bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Multiepitope Polyproteins in Serodiagnosis of Active Tuberculosis
Authors: Houghton Raymond L. , Lodes Michael J. , Dillon Davin C. , Reynolds Lisa D. , Day Craig H. , McNeill Patricia D. , Hendrickson Ronald C. , Skeiky Yasir A. W. , Sampaio Diana P. , Badaro Roberto , Lyashchenko Konstantin P. , Reed Steven G.
Published: July 2002

Screening of genomic expression libraries fromMycobacterium tuberculosiswith sera from tuberculosis (TB) patients or rabbit antiserum toM. tuberculosisled to the identification of novel antigens capable of detecting specific antibodies toM. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of ∼98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of ∼93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test forM. tuberculosisantibody detection.
Spectrum of Kaposi's Sarcoma-Associated Herpesvirus, or Human Herpesvirus 8, Diseases
Authors: Ablashi Dharam V. , Chatlynne Louise G. , Whitman, James E. , Cesarman Ethel
Published: July 2002

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), discovered in 1994, is a human rhadinovirus (gamma-2 herpesvirus). Unlike other human herpesviruses (herpes simplex virus, Epstein-Barr virus, varicella-zoster virus, cytomegalovirus, HHV-6, and HHV-7), it is not widespread in the general population and has many unique proteins. HHV-8 is strongly associated with all subtypes of Kaposi's sarcoma (KS), multicentric Castleman's disease, and a rare form of B-cell lymphoma, primary effusion lymphoma. In addition, HHV-8 DNA sequences have been found in association with other diseases, but the role of the virus in these diseases is largely unconfirmed and remains controversial. The seroprevalence of HHV-8, based on detection of latent and lytic proteins, is 2 to 5% in healthy donors except in certain geographic areas where the virus is endemic, 80 to 95% in classic KS patients, and 40 to 50% in HIV-1 patients without KS. This virus can be transmitted both sexually and through body fluids (e.g., saliva and blood). HHV-8 is a transforming virus, as evidenced by its presence in human malignancies, by the in vitro transforming properties of several of its viral genes, and by its ability to transform some primary cells in culture. It is not, however, sufficient for transformation, and other cofactors such as immunosuppressive cytokines are involved in the development of HHV-8-associated malignancies. In this article, we review the biology, molecular virology, epidemiology, transmission, detection methods, pathogenesis, and antiviral therapy of this newly discovered human herpesvirus.
Legionella
and Legionnaires' Disease: 25 Years of Investigation
Authors: Fields Barry S. , Benson Robert F. , Besser Richard E.
Published: July 2002

There is still a low level of clinical awareness regarding Legionnaires' disease 25 years after it was first detected. The causative agents, legionellae, are freshwater bacteria with a fascinating ecology. These bacteria are intracellular pathogens of freshwater protozoa and utilize a similar mechanism to infect human phagocytic cells. There have been major advances in delineating the pathogenesis of legionellae through the identification of genes which allow the organism to bypass the endocytic pathways of both protozoan and human cells. Other bacteria that may share this novel infectious process are
Coxiella burnetti
and
Brucella
spp. More than 40 species and numerous serogroups of legionellae have been identified. Most diagnostic tests are directed at the species that causes most of the reported human cases of legionellosis,
L. pneumophila
serogroup 1. For this reason, information on the incidence of human respiratory disease attributable to other species and serogroups of legionellae is lacking. Improvements in diagnostic tests such as the urine antigen assay have inadvertently caused a decrease in the use of culture to detect infection, resulting in incomplete surveillance for legionellosis. Large, focal outbreaks of Legionnaires' disease continue to occur worldwide, and there is a critical need for surveillance for travel-related legionellosis in the United States. There is optimism that newly developed guidelines and water treatment practices can greatly reduce the incidence of this preventable illness.
Up-Regulation of Fas (CD95) and Induction of Apoptosis in Intestinal Epithelial Cells by Nematode
-
Derived Molecules
Authors: Kuroda Akio , Uchikawa Ryuichi , Matsuda Shinji , Yamada Minoru , Tegoshi Tatsuya , Arizono Naoki
Published: July 2002

Infection by the intestinal nematode
Nippostrongylus brasiliensis
induces acceleration of apoptosis in the small intestinal villus epithelial cells in vivo. In the present study, we examined whether worm extract or excretory-secretory product induces apoptosis in the rat intestinal epithelial cell line IEC-6 in vitro. In the presence of worm extract or excretory-secretory product (≥6 μg/ml), IEC-6 cell growth was significantly suppressed, and there was a concomitant increase in the number of detached cells in culture dishes. Detached cells showed nuclear fragmentation, activation of caspase-3, and specific cleavage of poly(ADP-ribose) polymerase, suggesting that apoptosis was induced in these cells. Semiquantitative reverse transcription-PCR showed that expression of Fas (CD95) mRNA was up-regulated as early as 6 h after addition of excretory-secretory product, while Fas ligand expression and p53 expression were not up-regulated. Fluorescence-activated cell sorter analyses revealed a significant increase in Fas expression and a slight increase in FasL expression in IEC-6 cells cultured in the presence of excretory-secretory product, while control IEC-6 cells expressed neither Fas or FasL. These results indicated that
N. brasiliensis
worms produce and secrete biologically active molecules that trigger apoptosis in intestinal epithelial cells together with up-regulation of Fas expression, although the mechanism of induction of apoptosis remains to be elucidated.
LuxS-Mediated Quorum Sensing inBorrelia burgdorferi, the Lyme Disease Spirochete
Authors: Stevenson Brian , Babb Kelly
Published: July 2002

The establishment ofBorrelia burgdorferiinfection involves numerous interactions between the bacteria and a variety of vertebrate host and arthropod vector tissues. This complex process requires regulated synthesis of many bacterial proteins. We now demonstrate that these spirochetes utilize a LuxS/autoinducer-2 (AI-2)-based quorum-sensing mechanism to regulate protein expression, the first system of cell-cell communication to be described in a spirochete. TheluxSgene ofB. burgdorferiwas identified and demonstrated to encode a functional enzyme by complementation of anEscherichia coli luxSmutant. CulturedB. burgdorferiresponded to AI-2 by altering the expression levels of a large number of proteins, including the complement regulator factor H-binding Erp proteins. Through this mechanism, a population of Lyme disease spirochetes may synchronize production of specific proteins needed for infection processes.
Plasma Membrane Cholesterol Modulates Cellular Vacuolation Induced by the
Helicobacter pylori
Vacuolating Cytotoxin
Authors: Patel Hetal K. , Willhite David C. , Patel Rakhi M. , Ye Dan , Williams Christopher L. , Torres Eric M. , Marty Kent B. , MacDonald Robert A. , Blanke Steven R.
Published: July 2002

The
Helicobacter pylori
vacuolating cytotoxin (VacA) induces the degenerative vacuolation of mammalian cells both in vitro and in vivo. Here, we demonstrate that plasma membrane cholesterol is essential for vacuolation of mammalian cells by VacA. Vacuole biogenesis in multiple cell lines was completely blocked when cholesterol was extracted selectively from the plasma membrane by using β-cyclodextrins. Moreover, increasing plasma membrane cholesterol levels strongly potentiated VacA-induced vacuolation. In contrast, inhibiting de novo biosynthesis of cholesterol with lovastatin or compactin had no detectable effect on vacuolation. While depletion of plasma membrane cholesterol has been shown to interfere with both clathrin-mediated endocytosis and caveola-dependent endocytosis, neither of these two internalization pathways was found to be essential for vacuolation of cells by VacA. Depleting plasma membrane cholesterol attenuated the entry of VacA into HeLa cells. In addition, β-cyclodextrin reagents blocked vacuolation of cells that were either preloaded with VacA or had VacA directly expressed within the cytosol. Collectively, our results suggest that plasma membrane cholesterol is important for both the intoxication mechanism of VacA and subsequent vacuole biogenesis.
Generation and Characterization of Human Monoclonal scFv Antibodies against
Helicobacter pylori
Antigens
Authors: Reiche Nicole , Jung Andreas , Brabletz Thomas , Vater Tanja , Kirchner Thomas , Faller Gerhard
Published: July 2002

Infection with
Helicobacter pylori
is chronic despite a vigorous cellular and humoral immune response and causes severe pathology in some patients. In this study, phage display was used as a new approach in order to investigate the role of the host's humoral immune response in the pathogenesis of
H. pylori
gastritis. Human monoclonal single-chain Fv (scFv) antibody fragments against
H. pylori
cell lysate and the
H. pylori
urease were isolated from an immune phage display library, constructed from peripheral blood lymphocytes of an
H. pylori
-infected patient. After affinity selection, 23% of the clones tested showed binding activity against a lysate of the
H. pylori
Sydney strain in enzyme-linked immunosorbent assay (ELISA) and 9% bound the
H. pylori
urease. Further characterization by PCR-fingerprint analysis and sequencing revealed that two closely related
H. pylori
binders and one antiurease scFv could be isolated. The selected scFvs were highly specific as analyzed by ELISA and immunoblots using various bacterial lysates and recombinant proteins. Analysis of the humoral immune response following
H. pylori
infection using human monoclonal antibodies might contribute to a better understanding of the pathogenesis of the disease. Moreover, using immune phage display libraries, it might be possible for relevant epitopes of
H. pylori
antigens to be determined, which might be of use for vaccine development.
Role of Yops and Adhesins in Resistance ofYersinia enterocoliticato Phagocytosis
Authors: Grosdent Nadine , Maridonneau-Parini Isabelle , Sory Marie-Paule , Cornelis Guy R.
Published: July 2002

Yersinia enterocoliticais a pathogen endowed with two adhesins, Inv and YadA, and with the Ysc type III secretion system, which allows extracellular adherent bacteria to inject Yop effectors into the cytosol of animal target cells. We tested the influence of all of these virulence determinants on opsonic and nonopsonic phagocytosis by PU5-1.8 and J774 mouse macrophages, as well as by human polymorphonuclear leukocytes (PMNs). The adhesins contributed to phagocytosis in the absence of opsonins but not in the presence of opsonins. In agreement with previous results, YadA counteracted opsonization. In every instance, the Ysc-Yop system conferred a significant level of resistance to phagocytosis. Nonopsonized single-mutant bacteria lacking either YopE, -H, -T, or -O were phagocytosed significantly more by J774 cells and by PMNs. Opsonized bacteria were phagocytosed more than nonopsonized bacteria, and mutant bacteria lacking either YopH, -T, or -O were phagocytosed significantly more by J774 cells and by PMNs than were wild-type (WT) bacteria. Opsonized mutants lacking only YopE were phagocytosed significantly more than were WT bacteria by PMNs but not by J774 cells. Thus, YopH, -T, and -O were involved in all of the phagocytic processes studied here but YopE did not play a clear role in guarding against opsonic phagocytosis by J774. Mutants lacking YopP and YopM were, in every instance, as resistant as WT bacteria. Overexpression of YopE, -H, -T, or -O alone did not confer resistance to phagocytosis, although it affected the cytoskeleton. These results show that YopH, YopT, YopO, and, in some instances, YopE act synergistically to increase the resistance ofY. enterocoliticato phagocytosis by macrophages and PMNs.
Pathogenesis of Pneumococcal Pneumonia in Cyclophosphamide-Induced Leukopenia in Mice
Authors: Wang Erjian , Simard Marie , Ouellet Nathalie , Bergeron Yves , Beauchamp Denis , Bergeron Michel G.
Published: July 2002

Streptococcus pneumoniaepneumonia frequently occurs in leukopenic hosts, and most patients subsequently develop lung injury and septicemia. However, few correlations have been made so far between microbial growth, inflammation, and histopathology of pneumonia in specific leukopenic states. In the present study, the pathogenesis of pneumococcal pneumonia was investigated in mice rendered leukopenic by the immunosuppressor antineoplastic drug cyclophosphamide. Compared to the immunocompetent state, cyclophosphamide-induced leukopenia did not hamper interleukin-1 (IL-1), IL-6, macrophage inflammatory protein-1 (MIP-1), MIP-2, and monocyte chemotactic protein-1 secretion in infected lungs. Leukopenia did not facilitate bacterial dissemination into the bloodstream despite enhanced bacterial proliferation into lung tissues. Pulmonary capillary permeability and edema as well as lung injury were enhanced in leukopenic mice despite the absence of neutrophilic and monocytic infiltration into their lungs, suggesting an important role for bacterial virulence factors and making obvious the fact that neutrophils are ultimately not required for lung injury in this model. Scanning and transmission electron microscopy revealed extensive disruption of alveolar epithelium and a defect in surfactant production, which were associated with alveolar collapse, hemorrhage, and fibrin deposits in alveoli. These results contrast with those observed in immunocompetent animals and indicate that leukopenic hosts suffering from pneumococcal pneumonia are at a higher risk of developing diffuse alveolar damage.
Variation in Extracellular Protease Production among Clinical Isolates of
Staphylococcus aureus
Due to Different Levels of Expression of the Protease Repressor
sarA
Authors: Karlsson Anna , Arvidson Staffan
Published: July 2002

Staphylococcus aureus
produces four major extracellular proteases: staphylococcal serine protease (V8 protease; SspA), cysteine protease (SspB), metalloprotease (aureolysin; Aur), and staphopain (Scp). Several in vitro studies have suggested that these enzymes are important virulence factors. Here we analyzed the protease production of 92
S. aureus
strains from infected human soft tissue. Twenty-one strains produced variable zones of proteolysis on casein agar plates, while the remaining 71 strains appeared to be protease negative. The major protease genes were present in all protease-positive (
n
= 5) and protease-negative (
n
= 12) strains analyzed. Northern blotting showed that transcription of the protease genes was suppressed due to increased sigma factor B (SigB)-dependent expression of the protease repressor SarA. Other SigB-dependent traits such as pigmentation and expression of
asp
23 were also increased in protease-negative compared to protease-positive strains. Inactivation of
sarA
in three protease-negative strains resulted in increased transcription of all protease genes and increased protease production, while overexpression of
sarA
in a strain producing protease at high levels repressed protease production. Our results suggest that the protease genes are conserved among clinical
S. aureus
strains and that the level of SigB-dependent expression of the protease repressor
sarA
determines the level of protease production in each strain.
Induction of Cell-Mediated Immunity toStaphylococcus aureusin the Mouse Mammary Gland by Local Immunization with a Live Attenuated Mutant
Authors: Gómez Marisa I. , Sordelli Daniel O. , Buzzola Fernanda R. , García Verónica E.
Published: July 2002

The efficacy of intramammary (Ima) immunization with a live attenuated (la)Staphylococcus aureusmutant to protect the mouse mammary gland from infection has previously been established. The present study was aimed at evaluating whetherImaimmunization withla-S. aureuscan induce cell-mediated immune responses to the pathogen within the mammary gland. Mice were immunized byImaroute withla-S. aureus, and regional lymph node mononuclear cells were obtained thereafter. A higher expression of the interleukin-2 receptor was found on B and T cells from immunized mice when they were compared with control mice. Immunization withla-S. aureusinduced strong proliferative responses toS. aureus. Moreover, significantly increased levels of gamma interferon (IFN-γ) were produced by CD4+T cells when lymphocytes from immunized mice, but not from control mice, were cultured in the presence of staphylococcal antigens. Moreover, a significant increase in the percentage of IFN-γ-producing CD4+and CD8+T cells was observed afterS. aureus Imachallenge in immunized mice compared to challenged control mice. Our results demonstrated thatImaimmunization withla-S. aureusinduced primed lymphocyte populations capable of responding against staphylococcal antigens during in vitro stimulation, as well as during in vivo infection byS. aureus. CD4+and CD8+T cells appear to be the main lymphocyte subpopulations involved in this response. It is suggested that IFN-γ production induced byImaimmunization may play a pivotal role in the eradication of intracellular staphylococci.
Comparison of Alteration of Cell Surface Carbohydrates of the Chinchilla Tubotympanum and Colonial Opacity Phenotype of
Streptococcus pneumoniae
during Experimental Pneumococcal Otitis Media with or without an Antecedent Influenza A Virus Infection
Authors: Tong H. H. , Grants I. , Liu X. , DeMaria T. F.
Published: July 2002

Experimental and clinical studies suggest that influenza A virus promotes
Streptococcus pneumoniae
-induced otitis media; however, the mechanism underlying this synergistic interaction has not been completely defined. In this study, glycoconjugate expression patterns were evaluated on the cell surface in the chinchilla eustachian tube (ET) lumen of a cohort challenged intranasally (i.n.) with
S. pneumoniae
type 6A, which is predominantly transparent and a cohort with an antecedent influenza A virus infection, followed by i.n. inoculation with
S. pneumoniae
. The labeling patterns obtained with six lectin probes revealed that the binding of
Bandeiraea simplicifolia
lectin II, succinylated wheat germ agglutinin, and peanut agglutinin were significantly increased in the lumenal surface of the ET in the cohort infected with both pathogens compared to the cohort inoculated with only
S. pneumoniae
, which indicated that
N
-acetylglucosamine (GlcNAc) and
d
-galactose residues were exposed. A significant decreased labeling with
Sambucus nigra
agglutinin in the combined influenza A virus and pneumococcus infection cohort suggested that there were few sialic acid residues remaining in the ET epithelium. In addition, the colonial opacity of
S. pneumoniae
during the disease course was examined. The opaque phenotype was predominant among the pneumococcus isolates from the middle-ear fluid in the cohort infected with the both pathogens. Together, these data suggest that the synergic effect of influenza A virus and
S. pneumoniae
on the changes of the carbohydrate moieties in the ET epithelium and that the selection of the opaque variant may facilitate the pneumococcal invasion of the middle ear.
Inflammatory Cytokines Enhance the Interaction of
Mannheimia haemolytica
Leukotoxin with Bovine Peripheral Blood Neutrophils In Vitro
Authors: Leite F. , O'Brien S. , Sylte M. J. , Page T. , Atapattu D. , Czuprynski C. J.
Published: July 2002

Mannheimia
(
Pasteurella
)
haemolytica
A1 produces several virulence factors that play an important role in the pathogenesis of bovine pneumonic pasteurellosis. Foremost among these is a leukotoxin (LKT) that specifically kills ruminant leukocytes. Recent evidence suggests that
M. haemolytica
LKT binding to bovine leukocytes is mediated by the β
2
-integrin CD11a/CD18 (lymphocyte function-associated antigen 1 [LFA-1]), which subsequently induces activation and cytolysis of these cells. Inflammatory cytokines, which are released during viral and bacterial infection, are reported to increase LFA-1 expression and conformational activation. We investigated the effects of the inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) on the interaction of
M. haemolytica
LKT with bovine peripheral blood neutrophils (PMNs). In this study we demonstrated, by flow cytometry, that bovine PMNs increased their binding to an anti-bovine LFA-1 monoclonal antibody (BAT75A) following in vitro incubation with IL-1β, TNF-α, or IFN-γ. Incubation with cytokines also increased CD18 expression, as assessed by real-time PCR and by Western blotting. Increased LFA-1 expression by PMNs exposed to cytokines was associated with increased LKT binding and cytotoxicity. The latter represented, at least in part, enhanced PMN apoptosis, as assessed by propidium iodine staining and caspase-3 activation. The results of this study suggest that inflammatory cytokines may play an important role in enhancing the biological response of bovine PMNs to
M. haemolytica
LKT.
Flavohemoglobin Hmp ProtectsSalmonella entericaSerovar Typhimurium from Nitric Oxide-Related Killing by Human Macrophages
Authors: Stevanin Tânia M. , Poole Robert K. , Demoncheaux Eric A. G. , Read Robert C.
Published: July 2002

Survival of macrophage microbicidal activity is a prerequisite for invasive disease caused by the enteric pathogenSalmonella entericaserovar Typhimurium. Flavohemoglobins, such as those ofEscherichia coli,Salmonella, and yeast, play vital roles in protection of these microorganisms in vitro from nitric oxide (NO) and nitrosative stress. ASalmonella hmpmutant defective in flavohemoglobin (Hmp) synthesis exhibits growth that is hypersensitive to nitrosating agents. We found that respiration of this mutant exhibited increased inhibition by NO, whereas wild-type cells pregrown with sodium nitroprusside orS-nitrosoglutathione showed enhanced tolerance of NO. Most significantly,hmpmutants internalized by primary human peripheral monocyte-derived macrophages survived phagocytosis relatively poorly compared with similarly bound and internalized wild-type cells. That the enhanced sensitivity to macrophage microbicidal activity is due primarily to the failure ofSalmonellato detoxify NO was suggested by the ability ofl-NG-monomethyl arginine—an inhibitor of NO synthase—to eliminate the difference in killing between wild-type andhmpmutantSalmonellacells. These observations suggest thatSalmonellaHmp contributes to protection from NO-mediated inhibition by human macrophages.
Loss of Regulatory Protein RfaH Attenuates Virulence of Uropathogenic
Escherichia coli
Authors: Nagy Gábor , Dobrindt Ulrich , Schneider György , Khan A. Salam , Hacker Jörg , Emödy Levente
Published: July 2002

RfaH is a regulatory protein in
Escherichia coli
and
Salmonella enterica
serovar Typhimurium. Although it enhances expression of different factors that are proposed to play a role in bacterial virulence, a direct effect of RfaH on virulence has not been investigated so far. We report that inactivation of
rfaH
dramatically decreases the virulence of uropathogenic
E. coli
strain 536 in an ascending mouse model of urinary tract infection. The mortality rate caused by the wild-type strain in this assay is 100%, whereas that of its isogenic
rfaH
mutant does not exceed 18%. In the case of coinfection, the wild-type strain 536 shows higher potential to colonize the urinary tract even when it is outnumbered 100-fold by its
rfaH
mutant in the inoculum. In contrast to the wild-type strain, serum resistance of strain 536
rfaH
::
cat
is fully abolished. Furthermore, we give evidence that, besides a major decrease in the amount of hemin receptor ChuA (G. Nagy, U. Dobrindt, M. Kupfer, L. Emody, H. Karch, and J. Hacker, Infect. Immun. 69:1924-1928, 2001), loss of the RfaH protein results in an altered lipopolysaccharide phenotype as well as decreased expression of K15 capsule and alpha-hemolysin, whereas levels of other pathogenicity factors such as siderophores, flagella, Prf, and S fimbriae appear to be unaltered in strain 536
rfaH
::
cat
in comparison to the wild-type strain.
trans c
omplementation of the mutant strain with the
rfaH
gene restores wild-type levels of the affected virulence factors and consequently restitutes virulence in the mouse model of ascending urinary tract infection.
Interleukin-10 Controls the Onset of Irreversible Septic Shock
Authors: Latifi Samir Q. , O'Riordan Mary Ann , Levine Alan D.
Published: July 2002

Lethality from sepsis is believed to be mediated by a proinflammatory cytokine cascade, yet blocking the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1) fails to prevent mortality in human disease and a mouse model of sepsis induced by cecal ligation and puncture (CLP). The role of the antiinflammatory cytokine IL-10 in the CLP model of sepsis is unclear, with either protective or harmful effects demonstrated, depending upon the time of intervention. We therefore hypothesize that IL-10 functions as a temporal regulator of the transition from early reversible sepsis to the late phase of irreversible shock. Transition from reversible sepsis to irreversible shock in the CLP model was defined as the time when removal of the necrotic cecum by rescue surgery is no longer effective. We subjected IL-10-deficient (IL-10−/−) and wild-type (IL-10+/+) mice to CLP and monitored the progression of sepsis, the onset of irreversible shock, and mortality. Onset of lethality in IL-10−/−mice occurred significantly earlier than in IL-10+/+mice and was associated with 15-fold-higher serum levels of TNF-α and IL-6. Consistent with these findings, the efficacy of rescue surgery after lethal CLP is lost 10 h earlier in IL-10−/−mice than in IL-10+/+mice. Treatment with recombinant human IL-10 5 h after CLP significantly improved survival and lengthened the therapeutic window for rescue surgery in both strains of mice. These results demonstrate that IL-10 controls the onset of irreversible septic shock after CLP.
Autotransported Serine Protease A of
Neisseria meningitidis
: an Immunogenic, Surface-Exposed Outer Membrane, and Secreted Protein
Authors: Turner David P. J. , Wooldridge Karl G. , Ala'Aldeen Dlawer A. A.
Published: July 2002

Several autotransporter proteins have previously been identified in
Neisseria meningitidis
. Using molecular features common to most members of the autotransporter family of proteins, we have identified an additional novel ca. 112-kDa autotransporter protein in the meningococcal genomic sequence data. This protein, designated autotransported serine protease A (AspA), has significant N-terminal homology to the secreted serine proteases (subtilases) from several organisms and contains a serine protease catalytic triad. The amino acid sequence of AspA is well-conserved in serogroup A, B, and C meningococci. In
Neisseria gonorrhoeae
, the AspA homologue appears to be a pseudogene. The gene encoding AspA was cloned and expressed from meningococcal strain MC58 (B15:P1.16b). Anti-AspA antibodies were detected in patients' convalescent-phase sera, suggesting that AspA is expressed in vivo during infection and is immunogenic and cross-reactive. Rabbit polyclonal monospecific anti-AspA serum was used to probe whole-cell proteins from a panel of wild-type meningococcal strains and two AspA mutant strains. Expression of the ca. 112-kDa precursor polypeptide was detected in 12 of 20 wild-type meningococcal strains examined, suggesting that AspA expression is phase variable. Immunogold electron microscopy and cellular fractionation studies showed that the AspA precursor is transported to the outer membrane and remains surface exposed. Western blot experiments confirmed that smaller, ca. 68- or 70-kDa components of AspA (AspA68 and AspA70, respectively) are then secreted into the meningococcal culture supernatant. Site-directed mutagenesis of S426 abolished secretion of both rAspA68 and rAspA70 in
Escherichia coli
, confirming that AspA is an autocleaved autotransporter protein. In conclusion, we characterized a novel, surface-exposed and secreted, immunogenic, meningococcal autotransporter protein.
Tumor Necrosis Factor Alpha Stimulates Killing ofMycobacterium tuberculosisby Human Neutrophils
Authors: Kisich Kevin O. , Higgins Michael , Diamond Gill , Heifets Leonid
Published: July 2002

The ability of human neutrophils to aid in defense against pulmonary infection withMycobacterium tuberculosisis controversial. In this study, we have shown that neutrophils respond to and phagocytoseM. tuberculosisin human lesions. Neutrophils from healthy individuals were able to kill significant fractions of an inoculum ofM. tuberculosiswithin 1 h of phagocytosis, and this ability was enhanced by tumor necrosis factor alpha but not by gamma interferon. The mycobactericidal mechanism was nonoxidative, as inhibitors of reactive oxygen or reactive nitrogen intermediates did not interfere with killing. However, the mycobactericidal mechanism was associated with increased exposure of intracellularM. tuberculosisto neutrophil defensins. In vitro, human neutrophil peptides 1 to 3 were not able to kill the bacilli even at much higher levels. These studies support the concept that human neutrophils are directly involved in defense against infection withM. tuberculosis.
Acylation of the Lipooligosaccharide ofHaemophilus influenzaeand Colonization: anhtrBMutation Diminishes the Colonization of Human Airway Epithelial Cells
Authors: Swords W. Edward , Chance Deborah L. , Cohn Leah A. , Shao Jianqiang , Apicella Michael A. , Smith Arnold L.
Published: July 2002

Haemophilus influenzaeis a commensal and opportunistic pathogen of the human airways. A number of surface molecules contribute to colonization of the airways byH. influenzae, such as adhesins, including structures found in the lipooligosaccharide (LOS). A human bronchiolar xenograft model was employed to investigate the host-bacterial interactions involved in the colonization of the airway byH. influenzae. Differential display was used to identifyH. influenzaemRNA that reflect genes which were preferentially expressed in the xenograft compared to growth. Eleven mRNA fragments had consistent increased expression when the bacteria grew in xenografts. On sequencing these fragments, eight open reading frames were identified. Three of these had no match in the NCBI or the TIGR database, while an additional three were homologous to genes involved in heme or iron acquisition and utilization: two of the mRNAs encoded proteins homologous to enzymes involved in LOS biosynthesis: a heptosyl transferase (rfaF) involved in the synthesis of the LOS core and a ketodeoxyoctonate phosphate-dependent acyltransferase (htrB) that performs one of the late acylation reactions in lipid A synthesis. Inoculation of human bronchiolar xenografts revealed a significant reduction in colonization capacity byhtrBmutants. In vitro,htrBmutants elicited lesser degrees of cytoskeletal rearrangement and less stimulation of host cell signaling with 16HBE14o−cells and decreased intracellular survival. These results implicate acylation ofH. influenzaelipid A as playing a key role in the organisms' colonization of the normal airway.
Escherichia coli
Shiga-Like Toxins Induce Apoptosis and Cleavage of Poly(ADP-Ribose) Polymerase via In Vitro Activation of Caspases
Authors: Ching Joyce C. Y. , Jones Nicola L. , Ceponis Peter J. M. , Karmali Mohamed A. , Sherman Philip M.
Published: July 2002

Shiga-like toxin-producing
Escherichia coli
causes hemorrhagic colitis and hemolytic-uremic syndrome in association with the production of Shiga-like toxins, which induce cell death via either necrosis or apoptosis. However, the abilities of different Shiga-like toxins to trigger apoptosis and the sequence of intracellular signaling events mediating the death of epithelial cells have not been completely defined. Fluorescent dye staining with acridine orange and ethidium bromide showed that Shiga-like toxin 1 (Stx1) induced apoptosis of HEp-2 cells in a dose- and time-dependent manner. Stx2 also induced apoptosis in a dose-dependent manner. Apoptosis induced by Stx1 (200 ng/ml) and apoptosis induced by Stx2 (200 ng/ml) were maximal following incubation with cells for 24 h (94.3% ± 1.8% and 81.7% ± 5.2% of the cells, respectively). Toxin-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, cell shrinkage, and the formation of apoptotic bodies, as assessed by transmission electron microscopy. Stx2c induced apoptosis weakly even at a high dose (1,000 ng/ml for 24 h; 26.7% ± 1.3% of the cells), whereas Stx2e did not induce apoptosis of HEp-2 cells. Thin-layer chromatography confirmed that HEp-2 cells express the Stx1-Stx2-Stx2c receptor, globotriaosylceramide (Gb3), but not the Stx2e receptor, globotetraosylceramide (Gb4). Western blot analysis of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme, demonstrated that incubation with Stx1 and Stx2 induced cleavage, whereas incubation with Stx2e did not result in cleavage of PARP. A pan-caspase inhibitor (Z-VAD-FMK) and a caspase-8-specific inhibitor (Z-IETD-FMK) eliminated, in a dose-dependent fashion, the cleavage of PARP induced by Shiga-like toxins. Caspase-8 activation was confirmed by detection of cleavage of this enzyme by immunoblotting. Cleavage of caspase-9 and the proapoptotic member of the Bcl-2 family BID was also induced by Stx1, as determined by immunoblot analyses. We conclude that different Shiga-like toxins induce different degrees of apoptosis that correlates with toxin binding to the glycolipid receptor Gb3 and that caspases play an integral role in the signal transduction cascade leading to toxin-mediated programmed cell death.
Identification of
Klebsiella pneumoniae
Genes Involved in Intestinal Colonization and Adhesion Using Signature-Tagged Mutagenesis
Authors: Maroncle Nathalie , Balestrino Damien , Rich Chantal , Forestier Christiane
Published: July 2002

Klebsiella pneumoniae
is an opportunistic pathogen responsible for nosocomial infections that initially colonize the intestinal tract of patients. Signature-tagged mutagenesis was used to identify genes required for this function. A library of 2,200 mutants was analyzed for the inability of the mutants to survive in a murine model of intestinal colonization and to adhere to human intestinal cells (Int-407) in vitro. Twenty-nine attenuated mutants were selected for further analyses after competition assays against the wild-type strain. Whatever the screening model, most of the transposon insertions occurred in genes involved in metabolic pathways, membrane transport, DNA metabolism, transcriptional regulation, and unknown functions. Only one mutant was attenuated in both the murine colonization and the in vitro adhesion models, and the sequence disrupted by the transposon had homology to adhesin-encoding genes of
Haemophilus
sp.
Interleukin-8 Secretion fromMycobacterium tuberculosis-Infected Monocytes Is Regulated by Protein Tyrosine Kinases but Not by ERK1/2 or p38 Mitogen-Activated Protein Kinases
Authors: Ameixa Clara , Friedland Jon S.
Published: July 2002

Mycobacterium tuberculosisupregulates NF-κB binding and interleukin-8 (IL-8) gene expression and secretion in primary human monocytes. Inhibition of tyrosine protein kinases but not of ERK1/2 or p38 mitogen-activated protein kinases downregulates tuberculosis-induced IL-8 secretion. The inhibitor genistein decreased NF-κB nuclear translocation and IL-8 gene transcription in addition to acting on posttranscriptional processing.
Phase Variation Analysis of
Coxiella burnetii
during Serial Passage in Cell Culture by Use of Monoclonal Antibodies
Authors: Hotta Akitoyo , Kawamura Midori , To Ho , Andoh Masako , Yamaguchi Tsuyoshi , Fukushi Hideto , Hirai Katsuya
Published: July 2002

Antigenic changes in
Coxiella burnetii
Nine Mile strain phase I during serial passages in cell culture were analyzed with three groups of monoclonal antibodies (MAbs) against lipopolysaccharide. The MAbs of group 1 did not react with organisms that were passaged over five times, and the MAbs of group 2 did not react with organisms that were passaged over eight times. The MAbs of group 3 reacted with organisms passaged up to 15 times but did not react with phase II cells. These results suggest that
C. burnetii
could be differentiated into four phase states during phase variation.
Mice That Are Congenic for the
char2
Locus Are Susceptible to Malaria
Authors: Burt Rachel A. , Marshall Vikki M. , Wagglen Jamie , Rodda Fleur R. , Senyschen Danielle , Baldwin Tracey M. , Buckingham Lynn A. , Foote Simon J.
Published: July 2002

A major advance has been made towards the positional cloning of
char2
(a quantitative trait locus encoding resistance to
Plasmodium chabaudi
malaria). Mice congenic for the locus have been used to fine map the gene and to prove that
char2
plays a significant role in the outcome of malarial infection, independently of other resistance loci.
ASalmonella entericaSerovar TyphimuriumhemAMutant Is Highly Susceptible to Oxidative DNA Damage
Authors: Elgrably-Weiss Maya , Park Sunny , Schlosser-Silverman Eliana , Rosenshine Ilan , Imlay James , Altuvia Shoshy
Published: July 2002

The first committed step in the biosynthesis of heme, an important cofactor of two catalases and a number of cytochromes, is catalyzed by thehemAgene product.Salmonella entericaserovar TyphimuriumhemA26::Tn10d(hemA26) was identified in a genetic screen of insertion mutants that were sensitive to hydrogen peroxide. Here we show that thehemA26mutant respires at half the rate of wild-type cells and is highly susceptible to the effects of oxygen species. Exposure of thehemA26strain to hydrogen peroxide results in extensive DNA damage and cell death. The chelation of intracellular free iron fully abrogates the sensitivity of this mutant, indicating that the DNA damage results from the iron-catalyzed formation of hydroxyl radicals. The inactivation of heme synthesis does not change the amount of intracellular iron, but by diminishing the rate of respiration, it apparently increases the amount of reducing equivalents available to drive the Fenton reaction. We also report that hydrogen peroxide has opposite effects on the expression ofhemAandhemH, the first and last genes of heme biosynthesis pathway, respectively.hemAmRNA levels decrease, while the transcription ofhemHis induced by hydrogen peroxide, in anoxyR-dependent manner. TheoxyR-dependent induction is suppressed under conditions that accelerate the Fenton reaction by a mechanism that is not yet understood.
RelA Protein Is Involved in Induction of Genetic Competence in CertainBacillus subtilisStrains by Moderating the Level of Intracellular GTP
Authors: Inaoka Takashi , Ochi Kozo
Published: July 2002

We found that the ability to develop genetic competence of a certain relaxed (relA) aspartate-auxotrophic strain ofBacillus subtilisis significantly lower than that of the isogenic stringent (relA+) strain. Transcriptional fusion analysis utilizing alacZreporter gene indicated that the amount of the ComK protein, known as the key protein for competence development, is greatly reduced in the relaxed strain than in the stringent strain. We also found that the addition of decoyinine, a GMP synthetase inhibitor, induces expression of a competence gene (comG) in the relaxed strain, accompanied by a pronounced decrease in the level of intracellular GTP as measured by high-performance liquid chromatography. The transformation efficiency of the relaxed strain increased 100-fold when decoyinine was added att0(the transition point between exponential to stationary growth phase). Conversely, supplementation of guanosine together with decoyinine completely abolished the observed effect of adding decoyinine on competence development. Furthermore, the impaired ability of the relaxed strain for competence development was completely restored by disrupting thecodYgene, which is known to negatively controlcomKexpression. Our results indicate that the RelA protein plays an essential role in the induction of competence development at least under certain physiological conditions by reducing the level of intracellular GTP and overcoming CodY-mediated regulation.
Activation of Antibiotic Biosynthesis by Specified Mutations in the
rpoB
Gene (Encoding the RNA Polymerase β Subunit) of
Streptomyces lividans
Authors: Hu Haifeng , Zhang Qin , Ochi Kozo
Published: July 2002

We found that the biosynthesis of actinorhodin (Act), undecylprodigiosin (Red), and calcium-dependent antibiotic (CDA) are dramatically activated by introducing certain mutations into the
rpoB
gene that confer resistance to rifampin to
Streptomyces lividans
66, which produces less or no antibiotics under normal growth conditions. Activation of Act and/or Red biosynthesis by inducing mutations in the
rpoB
gene was shown to be dependent on the mutation's position and the amino acid species substituted in the β-subunit of the RNA polymerase. Mutation analysis identified 15 different kinds of point mutations, which are located in region I, II, or III of the
rpoB
gene and, in addition, two novel mutations (deletion of nucleotides 1287 to 1289 and a double substitution at nucleotides 1309 and 1310) were also found. Western blot analyses and S1 mapping analyses demonstrated that the expression of
act
II
-
ORF4 and
redD
, which are pathway-specific regulatory genes for Act and Red, respectively, was activated in the mutants able to produce Act and Red. The ActIV-ORF1 protein (an enzyme for Act biosynthesis) and the RedD protein were produced just after the upregulation of ActII-ORF4 and RedZ, respectively. These results indicate that the mutation in the
rpoB
gene of
S. lividans
, resulting in the activation of Act and/or Red biosynthesis, functions at the transcription level by activating directly or indirectly the key regulatory genes,
act
II
-
ORF4 and
redD
. We propose that the mutated RNA polymerase may function by mimicking the ppGpp-bound form in activating the onset of secondary metabolism in
Streptomyces
.
A Repressor Protein, PhaR, Regulates Polyhydroxyalkanoate (PHA) Synthesis via Its Direct Interaction with PHA
Authors: Maehara Akira , Taguchi Seiichi , Nishiyama Tatsuaki , Yamane Tsuneo , Doi Yoshiharu
Published: July 2002

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the
phaR
gene, which is located downstream of
phaP
in
Paracoccus denitrificans
, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of
phaP
and
phaR
, suggesting that PhaR plays a role in the regulation of
phaP
expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant
Escherichia coli
was able to rebind specifically to poly[(
R
)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that
phaP
expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.
Role of the Murein Precursor UDP-
N
-Acetylmuramyl-
l
-Ala-γ-
d
-Glu-
meso
-Diaminopimelic Acid-
d
-Ala-
d
-Ala in Repression of β-Lactamase Induction in Cell Division Mutants
Authors: Uehara Tsuyoshi , Park James T.
Published: July 2002

Certain β-lactam antibiotics induce the chromosomal
ampC
β-lactamase of many gram-negative bacteria. The natural inducer, though not yet unequivocally identified, is a cell wall breakdown product which enters the cell via the AmpG permease component of the murein recycling pathway. Surprisingly, it has been reported that β-lactamase is not induced by cefoxitin in the absence of FtsZ, which is required for cell division, or in the absence of penicillin-binding protein 2 (PBP2), which is required for cell elongation. Since these results remain unexplained, we examined an
ftsZ
mutant and other cell division mutants (
ftsA
,
ftsQ
, and
ftsI
) and a PBP2 mutant for induction of β-lactamase. In all mutants, β-lactamase was not induced by cefoxitin, which confirms the initial reports. The murein precursor, UDP-
N
-acetylmuramyl-
l
-Ala-γ-
d
-Glu-
meso
-diaminopimelic acid-
d
-Ala-
d
-Ala (UDP-MurNAc-pentapeptide), has been shown to serve as a corepressor with AmpR to repress β-lactamase expression in vitro. Our results suggest that β-lactamase is not induced because the
fts
mutants contain a greatly increased amount of corepressor which the inducer cannot displace. In the PBP2(Ts) mutant, in addition to accumulation of corepressor, cell wall turnover and recycling were greatly reduced so that little or no inducer was available. Hence, in both cases, a high ratio of repressor to inducer presumably prevents induction.
Detection and Differentiation of
Cryptosporidium
Parasites That Are Pathogenic for Humans by Real-Time PCR
Authors: Limor Josef R. , Lal Altaf A. , Xiao Lihua
Published: July 2002

Cryptosporidiosis is a significant cause of food-borne and waterborne outbreaks of diarrheal diseases. To better understand the route of transmission of
Cryptosporidium
parasites, a number of genotyping techniques have been developed, based on PCR-restriction fragment length polymorphism or sequencing analysis of antigen, structural, and housekeeping genes. In this study, a real-time assay for the detection of
Cryptosporidium
oocysts is described. This technique had a detection limit of five oocysts. By melting curve analysis of PCR products with fluorescence-labeled hybridization probes, this technique was able to differentiate five common
Cryptosporidium
parasites that are pathogenic for humans in a single PCR. We evaluated and validated the test using samples from presently known
Cryptosporidium
parasites that are pathogenic for humans. This technique provides an alternative molecular tool in epidemiologic studies of human cryptosporidiosis.