bluuebunny/crossref_metadata_2025 huggingface dataset:
Carboxy-Terminal Proteolytic Processing of
Helicobacter pylori
Vacuolating Toxin
Authors: Nguyen Viet Q. , Caprioli Richard M. , Cover Timothy L.
Published: July 2002

The
vacA
gene of
Helicobacter pylori
strain 60190 encodes a 1,287-amino-acid protoxin, which undergoes cleavage of a 33-amino-acid amino-terminal signal sequence and carboxy-terminal proteolytic processing to yield a mature secreted toxin. Several features of VacA suggest that it belongs to the autotransporter family of gram-negative bacterial secreted proteins. Based on matrix-assisted laser desorption ionization–time of flight mass spectrometric analysis, we calculate that the mature toxin has a mass of 88.2 ± 0.2 kDa and consists of approximately 821 amino acids.
Effect on Polymorphonuclear Cell Function of a Human-Specific Cytotoxin, Intermedilysin, Expressed by
Streptococcus intermedius
Authors: Macey M. G. , Whiley R. A. , Miller L. , Nagamune H.
Published: July 2002

Streptococcus intermedius
is a member of the normal flora of the mouth but is also an opportunistic pathogen associated with purulent infections at oral and nonoral sites. Intermedilysin (ILY) has been shown to be a cytolysin capable of generating pores in the cell membrane of erythrocytes demonstrable by electron microscopy. This effect has been shown to be specific for human cells. Since polymorphonuclear cells (PMNs) are the main cell involved in innate immunity we investigated the effect of purified intermedilysin from
Streptococcus intermedius
on PMN function. Active ILY at a concentration of 40 ng/μl caused a significant decrease in the number of intact PMNs after 60 min. The active cytolysin, when compared with heat-inactivated ILY, did not appear to be chemotactic for the PMNs but did cause an increase in intracellular calcium, with increased cell surface CD11b expression, metabolic burst, and phagocytosis of
Staphylococcus aureus
. These findings may have implications for the role of ILY in deep-seated abscesses.
Toxicity of Shiga Toxin 1 in the Central Nervous System of Rabbits
Authors: Fujii Jun , Kinoshita Yoshimasa , Yutsudo Takashi , Taniguchi Hatsumi , Obrig Tom , Yoshida Shin-Ichi
Published: July 2002

The action of Shiga toxin (Stx) on the central nervous system was examined in rabbits. Intravenous Stx1 was 44 times more lethal than Stx2 and acted more rapidly than Stx2. However, Stx1 accumulated more slowly in the cerebrospinal fluid than did Stx2. Magnetic resonance imaging demonstrated a predominance of Stx1-dependent lesions in the spinal cord. Pretreatment of the animals with anti-Stx1 antiserum intravenously completely protected against both development of brain lesions and mortality.
Interaction ofNeisseria meningitidiswith Human Dendritic Cells
Authors: Kolb-Mäurer Annette , Unkmeir Alexandra , Kämmerer Ulrike , Hübner Claudia , Leimbach Thomas , Stade Anne , Kämpgen Eckhart , Frosch Matthias , Dietrich Guido
Published: July 2002

Infection withNeisseria meningitidisserogroup B is responsible for fatal septicemia and meningococcal meningitis. The severity of disease directly correlates with the production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, and IL-8. However, the source of these cytokines has not been clearly defined yet. Since bacterial infection involves the activation of dendritic cells (DCs), we analyzed the interaction ofN. meningitidiswith monocyte-derived DCs. UsingN. meningitidisserogroup B wild-type and unencapsulated bacteria, we found that capsule expression significantly impaired neisserial adherence to DCs. In addition, phagocytic killing of the bacteria in the phagosome is reduced by at least 10- to 100-fold. However, all strains induced strong secretion of proinflammatory cytokines TNF-α, IL-6, and IL-8 by DCs (at least 1,000-fold at 20 h postinfection [p.i.]), with significantly increased cytokine levels being measurable by as early as 6 h p.i. Levels of IL-1β, in contrast, were increased only 200- to 400-fold at 20 h p.i. with barely measurable induction at 6 h p.i. Moreover, comparable amounts of cytokines were induced by bacterium-free supernatants ofNeisseriacultures containing neisserial lipooligosaccharide as the main factor. Our data suggest that activated DCs may be a significant source of high levels of proinflammatory cytokines in neisserial infection and thereby may contribute to the pathology of meningococcal disease.
Phase Variation among Major Surface Antigens of
Mycoplasma penetrans
Authors: Röske Kerstin , Blanchard Alain , Chambaud Isabelle , Citti Christine , Helbig Jürgen H. , Prevost Marie-Christine , Rosengarten Renate , Jacobs Enno
Published: July 2002

The pathogenicity and prevalence of
Mycoplasma penetrans
, a
Mycoplasma
species recently isolated from humans, are still debated. A major P35 antigen, which is used as target epitope in serological assays, was shown to be a phase-variable lipid-associated membrane protein (LAMP). In this study, we performed a comparative analysis of the LAMP patterns from five
M. penetrans
clinical isolates and from the type strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and immunoblots with sera serially collected from an
M. penetrans
-infected patient indicated that these strains expressed different LAMP repertoires. Furthermore, the intraclonal variation in the expression of LAMPs (P34A, P34B, P35, and P38) was monitored by immunoblot analysis with three specific monoclonal antibodies (MAbs) developed in this study and MAb 7 to P35. The phase variation of these LAMPs occurs in an independent manner, with frequencies of variation ranging from 10
−2
to 10
−4
per cell per generation. Consistent with their amphipathic nature, the P34B and P38 antigens were found exposed at the cell surface. The DNA sequence encoding the P38 antigen was defined and found to be related to those of the P35 gene and other putative LAMP-encoding genes, suggesting that these variable antigens are encoded by a family of related genes. Finally, the serum samples from an
M. penetrans
-infected patient contained antibodies that reacted with a P36 antigen expressed in different
M. penetrans
strains but not in the isolate recovered from this patient. This result suggested that in vivo phase variation of P36 occurred, which would support a role for these LAMP variations in avoiding the host's immune vigilance.
Schistosome Infection Stimulates Host CD4+T Helper Cell and B-Cell Responses against a Novel Egg Antigen, Thioredoxin Peroxidase
Authors: Williams David L. , Asahi Hiroko , Botkin Douglas J. , Stadecker Miguel J.
Published: July 2002

Egg granuloma formation during schistosome infections is mediated by CD4+T helper (Th) cells sensitized to egg antigens; however, most of the relevant sensitizing egg antigens are still unknown. Here we show that schistosome thioredoxin peroxidase (TPx)-1 is a novel T- and B-cell egg antigen in schistosome-infected mice. CD4+Th cell responses to fractionated egg components identified a significant response against a 26-kDa antigen; a partial amino acid sequence of this antigen was found to be identical to that ofSchistosoma mansoniTPx-1. The native TPx-1 elicited significant proliferative responses as well as gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-5 secretion in CD4+cells from 8.5-week-infected CBA and C57BL/6 mice. By comparison, recombinant TPx-1 elicited a smaller, more type 1-polarized response, with significant production of IFN-γ and IL-2, less IL-5, and essentially no IL-4. In C57BL/6 mice the responses to TPx-1 were relatively more prominent than that directed against the major egg antigen, Sm-p40, whereas in CBA mice the reverse was true. B-cell responses were also monitored in infected C57BL/6, C3H, CBA, and BALB/c mice. All strains had significant antibody levels against the TPx-1 protein, but the most significant antibody production ensued following parasite oviposition. TPx-1 was localized in eggs and shown to be secreted by eggs. The identification of egg antigens is important to understand the specific basis of granuloma formation in schistosome infections and may prove to be useful in strategies to ameliorate pathological responses.
Interactions of Surfactant Proteins A and D withSaccharomyces cerevisiaeandAspergillus fumigatus
Authors: Allen Martin J. , Voelker Dennis R. , Mason Robert J.
Published: July 2002

Surfactant proteins A (SP-A) and D (SP-D) are members of the collectin family of calcium-dependent lectins and are important pulmonary host defense molecules. Human SP-A and SP-D and rat SP-D bind toAspergillus fumigatusconidia, but the ligand remains unidentified. To identify a fungal ligand for SP-A and/or SP-D, we examined the interactions of the proteins withSaccharomyces cerevisiae. SP-D but not SP-A bound yeast cells, and EDTA inhibited the binding. SP-D also aggregated yeast cells and isolated yeast cell walls. Treating yeast cells to remove cell wall mannoprotein did not reduce SP-D binding, and SP-D failed to aggregate chitin. However, SP-D aggregated yeast glucan before and after treatment with a β(1→3)-glucanase, suggesting a specific interaction between the collectin and β(1→6)-glucan. In support of this idea, SP-D-induced yeast aggregation was strongly inhibited by pustulan [a β(1→6)-linked glucose homopolymer] but was not inhibited by laminarin [a β(1→3)-linked glucose homopolymer]. Additionally, pustulan but not laminarin strongly inhibited SP-D binding toA. fumigatus. The pustulan concentration for 50% inhibition of SP-D binding toA. fumigatusis 1.0 ± 0.3 μM glucose equivalents. Finally, SP-D showed reduced binding to the β(1→6)-glucan-deficientkre6yeast mutant. Taken together, these observations demonstrate that β(1→6)-glucan is an important fungal ligand for SP-D and that glycosidic bond patterns alone can determine if an extended carbohydrate polymer is recognized by SP-D.
Specific Antibodies to
Porphyromonas gingivalis
Lys-Gingipain by DNA Vaccination Inhibit Bacterial Binding to Hemoglobin and Protect Mice from Infection
Authors: Kuboniwa Masae , Amano Atsuo , Shizukuishi Satoshi , Nakagawa Ichiro , Hamada Shigeyuki
Published: July 2002

Lys-gingipain (KGP), a lysine-specific cysteine proteinase, is one of the major virulence factors of
Porphyromonas gingivalis
. Here we examined the involvement of the catalytic domain of KGP (KGP
cd
) in hemoglobin binding by
P. gingivalis
, using a specific immunoglobulin G (IgG) elicited by the administration of plasmid DNA encoding KGP
cd
or the catalytic domain of Arg-gingipain (RGP
cd
). The pSeq2A/
kgp
cd
and pSeq2B/
rgp
cd
plasmids were constructed by the ligation of
kgp
cd
and
rgp
cd
DNA fragments, respectively. Female BALB/c mice were immunized with each of these plasmids. pSeq2A/
kgp
cd
elicited a strong response to recombinant KGP
cd
(rKGP
cd
), as well as to comparably produced rRGP
cd
-reactive antibodies. The serum antibodies elicited by pSecTag2B/
rgp
cd
also cross-reacted with rKGP
cd
as well as rRGP
cd
. Anti-KGP
cd
IgG significantly inhibited hemoglobin binding by
P. gingivalis
. Furthermore, the inhibition of hemoglobin binding was markedly enhanced by a combination of anti-KGP
cd
and anti-fimbriae. Anti-RGP
cd
IgG showed a negligible inhibitory effect, while both anti-KGP
cd
and anti-RGP
cd
IgGs showed significant inhibitory effects on Lys- and Arg-specific proteolytic activities and on the growth of
P. gingivalis
under iron-restricted conditions where supplemented hemoglobin was the sole iron source. Immunized mice were challenged by intraperitoneal inoculation with
P. gingivalis
. All nonimmunized mice died within 72 h; however, vaccination with pSeq2A/
kgp
cd
and pSeq2B/
rgp
cd
prevented inflammatory responses and prolonged the survival rate of immunized mice by 43 and 27%, respectively. These results suggest that KGP
cd
acts as a hemoglobin-binding protein and can also be useful as an immunogen inducing a protective response to
P. gingivalis
infection.
Monoclonal Antibodies Specific forNeisseria meningitidisGroup B Polysaccharide and Their Peptide Mimotopes
Authors: Shin J. S. , Lin J. S. , Anderson P. W. , Insel R. A. , Nahm M. H.
Published: July 2002

From five mice immunized withEscherichia coliK1 bacteria, we produced 12 immunoglobulin M hybridomas secreting monoclonal antibodies (MAbs) that bind toNeisseria meningitidisgroup B (NMGB). The 12 MAbs also bound the capsular polysaccharide (PS) ofE. coliK1 [which, like NMGB, is α(2-8)-linked polysialic acid (PSA)] and bound to EV36, a nonpathogenicE. coliK-12 strain producing α(2-8) PSA. Except for HmenB5, which cross-reacted withN. meningitidisgroup C, none of the MAbs bound toN. meningitidisgroups A, C, and Y. Of the 12 MAbs, 6 were autoantibodies as defined by binding to CHP-134, a neuroblastoma cell line expressing short-chain α(2-8) PSA; five of these MAbs killed NMGB in the presence of rabbit complement, and two also killed NMGB with human complement. The other six MAbs, however, were nonautoreactive; all killed NMGB with rabbit complement, and five killed NMGB with human complement. To obtain peptide mimotopes of NMGB PS, four of the nonautoreactive MAbs (HmenB2, HmenB3, HmenB13, and HmenB14) were used to screen two types of phage libraries, one with a linear peptide of 7 amino acids and the other with a circular peptide of 7 amino acids inserted between two linked cysteines. We obtained 86 phage clones that bound to the screening MAb in the absence but not in the presence ofE. coliK1 PSA in solution. The clones contained 31 linear and 4 circular mimotopes expressing unique sequences. These mimotopes nonrandomly expressed amino acids and were different from previously described mimotopes for NMGB PS. The new mimotopes may be useful in producing a vaccine(s) capable of eliciting anti-NMGB antibodies not reactive with neuronal tissue.
Timing, Localization, and Persistence of Colonization by Segmented Filamentous Bacteria in the Neonatal Mouse Gut Depend on Immune Status of Mothers and Pups
Authors: Jiang Han-Qing , Bos Nicolaas A. , Cebra John J.
Published: July 2002

As a member of the indigenous gut mucosal microbiota, segmented filamentous bacteria (SFB) colonize the guts of a variety of vertebrates and invertebrates. They are potent microbial stimuli of the gut mucosal immune system. In the small intestines of mice and rats, it has been observed that SFB are absent during the suckling period and appear in high numbers shortly after weaning, then quickly retreat to the cecum and large intestine. In this study, we explored whether this microecological phenomenon resulted from the interaction between SFB and the passively acquired maternal mucosal immunity and/or the actively acquired mucosal immunity. We set up a mouse model by reciprocal crossings and backcrossings of SFB-monoassociated, formerly germ-free, immunocompetent (+/+) BALB/c mice and immunodeficient (scid/scid) mice to produce pups which are either immunocompetent (scid/+) or immunodeficient (scid/scid) and are born either to immunocompetent (scid/+) mothers or to immunodeficient (scid/scid) mothers. We monitored the number of SFB on the mucosa of the small intestine in the four different groups of mice after birth, as well as the level of passively acquired antibodies, the active gut mucosal immune responses, and immunoglobulin A (IgA) coating of SFB in the gut. The results showed that, irrespective of whether the pups were scid/scid or scid/+, SFB could be found earlier on the mucosa of the small intestine in pups born to scid/scid mothers, appearing from day 13 and rapidly reaching a climax around weaning time on day 28, compared to the significantly delayed colonization in the pups of scid/+ mothers, starting from day 16 and peaking around days 28 to 32. After the climax, SFB quickly declined to very low levels in the small intestines of scid/+ pups of either scid/scid mothers or scid/+ mothers, whereas they remained at high levels in scid/scid pups at least until day 70, the last observation time in this study. The dynamic changes in SFB colonization of the small intestines of the different groups of pups may be related to the dynamic changes in the levels of SFB coated with secretory IgA (sIgA), which resulted from the significantly different levels of sIgA obtained from the mothers' milk during the suckling period and, later, of self-produced sIgA in the small intestine. Nevertheless, it is evident that the timing, localization, and persistence of colonization of the neonatal gut by SFB depends on the immune status of both mothers and pups.
Presence ofLpsdMutation Influences Cytokine Regulation In Vivo by theMycoplasma arthritidisMitogen Superantigen and Lethal Toxicity in Mice Infected withM. arthritidis
Authors: Mu Hong-Hua , Sawitzke Allen D. , Cole Barry C.
Published: July 2002

TheMycoplasma arthritidismitogen (MAM) superantigen (SAg) is a potent activator of human and murine cells and is produced by an organism that is a cause of acute and chronic arthritis of rodents. It is phylogenetically unrelated to other bacterial SAgs and exhibits a number of unique features. We recently demonstrated that MAM differentially regulates the cytokine responses of different mouse strains following in vivo administration. Here we show that the presence in inbred C3H/HeJ mice of the mutantLpsdgene, which is associated with a defect in Toll-like receptor 4 (TLR4), influences MAM regulation of cytokine profiles in vivo.Whereas the levels of type 1 cytokines (interleukin-2 [IL-2], gamma interferon, IL-12, and tumor necrosis factor alpha) were depressed in cells from MAM-injected wild-type C3H/HeSnJ mice, they were elevated in cells from C3H/HeJ mice. Furthermore, the levels of type 2 cytokines (IL-4, IL-6, and IL-10) were elevated inLpsnC3H/HeSnJ mice but depressed inLpsdC3H/HeJ mice. The transcript for IL-12 p40 was highly expressed in C3H/HeJ but not C3H/HeSnJ mice. F1mice exhibited the same cytokine profile as C3H/HeJ mice, indicating that the mutant gene exhibited dominant-negative inheritance. In addition, C3H/HeJ mice were highly susceptible to toxic death in comparison with C3H/HeSnJ mice after injection with liveM. arthritidisorganisms. Our results suggest that MAM interacts with the lipopolysaccharide signaling pathway, possibly involving TLR4 or a combinatorial Toll complex.
Hyphae and Yeasts ofCandida albicansDifferentially Regulate Interleukin-12 Production by Human Blood Monocytes: Inhibitory Role ofC. albicansGermination
Authors: Liu Liming , Kang Kefei , Takahara Masakazu , Cooper Kevin D. , Ghannoum Mahmoud A.
Published: July 2002

The role ofCandida albicansyeast-to-hypha transition in interleukin-12 (IL-12) production by monocytes was investigated. GerminatingC. albicansnot only failed to induce IL-12 p70 but also suppressed IL-12 production induced by heat-killedC. albicans. Comparison of the abilities of germinatingC. albicansand agerminating mutants to inhibit IL-12 production showed that germination ofC. albicansplays a critical role in the inhibition of IL-12 production.
Assessment of
Helicobacter pylori
Gene Expression within Mouse and Human Gastric Mucosae by Real-Time Reverse Transcriptase PCR
Authors: Rokbi Bachra , Seguin Delphine , Guy Bruno , Mazarin Véronique , Vidor Emmanuel , Mion François , Cadoz Michel , Quentin-Millet Marie-José
Published: July 2002

Despite increasing knowledge on the biology of
Helicobacter pylori
, little is known about the expression pattern of its genome during infection. While mouse models of infection have been widely used for the screening of protective antigens, the reliability of the mouse model for gene expression analysis has not been assessed. In an attempt to address this question, we have developed a quantitative reverse transcriptase PCR (RT-PCR) that allowed the detection of minute amounts of mRNA within the gastric mucosa. The expression of four genes, 16S rRNA,
ureA
(encoding urease A subunit),
katA
(catalase), and
alpA
(an adhesin), was monitored during the course of a 6-month infection of mice and in biopsy samples from of 15 infected humans. We found that the selected genes were all expressed within both mouse and human infected mucosae. Moreover, the relative abundance of transcripts was the same (16S rRNA >
ureA
>
katA
>
alpA
), in the two models. Finally, results obtained with the mouse model suggest a negative effect of bacterial burden on the number of transcripts of each gene expressed per CFU (
P
< 0.05 for 16S rRNA,
alpA
, and
katA
). Overall, this study demonstrates that real-time RT-PCR is a powerful tool for the detection and quantification of
H. pylori
gene expression within the gastric mucosa and strongly indicates that mice experimentally infected with
H. pylori
provide a valuable model for the analysis of bacterial gene expression during infection.
Facilitated Intranasal Induction of Mucosal and Systemic Immunity to Mutans Streptococcal Glucosyltransferase Peptide Vaccines
Authors: Smith Daniel J. , King William F. , Barnes Leigh A. , Trantolo Debra , Wise Donald L. , Taubman Martin A.
Published: July 2002

Synthetic peptide vaccines which are derived from functional domains ofStreptococcus mutansglucosyltransferases (GTF) have been shown to induce protective immunity in Sprague-Dawley rats after subcutaneous injection in the salivary gland region. Since mucosal induction of salivary immunity would be preferable in humans, we explored methods to induce mucosal antibody in the rat to the GTF peptide vaccines HDS and HDS-GLU after intranasal administration. Several methods of facilitation of the immune response were studied: the incorporation of peptides in bioadhesive poly(d,l-lactide-coglycolide) (PLGA) microparticles, the use of monoepitopic (HDS) or diepitopic (HDS-GLU) peptide constructs, or the use of mucosal adjuvants. Salivary immunoglobulin A (IgA) responses were not detected after intranasal administration of diepitopic HDS-GLU peptide constructs in alum or after incorporation into PLGA microparticles. However, significant primary and secondary salivary IgA and serum IgG antibody responses to HDS were induced in all rats when cholera holotoxin (CT) or a detoxified mutantEscherichia coliheat-labile enterotoxin (R192G LT) were intranasally administered with HDS peptide constructs in PLGA. Coadministration of LT with HDS resulted in predominantly IgG2a responses in the serum, while coadministration with CT resulted in significant IgG1 and IgG2a responses to HDS. Serum IgG antibody, which was induced to the HDS peptide construct by coadministration with these adjuvants, also bound intact mutans streptococcal GTF in an enzyme-linked immunosorbent assay and inhibited its enzymatic activity. Thus, immune responses which are potentially protective for dental caries can be induced to peptide-based GTF vaccines after mucosal administration if combined with the CT or LT R192G mucosal adjuvant.
Immunogenicity of Well-Characterized SyntheticPlasmodium falciparumMultiple Antigen Peptide Conjugates
Authors: Joshi Manju B. , Gam Albert A. , Boykins Robert A. , Kumar Sanjai , Sacci John , Hoffman Stephen L. , Nakhasi Hira L. , Kenney Richard T.
Published: July 2002

Given the emerging difficulties with malaria drug resistance and vector control, as well as the persistent lack of an effective vaccine, new malaria vaccine development strategies are needed. We used a novel methodology to synthesize and fully characterize multiple antigen peptide (MAP) conjugates containing protective epitopes fromPlasmodium falciparumand evaluated their immunogenicity in four different strains of mice. A di-epitope MAP (T3-T1) containing two T-cell epitopes of liver stage antigen-1 (LSA-1), a di-epitope MAP containing T-cell epitopes from LSA-1 and from merozoite surface protein-1, and a tri-epitope MAP (T3-CS-T1) containing T3-T1 and a potent B-cell epitope from the circumsporozoite protein central repeat region were tested in this study. Mice of all four strains produced peptide-specific antibodies; however, the magnitude of the humoral response indicated strong genetic restriction between the different strains of mice. Anti-MAP antibodies recognized stage-specific proteins on the malaria parasites in an immunofluorescence assay. In addition, serum from hybrid BALB/cJ × A/J CAF1 mice that had been immunized with the tri-epitope MAP T3-CS-T1 successfully inhibited the malaria sporozoite invasion of hepatoma cells in vitro. Spleen cells from immunized mice also showed a genetically restricted cellular immune response when stimulated with the immunogen in vitro. This study indicates that well-characterized MAPs combining solid-phase synthesis and conjugation chemistries are potent immunogens and that this approach can be utilized for the development of subunit vaccines.
Brucella abortus
HtrA Functions as an Authentic Stress Response Protease but Is Not Required for Wild-Type Virulence in BALB/c Mice
Authors: Phillips Robert W. , Roop R. Martin
Published: July 2002

A second mutation has recently been identified in the previously described
Brucella abortus htrA
mutant PHE1. As a result of this finding, a new
B. abortus htrA
mutant, designated RWP11, was constructed to evaluate the biological function of the
Brucella
HtrA protease. RWP11 is more sensitive to oxidative killing in vitro and less resistant to killing by cultured murine neutrophils and macrophages than the virulent parental strain 2308 but is not attenuated in BALB/c mice through 4 weeks postinfection. The in vitro phenotype of
B. abortus
RWP11 is consistent with the proposed function of bacterial HtrA proteases as components of a secondary line of defense against oxidative damage. The in vivo phenotype of this mutant, however, indicates that, unlike the corresponding
Salmonella
and
Yersinia
proteins,
Brucella
HtrA does not play a critical role in virulence in the mouse model.
The Extracytoplasmic Sigma Factor, ς
E
, Is Required for Intracellular Survival of Nontypeable
Haemophilus influenzae
in J774 Macrophages
Authors: Craig Jane E. , Nobbs Angela , High Nicola J.
Published: July 2002

Nontypeable
Haemophilus influenzae
(NTHi) causes a wide variety of respiratory tract infections in humans. It is capable of invading and surviving in epithelial cells and has also been shown to persist in macrophage-like cell line J774A.1. To determine the molecular mechanisms which enable NTHi to survive in an intracellular environment, differential display reverse transcriptase PCR was used to identify genes which were either induced or upregulated by NTHi residing in macrophages. Using this approach, we identified one transcript which was consistently amplified from intracellular NTHi cDNA. Nucleotide sequence analysis of this product revealed that it spanned the 3′ and 5′ ends of
rpoE
and
rseB
, respectively, which form part of the extracytoplasmic stress operon that encodes and regulates expression of alternate sigma factor sigma E (ς
E
). To confirm that expression of
rpoE
was upregulated following uptake of NTHi by macrophages, an
rpoE-lacZ
transcriptional fusion was constructed, and expression of β-galactosidase activity in broth-grown NTHi was compared with expression of β-galactosidase activity in intracellular NTHi. The level of β-galactosidase activity in NTHi 4 h after phagocytosis by macrophages was found to be 100-fold higher than that of broth-grown organisms, suggesting that genes of the ς
E
regulon may be important for persistence of NTHi in mammalian cells. The hypothesis that ς
E
plays a role in the intracellular survival of NTHi was subsequently confirmed by the decreased ability of an
rpoE
insertion mutant to survive in macrophages.
Nitric Oxide and Apoptosis Induced in Peyer's Patches by Attenuated Strains ofSalmonella entericaSerovar Enteritidis
Authors: Cerquetti M. C. , Goren N. B. , Ropolo A. J. , Grasso D. , Giacomodonato M. N. , Vaccaro M. I.
Published: July 2002

Nitric oxide (NO) is a toxic molecule of the immune system which contributes to the control of microbial pathogens. Additional functions of NO in innate and adaptive immunity have recently been described; these functions include the modulation of the cytokine response of lymphocytes and the regulation of immune cell apoptosis. In addition to direct microbicidal actions, NO has immunoregulatory effects relevant to the control of infections. In turn, infected macrophages and macrophage-regulating lymphocytes may undergo apoptosis during infection bySalmonellaspp. In this work we investigated the ability of attenuated strains ofSalmonella entericaserovar Enteritidis with different protective capacities to induce intestinal inducible nitric oxide synthase (iNOS) and apoptosis in Peyer's patches (PP) in mice. Results showed that the intestinal iNOS activity correlated with increased apoptosis in PP. Furthermore, the ability to induce intestinal NO production and apoptosis within the first few hours after immunization seemed to correlate with the protective capacity of mutant E/1/3 ofS. entericaserovar Enteritidis. It was found that nonprotective mutant C/2/2, which was unable to induce intestinal NO production, also failed to induce apoptosis in PP. Moreover, aminoguanidine treatment at the time of immunization resulted in inhibition of the NO production and apoptosis induced by protective mutant E/1/3 and completely abolished protection against challenge. These results suggest that the induction of iNOS in the intestinal mucosa by attenuated mutant E/1/3 ofS. entericaserovar Enteritidis at the time of immunization is necessary to generate a protective immune response.
Contribution of Fibronectin-Binding Protein to Pathogenesis of
Streptococcus suis
Serotype 2
Authors: de Greeff Astrid , Buys Herma , Verhaar Robin , Dijkstra Janny , van Alphen Loek , Smith Hilde E.
Published: July 2002

In the present study we investigated the role of the fibronectin (FN)- and fibrinogen (FGN)-binding protein (FBPS) in the pathogenesis of
Streptococcus suis
serotype 2 in piglets. The complete gene encoding FBPS from
S. suis
serotype 2 was cloned in
Escherichia coli
and sequenced. The occurrence of the gene in various serotypes was analyzed by hybridization studies. The FBPS protein was expressed in
E. coli
and purified, and binding to human FN and FGN was demonstrated. The induction of antibodies in piglets was studied upon infection. An isogenic mutant unable to produce FBPS was constructed, and the levels of virulence of the wild-type and mutant strains were compared in a competitive infection model in young piglets. Organ cultures showed that FBPS was not required for colonization of the tonsils but that FBPS played a role in the colonization of the specific organs involved in an
S. suis
infection. Therefore, the FBPS mutant was considered as an attenuated mutant.
Analyses of the Evolutionary Distribution of
Salmonella
Translocated Effectors
Authors: Hansen-Wester Imke , Stecher Bärbel , Hensel Michael
Published: July 2002

The type III secretion system encoded by
Salmonella
pathogenicity island 2 (SPI2) translocates
Salmonella
translocated effectors (STE) into host cells. STE are encoded by genes outside of SPI2. The distribution of STE loci within the salmonellae was investigated. In contrast to the SPI2 locus that is conserved within
Salmonella enterica
, STE loci show a variable distribution. In addition to other virulence determinants, the possession of various sets of STE loci may contribute to the different host ranges and pathogenic potentials of
S. enterica
serovars.
Safety and Shedding of an Attenuated Strain ofListeria monocytogeneswith a Deletion ofactA/plcBin Adult Volunteers: a Dose Escalation Study of Oral Inoculation
Authors: Angelakopoulos Haroula , Loock Katharina , Sisul David M. , Jensen Eric R. , Miller Jeff F. , Hohmann Elizabeth L.
Published: July 2002

Listeria monocytogenesis an intracellular bacterial pathogen which causes bacteremia and has a tropism for the central nervous system and a propensity to cause maternofetal infection.L. monocytogeneshas been shown to be an effective prophylactic and a therapeutic vaccine vector for viral and tumor antigens in animal models.L. monocytogenesmutants lacking the ActA protein, which is essential for intracellular movement, are attenuated but retain immunogenicity in mice. Given the pathogenic potential ofL. monocytogenes, we created an attenuated mutant strain bearing double deletions in theactAandplcBvirulence genes for an initial clinical safety study of a prototypeL. monocytogenesvector in adults. Twenty healthy volunteers received single escalating oral doses (106to 109CFU, 4 volunteers per dose cohort) of this attenuatedL. monocytogenes, designated LH1169. Volunteers were monitored in the hospital for 14 days with frequent clinical checks and daily blood and stool cultures, and they were monitored for six additional weeks as outpatients. There were no positive blood cultures and no fevers attributable to the investigational inoculation. Most volunteers shed vaccine bacteria for 4 days or less, without diarrhea. One volunteer had a late positive stool culture during outpatient follow-up. Three volunteers had abnormal liver function test results temporally associated with inoculation; one could be reasonably attributed to another cause. In the highest-dose cohort, humoral, mucosal, and cellular immune responses to the investigational organism were detected in individual volunteers. AttenuatedL. monocytogenescan be studied in adult volunteers without serious long-term health sequelae.
Transcriptional Control of Expression of Genes for Photosynthetic Reaction Center and Light-Harvesting Proteins in the Purple Bacterium
Rhodovulum sulfidophilum
Authors: Masuda Shinji , Nagashima Kenji V. P. , Shimada Keizo , Matsuura Katsumi
Published: July 2002

The purple photosynthetic bacterium
Rhodovulum sulfidophilum
synthesizes photosynthetic apparatus even under highly aerated conditions in the dark. To understand the oxygen-independent expression of photosynthetic genes, the expression of the
puf
operon coding for the light-harvesting 1 and reaction center proteins was analyzed. Northern blot hybridization analysis showed that
puf
mRNA synthesis was not significantly repressed by oxygen in this bacterium. High-resolution 5′ mapping of the
puf
mRNA transcriptional initiation sites and DNA sequence analysis of the
puf
upstream regulatory region indicated that there are three possible promoters for the
puf
operon expression, two of which have a high degree of sequence similarity with those of
Rhodobacter capsulatus
, which shows a high level of oxygen repression of photosystem synthesis. Deletion analysis showed that the third promoter is oxygen independent, but the activity of this promoter was not enough to explain the aerobic level of mRNA. The posttranscriptional
puf
mRNA degradation is not significantly influenced by oxygen in
R. sulfidophilum
. From these results, we conclude that
puf
operon expression in
R. sulfidophilum
is weakly repressed by oxygen, perhaps as a result of the following: (i) there are three promoters for
puf
operon transcription, at least one of which is oxygen independent; (ii) readthrough transcripts which may not be affected by oxygen may be significant in maintaining the
puf
mRNA levels; and (iii) the
puf
mRNA is fairly stable even under aerobic conditions.
Rapid Turnover of FlhD and FlhC, the Flagellar Regulon Transcriptional Activator Proteins, during
Proteus
Swarming
Authors: Claret Laurent , Hughes Colin
Published: July 2002

The enterobacterial
flhDC
master operon activates expression of the flagellar biogenesis gene hierarchy and also represses cell division. During
Proteus mirabilis
differentiation into elongated hyperflagellated swarm cells,
flhDC
transcription is strongly but transiently increased. We show that concentration of the FlhD and FlhC proteins is also tightly controlled at the posttranslational level. This is achieved by protein degradation, which is most severe after differentiation when the half-life of both proteins is ca. 2 min. Degradation is energy dependent and putatively involves the Lon protease.
Cloning and Characterization of 1-Deoxy-
d
-Xylulose 5-Phosphate Synthase from
Streptomyces
sp. Strain CL190, Which Uses both the Mevalonate and Nonmevalonate Pathways for Isopentenyl Diphosphate Biosynthesis
Authors: Kuzuyama Tomohisa , Takagi Motoki , Takahashi Shunji , Seto Haruo
Published: July 2002

In addition to the ubiquitous mevalonate pathway,
Streptomyces
sp. strain CL190 utilizes the nonmevalonate pathway for isopentenyl diphosphate biosynthesis. The initial step of this nonmevalonate pathway is the formation of 1-deoxy-
d
-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate catalyzed by DXP synthase. The corresponding gene,
dxs
, was cloned from CL190 by using PCR with two oligonucleotide primers synthesized on the basis of two highly conserved regions among
dxs
homologs from six genera. The
dxs
gene of CL190 encodes 631 amino acid residues with a predicted molecular mass of 68 kDa. The recombinant enzyme overexpressed in
Escherichia coli
was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 130 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of 9.0, with a
V
max
of 370 U per mg of protein and

K
m

s of 65 μM for pyruvate and 120 μM for
d
-glyceraldehyde 3-phosphate. The purified enzyme catalyzed the formation of 1-deoxyxylulose by condensation of pyruvate and glyceraldehyde as well, with a

K
m

value of 35 mM for
d
-glyceraldehyde. To compare the enzymatic properties of CL190 and
E. coli
DXP synthases, the latter enzyme was also overexpressed and purified. Although these two enzymes had different origins, they showed the same enzymatic properties.
The Right End of the
vir
Region of an Octopine-Type Ti Plasmid Contains Four New Members of the
vir
Regulon That Are Not Essential for Pathogenesis
Authors: Kalogeraki Virginia S. , Zhu Jun , Stryker Joel L. , Winans Stephen C.
Published: July 2002

We sequenced the
virD-virE
,
virE-virF
, and
virF
–T-DNA intergenic regions of an octopine Ti plasmid. Four newly described genes were induced by the
vir
gene inducer acetosyringone, two of which are conserved in the nopaline-type Ti plasmid pTiC58. One gene resembles a family of phosphatase genes. Each of these genes is dispensible for tumorigenesis.
Analysis of thebmpGene Family inBorrelia burgdorferiSensu Lato
Authors: Gorbacheva Victoria Y. , Godfrey Henry P. , Cabello Felipe C.
Published: July 2002

BmpA, BmpB, BmpC, and BmpD are homologousBorrelia burgdorferilipoproteins of unknown functions, encoded by thebmpgenes of paralogous chromosomal gene family 36. At least some of the Bmp proteins are immunogens in infected vertebrate hosts. The genetic organization of thebmpregion has been characterized for a variety ofB. burgdorferisensu lato strains by Southern hybridization, PCR amplification, and DNA sequencing. All fourbmpgenes were present in the same relative order in allB. burgdorferisensu lato low- and high-passage-number isolates. While there were no differences in the relative orders of thebmpgenes in these species, variations in DNA sequence in thebmpD-bmpCandbmpC-bmpAintergenic regions were significantly more common than in the corresponding 3′bmpDandbmpCcoding regions. The genetic structure of the chromosomal region containing thebmpgenes thus appears to be well conserved across different species ofB. burgdorferi, but variations in DNA fine structure that prevent PCR primer annealing may occur in this region and make Southern hybridization much more reliable than PCR for detection of the presence of these genes. Our results also suggest thatbmpgene products may be used as reagents in the preparation of vaccines and diagnostic assays to protect against and diagnose Lyme disease produced byB. burgdorferisensu lato.
Insertion Mutagenesis and Membrane Topology Model of the
Pseudomonas aeruginosa
Outer Membrane Protein OprM
Authors: Wong Kendy K. Y. , Hancock Robert E. W.
Published: July 2002

Pseudomonas aeruginosa
OprM is a protein involved in multiple-antibiotic resistance as the outer membrane component for the MexA-MexB-OprM efflux system. Planar lipid bilayer experiments showed that OprM had channel-forming activity with an average single-channel conductance of only about 80 pS in 1 M KCl. The gene encoding OprM was subjected to insertion mutagenesis by cloning of a foreign epitope from the circumsporozoite form of the malarial parasite
Plasmodium falciparum
into 11 sites. In
Escherichia coli
, 8 of the 11 insertion mutant genes expressed proteins at levels comparable to those obtained with the wild-type gene and the inserted malarial epitopes were surface accessible as assessed by indirect immunofluorescence. When moved to a
P. aeruginosa
OprM-deficient strain, seven of the insertion mutant genes expressed proteins at variable levels comparable to that of wild-type OprM and three of these reconstituted MIC profiles resembling those of the wild-type protein, while the other mutant forms showed variable MIC results. Utilizing the data from these experiments, in conjunction with multiple sequence alignments and structure predictions, an OprM topology model with 16 β strands was proposed.
Quorum Sensing inVibrio fischeri: Analysis of the LuxR DNA Binding Region by Alanine-Scanning Mutagenesis
Authors: Egland Kristi A. , Greenberg E. P.
Published: July 2002

LuxR is the transcriptional activator for quorum-sensing control of luminescence inVibrio fischeri. A series of alanine-scanning mutants spanning a predicted helix-turn-helix region in the DNA binding domain of LuxR was constructed, and the activity of each of the LuxR mutant proteins in recombinantEscherichia coliwas investigated. The region covered by the mutagenesis spanned residues 190 to 224. About half of the alanine-scanning mutants showed activities similar to that of the wild-type LuxR: at least two were positive-control mutants, four appeared to be defective in DNA binding, and several others were characterized as DNA binding affinity mutants. This analysis, taken together with information about other bacterial transcription factors, provides insights into amino acid residues in LuxR that are involved in DNA binding and transcriptional activation.
Role for
hetC
in the Transition to a Nondividing State during Heterocyst Differentiation in
Anabaena
sp
Authors: Xu Xudong , Wolk C. Peter
Published: July 2002

Nitrogen-deprived filaments of wild-type or
hetC Anabaena
sp. produce respectively, at semiregular intervals, heterocysts and weakly fluorescent cells. Unlike heterocysts, the latter cells can divide and elongate, producing a pattern of spaced series of small cells. Because a
hetR
::
gfp
fusion is expressed most strongly in the small cells, we propose that these small cells represent a very early stage of heterocyst differentiation.
hetC
::
gfp
is expressed most strongly in proheterocysts and heterocysts.
Engineering a Homo-Ethanol Pathway in
Escherichia coli
: Increased Glycolytic Flux and Levels of Expression of Glycolytic Genes during Xylose Fermentation
Authors: Tao Han , Gonzalez Ramon , Martinez Alfredo , Rodriguez Maria , Ingram L. O. , Preston J. F. , Shanmugam K. T.
Published: July 2002

Replacement of the native fermentation pathway in
Escherichia coli
B with a homo-ethanol pathway from
Zymomonas mobilis
(
pdc
and
adhB
genes) resulted in a 30 to 50% increase in growth rate and glycolytic flux during the anaerobic fermentation of xylose. Gene array analysis was used as a tool to investigate differences in expression levels for the 30 genes involved in xylose catabolism in the parent (strain B) and the engineered strain (KO11). Of the 4,290 total open reading frames, only 8% were expressed at a significantly higher level in KO11 (
P
< 0.05). In contrast, over half of the 30 genes involved in the catabolism of xylose to pyruvate were expressed at 1.5-fold- to 8-fold-higher levels in KO11. For 14 of the 30 genes, higher expression was statistically significant at the 95% confidence level (
xylAB, xylE, xylFG, xylR, rpiA, rpiB, pfkA, fbaA, tpiA, gapA, pgk,
and
pykA
) during active fermentation (6, 12, and 24 h). Values at single time points for only four of these genes (
eno, fbaA, fbaB,
and
talA
) were higher in strain B than in KO11. The relationship between changes in mRNA (cDNA) levels and changes in specific activities was verified for two genes (
xylA
and
xylB
) with good agreement. In KO11, expression levels and activities were threefold higher than in strain B for xylose isomerase (
xylA
) and twofold higher for xylulokinase (
xylB
). Increased expression of genes involved in xylose catabolism is proposed as the basis for the increase in growth rate and glycolytic flux in ethanologenic KO11.
Forespore-Specific Transcription of the
lonB
Gene during Sporulation in
Bacillus subtilis
Authors: Serrano Monica , Hövel Sven , Moran Charles P. , Henriques Adriano O. , Völker Uwe
Published: July 2002

The
Bacillus subtilis
genome encodes two members of the Lon family of prokaryotic ATP-dependent proteases. One, LonA, is produced in response to temperature, osmotic, and oxidative stress and has also been implicated in preventing ς
G
activity under nonsporulation conditions. The second is encoded by the
lonB
gene, which resides immediately upstream from
lonA
. Here we report that transcription of
lonB
occurs during sporulation under ς
F
control and thus is restricted to the prespore compartment of sporulating cells. First, expression of a
lonB-lacZ
transcriptional fusion was abolished in strains unable to produce ς
F
but remained unaffected upon disruption of the genes encoding the early and late mother cell regulators ς
E
and ς
K
or the late forespore regulator ς
G
. Second, the fluorescence of strains harboring a
lonB-gfp
fusion was confined to the prespore compartment and depended on ς
F
production. Last, primer extension analysis of the
lonB
transcript revealed −10 and −35 sequences resembling the consensus sequence recognized by ς
F
-containing RNA polymerase. We further show that the
lonB
message accumulated as a single monocistronic transcript during sporulation, synthesis of which required ς
F
activity. Disruption of the
lonB
gene did not confer any discernible sporulation phenotype to otherwise wild-type cells, nor did expression of
lonB
from a multicopy plasmid. In contrast, expression of a fusion of the
lonB
promoter to the
lonA
gene severely reduced expression of the ς
G
-dependent
sspE
gene and the frequency of sporulation. In confirmation of earlier observations, we found elevated levels of ς
F
-dependent activity in a
spoIIIE47
mutant, in which the
lonB
region of the chromosome is not translocated into the prespore. Expression of either
lonB
or the P

lonB

-lonA
fusion from a plasmid in the
spoIIIE47
mutant reduced ς
F
-dependent activity to wild-type levels. The results suggest that both LonA and LonB can prevent abnormally high ς
F
activity but that only LonA can negatively regulate ς
G
.