bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions among Plant Species and Microorganisms in Salt Marsh Sediments
Authors: Burke David J. , Hamerlynck Erik P. , Hahn Dittmar
Published: July 2002

The interactions among
Spartina patens
and sediment microbial populations and the interactions among
Phragmites australis
and sediment microbial populations were studied at monotypic sites in Piermont Marsh, a salt marsh of the Hudson River north of New York, N.Y., at key times during the growing season. Arbuscular mycorrhizal fungi (AMF) effectively colonized
S. patens
but not
P. australis
, and there were seasonal increases and decreases that coincided with plant growth and senescence (17 and 6% of the
S. patens
root length were colonized, respectively). In sediment samples from the
Spartina
site, the microbial community and specific bacterial populations were at least twice as large in terms of number and biomass as the microbial community and specific bacterial populations in sediment samples from the
Phragmites
site, and peak values occurred during reproduction. Members of the domain
Bacteria
, especially members of the α-, γ-, and δ-subdivisions of the
Proteobacteria
, were the most abundant organisms at both sites throughout the growing season. The populations were generally more dynamic in samples from the
Spartina
site than in samples from the
Phragmites
site. No differences between the two sites and no differences during the growing season were observed when restriction fragment length polymorphism analyses of
nifH
amplicons were performed in an attempt to detect shifts in the diversity of nitrogen-fixing bacteria. Differences were observed only in the patterns generated by PCR or reverse transcription-PCR for samples from the
Spartina
site, suggesting that there were differences in the overall and active populations of nitrogen-fixing bacteria. Regression analyses indicated that there was a positive interaction between members of the δ-subdivision of the
Proteobacteria
and root biomass but not between members of the δ-subdivision of the
Proteobacteria
and macroorganic matter at both sites. In samples from the
Spartina
site, there were indications that there were bacterium-fungus interactions since populations of members of the α-subdivision of the
Proteobacteria
were negatively associated with AMF colonization and populations of members of the γ-subdivision of the
Proteobacteria
were positively associated with AMF colonization.
Identification of Different States of Hepatitis B Virus Infection with a Quantitative PCR Assay
Authors: Kessler Harald H. , Preininger Sabine , Stelzl Evelyn , Daghofer Elisabeth , Santner Brigitte I. , Marth Egon , Lackner Herwig , Stauber Rudolf E.
Published: July 2002

The level of hepatitis B virus (HBV) DNA in serum reflects the replicative activity of HBV. To compare serum HBV DNA levels in different states of hepatitis B, 47 sera of patients with HBeAg-positive chronic hepatitis B, 4 sera of patients with HBeAg-negative chronic hepatitis B, 40 samples of patients after HBeAg seroconversion during alpha interferon treatment, 57 sera of inactive HBsAg carriers, and 42 sera of patients who had recovered from chronic hepatitis B more than 12 months prior to blood collection were checked for the presence of HBV DNA with the Amplicor HBV Monitor Test. In patients with HBeAg-positive chronic hepatitis B, the median of serum HBV DNA levels (8.3 × 10
8
copies/ml) was significantly higher than that for patients after HBeAg seroconversion (6.2 × 10
3
copies/ml) and than that for inactive HBsAg carriers (5.6 × 10
3
copies/ml). None of the patients who had recovered from hepatitis B had detectable HBV DNA in serum. Quantitative PCR proved to be a valuable tool for identification of different states of HBV infection. This technique was found to be a good method for determination of serum HBV DNA levels both for patients with HBeAg seroconversion and for inactive carriers who showed low viremia not detectable by conventional hybridization assays.
Evaluation of Human FcγRIIA (CD32) and FcγRIIIB (CD16) Polymorphisms in Caucasians and African-Americans Using Salivary DNA
Authors: van Schie Rob C. A. A. , Wilson Mark E.
Published: July 2002

Two classes of low-affinity receptors for the Fc region of immunoglobulin G (IgG) (FcγR) are constitutively expressed on resting human neutrophils. These receptors, termed FcγRIIa (CD32) and FcγRIIIb (CD16), display biallelic polymorphisms which have functional consequences with respect to binding and/or ingestion of targets opsonized by human IgG subclass antibodies. The H131-R131 polymorphism of CD32 influences binding of human IgG2 and, to a lesser extent, human IgG3 to neutrophils. The neutrophil antigen (NA1-NA2) polymorphism of CD16 influences the efficiency of phagocytosis of bacteria opsonized by human IgG1 and IgG3. These polymorphisms may influence host susceptibility to certain infectious and/or autoimmune diseases, prompting interest in the development of facile methods for determination of CD32 and CD16 genotype in various clinical settings. We previously reported that genomic DNA from saliva is a suitable alternative to DNA from blood in PCR-based analyses of CD32 and CD16 polymorphisms. In the present study, we utilized for the first time this salivary DNA-based methodology to define CD32 and CD16 genotypes in 271 Caucasian and 118 African-American subjects and to investigate possible linkage disequilibrium between certain CD32 and CD16 genotypes in these two ethnic groups. H131 and R131 gene frequencies were 0.45 and 0.55, respectively, among Caucasians and 0.59 among African-Americans. NA1 and NA2 gene frequencies were 0.38 and 0.62 among Caucasians and 0.39 and 0.61 among African-Americans. Since FcγRIIa and FcγRIIIb synergize in triggering neutrophils, we also assessed the frequency of different CD32 and CD16 genotype combinations in these two groups. In both groups, the R/R131-NA2/NA2 genotype combination was more common than the H/H131-NA1/NA1 combination (threefold for Caucasians versus sevenfold for African-Americans). Whether individuals with the combined R/R131-NA2/NA2 genotype are at greater risk for development of infectious and/or autoimmune diseases requires further investigation, which can be conveniently performed using DNA from saliva rather than blood.
Shipment Impairs Lymphocyte Proliferative Responses to Microbial Antigens
Authors: Betensky Rebecca A. , Connick Elizabeth , Devers Jennifer , Landay Alan L. , Nokta Mostafa , Plaeger Susan , Rosenblatt Howard , Schmitz John L. , Valentine Fred , Wara Diane , Weinberg Adriana , M. Lederman Howard
Published: July 2002

Lymphocyte proliferation assays (LPAs) are widely used to assess T-lymphocyte function of patients with human immunodeficiency virus infection and other primary and secondary immunodeficiency disorders. Since these assays require expertise not readily available at all clinical sites, specimens may be shipped to central labs for testing. We conducted a large multicenter study to evaluate the effects of shipping on assay performance and found significant loss of LPA activity. This may lead to erroneous results for individual subjects and introduce bias into multicenter trials.
Evaluation of Primary Binding Assays for Presumptive Serodiagnosis of Swine Brucellosis in Argentina
Authors: Paulo P. Silva , Vigliocco A. M. , Ramondino R. F. , Marticorena D. , Bissi E. , Briones G. , Gorchs C. , Gall D. , Nielsen K.
Published: July 2002

An indirect enzyme-linked immunosorbent assay (IELISA), a competitive ELISA (CELISA), and a fluorescence polarization assay (FPA) for the presumptive serological diagnosis of swine brucellosis were evaluated using two populations of swine sera: sera from brucellosis-free Canadian herds and sera from Argentina selected based on positive reactions in the buffered antigen plate agglutination test (BPAT) and the 2-mercaptoethanol (2-ME) test. In addition, sera from adult swine from which
Brucella suis
was isolated at least once for each farm of origin were evaluated. The IELISA, CELISA, and FPA specificity values were 99.9, 99.5, and 98.3%, respectively, and the IELISA, CELISA, and FPA sensitivity values relative to the BPAT and the 2-ME test were 98.9, 96.6, and 93.8%, respectively. Actual sensitivity was assessed by using 37 sera from individual pigs from which
B. suis
was cultured, and the values obtained were as follows: BPAT, 86.5%; 2-ME test, 81.1%; IELISA, 86.5%; CELISA, 78.5%; and FPA, 80.0%.
Anti-Trypanosoma cruziImmunoglobulin G1 Can Be a Useful Tool for Diagnosis and Prognosis of Human Chagas' Disease
Authors: Cordeiro Flávia Drumond , Martins-Filho Olindo Assis , Da Costa Rocha Manoel Otávio , Adad Sheila Jorge , Corrêa-Oliveira Rodrigo , Romanha Alvaro José
Published: July 2002

Two functionally distinct antibodies, categorized as conventional serology antibodies (CSA) and lytic antibodies (LA) have been described in Chagas' disease, based on their ability to bind to fixed epimastigotes (EPI) or live trypomastigotes (TRYPO), respectively. In this study, the profile of immunoglobulin G (IgG) subclasses of CSA and LA were analyzed by flow cytometry using serum samples from chronic chagasic patients with the indeterminate (IND), cardiac (CARD), and digestive (DIG) clinical forms of the disease. The results were expressed as percentage of positive fluorescent parasites (PPFP) for each sample. CSA showed a higher PPFP than LA for all samples. At serum dilutions between 1:256 and 1:2,048, IgG1 anti-EPI was able to distinguish chagasic from nonchagasic individuals. Different profiles of IgG subclasses were observed for CSA and LA. IgG1 and IgG2 were the main subclasses in CSA, whereas IgG1 and IgG3 were the predominant ones in LA. The reactivity of IgG2 anti-EPI was greater in IND and CARD than in DIG patients. Furthermore, a low level of IgG1 and IgG3 LA was associated with most of the CARD patients. On the other hand, a high level of IgG1 LA was associated with most of the IND patients. In summary, our findings indicate the potential of IgG1 anti-EPI for serological diagnosis of Chagas' disease, providing further evidence for a protective role of LA, and show that IgG1 anti-liveTrypanosoma cruziTRYPO may be used to predict the risk of cardiac damage in Chagas' disease.
Effects of Calorie Restriction on Polymicrobial Peritonitis Induced by Cecum Ligation and Puncture in Young C57BL/6 Mice
Authors: Sun Dongxu , Muthukumar Alagar Raju , Lawrence Richard A. , Fernandes Gabriel
Published: July 2002

Calorie restriction (CR) is known to prolong the life span and maintain an active immune function in aged mice, but it is still not known if rodents under CR can respond optimally to bacterial infection. We report here on the influence of CR on the response of peritoneal macrophages to lipopolysaccharide, splenic NF-κB and NF–interleukin-6 (IL-6) activities, and mortality in polymicrobial sepsis induced by cecal ligation and puncture (CLP). Macrophages from 6-month-old C57BL/6 mice on a calorie-restricted diet were less responsive to lipopolysaccharide, as evidenced by lower levels of IL-12 and IL-6 protein and mRNA expression. Furthermore, in vitro lipopolysaccharide-stimulated macrophages from mice under CR also expressed decreased lipopolysaccharide receptor CD14 levels as well as Toll-like receptor 2 (TLR2) and TLR4 mRNA levels. In addition, the phagocytic capacity and class II (I-Ab) expression of macrophages were also found to be significantly lower in mice under CR. Mice under CR died earlier (P< 0.005) after sepsis induced by CLP, which appeared to be a result of increased levels in serum of the proinflammatory cytokines tumor necrosis factor alpha and IL-6 and splenic NF-κB and NF–IL-6 activation 4 h after CLP. However, mice under CR survived significantly (P< 0.005) longer than mice fed ad libitum when injected with paraquat, a free radical-inducing agent. These data suggest that young mice under CR may be protected against oxidative stress but may have delayed maturation of macrophage function and increased susceptibility to bacterial infection.
Identification of thepsaAGene, Coding for Pneumococcal Surface Adhesin A, in Viridans Group Streptococci other thanStreptococcus pneumoniae
Authors: Jado Isabel , Fenoll Asunción , Casal Julio , Pérez Amalia
Published: July 2002

The gene encoding the pneumococcal surface adhesin A (PsaA) protein has been identified in three different viridans group streptococcal species. Comparative studies of thepsaAgene identified in different pneumococcal isolates by sequencing PCR products showed a high degree of conservation among these strains. PsaA is encoded by an open reading frame of 930 bp. The analysis of this fragment inStreptococcus mitis, Streptococcus oralis, andStreptococcus anginosusstrains revealed a sequence identity of 95, 94, and 90%, respectively, to the corresponding open reading frame of the previously reportedStreptococcus pneumoniaeserotype 6B strain. Our results confirm thatpsaAis present and detectable in heterologous bacterial species. The possible implications of these results for the suitability and potential use of PsaA in the identification and diagnosis of pneumococcal diseases are discussed.
Toxin Genes and Other Characteristics of
Staphylococcus aureus
Isolates from Milk of Cows with Mastitis
Authors: Akineden Ö. , Annemüller C. , Hassan A. A. , Lämmler C. , Wolter W. , Zschöck M.
Published: July 2002

In the present study, 103
Staphylococcus aureus
strains isolated from milk samples from 60 cows with mastitis from eight different farms in seven different locations in one region of Germany were compared pheno- and genotypically and by identification of various toxins. On the basis of culture and hemolytic properties and by determination of the tube coagulase reaction, all of the isolates could be identified as
S. aureus
. This could be confirmed by PCR amplification of species-specific parts of the gene encoding the 23S rRNA. In addition, all of the
S. aureus
isolates harbored the genes encoding staphylococcal coagulase and clumping factor and the genes encoding the X region and the immunoglobulin G binding region of protein A. These four genes displayed size polymorphisms. By PCR amplification, the genes for the toxins staphylococcal enterotoxin A (SEA), SEC, SED, SEG, SEI, SEJ, and TSST-1 but not those for SEB, SEE, SEH, and the exfoliative toxins ETA and ETB could be detected. To analyze the epidemiological relationships, the isolates were subjected to DNA fingerprinting by macrorestriction analysis of their chromosomal DNAs. According to the observed gene polymorphisms, the toxin patterns, and the information given by macrorestriction analysis of the isolates by pulsed-field gel electrophoresis, a limited number of clones seemed to be responsible for the cases of bovine mastitis on the various farms.
Use of Recombinant Envelope Proteins for Serological Diagnosis of Dengue Virus Infection in an Immunochromatographic Assay
Authors: Cuzzubbo Andrea J. , Endy Timothy P. , Nisalak Ananda , Kalayanarooj Siripen , Vaughn David W. , Ogata Steven A. , Clements David E. , Devine Peter L.
Published: July 2002

An immunochromatographic test that incorporates recombinant antigens (Dengue Duo Rapid Strip Test; PanBio, Brisbane, Australia) has recently become commercially available. This assay is performed in 15 min and detects both immunoglobulin M (IgM) and IgG in a capture format. The four recombinant proteins used represent the N-terminal 80% of the viral envelope glycoproteins of dengue viruses 1, 2, 3, and 4, respectively. The sensitivity and specificity of the recombinant-antigen-based assay were 90 and 86%, respectively. The similar diagnostic performance of these antigens to that of enzyme-linked immunosorbent assays using whole dengue virus suggests that they mimic whole dengue viruses in primary structure and epitope conformation. These results suggest that recombinant proteins can be used in diagnostic assays for dengue to overcome safety issues associated with the use of whole virus.
Construction and Characterization of aMycobacterium tuberculosisMutant Lacking the Alternate Sigma Factor Gene,sigF
Authors: Chen Ping , Ruiz Rafael E. , Li Qing , Silver Richard F. , Bishai William R.
Published: July 2002

The alternate RNA polymerase sigma factor gene,sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain ofMycobacterium tuberculosis. The growth rate of the ΔsigFmutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the ΔsigFmutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-typesigFgene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the ΔsigFmutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P< 0.001). These data indicate thatM. tuberculosis sigFis a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the ΔsigFmutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.
Transforming Growth Factor β1 Ameliorates Intestinal Epithelial Barrier Disruption byCryptosporidium parvumIn Vitro in the Absence of Mucosal T Lymphocytes
Authors: Roche James K. , Martins Clovis A. P. , Cosme Rosana , Fayer Ronald , Guerrant Richard L.
Published: July 2002

Exposure to oocysts of the protozoanCryptosporidium parvumcauses intestinal epithelial cell dysfunction in vivo and in vitro, but effective means by which mucosal injury might be prevented remain unclear. We examined the ability of transforming growth factor β1 (TGF-β1)—a cytokine synthesized and released by cells in the intestine—to preserve the barrier function of human colonic epithelia when challenged withC. parvumoocysts and then studied the mechanisms involved. Epithelial barrier function was monitored electrophysiologically, receptors for TGF-β1 were localized by confocal microscopy, and TGF-β1-induced protein kinase C activation was detected intracellularly by translocation of its α isozyme. TGF-β1 alone enhanced intestinal epithelial barrier function, while exposure toC. parvumoocysts (≥105/monolayer) markedly reduced barrier function to ≤40% of that of the control. When epithelial monolayers were pretreated with TGF-β1 at 5.0 ng/ml, the barrier-disrupting effect ofC. parvumoocysts was almost completely abrogated for 96 h. Further investigation showed that (i) the RI and RII receptors for TGF-β1 were present on 55 and 65% of human epithelial cell line cells, respectively, over a 1-log-unit range of receptor protein expression, as shown by flow cytometry and confirmed by confocal microscopy; (ii) only basolateral and not apical TGF-β1 exposure of the polarized epithelial monolayer resulted in a protective effect; and (iii) TGF-β1 had no direct effect on the organism in reducing its tissue-disruptive effects. In exploring mechanisms to account for the barrier-preserving effects of TGF-β1 on epithelium, we found that the protein kinase C pathway was activated, as shown by translocation of its 80-kDa α isozyme within 30 s of epithelial exposure to TGF-β1; the permeability of epithelial monolayers to passage of macromolecules was reduced by 42% with TGF-β1, even in the face of active protozoal infection; and epithelial cell necrosis monitored by lactate dehydrogenase release was decreased by 50% 70 h after oocyst exposure. Changes in epithelial function, initiated through an established set of surface receptors, likely accounts for the remarkable barrier-sparing effect of nanogram-per-milliliter concentrations of TGF-β1 when human colonic epithelium is exposed to an important human pathogen,C. parvum.
Systemic and Mucosal Immune Responses in Mice after Mucosal Immunization with Group B Streptococcus Type III Capsular Polysaccharide-Cholera Toxin B Subunit Conjugate Vaccine
Authors: Shen Xuzhuang , Lagergård Teresa , Yang Yonghong , Lindblad Marianne , Fredriksson Margareta , Holmgren Jan
Published: July 2002

Group B streptococci (GBS) colonize the female genital and rectal tracts and can cause invasive infection in susceptible newborns. An optimally effective GBS vaccine should induce mucosal and systemic immunity. In this study, we investigate the local and systemic immune responses to GBS type III capsular polysaccharide (CPS) after mucosal vaccination of mice via intranasal, peroral, rectal, and vaginal routes, with GBS type III CPS conjugated with recombinant cholera toxin B subunit (GBS III CPS-rCTB). Cholera toxin (CT) was added as an adjuvant. Immunoglobulin G (IgG) and IgA antibodies to the CPS were tested in serum, lungs, and intestinal, rectal, and vaginal extracts by enzyme-linked immunosorbent assay. The conjugated CPS administered by intranasal, peroral, rectal, and vaginal routes was much more effective at inducing both mucosal and systemic antibody responses to GBS III CPS than was unconjugated CPS. The CPS-specific immune responses in various organs were dependent on the route of immunization. Generally, the highest levels of IgA and IgG were generated in the regions or sites of the conjugate exposure. Thus, intranasal vaccination elicited the highest anti-CPS IgA and IgG antibody levels in the lungs, whereas peroral administration in the intestinal site and vaginal vaccination elicited the highest antibody levels in the vagina. Rectal vaccination was superior to the other routes in inducing high antibody levels in the rectum. The four routes of mucosal vaccination also induced distant antibody responses to CPS. Rectal vaccination induced high specific IgA levels in the vagina and intestine, and oral administration induced high specific IgA levels in the lungs and rectum. All four routes of vaccination with the conjugate elicited similarly high levels of anti-CPS IgG in serum. Intranasal vaccination with different doses of the conjugate (10, 30, and 80 μg of CPS) did not have a significant influence on the anti-CPS specific antibody responses. Intranasal immunization induced better antibody responses when one dose of the conjugate was divided and given on three consecutive days compared to administration of the full dose on one occasion. In conclusion, rectal and vaginal vaccination may be the best way of stimulating anti-CPS immune responses in the rectal and vaginal tracts, while high levels of anti-CPS antibodies in the lungs can be achieved after intranasal administration. The vaccination regimen thus might influence the mucosal immune response to CPS. This conjugate may serve as an effective mucosal vaccine for preventing mucosal colonization and invasive infection caused by GBS.
Carbohydrate Biopolymers Enhance Antibody Responses to Mucosally Delivered Vaccine Antigens
Authors: Bacon A. , Makin J. , Sizer P. J. , Jabbal-Gill I. , Hinchcliffe M. , Illum L. , Chatfield S. , Roberts M.
Published: July 2002

We have evaluated the ability of two carbohydrate biopolymers, chitosan and gellan, to enhance antibody responses to subunit influenza virus vaccines delivered to the respiratory tracts of mice. Groups of mice were vaccinated three times intranasally (i.n.) with 10 μg of purified influenza B/Panama virus surface antigens (PSAs), which consist of hemagglutinin (HA) and neuraminidase (NA), either alone or admixed with chitosan or gellan solutions. Separate groups were vaccinated subcutaneously (s.c.) with PSAs adsorbed to Alhydrogel or chitosan or gellan alone i.n. Serum antibody responses were determined by enzyme-linked immunosorbent assay (ELISA) for influenza virus-specific immunoglobulin G (IgG) and by HA inhibition (HAI) and NA inhibition (NAI) assays. The local respiratory immune response was measured by assaying for influenza virus-specific IgA antibody in nasal secretions and by enumerating nasal and pulmonary lymphocytes secreting IgA, IgG, and IgM anti-influenza virus-specific antibodies by enzyme-linked immunospotting (ELISPOT). When administered alone i.n., B/Panama PSA was poorly immunogenic. Parenteral immunization with B/Panama PSA with Alhydrogel elicited high titers of anti-B/Panama antibodies in serum but a very poor respiratory anti-B/Panama IgA response. In contrast, i.n. immunization with PSA plus chitosan stimulated very strong local and systemic anti-B/Panama responses. Gellan also enhanced the local and serum antibody responses to i.n. PSA but not to the same extent as chitosan. The ability of chitosan to augment the immunogenicity of influenza vaccines given i.n. was confirmed using PSA prepared from an influenza A virus (A/Texas H1N1).
Regulation of
Salmonella enterica
Serovar Typhimurium Invasion Genes by
csrA
Authors: Altier Craig , Suyemoto Mitsu , Lawhon Sara D.
Published: July 2002

Penetration of intestinal epithelial cells by
Salmonella enterica
serovar Typhimurium requires the expression of invasion genes, found in
Salmonella
pathogenicity island 1 (SPI1), that encode components of a type III secretion apparatus. These genes are controlled in a complex manner by regulators within SPI1, including HilA and InvF, and those outside SPI1, such as the two-component regulators PhoP/PhoQ and BarA/SirA. We report here that epithelial cell invasion requires the serovar Typhimurium homologue of
Escherichia coli csrA
, which encodes a regulator that alters the stability of specific mRNA targets. A deletion mutant of
csrA
was unable to efficiently invade cultured epithelial cells and showed reduced expression of four tested SPI1 genes,
hilA, invF, sipC
, and
prgH
. Overexpression of
csrA
from an induced
araBAD
promoter also negatively affected the expression of these genes, indicating that CsrA can act as both a positive and a negative regulator of SPI1 genes and suggesting that the bacterium must tightly control the level or activity of CsrA to achieve maximal invasion. We found that CsrA affected
hilA
, a regulator of the other three genes we tested, probably by controlling one or more genetic elements that regulate
hilA
. We also found that both the loss and the overexpression of
csrA
reduced the expression of two regulators of
hilA, hilC
and
hilD
, suggesting that
csrA
exerts its control of
hilA
through one or both of these regulators. We further found, however, that CsrA could affect the expression of both
invF
and
sipC
independent of its effects on
hilA
. One additional striking phenotype of the
csrA
mutant, not observed in a comparable
E. coli
mutant, was its slow growth. Phenotypic revertants that had normal growth rates, while maintaining the
csrA
mutation, were common. These suppressed strains, however, did not recover the ability to invade cultured cells, indicating that the
csrA
-mediated loss of invasion cannot be attributed simply to poor growth and that the growth and invasion deficits of the
csrA
mutant arise from effects of CsrA on different targets.
Role of Listeriolysin O in Cell-to-Cell Spread of
Listeria monocytogenes
Authors: Gedde Margaret M. , Higgins Darren E. , Tilney Lewis G. , Portnoy Daniel A.
Published: July 2002

Listeria monocytogenes
is a facultative intracellular bacterial pathogen that escapes from a host vacuolar compartment and grows rapidly in the cytosol. Listeriolysin O (LLO) is a secreted pore-forming protein essential for the escape of
L. monocytogenes
from the vacuole formed upon initial internalization. However, its role in intracellular growth and cell-to-cell spread events has not been testable by a genetic approach. In this study, purified six-His-tagged LLO (HisLLO) was noncovalently coupled to the surface of nickel-treated LLO-negative mutants. Bound LLO mediated vacuolar escape in approximately 2% of the mutants. After 5.5 h of growth, cytosolic bacteria were indistinguishable from wild-type bacteria with regard to formation of pseudopod-like extensions, here termed listeriopods, and spread to adjacent cells. However, bacteria in adjacent cells failed to multiply and were found in double-membrane vacuoles. Addition of bound LLO to mutants lacking LLO and two distinct phospholipases C (PLCs) also resulted in spread to adjacent cells, but these triple mutants became trapped in multiple-membrane vacuoles that are reminiscent of autophagocytic vacuoles. These studies show that neither LLO nor the PLCs are necessary for listeriopod formation and uptake of bacteria into neighboring cells but that LLO is required for the escape of
L. monocytogenes
from the double-membrane vacuole that forms upon cell-to-cell spread.
Chlamydial Development Is Adversely Affected by Minor Changes in Amino Acid Supply, Blood Plasma Amino Acid Levels, and Glucose Deprivation
Authors: Harper Angela , Pogson Christopher I. , Jones Meirion L. , Pearce John H.
Published: July 2002

This study has demonstrated the extreme sensitivity of
Chlamydia trachomatis
growing in McCoy cells to small changes in external amino acid supply. In the absence of cycloheximide, a decrease in the amino acid concentration of medium to 75% of control values was sufficient to induce the growth of enlarged chlamydial forms of reduced infectivity. Morphology became more distorted and the yield of infectious particles from inclusions declined as medium amino acid levels were further reduced. These events correlated with a general decline in intracellular amino acids, as measured by high-performance liquid chromatography, suggesting that chlamydiae require a minimum concentration of each amino acid for normal development. Cycloheximide enhanced the production of normal organisms and increased infectivity yield in media, suggesting that the drug increased the available pool of amino acids. This was supported by intracellular amino acid analyses. Aberrant forms with reduced infectivity were also induced during supply of infected cell cultures with medium containing blood plasma amino acid concentrations, supporting the proposal that nutrient levels in vivo could promote abnormal chlamydial development. Markedly abnormal forms were also observed during glucose deprivation, providing further evidence that aberrant development is a general stress-related response.
Cellular Changes and Apoptosis in the Spleens and Peripheral Blood of Mice Infected with Blood-StagePlasmodium chabaudi chabaudiAS
Authors: Helmby Helena , Jönsson Gun , Troye-Blomberg Marita
Published: July 2002

Infection with blood-stagePlasmodium chabaudi chabaudiAS results in splenomegaly, peripheral leukocytosis, and a major activation of the immune system. The frequencies and absolute numbers of T-cell, B-cell, and macrophage populations in spleen and peripheral blood fromP. chabaudi-infected BALB/c mice were compared and found to be significantly altered during acute infection. The kinetics of the redistribution of the different cell types in spleen and peripheral blood were different, with T and B cells appearing in the blood when their frequencies and absolute numbers in the spleen were low. The frequency and absolute number of apoptotic cells in the spleen were increased during acuteP. chabaudiinfection and involved both T cells, B cells, and macrophages. Both Fas and Fas-ligand expression were increased in the spleen. Taken together, our data provide new information on the complex cellular interactions that take place in the immune system during blood-stage malaria infection in a mouse model.
Role of Adhesins and Toxins in Invasion of Human Tracheal Epithelial Cells by
Bordetella pertussis
Authors: Bassinet Laurence , Gueirard Pascale , Maitre Bernard , Housset Bruno , Gounon Pierre , Guiso Nicole
Published: July 2002

Bordetella pertussis
, the agent of whooping cough, can invade and survive in several types of eukaryotic cell, including CHO, HeLa 229, and HEp-2 cells and macrophages. In this study, we analyzed bacterial invasiveness in nonrespiratory human HeLa epithelial cells and human HTE and HAE0 tracheal epithelial cells. Invasion assays and transmission electron microscopy analysis showed that
B. pertussis
strains invaded and survived, without multiplying, in HTE or HAE0 cells. This phenomenon was
bvg
regulated, but invasive properties differed between
B. pertussis
strains and isolates and the
B. pertussis
reference strain. Studies with
B. pertussis
mutant strains demonstrated that filamentous hemagglutinin, the major adhesin, was involved in the invasion of human tracheal epithelial cells by bacteria but not in that of HeLa cells. Fimbriae and pertussis toxin were not found to be involved. However, we found that the production of adenylate cyclase-hemolysin prevents the invasion of HeLa and HTE cells by
B. pertussis
because an adenylate cyclase-hemolysin-deficient mutant was found to be more invasive than the parental strain. The effect of adenylate cyclase-hemolysin was mediated by an increase in the cyclic AMP concentration in the cells. Pertactin (PRN), an adhesin, significantly inhibited the invasion of HTE cells by bacteria, probably via its interaction with adenylate cyclase-hemolysin. Isolates producing different PRNs were taken up similarly, indicating that the differences in the sequences of the PRNs produced by these isolates do not affect invasion. We concluded that filamentous hemagglutinin production favored invasion of human tracheal cells but that adenylate cyclase-hemolysin and PRN production significantly inhibited this process.
Effect ofChlamydia trachomatisInfection and Subsequent Tumor Necrosis Factor Alpha Secretion on Apoptosis in the Murine Genital Tract
Authors: Perfettini Jean-Luc , Darville Toni , Gachelin Gabriel , Souque Philippe , Huerre Michel , Dautry-Varsat Alice , Ojcius David M.
Published: July 2002

The pathology observed duringChlamydiainfection is due initially to localized tissue damage caused by the infection itself, followed by deleterious host inflammatory responses that lead to permanent scarring. We have recently reported that the infection byChlamydiain vitro results in apoptosis of epithelial cells and macrophages and that infected monocytes secrete the proinflammatory cytokine interleukin-1β. At the same time, proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) can also trigger apoptosis of susceptible cells. To study the possible relationship betweenChlamydia trachomatisinfection and apoptosis in vivo, we used the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling technique to determine whether infection may cause apoptosis in the genital tract of mice and, conversely, whether cytokines produced during the inflammatory response may modulate the level of apoptosis. Our results demonstrate that infected cells in the endocervix at day 2 or 7 after infection are sometimes apoptotic, although there was not a statistically significant change in the number of apoptotic cells in the endocervix. However, large clumps of apoptotic infected cells were observed in the lumen, suggesting that apoptotic cells may be shed from the endocervix. Moreover, there was a large increase in the number of apoptotic cells in the uterine horns and oviducts after 2 or 7 days of infection, which was accompanied by obvious signs of upper tract pathology. Interestingly, depletion of TNF-α led to a decrease in the level of apoptosis in the uterine horns and oviducts of animals infected for 7 days, suggesting that the inflammatory cytokines may exert part of their pathological effect via apoptosis in infected tissues.
Purification, Characterization, and Molecular Analysis of the Gene Encoding Glucosyltransferase from
Streptococcus oralis
Authors: Fujiwara Taku , Hoshino Tomonori , Ooshima Takashi , Sobue Shizuo , Hamada Shigeyuki
Published: July 2002

Streptococcus oralis
is a member of the oral streptococcal family and an early-colonizing microorganism in the oral cavity of humans.
S. oralis
is known to produce glucosyltransferase (GTase), which synthesizes glucans from sucrose. The enzyme was purified chromatographically from a culture supernatant of
S. oralis
ATCC 10557. The purified enzyme, GTase-R, had a molecular mass of 173 kDa and a pI of 6.3. This enzyme mainly synthesized water-soluble glucans with no primer dependency. The addition of GTase markedly enhanced the sucrose-dependent resting cell adhesion of
Streptococcus mutans
at a level similar to that found in growing cells of
S. mutans
. The antibody against GTase-R inhibited the glucan-synthesizing activities of
Streptococcus gordonii
and
Streptococcus sanguis
, as well as
S. oralis
. The N-terminal amino acid sequence of GTase-R exhibited no similarities to known GTase sequences of oral streptococci. Using degenerate PCR primers, an 8.1-kb DNA fragment, carrying the gene (
gtfR
) coding for GTase-R and its regulator gene (
rgg
), was cloned and sequenced. Comparison of the deduced amino acid sequence revealed that the
rgg
genes of
S. oralis
and
S. gordonii
exhibited a close similarity. The
gtfR
gene was found to possess a species-specific nucleotide sequence corresponding to the N-terminal 130 amino acid residues. Insertion of
erm
or
aphA
into the
rgg
or
gtfR
gene resulted in decreased GTase activity by the organism and changed the colony morphology of these transformants. These results indicate that
S. oralis
GTase may play an important role in the subsequent colonizing of mutans streptoccoci.
Cytophilic Immunoglobulin Responses toPlasmodium falciparumGlutamate-Rich Protein Are Correlated with Protection against Clinical Malaria in Dielmo, Senegal
Authors: Oeuvray Claude , Theisen Michael , Rogier Christophe , Trape Jean-Francois , Jepsen Søren , Druilhe Pierre
Published: July 2002

The goal of this study was to analyze antibody responses toPlasmodium falciparumglutamate-rich protein (GLURP) using clinical data and plasma samples obtained from villagers of Dielmo, Senegal. This molecule was chosen because it is targeted by human antibodies which induce parasite growth inhibition in antibody-dependent cellular inhibition (ADCI) assays. The results showed a strong correlation between protection against malaria attacks and levels of immunoglobulin G2 (IgG2) and IgG3 against GLURP94–489(R0) and IgG3 against GLURP705–1178(R2) when corrected for the confounding effect of age-related exposure to malaria. Thus, GLURP may play a role in the induction of protective immunity againstP. falciparummalaria.
Pyrogenic Toxin Superantigen Site Specificity in Toxic Shock Syndrome and Food Poisoning in Animals
Authors: Schlievert Patrick M. , Jablonski Lynn M. , Roggiani Manuela , Sadler Ingrid , Callantine Scott , Mitchell David T. , Ohlendorf Douglas H. , Bohach Gregory A.
Published: July 2002

Staphylococcus aureusandStreptococcus pyogenesexpress pyrogenic toxin superantigens (PTSAgs) that are associated with toxic shock syndrome (TSS) and staphylococcal food poisoning (SFP). Most PTSAgs cause TSS in deep-tissue infections, whereas only TSS toxin 1 (TSST-1) is associated with menstrual, vaginal TSS. In contrast, SFP has been linked only with staphylococcal enterotoxins (SEs). Because of the differential abilities of PTSAgs to cause systemic or localized symptoms in a site-dependent manner, the present study was undertaken to assess the toxins' abilities to cross mucosal barriers. The activity of three PTSAgs when delivered orally, vaginally, or intravenously to rabbits and orally to monkeys was investigated. TSST-1 induced shock via all three routes in rabbits. Although active when administered intravenously, SEC1 and streptococcal pyrogenic exotoxin A (SPEA) did not cause symptoms when administered orally or vaginally. Only SEC1 induced emesis in the monkey feeding assay. TSST-1, albeit less stable than SEC1 and SPEA to pepsin, induced diarrhea in monkeys. Our results may explain the unique association of TSST-1 with menstrual TSS and why SPEA is only rarely associated with TSS after pharyngitis, despite being highly associated with TSS after subcutaneous infections. Finally, our studies indicate that enterotoxicity in SFP is not the result of superantigenicity.
Constitutive Mutations of the
Salmonella enterica
Serovar Typhimurium Transcriptional Virulence Regulator
phoP
Authors: Gunn John S. , Ernst Robert K. , McCoy Andrea J. , Miller Samuel I.
Published: July 2002

The PhoP-PhoQ two-component system is necessary for the virulence of
Salmonella
spp. and is responsible for regulating several modifications of the lipopolysaccharide (LPS). Mutagenesis of the transcriptional regulator
phoP
resulted in the identification of a mutant able to activate transcription of regulated genes ∼100-fold in the absence of PhoQ. Sequence analysis showed two single-base alterations resulting in amino acid changes at positions 93 (S93N) and 203 (Q203R). These mutations were individually created, and although each resulted in a constitutive phenotype, the double mutant displayed a synergistic effect both in the induction of PhoP-activated gene expression and in resistance to antimicrobial peptides. The constitutive
phoP
gene was placed under the control of an arabinose-inducible promoter to examine the kinetics of PhoP-activated gene induction and the resultant modifications of LPS. Gene induction and 2-hydroxymyristate modification of the lipid A were shown to occur within minutes of the addition of arabinose and to peak at 4 h. As the first constitutive mutant of
phoP
identified, this allele will be invaluable to future genetic and biochemical studies of this and likely other regulatory systems.
Natural History of
Streptococcus sanguinis
in the Oral Cavity of Infants: Evidence for a Discrete Window of Infectivity
Authors: Caufield Page W. , Dasanayake Ananda P. , Li Yihong , Pan Yaping , Hsu Jay , Hardin J. Michael
Published: July 2002

The heterogeneous group of oral bacteria within the sanguinis (sanguis) streptococci comprise members of the indigenous biota of the human oral cavity. While the association of
Streptococcus sanguinis
with bacterial endocarditis is well described in the literature,
S. sanguinis
is thought to play a benign, if not a beneficial, role in the oral cavity. Little is known, however, about the natural history of
S. sanguinis
and its specific relationship with other oral bacteria. As part of a longitudinal study concerning the transmission and acquisition of oral bacteria within mother-infant pairs, we examined the initial acquisition of
S. sanguinis
and described its colonization relative to tooth emergence and its proportions in plaque and saliva as a function of other biological events, including subsequent colonization with mutans streptococci. A second cohort of infants was recruited to define the taxonomic affiliation of
S. sanguinis
. We found that the colonization of the
S. sanguinis
occurs during a discrete “window of infectivity” at a median age of 9 months in the infants. Its colonization is tooth dependent and correlated to the time of tooth emergence; its proportions in saliva increase as new teeth emerge. In addition, early colonization of
S. sanguinis
and its elevated levels in the oral cavity were correlated to a significant delay in the colonization of mutans streptococci. Underpinning this apparent antagonism between
S. sanguinis
and mutans streptococci is the observation that after mutans streptococci colonize the infant, the levels of
S. sanguinis
decrease. Children who do not harbor detectable levels of mutans streptococci have significantly higher levels of
S. sanguinis
in their saliva than do children colonized with mutans streptococci. Collectively, these findings suggest that the colonization of
S. sanguinis
may influence the subsequent colonization of mutans streptococci, and this in turn may suggest several ecological approaches toward controlling dental caries.
High Intracellular Level of Guanosine Tetraphosphate in
Mycobacterium smegmatis
Changes the Morphology of the Bacterium
Authors: Ojha Anil K. , Mukherjee Tapan K. , Chatterji Dipankar
Published: July 2002

Almost one-third of the world population today harbors the tubercle bacillus asymptomatically. It is postulated that the morphology and staining pattern of the long-term persistors are different from those of actively growing culture. Interestingly, it has been found that the morphology and staining pattern of the starved in vitro population of mycobacteria is similar to the persistors obtained from the lung lesions. In order to delineate the biochemical characteristics of starved mycobacteria,
Mycobacteria smegmatis
was grown in 0.2% glucose as a sole carbon source along with an enriched culture in 2% glucose. Accumulation of the stringent factor guanosine tetraphosphate (ppGpp) with a concomitant change in morphology was observed for
M. smegmatis
under carbon-deprived conditions. In addition,
M. smegmatis
assumed a coccoid morphology when ppGpp was ectopically produced by overexpressing
Escherichia coli relA
, even in an enriched medium. The
Mycobacterium tuberculosis relA
and
spoT
homologue, when induced in
M. smegmatis
, also resulted in the overproduction of ppGpp with a change in the bacterium's growth characteristics.
CD4+Cells Are Indispensable for Ulcer Development in Murine Cutaneous Leishmaniasis
Authors: Terabe Masaki , Kuramochi Takashi , Ito Mamoru , Hatabu Toshimitsu , Sanjoba Chizu , Chang Kwang-Poo , Onodera Takashi , Matsumoto Yoshitsugu
Published: July 2002

One of the most characteristic clinical features in cutaneous leishmaniasis is the development of nodules followed by ulcerations at the site of infection.Leishmania amazonensis-infected mice show similar ulcerative lesions.Leishmania-infected severe combined immunodeficiency (SCID) mice, however, have been shown to develop nonulcerative nodules. In the present study, the roles of T cells in ulceration were examined using SCID mice in cell reconstitution experiments. After development of nonulcerative nodules, SCID mice were inoculated with splenocytes from eitherLeishmania-infected or naive immunocompetent mice, resulting in ulceration in all mice. When naive splenocytes were depleted of CD4+, CD8+, or B220+cell populations and the remaining cells were injected intoLeishmania-infected SCID mice after the development of nodules, only SCID mice inoculated with splenocytes depleted of CD4+cells did not show ulceration. The evidence obtained in this study clearly shows that the CD4+cell population is indispensable for ulceration in leishmaniasis lesions of SCID mice.