once the culture enters stationary phase, transcripts rapidly return to basal levels.
A DNA Ligase from a Hyperthermophilic Archaeon with Unique Cofactor Specificity
Authors: Nakatani Masaru , Ezaki Satoshi , Atomi Haruyuki , Imanaka Tadayuki
Published: July 2002

A gene encoding DNA ligase (

lig
Tk

) from a hyperthermophilic archaeon,
Thermococcus kodakaraensis
KOD1, has been cloned and sequenced, and its protein product has been characterized.

lig
Tk

consists of 1,686 bp, corresponding to a polypeptide of 562 amino acids with a predicted molecular mass of 64,079 Da. Sequence comparison with previously reported DNA ligases and the presence of conserved motifs suggested that Lig

Tk

was an ATP-dependent DNA ligase. Phylogenetic analysis indicated that Lig

Tk

was closely related to the ATP-dependent DNA ligase from
Methanobacterium thermoautotrophicum
ΔH, a moderate thermophilic archaeon, along with putative DNA ligases from
Euryarchaeota
and
Crenarchaeota
. We expressed

lig
Tk

in
Escherichia coli
and purified the recombinant protein. Recombinant Lig

Tk

was monomeric, as is the case for other DNA ligases. The protein displayed DNA ligase activity in the presence of ATP and Mg
2+
. The optimum pH of Lig

Tk

was 8.0, the optimum concentration of Mg
2+
, which was indispensable for the enzyme activity, was 14 to 18 mM, and the optimum concentration of K
+
was 10 to 30 mM. Lig

Tk

did not display single-stranded DNA ligase activity. At enzyme concentrations of 200 nM, we observed significant DNA ligase activity even at 100°C. Unexpectedly, Lig

Tk

displayed a relatively small, but significant, DNA ligase activity when NAD
+
was added as the cofactor. Treatment of NAD
+
with hexokinase did not affect this activity, excluding the possibility of contaminant ATP in the NAD
+
solution. This unique cofactor specificity was also supported by the observation of adenylation of Lig

Tk

with NAD
+
. This is the first biochemical study of a DNA ligase from a hyperthermophilic archaeon.
Evolutionary Conservation of Methyl-Accepting Chemotaxis Protein Location in
Bacteria
and
Archaea
Authors: Gestwicki Jason E. , Lamanna Allison C. , Harshey Rasika M. , McCarter Linda L. , Kiessling Laura L. , Adler Julius
Published: July 2002

The methyl-accepting chemotaxis proteins (MCPs) are concentrated at the cell poles in an evolutionarily diverse panel of bacteria and an archeon. In elongated cells, the MCPs are located both at the poles and at regions along the length of the cells. Together, these results suggest that MCP location is evolutionarily conserved.
Molecular Analysis of the
tagF
Gene, Encoding CDP-Glycerol:Poly(glycerophosphate) Glycerophosphotransferase of
Staphylococcus epidermidis
ATCC 14990
Authors: Fitzgerald Stephen N. , Foster Timothy J.
Published: July 2002

Staphylococcus epidermidis
ATCC 14990 produces a wall-associated glycerol teichoic acid which is chemically identical to the major wall-associated teichoic acid of
Bacillus subtilis
168. The
S. epidermidis tagF
gene was cloned from genomic DNA and sequenced. When introduced on a plasmid vector into
B. subtilis
1A486 carrying the conditionally lethal temperature-sensitive mutation
tagF1
(
rodC1
), it expressed an 85-kDa protein which allowed colonies to grow at the restrictive temperature. This showed that the cloned
S. epidermidis
gene encodes a functional CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase. An amino acid substitution at residue 616 in the recombinant TagF protein eliminated complementation. Unlike
B. subtilis
, where the
tagF
gene is part of the
tagDEF
operon, the
tagF
gene of
S. epidermidis
is not linked to any other
tag
genes. We attempted to disrupt the chromosomal
tagF
gene in
S. epidermidis
TU3298 by directed integration of a temperature-sensitive plasmid but this failed, whereas a control plasmid containing the 5′ end of
tagF
on a similarly sized DNA fragment was able to integrate. This suggests that the
tagF
gene is essential and that the TagF and other enzymes involved in teichoic acid biosynthesis could be targets for new antistaphylococcal drugs.
Utilization of Subsidiary Chromosomal Replication Terminators in
Bacillus subtilis
Authors: Griffiths A. A. , Wake R. G.
Published: July 2002

The
Bacillus subtilis
merodiploid strain GSY1127 contains a large nontandem duplication of a portion of its chromosome within its left (anticlockwise) replication segment. This causes displacement of the replication terminus region to a noticeably asymmetric location relative to
oriC
. The utilization of the subsidiary replication terminators,
TerIII
and
TerV
, in the merodiploid strain has been compared with that in
B. subtilis
168. It is shown that
TerIII
is utilized to a significant extent in GSY1127 and that
TerV
is used only marginally at the most. Neither of these terminators is used to a measurable extent in the 168 strain. It is concluded that
TerIII
and
TerV
do indeed function as backups to the major terminator
TerI
, as has been generally thought. It is further concluded that, in the 168 strain, the vast majority of clockwise forks are arrested at the highly efficient
TerI
terminator, with fork fusion between the approaching forks occurring frequently while the clockwise fork is stationary at
TerI
.
A Single Amino Acid Substitution in a Mannosyltransferase, WbdA, Converts the
Escherichia coli
O9 Polysaccharide into O9a: Generation of a New O-Serotype Group
Authors: Kido Nobuo , Kobayashi Hidemitsu
Published: July 2002

wbdA
is a mannosyltransferase gene that is involved in synthesis of the
Escherichia coli
O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1. The equivalent structural O polysaccharide in the
E. coli
O9 and
Klebsiella
O3 strains is 2-αMan-1,2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1, with an excess of one mannose in the 1,2 linkage. We have cloned
wbdA
genes from these O9 and O3 strains and shown by genetic and functional studies that
wbdA
is the only gene determining the O-polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we showed that a single amino acid substitution, C55R, in WbdA of
E. coli
O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to be conserved in other
E. coli
O9a strains. The reverse substitution, R55C, in WbdA of
E. coli
O9a resulted in lipopolysaccharide synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in WbdA is required for conversion from O9a to O9.
A Novel Sphingoglycolipid Containing Galacturonic Acid and 2-Hydroxy Fatty Acid in Cellular Lipids of
Sphingomonas yanoikuyae
Authors: Naka Takashi , Fujiwara Nagatoshi , Yabuuchi Eiko , Doe Matsumi , Kobayashi Kazuo , Kato Yoshiko , Yano Ikuya
Published: July 2002

A novel sphingoglycolipid was isolated from
Sphingomonas yanoikuyae
, and its structure was identified as a galacturonosyl-β (1→1)-ceramide. This was a characteristic sphingoglycolipid present in
S. yanoikuyae
and certain other species of
Sphingomonas
, such as
Sphingomonas mali
,
Sphingomonas terrae
, and
Sphingomonas macrogoltabidus
, but not in the type species of
Sphingomonas
,
Sphingomonas paucimobilis
.
The N Terminus of FliM Is Essential To Promote Flagellar Rotation in
Rhodobacter sphaeroides
Authors: Poggio Sebastian , Osorio Aurora , Corkidi Gabriel , Dreyfus Georges , Camarena Laura
Published: July 2002

FliM is part of the flagellar switch complex. Interaction of this protein with phospho-CheY (CheY-P) through its N terminus constitutes the main information relay point between the chemotactic system and the flagellum. In this work, we evaluated the role of the N terminus of FliM in the swimming behavior of
Rhodobacter sphaeroides
. Strains expressing the FliM protein with substitutions in residues previously reported in
Escherichia coli
as being important for interaction with CheY showed an increased stop frequency compared with wild-type cells. In accordance, we observed that
R. sphaeroides
cells expressing FliM lacking either the first 13 or 20 amino acids from the N terminus showed a stopped phenotype. We show evidence that FliMΔ13 and FliMΔ20 are stable proteins and that cells expressing them allow flagellin export at levels indistinguishable from those detected for the wild-type strain. These results suggest that the N-terminal region of FliM is required to promote swimming in this bacterium. The role of CheY in controlling flagellar rotation in this organism is discussed.
Autoamplification of a Two-Component Regulatory System Results in “Learning” Behavior
Authors: Hoffer Sally M. , Westerhoff Hans V. , Hellingwerf Klaas J. , Postma Pieter W. , Tommassen Jan
Published: July 2002

We have tested the hypothesis that the autoamplification of two-component regulatory systems results in “learning” behavior, i.e., that bacteria respond faster or more extensively to a signal when a similar signal has been perceived in the past. Indeed, the induction of alkaline phosphatase activity upon phosphate limitation was faster if the cultures had been limited for phosphate previously, and this faster response correlated with the autoamplification of the cognate two-component system.
Roles of FrxA and RdxA Nitroreductases of
Helicobacter pylori
in Susceptibility and Resistance to Metronidazole
Authors: Jeong Jin-Yong , Mukhopadhyay Asish K. , Akada Junko K. , Dailidiene Daiva , Hoffman Paul S. , Berg Douglas E.
Published: July 2002

The relative importance of the
frxA
and
rdxA
nitroreductase genes of
Helicobacter pylori
in metronidazole (MTZ) susceptibility and resistance has been controversial. Jeong et al. (J. Bacteriol. 182:5082–5090, 2000) had interpreted that Mtz
s
H. pylori
were of two types: type I, requiring only inactivation of
rdxA
to became resistant, and type II, requiring inactivation of both
rdxA
and
frxA
to become resistant;
frxA
inactivation by itself was not sufficient to confer resistance. In contrast, Kwon et al. (Antimicrob. Agents Chemother. 44:2133–2142, 2000) had interpreted that resistance resulted from inactivation either of
frxA
or
rdxA
. These two interpretations were tested here. Resistance was defined as efficient colony formation by single cells from diluted cultures rather than as growth responses of more dense inocula on MTZ-containing medium. Tests of three of Kwon's Mtz
s
strains showed that each was type II, requiring inactivation of both
rdxA
and
frxA
to become resistant. In additional tests, derivatives of
frxA
mutant strains recovered from MTZ-containing medium were found to contain new mutations in
rdxA
, and
frxA
inactivation slowed MTZ-induced killing of Mtz
s
strains. Northern blot analyses indicated that
frxA
mRNA, and perhaps also
rdxA
mRNA, were more abundant in type II than in type I strains. We conclude that development of MTZ resistance in
H. pylori
requires inactivation of
rdxA
alone or of both
rdxA
and
frxA
, depending on bacterial genotype, but rarely, if ever, inactivation of
frxA
alone, and that
H. pylori
strains differ in regulation of nitroreductase gene expression. We suggest that such regulatory differences may be significant functionally during human infection.