bluuebunny/crossref_metadata_2025 huggingface dataset:
Salmonella entericaSerovar-Host Specificity Does Not Correlate with the Magnitude of Intestinal Invasion in Sheep
Authors: Uzzau Sergio , Leori Guido S. , Petruzzi Valentino , Watson Patricia R. , Schianchi Giuseppe , Bacciu Donatella , Mazzarello Vittorio , Wallis Timothy S. , Rubino Salvatore
Published: July 2002

The colonization of intestinal and systemic tissues bySalmonella entericaserovars with different host specificities was determined 7 days after inoculation of 1 to 2-month-old lambs. Following oral inoculation,S. entericaserovars Abortusovis, Dublin, and Gallinarum were recovered in comparable numbers from the intestinal mucosa, but serovar Gallinarum was recovered in lower numbers than the other serovars from systemic sites. The pattern of bacterial recovery from systemic sites following intravenous inoculation was similar. The magnitude of intestinal invasion was evaluated in ovine ligated ileal loops in vivo. Serovars Dublin and Gallinarum and the broad-host-rangeSalmonellaserovar Typhimurium were recovered in comparable numbers from ileal mucosa 3 h after loop inoculation, whereas the recovery of serovar Abortusovis was approximately 10-fold lower. Microscopic analysis of intestinal mucosae infected with serovars Typhimurium and Dublin showed dramatic morphological changes and infiltration of inflammatory cells, whereas mucosae infected with serovars Abortusovis and Gallinarum were indistinguishable from uninfected mucosae. Together these data suggest thatSalmonellaserovar specificity in sheep correlates with bacterial persistence at systemic sites. Intestinal invasion and avoidance of the host's intestinal inflammatory response may contribute to but do not determine the specificity of serovar Abortosovis for sheep. Intestinal invasion by serovar Abortusovis was significantly reduced after mutation ofinvHbut was not reduced following curing of the virulence plasmid, suggesting that theSalmonellapathogenicity island 1 influences but the virulence plasmid genes do not influence the ability of serovar Abortusovis to invade the intestinal mucosa in sheep.
Activation of Interleukin-1 Receptor-Associated Kinase by Gram-Negative Flagellin
Authors: Moors Marlena A. , Li Liwu , Mizel Steven B.
Published: July 2002

Flagellin from various species of gram-negative bacteria activates monocytes to produce proinflammatory cytokines. We have analyzed the pathway by whichSalmonella enteritidisflagellin (FliC) activates murine and human monocyte/macrophage-like cell lines. Since lipopolysaccharide (LPS), the principal immune stimulatory component of gram-negative bacteria, is known to signal through Toll-like receptor 4 (TLR4), we tested the possibility that FliC also signals via TLR4. When murine HeNC2 cells were stimulated with LPS in the presence of a neutralizing anti-TLR4 monoclonal antibody, tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production were markedly reduced. In contrast, FliC-mediated TNF-α and NO production were minimally affected by the anti-TLR4 antibody. Furthermore, FliC, unlike LPS, stimulated TNF-α production in the TLR4 mutant cell line, GG2EE, indicating that TLR4 is not essential for FliC-mediated signaling. To test the possibility that FliC signals via another TLR, we measured FliC-mediated activation of interleukin-1 (IL-1) receptor-associated kinase (IRAK), a central component in IL-1R/TLR signaling. FliC induced IRAK activation in HeNC2 and GG2EE cells as well as in the human promonocytic cell line THP-1. IRAK activation was rapid in HeNC2 cells, with maximal activity observed after 5 min of treatment with FliC. In addition, FliC-mediated IRAK activation exhibited the same concentration dependence as was demonstrated for the induction of TNF-α. These results represent the first demonstration of IRAK activation by a purified bacterial protein and strongly suggest that a TLR distinct from TLR4 is involved in the macrophage inflammatory response to FliC.
FlhA, a Component of the Flagellum Assembly Apparatus of
Pseudomonas aeruginosa
, Plays a Role in Internalization by Corneal Epithelial Cells
Authors: Fleiszig Suzanne M. J. , Arora Shiwani K. , Van Rajana , Ramphal Reuben
Published: July 2002

Pseudomonas aeruginosa
invades various epithelial cell types in vitro and in vivo. The
P. aeruginosa
genome possesses a gene (
flhA
) which encodes a protein that is believed to be part of the export apparatus for flagellum assembly and which is homologous to
invA
of
Salmonella
spp. Because
invA
is required for invasion of
Salmonella
spp., a role for
flhA
in
P. aeruginosa
invasion was explored using cultured rabbit corneal epithelial cells. An
flhA
mutant of
P. aeruginosa
strain PAO1 was constructed and was shown to be nonmotile. Complementation with
flhA
in
trans
restored motility. Corneal cells were infected for 3 h with the wild type (PAO1), the
flhA
mutant, the
flhA
mutant complemented with
flhA
in
trans
, an
flhA
mutant containing the plasmid vector control, or an
fliC
mutant (nonmotile mutant control). Invasion was quantified by amikacin exclusion assays. Both the
flhA
and the
fliC
mutants invaded at a lower level than the wild-type strain did, suggesting that both
fliC
and
flhA
played roles in invasion. However, loss of motility was not sufficient to explain the reduced invasion by
flhA
mutants, since centrifugation of bacteria onto cells did not restore invasion to wild-type levels. Unexpectedly, the
flhA
mutant adhered significantly better to corneal epithelial cells than wild-type bacteria or the
fliC
mutant did. The percentage of adherent bacteria that invaded was reduced by ∼80% for the
flhA
mutant and ∼50% for the
fliC
mutant, showing that only part of the role of
flhA
in invasion involves
fliC
. Invasion was restored by complementing the
flhA
mutant with
flhA
in
trans
but not by the plasmid vector control. Intracellular survival assays, in which intracellular bacteria were enumerated after continued incubation in the presence of antibiotics, showed that although
flhA
and
fliC
mutants had a reduced capacity for epithelial cell entry, they were not defective in their ability to survive within those cells after entry. These results suggest that the flagellum assembly type III secretion system plays a role in
P. aeruginosa
invasion of epithelial cells. Since the
flhA
mutants were not defective in their ability to adhere to corneal epithelial cells, to retain viability at the cell surface, or to survive inside epithelial cells after entry, the role of
flhA
in invasion of epithelial cells is likely to occur during the process of bacterial internalization.
Enhanced Susceptibility to Pulmonary Infection withBurkholderia cepaciainCftr−/−Mice
Authors: Sajjan Uma , Thanassoulis George , Cherapanov Vera , Lu Annie , Sjolin Carola , Steer Brent , Wu Yi Jun , Rotstein Ori D. , Kent Geraldine , McKerlie Colin , Forstner Janet , Downey Gregory P.
Published: July 2002

Progressive pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the molecular basis for this susceptibility remains incompletely understood. To study this problem, we developed a model of chronic pneumonia by repeated instillation of a clinical isolate ofBurkholderia cepacia(genomovar III, ET12 strain), an opportunistic gram-negative bacterium, from a case of CF into the lungs ofCftrm1unc−/−(Cftr−/−) and congenicCftr+/+controls. Nine days after the last instillation, the CF transmembrane regulator knockout mice showed persistence of viable bacteria with chronic severe bronchopneumonia while wild-type mice remained healthy. The histopathological changes in the lungs of the susceptibleCftr−/−mice were characterized by infiltration of a mixed inflammatory-cell population into the peribronchiolar and perivascular spaces, Clara cell hyperplasia, mucus hypersecretion in airways, and exudation into alveolar airspaces by a mixed population of macrophages and neutrophils. An increased proportion of neutrophils was observed in bronchoalveolar lavage fluid from theCftr−/−mice, which, despite an increased bacterial load, demonstrated minimal evidence of activation. Alveolar macrophages fromCftr−/−mice also demonstrated suboptimal activation. These observations suggest that the pulmonary host defenses are compromised in lungs from animals with CF, as manifested by increased susceptibility to bacterial infection and lung injury. This murine model of chronic pneumonia thus reflects, in part, the situation in human patients and may help elucidate the mechanisms leading to defective host defense in CF.
Comparison of Virulence of
Legionella longbeachae
Strains in Guinea Pigs and U937 Macrophage-Like Cells
Authors: Doyle Robyn M. , Cianciotto Nicholas P. , Banvi Shaila , Manning Paul A. , Heuzenroeder Michael W.
Published: July 2002

A guinea pig model of experimental legionellosis was established for assessment of virulence of isolates of
Legionella longbeachae
. The results showed that there were distinct virulence groupings of
L. longbeachae
serogroup 1 strains based on the severity of disease produced in this model. Statistical analysis of the animal model data suggests that Australian isolates of
L. longbeachae
may be inherently more virulent than non-Australian strains. Infection studies performed with U937 cells were consistent with the animal model studies and showed that isolates of this species were capable of multiplying within these phagocytic cells. Electron microscopy studies of infected lung tissue were also undertaken to determine the intracellular nature of
L. longbeachae
serogroup 1 infection. The data showed that phagosomes containing virulent
L. longbeachae
serogroup 1 appeared bloated, contained cellular debris and had an apparent rim of ribosomes while those containing avirulent
L. longbeachae
serogroup 1 were compact, clear and smooth.
Cellular and Humoral Immune Responses and Protection against Schistosomes Induced by a Radiation-Attenuated Vaccine in Chimpanzees
Authors: Eberl Matthias , Langermans Jan A. M. , Frost Patrice A. , Vervenne Richard A. , van Dam Govert J. , Deelder André M. , Thomas Alan W. , Coulson Patricia S. , Wilson R. Alan
Published: July 2002

The radiation-attenuatedSchistosoma mansonivaccine is highly effective in rodents and primates but has never been tested in humans, primarily for safety reasons. To strengthen its status as a paradigm for a human recombinant antigen vaccine, we have undertaken a small-scale vaccination and challenge experiment in chimpanzees (Pan troglodytes). Immunological, clinical, and parasitological parameters were measured in three animals after multiple vaccinations, together with three controls, during the acute and chronic stages of challenge infection up to chemotherapeutic cure. Vaccination induced a strong in vitro proliferative response and early gamma interferon production, but type 2 cytokines were dominant by the time of challenge. The controls showed little response to challenge infection before the acute stage of the disease, initiated by egg deposition. In contrast, the responses of vaccinated animals were muted throughout the challenge period. Vaccination also induced parasite-specific immunoglobulin M (IgM) and IgG, which reached high levels at the time of challenge, while in control animals levels did not rise markedly before egg deposition. The protective effects of vaccination were manifested as an amelioration of acute disease and overall morbidity, revealed by differences in gamma-glutamyl transferase level, leukocytosis, eosinophilia, and hematocrit. Moreover, vaccinated chimpanzees had a 46% lower level of circulating cathodic antigen and a 38% reduction in fecal egg output, compared to controls, during the chronic phase of infection.
Characterization ofHaemophilus ducreyi cdtA, cdtB, andcdtCMutants in In Vitro and In Vivo Systems
Authors: Lewis David A. , Stevens Marla K. , Latimer Jo L. , Ward Christine K. , Deng Kaiping , Blick Robert , Lumbley Sheryl R. , Ison Catherine A. , Hansen Eric J.
Published: July 2002

Haemophilus ducreyiexpresses a soluble cytolethal distending toxin (CDT) that is encoded by thecdtABCgene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. ThecdtA, cdtB, andcdtCgenes ofH. ducreyiwere cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in anEscherichia colibackground. All three gene products had to be expressed in order forE. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. IsogenicH. ducreyi cdtAandcdtBmutants were constructed and used in combination with the wild-type parent strain and a previously describedH. ducreyi cdtCmutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900–3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary forH. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from thecdtBandcdtCmutants had no effect on HeLa cells, whereas these same fractions from acdtAmutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with theH. ducreyicell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both thecdtAmutant and thecdtBmutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.
Fas/Fas Ligand System Mediates Epithelial Injury, but Not Pulmonary Host Defenses, in Response to Inhaled Bacteria
Authors: Matute-Bello Gustavo , Frevert Charles W. , Liles W. Conrad , Nakamura Morio , Ruzinski John T. , Ballman Kimberly , Wong Venus A. , Vathanaprida Charie , Martin Thomas R.
Published: July 2002

The Fas/Fas ligand (FasL) system has been implicated in alveolar epithelial cell apoptosis during pulmonary fibrosis and acute respiratory distress syndrome. However, Fas ligation can also lead to cell activation and cytokine production. The goal of this study was to determine the role of the Fas/FasL system in host defenses againstEscherichia coli,Staphylococcus aureus,andStreptococcus pneumoniae. We administered bacteria by aerosolization into the lungs of Fas-deficient (lpr) mice and wild-type (C57BL/6) mice and measured bacterial clearance at 6 and 12 h. One hour prior to euthanasia, the mice received an intraperitoneal injection of human serum albumin (HSA) for alveolar permeability determinations. At all times after bacterial challenges, the lungs of thelprmice contained similar or lower numbers of bacteria than those of the C57BL/6 mice. Alveolar permeability changes, as determined by bronchoalveolar lavage fluid HSA concentrations, were less severe in thelprmice 6 h after the challenges. In response toE. coli, thelprmice had significantly more polymorphonuclear leukocytes (PMN) and macrophage inflammatory protein 2 in the lungs, whereas histopathologic changes were less severe. In contrast, in response to the gram-positive cocci, thelpranimals had similar or lower numbers of PMN. We conclude that the Fas/FasL system contributes to the development of permeability changes and tissue injury during-gram negative bacterial pneumonia. The Fas/FasL system did not have a major role in the clearance of aerosolized bacteria from the lungs at the bacterial doses tested.
In Vivo and In Vitro Studies of Cytosolic Phospholipase A2Expression inHelicobacter pyloriInfection
Authors: Nardone Gerardo , Holicky Eileen L. , Uhl James R. , Sabatino Lina , Staibano Stefania , Rocco Alba , Colantuoni Vittorio , Manzo Barbara A. , Romano Marco , Budillon Gabriele , Cockerill Franklin R. , Miller Laurence J.
Published: July 2002

Modifications of mucosal phospholipids have been detected in samples from patients withHelicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2expression and PLA2activity in the gastric mucosae of patients with and withoutH. pyloriinfection. In gastric biopsies from 10H. pylori-positive patients, cPLA2levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2activity were higher than in 10H. pylori-negative gastritis patients. To clarify whetherH. pylorihad a direct effect on the cellular expression of cPLA2, we studied cPLA2expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with differentH. pyloristrains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2protein expression was observed in AGS or MKN 28 cells treated with wild-typeH. pylori. In conclusion, our study shows increased cPLA2expression and PLA2activity in the gastric mucosae of patients withH. pyloriinfection and no change in epithelial cell lines exposed toH. pylori.
Isolation and Characterization of a
Vibrio vulnificus
Mutant Deficient in Both Extracellular Metalloprotease and Cytolysin
Authors: Fan Jong-Jin , Shao Chung-Ping , Ho Ya-Chi , Yu Chun-Keung , Hor Lien-I
Published: July 2002

We isolated a
Vibrio vulnificus
mutant that was deficient in both metalloprotease and cytolysin by allelic exchange. The virulence of this mutant in mice and its cytotoxicity for HEp-2 cells were comparable to those of the wild-type strain, indicating that neither factor was essential for these properties. The cytolysin, but not the protease, seemed to be important for causing damage in the alimentary tract of the mice.
Neisserial Immunoglobulin A1 Protease Induces Specific T-Cell Responses in Humans
Authors: Tsirpouchtsidis Anastasios , Hurwitz Robert , Brinkmann Volker , Meyer Thomas F. , Haas Gaby
Published: July 2002

We have previously shown that immunoglobulin A1 (IgA1) protease, an exoenzyme of pathogenic neisseriae, can trigger the release of proinflammatory cytokines from human monocytic subpopulations. Here, we demonstrate a dose-dependent T-cell response to recombinant gonococcal IgA1 protease (strain MS11) in healthy human blood donors. This response was delayed in comparison to the immune response against tetanus toxoid. Stimulation with IgA1 protease led to the activation of CD4+and CD8+T cells, as well as CD19+B cells and CD56+NK cells, indicated by de novo expression of CD69. Only CD4+T cells proliferated and stained positive for intracellular gamma interferon (IFN-γ). Both proliferation and IFN-γ production were dependent on antigen presentation via major histocompatibility complex class II. Peripheral blood mononuclear cells stimulated with IgA1 protease produce IFN-γ and tumor necrosis factor alpha but no, or very low amounts of, interleukin-10 (IL-10) or IL-4, indicating a Th1-based proinflammatory immune response. These findings support the significance of IgA1 protease as a virulence determinant of bacterial meningitis and its function as a dominant proinflammatory T-cell antigen.
Variations in the
csgD
Promoter of
Escherichia coli
O157:H7 Associated with Increased Virulence in Mice and Increased Invasion of HEp-2 Cells
Authors: Uhlich Gaylen A. , Keen James E. , Elder Robert O.
Published: July 2002

Promoter alterations in the
csgD
gene of
Escherichia coli
O157:H7 strains ATCC 43894 and ATCC 43895 are associated with variations in curli expression and the ability to bind Congo red dye. Red variants of each strain were more invasive for cultured HEp-2 cells than were white variants. An ATCC 43895 red variant was more virulent than a white variant in a mouse model. However, there were no differences in Shiga toxin production between red and white variants.
Porphyromonas gingivalis
Fimbriae Bind to Cytokeratin of Epithelial Cells
Authors: Sojar Hakimuddin T. , Sharma Ashu , Genco Robert J.
Published: July 2002

The adherence of
Porphyromonas gingivalis
to host cells is likely a prerequisite step in the pathogenesis of
P. gingivalis
-induced periodontal disease.
P. gingivalis
binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of
P. gingivalis
fimbriae. Using an overlay assay with purified
P. gingivalis
fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which
P. gingivalis
fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that
P. gingivalis
fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified
P. gingivalis
fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial cytokeratins may act as receptor(s) for
P. gingivalis
fimbriae. We hypothesize that adherence of
P. gingivalis
fimbriae to cytokeratin may be important for colonization of oral mucous membranes and possibly also for activation of epithelial cells.
Autocrine and Exocrine Regulation of Interleukin-10 Production in THP-1 Cells Stimulated withBorrelia burgdorferiLipoproteins
Authors: Giambartolomei Guillermo H. , Dennis Vida A. , Lasater Barbara L. , Murthy P. K. , Philipp Mario T.
Published: July 2002

We have recently demonstrated that interleukin-10 (IL-10), produced by THP-1 monocytes in response toBorrelia burgdorferilipoproteins, dampens the production of concomitantly elicited inflammatory cytokines. Thus, IL-10 could potentially down-regulate inflammatory and microbicidal effector mechanisms of the innate immune response to aB. burgdorferiinfection, facilitating the establishment of the spirochete. To understand the mechanism(s) implicated in the regulation of the synthesis and release of IL-10 during early infection, we investigated the autocrine effects of IL-6, IL-12, tumor necrosis factor alpha (TNF-α), and IL-10 itself, as well as the exocrine effect of IFN-γ on the production of macrophage-derived IL-10 with lipoprotein as a stimulant. In addition, in view of the differences in the receptor and signal transduction pathways of lipopolysaccharide (LPS) and bacterial lipoproteins, we also investigated the effects described above with LPS as a stimulant. The THP-1 human monocytic cell line and purified recombinant lipidated OspA (L-OspA) were used as the model target cell and stimulant, respectively. TNF-α increased the production of IL-10, as elicited by lipoproteins. The production of IL-10 by THP-1 cells stimulated with L-OspA was autoregulated by a negative feedback mechanism involving the IL-10 receptor (IL-10R). Exogenous IFN-γ significantly inhibited the production of IL-10. Both autocrine (IL-10) and exocrine (IFN-γ) inhibition of IL-10 production resulted in an increase in the production of the proinflammatory cytokines IL-6 and IL-12. The same results were obtained when the stimulant was LPS. The results further illustrate that IL-10 may play a pivotal role in Lyme disease pathogenesis. Moreover, the regulation of its production with lipoprotein as a stimulant is indistinguishable from that observed when LPS acts as a stimulant.
Inhibition of Apoptosis by Gamma Interferon in Cells and Mice Infected withChlamydia muridarum(the Mouse Pneumonitis Strain ofChlamydia trachomatis)
Authors: Perfettini Jean-Luc , Darville Toni , Dautry-Varsat Alice , Rank Roger G. , Ojcius David M.
Published: July 2002

The effect of gamma interferon (IFN-γ) on apoptosis due to infection byChlamydia muridarum(the mouse pneumonitis strain ofChlamydia trachomatis) was studied in epithelial cells in culture and in the genital tracts of mice. IFN-γ concentrations that induce the formation of aberrant, persistent chlamydiae inhibit apoptosis due toC. muridaruminfection. In cells treated with an IFN-γ concentration that leads to the development of a heterogenous population of normal and aberrantChlamydiavacuoles, apoptosis was inhibited preferentially in cells that contained the aberrant vacuoles. The inhibitory effect of IFN-γ appears to be due in part to expression of host cell indoleamine 2,3-dioxygenase activity, since inhibition of apoptosis could be partially reversed through coincubation with exogenous tryptophan. Apoptotic cells were observed in the genital tracts of wild-type mice infected withC. muridarum, and a significantly larger number of apoptotic cells was detected in infected IFN-γ-deficient mice. These results suggest that IFN-γ may contribute to pathogenesis of persistentChlamydiainfections in vivo by preventing apoptosis of infected cells.
Relationship of
Yersinia pseudotuberculosis
O Antigens IA, IIA, and IVB: the IIA Gene Cluster Was Derived from That of IVB
Authors: Pacinelli Elvia , Wang Lei , Reeves Peter R.
Published: July 2002

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria and is highly polymorphic. In this study, we obtained sequences of the O-antigen gene clusters for the
Yersinia pseudotuberculosis
antigens IA, IIA, and IVB. We propose that the IIA gene cluster was derived from the IVB cluster, one of the very few cases in which a parent gene cluster is identified, and that the IA gene cluster could be a hybrid of the IVB and IB gene clusters. All three O antigens contain 6-deoxy
-d-
mannoheptose, and we identified six genes for the biosynthetic pathway for the precursor of this sugar, GDP-6-deoxy
-d-
mannoheptose.
Immunogenicity and Protective Efficacy of Neonatal Vaccination against Bordetella pertussis in a Murine Model: Evidence for Early Control of Pertussis
Authors: Roduit Caroline , Bozzotti Paola , Mielcarek Nathalie , Lambert Paul-Henri , del Giudice Giuseppe , Locht Camille , Siegrist Claire-Anne
Published: July 2002

A significant resurgence of early cases of pertussis is being observed in infants too young to have yet completed their three-dose vaccination schedule. In this study, murine models of immunization andBordetella pertussischallenge were adapted to early life. This allowed comparative evaluation of immunogenicity and protective efficacy of immunization initiated in the neonatal period (7-day-old mice) or in infancy (3-week-old mice) with diphtheria-tetanus-whole-cell pertussis (DTPw) and diphtheria-tetanus-acellular pertussis (DTPa) vaccines. Neonatal DTPa vaccination induced strong pertussis-specific antibody and memory responses. Patterns of bacterial clearance were similar in both age groups. In contrast, as observed in human neonates, neonatal DTPw priming did not induce significant antibody responses to pertussis toxin (PT) and filamentous hemagglutinin (FHA) and even interfered with subsequent antibody responses. However, this did not reflect induction of permanent neonatal tolerance, as antigen-specific antibodies could be elicited by subsequent exposure to DTPa. Furthermore, despite these blunted PT and FHA antibody responses, the protective efficacy of DTPw in neonatal mice proved similar to that in infant mice, resulting in complete bacterial clearance at day 8 afterB. pertussischallenge. Thus, neonatal priming with antipertussis vaccines should be considered to reduce the window of vulnerability to pertussis at the time of its greatest severity.
TheSalmonella entericaSerotype Typhimurium Effector Proteins SipA, SopA, SopB, SopD, and SopE2 Act in Concert To Induce Diarrhea in Calves
Authors: Zhang Shuping , Santos Renato L. , Tsolis Renee M. , Stender Silke , Hardt Wolf-Dietrich , Bäumler Andreas J. , Adams L. Garry
Published: July 2002

Salmonella entericaserotype Typhimurium requires a functional type III secretion system encoded bySalmonellapathogenicity island 1 (SPI1) to cause diarrhea. We investigated the role of genes encoding secreted target proteins of the SPI1-associated type III secretion system for enteropathogenicity in calves.Salmonellaserotype Typhimurium strains having mutations insptP,avrA,sspH1, orslrPinduced fluid secretion in the bovine ligated ileal loop model at levels similar to that of the wild type. In contrast, mutations insipA,sopA,sopB,sopD, orsopE2significantly reduced fluid accumulation in bovine ligated ileal loops at 8 h postinfection. A strain carrying mutations insipA,sopA,sopB,sopD, andsopE2(sipA sopABDE2mutant) caused the same level of fluid accumulation in bovine ligated ileal loops as a strain carrying a mutation insipB, a SPI1 gene required for the translocation of effector proteins into host cells. A positive correlation was observed between the severity of histopathological lesions detected in the ileal mucosa and the levels of fluid accumulation induced by the different mutants. After oral infection of calves, theSalmonellaserotype TyphimuriumsipAsopABDE2mutant caused only mild diarrhea and was more strongly attenuated than strains having only single mutations. These data demonstrate that SipA, SopA, SopB, SopD, and SopE2 are major virulence factors responsible for diarrhea duringSalmonellaserotype Typhimurium infection of calves.
Haloalkane-UtilizingRhodococcusStrains Isolated from Geographically Distinct Locations Possess a Highly Conserved Gene Cluster Encoding Haloalkane Catabolism
Authors: Poelarends Gerrit J. , Zandstra Marjan , Bosma Tjibbe , Kulakov Leonid A. , Larkin Michael J. , Marchesi Julian R. , Weightman Andrew J. , Janssen Dick B.
Published: July 2002

The sequences of the 16S rRNA and haloalkane dehalogenase (dhaA) genes of five gram-positive haloalkane-utilizing bacteria isolated from contaminated sites in Europe, Japan, and the United States and of the archetypal haloalkane-degrading bacteriumRhodococcussp. strain NCIMB13064 were compared. The 16S rRNA gene sequences showed less than 1% sequence divergence, and all haloalkane degraders clearly belonged to the genusRhodococcus. All strains shared a completely conserveddhaAgene, suggesting that thedhaAgenes were recently derived from a common ancestor. The genetic organization of thedhaAgene region in each of the haloalkane degraders was examined by hybridization analysis and DNA sequencing. Three different groups could be defined on the basis of the extent of the conserveddhaAsegment. The minimal structure present in all strains consisted of a conserved region of 12.5 kb, which included the haloalkane-degradative gene cluster that was previously found in strain NCIMB13064. Plasmids of different sizes were found in all strains. Southern hybridization analysis with adhaAgene probe suggested that all haloalkane degraders carry thedhaAgene region both on the chromosome and on a plasmid (70 to 100 kb). This suggests that an ancestral plasmid was transferred between theseRhodococcusstrains and subsequently has undergone insertions or deletions. In addition, transposition events and/or plasmid integration may be responsible for positioning thedhaAgene region on the chromosome. The data suggest that the haloalkane dehalogenase gene regions of these gram-positive haloalkane-utilizing bacteria are composed of a single catabolic gene cluster that was recently distributed worldwide.
In Vivo Analysis of the Mechanisms for Oxidation of Cytosolic NADH by
Saccharomyces cerevisiae
Mitochondria
Authors: Overkamp Karin M. , Bakker Barbara M. , Kötter Peter , van Tuijl Arjen , de Vries Simon , van Dijken Johannes P. , Pronk Jack T.
Published: July 2002

During respiratory glucose dissimilation, eukaryotes produce cytosolic NADH via glycolysis. This NADH has to be reoxidized outside the mitochondria, because the mitochondrial inner membrane is impermeable to NADH. In
Saccharomyces cerevisiae
, this may involve external NADH dehydrogenases (Nde1p or Nde2p) and/or a glycerol-3-phosphate shuttle consisting of soluble (Gpd1p or Gpd2p) and membrane-bound (Gut2p) glycerol-3-phosphate dehydrogenases. This study addresses the physiological relevance of these mechanisms and the possible involvement of alternative routes for mitochondrial oxidation of cytosolic NADH. Aerobic, glucose-limited chemostat cultures of a
gut2Δ
mutant exhibited fully respiratory growth at low specific growth rates. Alcoholic fermentation set in at the same specific growth rate as in wild-type cultures (0.3 h
−1
). Apparently, the glycerol-3-phosphate shuttle is not essential for respiratory glucose dissimilation. An
nde1Δ nde2Δ
mutant already produced glycerol at specific growth rates of 0.10 h
−1
and above, indicating a requirement for external NADH dehydrogenase to sustain fully respiratory growth. An
nde1Δ nde2Δ gut2Δ
mutant produced even larger amounts of glycerol at specific growth rates ranging from 0.05 to 0.15 h
−1
. Apparently, even at a low glycolytic flux, alternative mechanisms could not fully replace the external NADH dehydrogenases and glycerol-3-phosphate shuttle. However, at low dilution rates, the
nde1Δ nde2Δ gut2Δ
mutant did not produce ethanol. Since glycerol production could not account for all glycolytic NADH, another NADH-oxidizing system has to be present. Two alternative mechanisms for reoxidizing cytosolic NADH are discussed: (i) cytosolic production of ethanol followed by its intramitochondrial oxidation and (ii) a redox shuttle linking cytosolic NADH oxidation to the internal NADH dehydrogenase.
Secretion of the
Caulobacter crescentus
S-Layer Protein: Further Localization of the C-Terminal Secretion Signal and Its Use for Secretion of Recombinant Proteins
Authors: Bingle Wade H. , Nomellini John F. , Smit John
Published: July 2002

The secretion signal of the
Caulobacter crescentus
S-layer protein (RsaA) was localized to the C-terminal 82 amino acids of the molecule. Protein yield studies showed that 336 or 242 C-terminal residues of RsaA mediated secretion of >50 mg of a cellulase passenger protein per liter to the culture fluids.
The Dual-Specificity Protein Phosphatase Yvh1p Regulates Sporulation, Growth, and Glycogen Accumulation Independently of Catalytic Activity in
Saccharomyces cerevisiae
via the Cyclic AMP-Dependent Protein Kinase Cascade
Authors: Beeser Alexander E. , Cooper Terrance G.
Published: July 2002

Yvh1p, a dual-specific protein phosphatase induced specifically by nitrogen starvation, regulates cell growth as well as initiation and completion of sporulation. We demonstrate that
yvh1
disruption mutants are also unable to accumulate glycogen in stationary phase. A catalytically inactive variant of
yvh1
(C117S) and a DNA fragment encoding only the Yvh1p C-terminal 159 amino acids (which completely lacks the phosphatase domain) complement all three phenotypes as well as the wild-type allele; no complementation occurs with a fragment encoding only the C-terminal 74 amino acids. These observations argue that phosphatase activity is not required for the Yvh1p functions we measured. Mutations which decrease endogenous cyclic AMP (cAMP) levels partially suppress the sporulation and glycogen accumulation defects. In addition, reporter gene expression supported by a
DRR2
promoter fragment, containing two stress response elements known to respond to cAMP-protein kinase A, decreases in a
yvh1
disruption mutant. Therefore, our results identify three cellular processes that both require Yvh1p and respond to alterations in cAMP, and they lead us to suggest that Yvh1p may be a participant in and/or a contributor to regulation of the cAMP-dependent protein kinase cascade. The fact that decreasing the levels of cAMP alleviates the need for Yvh1p function supports this suggestion.
Reduction of Cob(III)alamin to Cob(II)alamin in
Salmonella enterica
Serovar Typhimurium LT2
Authors: Fonseca Maris V. , Escalante-Semerena Jorge C.
Published: July 2002

Reduction of the cobalt ion of cobalamin from the Co(III) to the Co(I) oxidation state is essential for the synthesis of adenosylcobalamin, the coenzymic form of this cofactor. A cob(II)alamin reductase activity in
Salmonella enterica
serovar Typhimurium LT2 was isolated to homogeneity. N-terminal analysis of the homogeneous protein identified NAD(P)H:flavin oxidoreductase (Fre) (EC
1.6.8.1
) as the enzyme responsible for this activity. The
fre
gene was cloned, and the overexpressed protein, with a histidine tag at its N terminus, was purified to homogeneity by nickel affinity chromatography. His-tagged Fre reduced flavins (flavin mononucleotide [FMN] and flavin adenine dinucleotide [FAD]) and cob(III)alamin to cob(II)alamin very efficiently. Photochemically reduced FMN substituted for Fre in the reduction of cob(III)alamin to cob(II)alamin, indicating that the observed cobalamin reduction activity was not Fre dependent but FMNH
2
dependent. Enzyme-independent reduction of cob(III)alamin to cob(II)alamin by FMNH
2
occurred at a rate too fast to be measured. The thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin was detectable by alkylation of the cob(I)alamin nucleophile with iodoacetate. Detection of the product, caboxymethylcob(III)alamin, depended on the presence of FMNH
2
in the reaction mixture. FMNH
2
failed to substitute for potassium borohydride in in vitro assays for corrinoid adenosylation catalyzed by the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme, even under conditions where Fre and NADH were present in the reaction mixture to ensure that FMN was always reduced. These results were interpreted to mean that Fre was not responsible for the generation of cob(I)alamin in vivo. Consistent with this idea, a
fre
mutant displayed wild-type cobalamin biosynthetic phenotypes. It is proposed that
S. enterica
serovar Typhimurium LT2 may not have a cob(III)alamin reductase enzyme and that, in vivo, nonadenosylated cobalamin and other corrinoids are maintained as co(II)rrinoids by reduced flavin nucleotides generated by Fre and other flavin oxidoreductases.
In Vivo and In Vitro Effects of (p)ppGpp on the ς
54
Promoter
Pu
of the TOL Plasmid of
Pseudomonas putida
Authors: Carmona Manuel , Rodríguez Maria J. , Martínez-Costa Óscar , de Lorenzo Víctor
Published: July 2002

The connection between the physiological control of the ς
54
-dependent
Pu
promoter of the TOL plasmid pWW0 of
Pseudomonas putida
and the stringent response mediated by the alarmone (p)ppGpp has been examined in vivo an in vitro. To this end, the key regulatory elements of the system were faithfully reproduced in an
Escherichia coli
strain and assayed as
lacZ
fusions in various genetic backgrounds lacking (p)ppGpp or overexpressing
relA
. Neither the responsiveness of
Pu
to 3-methyl benzylalcohol mediated by its cognate activator XylR nor the down-regulation of the promoter by rapid growth were affected in
relA/spoT
strains to an extent which could account for the known physiological control that governs this promoter. Overexpression of the
relA
gene [predicted to increase intracellullar (p)ppGpp levels] did, however, cause a significant gain in
Pu
activity. Since such a gain might be the result of indirect effects, we resorted to an in vitro transcription system to assay directly the effect of ppGpp on the transcriptional machinery. Although we did observe a significant increase in
Pu
performance through a range of ς
54
-RNAP concentrations, such an increase never exceeded twofold. The difference between these results and the behavior of the related
Po
promoter of the phenol degradation plasmid pVI150 could be traced to the different promoter sequences, which may dictate the type of metabolic signals recruited for the physiological control of ς
54
-systems.
Characterization of the Operon Encoding the Alternative ς
B
Factor from
Bacillus anthracis
and Its Role in Virulence
Authors: Fouet Agnès , Namy Olivier , Lambert Guillaume
Published: July 2002

The operon encoding the general stress transcription factor ς
B
and two proteins of its regulatory network, RsbV and RsbW, was cloned from the gram-positive bacterium
Bacillus anthracis
by PCR amplification of chromosomal DNA with degenerate primers, by inverse PCR, and by direct cloning. The gene cluster was very similar to the
Bacillus subtilis sigB
operon both in the primary sequences of the gene products and in the order of its three genes. However, the deduced products of sequences upstream and downstream from this operon showed no similarity to other proteins encoded by the
B. subtilis sigB
operon. Therefore, the
B. anthracis sigB
operon contains three genes rather than eight as in
B. subtilis
. The
B. anthracis
operon is preceded by a ς
B
-like promoter sequence, the expression of which depends on an intact ς
B
transcription factor in
B. subtilis
. It is followed by another open reading frame that is also preceded by a promoter sequence similarly dependent on
B. subtilis
ς
B
. We found that in
B. anthracis
, both these promoters were induced during the stationary phase and induction required an intact
sigB
gene. The
sigB
operon was induced by heat shock. Mutants from which
sigB
was deleted were constructed in a toxinogenic and a plasmidless strain. These mutants differed from the parental strains in terms of morphology. The toxinogenic
sigB
mutant strain was also less virulent than the parental strain in the mouse model.
B. anthracis
ς
B
may therefore be a minor virulence factor.
Essentiality, Expression, and Characterization of the Class II 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase of
Staphylococcus aureus
Authors: Wilding E. Imogen , Kim Dong-Yul , Bryant Alexander P. , Gwynn Michael N. , Lunsford R. Dwayne , McDevitt Damien , Myers Joseph E. , Rosenberg Martin , Sylvester Daniel , Stauffacher Cynthia V. , Rodwell Victor W.
Published: July 2002

Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen
Staphylococcus aureus
. In this study we showed through genetic disruption experiments that
mvaA
, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of
S. aureus
. Supplementation of media with mevalonate permitted isolation of an auxotrophic
mvaA
null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The
mvaA
gene was cloned from
S. aureus
DNA and expressed with an N-terminal His tag in
Escherichia coli
. The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the
S. aureus
enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of
Pseudomonas mevalonii
HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the
S. aureus
enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a

K
i

of 320 μM, over 10
4
higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.
Mutational Analysis of Genes Encoding the Early Flagellar Components of
Helicobacter pylori
: Evidence for Transcriptional Regulation of Flagellin A Biosynthesis
Authors: Allan Elaine , Dorrell Nick , Foynes Susan , Anyim Martin , Wren Brendan W.
Published: July 2002

We investigated the roles of
fliF
,
fliS
,
flhB
,
fliQ
,
fliG
, and
fliI
of
Helicobacter pylori
, predicted by homology to encode structural components of the flagellar basal body and export apparatus. Mutation of these genes resulted in nonmotile, nonflagellate strains. Western blot analysis showed that all the mutants had considerably reduced levels of both flagellin subunits and of FlgE, the flagellar hook protein. RNA slot blot hybridization showed reduced levels of
flaA
mRNA, indicating that transcription of the major flagellin gene is inhibited in the absence of the early components of the flagellar-assembly pathway. This is the first demonstration of a checkpoint in
H. pylori
flagellar assembly.
Pathogenic Neisseriae Can Use Hemoglobin, Transferrin, and Lactoferrin Independently of the
tonB
Locus
Authors: Desai Pragnya Jasvantrai , Garges Eric , Genco Caroline Attardo
Published: July 2002

Redundant TonB systems which function in iron transport from TonB-dependent ligands have recently been identified in several gram-negative bacteria. We demonstrate here that in addition to the previously described
tonB
locus, an alternative system exists for the utilization of iron from hemoglobin, transferrin, or lactoferrin in
Neisseria meningitidis
and
Neisseria gonorrhoeae
. Following incubation on media containing hemoglobin,
N. meningitidis
IR3436 (
tonB exbB exbD
deletion mutant) and
N. gonorrhoeae
PD3401 (
tonB
insertional mutant) give rise to colonies which can grow with hemoglobin. Transfer of Hb
+
variants (PD3437 or PD3402) to media containing hemoglobin, transferrin, and/or lactoferrin as sole iron sources resulted in growth comparable to that observed for the wild-type strains. Transformation of
N. meningitidis
IR3436 or
N. gonorrhoeae
PD3401 with chromosomal DNA from the Hb
+
variants yielded transformants capable of growth with hemoglobin. When we inactivated the TonB-dependent outer membrane hemoglobin receptors (HmbR or HpuB) in the
Neisseria
Hb
+
variants, these strains could not grow with hemoglobin; however, growth was observed with transferrin and/or lactoferrin. These results demonstrate that accumulation of iron from hemoglobin, transferrin, and lactoferrin in the pathogenic neisseriae can occur via a system that is independent of the previously described
tonB
locus.