bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacterial Endophthalmitis: Epidemiology, Therapeutics, and Bacterium-Host Interactions
Authors: Callegan Michelle C. , Engelbert Michael , Parke David W. , Jett Bradley D. , Gilmore Michael S.
Published: July 2002

Endophthalmitis is a severe inflammation of the interior of the eye caused by the introduction of contaminating microorganisms following trauma, surgery, or hematogenous spread from a distant infection site. Despite appropriate therapeutic intervention, bacterial endophthalmitis frequently results in visual loss, if not loss of the eye itself. Although the pathogenicity of bacterial endophthalmitis has historically been linked with toxin production during infection, a paucity of information exists as to the exact mechanisms of retinal toxicity and the triggers for induction of the intraocular immune response. Recently, research has begun to examine the bacterial and host molecular and cellular events that contribute to ocular damage during endophthalmitis. This review focuses on the causative agents and therapeutic challenges of bacterial endophthalmitis and provides current data from the analysis of the role of bacterial virulence factors and host inflammatory interactions in the pathogenesis of eye infections. Based on these and related studies, a hypothetical model for the molecular pathogenesis of bacterial endopthalmitis is proposed. Identifying and understanding the basic mechanisms of these bacterium-host interactions will provide the foundation for which novel, information-based therapeutic agents are developed in order to prevent vision loss during endophthalmitis.
Immunology of Diseases Associated withMalasseziaSpecies
Authors: Ashbee H. Ruth , Evans E. Glyn V.
Published: July 2002

Malassezia species are members of the human cutaneous commensal flora, in addition to causing a wide range of cutaneous and systemic diseases in suitably predisposed individuals. Studies examining cellular and humoral immune responses specific to Malassezia species in patients with Malassezia-associated diseases and healthy controls have generally been unable to define significant differences in their immune response. The use of varied antigenic preparations and strains from different Malassezia classifications may partly be responsible for this, although these problems can now be overcome by using techniques based on recent work defining some important antigens and also a new taxonomy for the genus. The finding that the genus Malassezia is immunomodulatory is important in understanding its ability to cause disease. Stimulation of the reticuloendothelial system and activation of the complement cascade contrasts with its ability to suppress cytokine release and downregulate phagocytic uptake and killing. The lipid-rich layer around the yeast appears to be pivotal in this alteration of phenotype. Defining the nonspecific immune response to Malassezia species and the way in which the organisms modulate it may well be the key to understanding how Malassezia species can exist as both commensals and pathogens.
Gamma Interferon Influences Intestinal Epithelial Hyperplasia Caused byLawsonia intracellularisInfection in Mice
Authors: Smith David G. E. , Mitchell Suzanne C. , Nash Tony , Rhind Susan
Published: July 2002

Lawsonia intracellularisis a recently identified bacterial pathogen which causes disease in a broad range of animals. Invasion of intestinal epithelial cells and the resultant hyperplasia of infected cells are central processes in disease pathogenesis. In this study, we aimed to establish whether immunocompetent mice were susceptible to infection and whether gamma interferon (IFN-γ) contributed to the pathogenesis of infection. Wild-type 129-Sv-Ev mice (129 mice) and IFN-γ receptor knockout mice based on the 129 background (IFN-γR−) were challenged orally with approximately 5.5 × 107L. intracellulariscells. Both 129 and IFN-γR−mice became infected, although the extent of infection (as determined by the proportion of infected crypts) was substantially lower in 129 mice than in IFN-γR−mice. Despite these differences, infected crypts showed characteristics typical of proliferative enteropathies of other animals, i.e., intracellular colonization of epithelial cells byL. intracellulariswith resultant epithelial hyperplasia. Infection in 129 mice was cleared between days 21 and 28 postchallenge, whereas infection in IFN-γR−mice was evident in 100% of animals from day 21 onward. Additionally, in IFN-γR−mice the infection was so extensive that fatalities resulted. IFN-γ therefore plays a significant role in limiting intracellular infection and increased cellular proliferation associated withL. intracellularis. L. intracellularisinfection is generally associated with modest cellular infiltration; therefore, further comparative examinations will be necessary to determine pathogenicity factors and define the role of IFN-γ in controlling this infection.
Neutrophils Play a Protective Nonphagocytic Role in SystemicMycobacterium tuberculosisInfection of Mice
Authors: Pedrosa Jorge , Saunders Bernadette M. , Appelberg Rui , Orme Ian M. , Silva Manuel T. , Cooper Andrea M.
Published: July 2002

Evidence showing that neutrophils play a protective role in the host response to infection by different intracellular parasites has been published in the past few years. We assessed this issue with regard to the infection of mice withMycobacterium tuberculosis. We found a chronic recruitment of neutrophils to the infection foci, namely, to the peritoneal cavity after intraperitoneal infection and to the spleen and liver after intravenous inoculation of the mycobacteria. However, bacilli were never found associated with the recruited neutrophils but rather were found inside macrophages. The intravenous administration of the antineutrophil monoclonal antibody RB6-8C5 during the first week of infection led to selective and severe neutropenia associated with an enhancement of bacillary growth in the target organs of the mice infected by the intravenous route. The neutropenia-associated exacerbation of infection was most important in the liver, where a bacterial load 10-fold higher than that in nonneutropenic mice was found; the exacerbation in the liver occurred both during and after the neutropenic period. Early in infection byM. tuberculosis, neutropenic mice expressed lower levels of mRNAs for gamma interferon and inducible nitric oxide synthase in the liver compared to nondepleted mice. These results point to a protective role of neutrophils in the host defense mechanisms againstM. tuberculosis, which occurs early in the infection and is not associated with the phagocytic activity of neutrophils but may be of an immunomodulatory nature.
Immunogenicity and Tolerance of a 7-Valent Pneumococcal Conjugate Vaccine in Nonresponders to the 23-Valent Pneumococcal Vaccine
Authors: Zielen S. , Bühring I. , Strnad N. , Reichenbach J. , Hofmann D.
Published: July 2002

There is still a lack of effective vaccination strategies for patients with a deficient antibody response to bacterial polysaccharide antigens. In an open trial, we evaluated the immunogenicity and tolerance of a new 7-valent pneumococcal conjugate vaccine in 22 infection-prone nonresponders to pneumococcal polysaccharide vaccine and 21 controls. In the patient group, nonresponsiveness was confirmed by repeated vaccination with a 23-valent pneumococcal polysaccharide vaccine. The study protocol provided two doses of the pneumococcal conjugate vaccine, given 4 to 6 weeks apart, for both groups. The antibody response was determined before each vaccination and on follow-up by an enzyme-linked immunosorbent assay and compared to the response in a functional opsonophagocytosis assay. Patients showed a significantly lower postvaccination immune response for all serotypes than did controls. The postvaccination response was serotype dependent. A median titer of >1 μg/ml in patients was recorded only for serotypes 4, 9V, 14, and 19F, which are known to be more immunogenic than serotypes 6B, 18C, and 23F. In the patient group, 70% responded to serotype 19F (Pnc 19F), 65% responded to Pnc 14 and 4, 60% responded to Pnc 9V, 55% responded to Pnc 18C, 50% responded to Pnc 23F, and 25% responded to Pnc 6B. In the control group >95% of individuals showed a titer of >1 μg/ml to every serotype. The vaccine was tolerated well, and no major side effects have been reported. The new pneumococcal conjugate vaccine is clearly more immunogenic in previous nonresponders than is the 23-valent pneumococcal vaccine. Immunization with a pneumococcal conjugate vaccine should be considered as a strategy to protect high-risk patients.
The Virulence Regulatory Protein ToxR Mediates Enhanced Bile Resistance in
Vibrio cholerae
and Other Pathogenic
Vibrio
Species
Authors: Provenzano Daniele , Schuhmacher Darren A. , Barker Justin L. , Klose Karl E.
Published: July 2002

The transmembrane regulatory protein ToxR is required for expression of virulence factors in the human diarrheal pathogen
Vibrio cholerae
, including cholera toxin (CT) and the toxin coregulated pilus (TCP). ToxR is necessary for transcription of the gene encoding a second regulatory protein, ToxT, which is the direct transcriptional activator of CT and TCP genes. However, ToxR, independent of ToxT, directly activates and represses transcription of the outer membrane porins OmpU and OmpT, respectively. The genes encoding TCP and CT (and including ToxT) lie on horizontally acquired genetic elements, while the
toxR
,
ompU
, and
ompT
genes are apparently in the ancestral
Vibrio
chromosome. The contribution of ToxR-dependent modulation of outer membrane porins to cholera pathogenesis has remained unknown. We demonstrate that ToxR mediates enhanced bile resistance in a ToxT-independent manner. In both classical and El Tor biotypes of
V. cholerae
, a
toxR
mutant strain has a reduced minimum bactericidal concentration (MBC) of bile, the bile component deoxycholate (DC), and the anionic detergent sodium dodecyl sulfate (SDS) compared to both wild-type and
toxT
mutant strains. Classical and El Tor
toxR
mutant strains also exhibit reduced growth rates at subinhibitory concentrations of DC and SDS. Growth of either
V. cholerae
biotype in subinhibitory concentrations of bile or DC induces increased ToxR-dependent production of a major 38-kDa outer membrane protein, which was confirmed to be OmpU by Western blot. Measurement of transcription of a
ompU
p-
lacZ
fusion in both biotypes reveals stimulation (about two- to threefold) of ToxR-dependent
ompU
transcription by the presence of bile or DC, suggesting that ToxR may respond to the presence of bile. The
toxR
mutant strains of three additional human intestinal pathogenic
Vibrio
species,
V. mimicus
,
V. fluvialis
, and
V. parahaemolyticus
, display lower MBCs of bile, DC, and SDS and have altered outer membrane protein profiles compared to the parental wild-type strains. Our results demonstrate a conserved role for ToxR in the modulation of outer membrane proteins and bile resistance of pathogenic
Vibrio
species and suggest that these ToxR-dependent outer membrane proteins may mediate enhanced resistance to bile. We speculate that ToxR-mediated bile resistance was an early step in the evolution of
V. cholerae
as an intestinal pathogen.
Phase 1 and Phase 2 Studies ofSalmonella entericaSerovar Paratyphi A O-Specific Polysaccharide-Tetanus Toxoid Conjugates in Adults, Teenagers, and 2- to 4-Year-Old Children in Vietnam
Authors: Konadu Edward Y. , Lin Feng-Ying C. , Hó Vô Anh , Thuy Nguyen Thi Thanh , Van Bay Phan , Thanh Tran Cong , Khiem Ha Ba , Trach Dang Duc , Karpas Arthur B. , Li Jianping , Bryla Dolores A. , Robbins John B. , Szu Shousun C.
Published: July 2002

Salmonella entericaserovar Paratyphi A O-specific polysaccharide (O-SP) was activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) and bound to tetanus toxoid (TT) with adipic acid dihydrazide as a linker (SPA-TT1) or directly (SPA-TT2). In mice, these two conjugates elicited high levels of immunoglobulin G (IgG) anti-lipopolysaccharide (LPS) in serum with bactericidal activity (E. Konadu, J. Shiloach, D. A. Bryla, J. B. Robbins, and S. C. Szu, Infect. Immun. 64:2709–2715, 1996). The safety and immunogenicity of the two conjugates were then evaluated sequentially in Vietnamese adults, teenagers, and 2- to 4-year-old children. None of the vaccinees experienced significant side effects, and all had preexisting LPS antibodies. At 4 weeks after injection, there were significant increases of the geometric mean IgG and IgM anti-LPS levels in the adults and teenagers: both conjugates elicited a greater than fourfold rise in the IgG anti-LPS level in serum in ≥80% of the volunteers. SPA-TT2elicited slightly higher, though not statistically significantly, levels of IgG anti-LPS than did SPA-TT1in these age groups. Accordingly, only SPA-TT2was evaluated in the 2- to 4-year-old children. On a random basis, one or two injections were administered 6 weeks apart to the children. No significant side effects were observed, and the levels of preexisting anti-LPS in serum were similar in children of all ages. A significant rise in the IgG anti-LPS titer was elicited by the first injection (P= 0.0001); a second injection did not elicit a booster response. Representative sera from all groups had bactericidal activity that could be adsorbed byS. entericaserovar Paratyphi A LPS.
The Leptospiral Major Outer Membrane Protein LipL32 Is a Lipoprotein Expressed during Mammalian Infection
Authors: Haake David A. , Chao Garlo , Zuerner Richard L. , Barnett Jeanne K. , Barnett Dean , Mazel Mary , Matsunaga James , Levett Paul N. , Bolin Carole A.
Published: July 2002

We report the cloning of the gene encoding the 32-kDa lipoprotein, designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containingEcoRI fragments ofLeptospira kirschneriDNA was screened, and a 5.0-kb DNA fragment which contained the entire structurallipL32gene was identified. Several lines of evidence indicate that LipL32 is lipid modified in a manner similar to that of other procaryotic lipoproteins. The deduced amino acid sequence of LipL32 would encode a 272-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by a lipoprotein signal peptidase cleavage site. LipL32 is intrinsically labeled during incubation ofL. kirschneriin media containing [3H]palmitate. The linkage of palmitate and the amino-terminal cysteine of LipL32 is acid labile. LipL32 is completely solubilized by Triton X-114 extraction ofL. kirschneri; phase separation results in partitioning of LipL32 exclusively into the hydrophobic, detergent phase, indicating that it is a component of the leptospiral outer membrane. CaCl2(20 mM) must be present during phase separation for recovery of LipL32. LipL32 is expressed not only during cultivation but also during mammalian infection. Immunohistochemistry demonstrated intense LipL32 reactivity withL. kirschneriinfecting proximal tubules of hamster kidneys. LipL32 is also a prominent immunogen during human leptospirosis. The sequence and expression of LipL32 is highly conserved among pathogenicLeptospiraspecies. These findings indicate that LipL32 may be important in the pathogenesis, diagnosis, and prevention of leptospirosis.
RecombinantStaphylococcus aureusExfoliative Toxins Are Not Bacterial Superantigens
Authors: Plano Lisa R. W. , Gutman Delia M. , Woischnik Markus , Collins Carleen M.
Published: July 2002

Staphylococcal scalded-skin syndrome is an exfoliative dermatitis characterized by the separation of the epidermis at the stratum granulosum. This disruption is mediated by one of twoStaphylococcus aureusexotoxins, exfoliative toxins A and B (ETA and ETB). Both ETA and ETB have been reported to be bacterial superantigens. A controversy exists, however, as other data indicate that these exotoxins are not superantigens. Here we demonstrate that recombinant exfoliative toxins produced inEscherichia colido not act as T-cell mitogens and thus are not bacterial superantigens. These data fit the clinical profile of the disease, which is not associated with the classic symptoms of a superantigen-mediated syndrome.
Brucella abortusStrain RB51 as a Vector for Heterologous Protein Expression and Induction of Specific Th1 Type Immune Responses
Authors: Vemulapalli Ramesh , He Yongqun , Boyle Stephen M. , Sriranganathan Nammalwar , Schurig Gerhardt G.
Published: July 2002

Brucella abortusstrain RB51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Our ultimate goal is to develop strain RB51 as a preferential vector for the delivery of protective antigens of other intracellular pathogens to which the induction of a strong Th1 type of immune response is needed for effective protection. As a first step in that direction, we studied the expression of a foreign reporter protein, β-galactosidase ofEscherichia coli, and the 65-kDa heat shock protein (HSP65) ofMycobacterium bovisin strain RB51. We cloned the promoter sequences ofBrucella sodCandgroEgenes in pBBR1MCS to generate plasmids pBBSODpro and pBBgroE, respectively. The genes for β-galactosidase (lacZ) and HSP65 were cloned in these plasmids and used to transform strain RB51. An enzyme assay in the recombinant RB51 strains indicated that the level of β-galactosidase expression is higher under thegroEpromoter than under thesodCpromoter. In strain RB51 containing pBBgroE/lacZ, but not pBBSODpro/lacZ, increased levels of β-galactosidase expression were observed after subjecting the bacteria to heat shock or following internalization into macrophage-like J774A.1 cells. Mice vaccinated with either of the β-galactosidase-expressing recombinant RB51 strains developed specific antibodies of predominantly the immunoglobulin G2a (IgG2a) isotype, and in vitro stimulation of their splenocytes with β-galactosidase induced the secretion of gamma interferon (IFN-γ), but not interleukin-4 (IL-4). A Th1 type of immune response to HSP65, as indicated by the presence of specific serum IgG2a, but not IgG1, antibodies, and IFN-γ, but not IL-4, secretion by the specific-antigen-stimulated splenocytes, was also detected in mice vaccinated with strain RB51 containing pBBgroE/hsp65. Studies with mice indicated that expression of β-galactosidase or HSP65 did not alter either the attenuation characteristics of strain RB51 or its vaccine efficacy againstB. abortus2308 challenge.
Clostridium perfringens
Iota Toxin: Binding Studies and Characterization of Cell Surface Receptor by Fluorescence-Activated Cytometry
Authors: Stiles Bradley G. , Hale Martha L. , Marvaud Jean-Christophe , Popoff Michel R.
Published: July 2002

The binding characteristics of iota toxin, a binary enterotoxin produced by
Clostridium perfringens
type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37°C for 10 min. The binding of Ib was inhibited by antisera against
C. perfringens
type E or
Clostridium spiroforme
culture supernatants, but not
C. perfringens
types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37°C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane.
Complementation ofBrucella abortusRB51 with a FunctionalwboAGene Results in O-Antigen Synthesis and Enhanced Vaccine Efficacy but No Change in Rough Phenotype and Attenuation
Authors: Vemulapalli Ramesh , He Yongqun , Buccolo Larissa S. , Boyle Stephen M. , Sriranganathan Nammalwar , Schurig Gerhardt G.
Published: July 2002

Brucella abortusRB51 is a stable rough, attenuated mutant vaccine strain derived from the virulent strain 2308. Recently, we demonstrated that thewboAgene in RB51 is disrupted by an IS711element (R. Vemulapalli, J. R. McQuiston, G. G. Schurig, N. Srirauganathan, S. M. Halling, and S. M. Boyle, Clin. Diagn. Lab. Immunol. 6:760–764, 1999). Disruption of thewboAgene in smooth, virulentB. abortus,Brucella melitensis, andBrucella suisresults in rough, attenuated mutants which fail to produce the O polysaccharide (O antigen). In this study, we explored whether thewboAgene disruption is responsible for the rough phenotype of RB51. We complemented RB51 with a functionalwboAgene, and the resulting strain was designated RB51WboA. Colony and Western blot analyses indicated that RB51WboA expressed the O antigen; immunoelectron microscopy revealed that the O antigen was present in the cytoplasm. Crystal violet staining, acryflavin agglutination, and polymyxin B sensitivity studies indicated that RB51WboA had rough phenotypic characteristics similar to those of RB51. Bacterial clearance studies of BALB/c mice indicated no increase in the survival ability of RB51WboA in vivo compared to that of RB51. Vaccination of mice with live RB51WboA induced antibodies to the O antigen which were predominantly of the immunoglobulin G2a (IgG2a) and IgG3 isotypes. After in vitro stimulation of splenocytes with killed bacterial cells, quantitation of gamma interferon in the culture supernatants indicated that RB51WboA immunization induced higher levels of gamma interferon than immunization with RB51. Mice vaccinated with RB51WboA were better protected against a challenge infection with the virulent strain 2308 than those vaccinated with RB51. These studies indicate that in addition to the disruption of thewboAgene there is at least one other mutation in RB51 responsible for its rough phenotype. These studies also suggest that the expressed O antigen in RB51WboA is responsible either directly or indirectly for the observed enhancement in the T-cell response.
Molecular Cloning and Expression of a Gene EncodingCryptosporidium parvumGlycoproteins gp40 and gp15
Authors: Cevallos Ana Maria , Zhang Xiaoping , Waldor Matthew K. , Jaison Smitha , Zhou Xiaoyin , Tzipori Saul , Neutra Marian R. , Ward Honorine D.
Published: July 2002

Cryptosporidium parvumis a significant cause of diarrheal disease worldwide. The specific molecules that mediateC. parvum-host cell interactions and the molecular mechanisms involved in the pathogenesis of cryptosporidiosis are unknown. In this study we have shown that gp40, a mucin-like glycoprotein, is localized to the surface and apical region of invasive stages of the parasite and is shed from its surface. gp40-specific antibodies neutralize infection in vitro, and native gp40 binds specifically to host cells, implicating this glycoprotein inC. parvumattachment to and invasion of host cells. We have cloned and sequenced a gene designatedCpgp40/15that encodes gp40 as well as gp15, an antigenically distinct, surface glycoprotein also implicated inC. parvum-host cell interactions. Analysis of the deduced amino acid sequence of the 981-bpCpgp40/15revealed the presence of an N-terminal signal peptide, a polyserine domain, multiple predictedO-glycosylation sites, a single potentialN-glycosylation site, and a hydrophobic region at the C terminus, a finding consistent with what is required for the addition of a GPI anchor. There is a single copy ofCpgp40/15in theC. parvumgenome, and this gene does not contain introns. Our data indicate that the twoCpgp40/15-encoded proteins, gp40 and gp15, are products of proteolytic cleavage of a 49-kDa precursor protein which is expressed in intracellular stages of the parasite. The surface localization of gp40 and gp15 and their involvement in the host-parasite interaction suggest that either or both of these glycoproteins may serve as effective targets for specific preventive or therapeutic measures for cryptosporidiosis.
Cloning and Sequence Analysis of a Highly PolymorphicCryptosporidium parvumGene Encoding a 60-Kilodalton Glycoprotein and Characterization of Its 15- and 45-Kilodalton Zoite Surface Antigen Products
Authors: Strong William B. , Gut Jiri , Nelson Richard G.
Published: July 2002

The apicomplexan parasiteCryptosporidium parvumis a major cause of serious diarrheal disease in both humans and animals. No efficacious chemo- or immunotherapies have been identified for cryptosporidiosis, but certain antibodies directed against zoite surface antigens and/or proteins shed by gliding zoites have been shown to neutralize infectivity in vitro and/or to passively protect against, or ameliorate, disease in vivo. We previously used monoclonal antibody 11A5 to identify a 15-kDa surface glycoprotein that was shed behind motile sporozoites and was recognized by several lectins that neutralized parasite infectivity for cultured epithelial cells. Here we report the cloning and sequence analysis of the gene encoding this 11A5 antigen. Surprisingly, the gene encoded a 330-amino-acid, mucin-like glycoprotein that was predicted to contain an N-terminal signal peptide, a homopolymeric tract of serine residues, 36 sites of O-linked glycosylation, and a hydrophobic C-terminal peptide specifying attachment of a glycosylphosphatidylinositol anchor. The single-copy gene lacked introns and was expressed during merogony to produce a 60-kDa precursor which was proteolytically cleaved to 15- and 45-kDa glycoprotein products that both localized to the surface of sporozoites and merozoites. The gp15/45/60 gene displayed a very high degree of sequence diversity amongC. parvumisolates, and the numerous single-nucleotide and single-amino-acid polymorphisms defined five to six allelic classes, each characterized by additional intra-allelic sequence variation. The gp15/45/60 single-nucleotide polymorphisms will prove useful for haplotyping and fingerprinting isolates and for establishing meaningful relationships betweenC. parvumgenotype and phenotype.
Genetic Characterization of a
Streptococcus mutans
LraI Family Operon and Role in Virulence
Authors: Kitten Todd , Munro Cindy L. , Michalek Suzanne M. , Macrina Francis L.
Published: July 2002

Proteins belonging to the LraI (for “lipoprotein receptor antigen”) family function as adhesins in several streptococci, as a virulence factor for endocarditis in at least one of these species, and potentially as metal transporters in many bacteria. We have identified and characterized the chromosomal locus containing the LraI family gene (designated
sloC
) from
Streptococcus mutans
, an agent of dental caries and endocarditis in humans. Northern blot analysis indicated that
sloC
is cotranscribed with three other genes. As with other LraI operons, the
sloA
and
sloB
genes apparently encode components of an ATP-binding cassette transport system. The product of the fourth gene,
sloR
, has homology to the metal-dependent regulator from
Corynebacterium diphtheriae
, DtxR. A potential binding site for SloR was identified upstream from the
sloABCR
operon and was conserved upstream from LraI operons in several other streptococci. Potential SloR homologs were identified in the unfinished genomic sequences from two of these,
S. pneumoniae
and
S. pyogenes
. Mutagenesis of
sloC
in
S. mutans
resulted in apparent loss of expression of the entire operon as assessed by Northern blot analysis. The
sloC
mutant was indistinguishable from its wild-type parent in a gnotobiotic rat model of caries but was significantly less virulent in a rat model of endocarditis. Virulence for endocarditis was restored by correction of the
sloC
mutation but not by provision of the
sloC
gene in
trans
, suggesting that virulence requires the expression of other genes in the
sloC
operon.
Invasion of Epithelial Cells byYersinia pestis: Evidence for aY. pestis-Specific Invasin
Authors: Cowan Clarissa , Jones Heather A. , Kaya Yasemin H. , Perry Robert D. , Straley Susan C.
Published: July 2002

The causative agent of plague,Yersinia pestis, is regarded as being noninvasive for epithelial cells and lacks the major adhesins and invasins of its enteropathogenic relativesYersinia enterocoliticaandYersinia pseudotuberculosis. However, there are studies indicating thatY. pestisinvades and causes systemic infection from ingestive and aerogenic routes of infection. Accordingly, we developed a gentamicin protection assay and reexamined invasiveness ofY. pestisfor HeLa cells. By optimizing this assay, we discovered thatY. pestisis highly invasive. Several factors, including the presence of fetal bovine serum, the configuration of the tissue culture plate, the temperature at which the bacteria are grown, and the presence of the plasminogen activator protease Pla-encoding plasmid pPCP1, were found to influence invasiveness strongly. Suboptimal combinations of these factors may have contributed to negative findings by previous studies attempting to demonstrate invasion byY. pestis. Invasion of HeLa cells was strongly inhibited by cytochalasin D and modestly inhibited by colchicine, indicating strong and modest respective requirements for microfilaments and microtubules. We found no significant effect of the iron status of yersiniae or of the pigmentation locus on invasion and likewise no significant effect of the Yops regulon. However, an unidentified thermally induced property (possibly theY. pestis-specific capsular protein Caf1) did inhibit invasiveness significantly, and the plasmid pPCP1, unique toY. pestis, was essential for highly efficient invasion. pPCP1 encodes an invasion-promoting factor and not just an adhesin, becauseY. pestislacking this plasmid still adhered to HeLa cells. These studies have enlarged our picture ofY. pestisbiology and revealed the importance of properties that are unique toY. pestis.
Expression of ExsA in
trans
Confers Type III Secretion System-Dependent Cytotoxicity on Noncytotoxic
Pseudomonas aeruginosa
Cystic Fibrosis Isolates
Authors: Dacheux Denis , Attree Ina , Toussaint Bertrand
Published: July 2002

Twelve
Pseudomonas aeruginosa
cystic fibrosis isolates that are not able to exert a type III secretion system (TTSS)-dependent cytotoxicity towards phagocytes have been further studied. The strains, although possessing TTSS genes and
exsA
, which encodes a positive regulator of the TTSS regulon, showed no transcriptional activation of the
exsCBA
regulatory operon. The expression of
exsA
in trans restored the in vitro secretion of TTSS proteins and ex vivo cytotoxicity.
Mimicry of a G Protein Mutation by Pertussis Toxin Expression in Transgenic
Caenorhabditis elegans
Authors: Darby Creg , Falkow Stanley
Published: July 2002

Pathogens produce virulence factors that interact directly with host molecules, but in many cases the host targets are unknown. The genetic and molecular identification of these orphan targets is often not feasible with mammalian experimental models. However, a substantial number of known targets are molecules and pathways that are conserved among eukaryotes, and therefore the use of nonmammalian model hosts to identify orphan targets may prove useful. To demonstrate the feasibility of this approach, we transformed the nematode
Caenorhabditis elegans
with a gene encoding the catalytic subunit of pertussis toxin (PTX), which in mammals inactivates G
o/i
α proteins. Expression of PTX in
C. elegans
produced phenotypes almost identical to those of a null mutation in the nematode gene encoding G
o/i
α. Furthermore, PTX suppressed the phenotype of a constitutively active form of nematode G
o/i
α protein. These results indicate that PTX is functional in nematodes and acts specifically on the
C. elegans
homologue of the mammalian target.
Transient Transgenic Expression of Gamma Interferon PromotesLegionella pneumophilaClearance in Immunocompetent Hosts
Authors: Deng Jane C. , Tateda Kazuhiro , Zeng Xianying , Standiford Theodore J.
Published: July 2002

Gamma interferon (IFN-γ) and T1-phenotype immune responses are important components of host defense against a variety of intracellular pathogens, includingLegionella pneumophila. The benefit of intrapulmonary adenovirus-mediated IFN-γ gene therapy was investigated in a nonlethal murine model of experimentalL. pneumophilapneumonia. Intratracheal (i.t.) administration of 106CFU ofL. pneumophilainduced the expression of T1 phenotype cytokines, such as IFN-γ and interleukin-12 (IL-12). Natural killer cells were identified as the major cellular source of IFN-γ. To determine if enhanced expression of IFN-γ in the lung could promote pulmonary clearance ofL. pneumophila, we i.t. administered 5 × 108PFU of a recombinant adenovirus vector containing the murine IFN-γ cDNA (AdmIFN-γ) concomitant withL. pneumophila. We observed a 10-fold decrease in lung bacterial CFU at day 2 in the AdmIFN-γ-treated group compared to controls (P< 0.01). Alveolar macrophages isolated from AdmIFN-γ-treated animals displayed enhanced killing of intracellularL. pneumophilaorganisms ex vivo. Similar improvements in bacterial clearance were observed with i.t. recombinant IFN-γ treatment. The transient transgenic expression of IL-12, a known inducer of IFN-γ and promoter of T1-type immune responses, resulted in more modest improvement in bacterial clearance (sixfold reduction;P< 0.05). These results demonstrate that, even in immunocompetent hosts, exogenous administration or transient transgenic expression of IFN-γ, and to a lesser extent IL-12, may be of potential therapeutic benefit in the treatment of patients withLegionellapneumonia.
Bartonella henselae-Specific Cell-Mediated Immune Responses Display a Predominantly Th1 Phenotype in Experimentally Infected C57BL/6 Mice
Authors: Arvand Mardjan , Ignatius Ralf , Regnath Thomas , Hahn Helmut , Mielke Martin E. A.
Published: July 2002

Immune responses of the immunocompetent host toBartonella henselaeinfection were investigated in the murine infection model using C57BL/6 mice. Following intraperitoneal infection with human-derivedB. henselaestrain Berlin-1, viable bacteria could be recovered from livers and spleens during the first week postinfection, whileBartonellaDNA remained detectable by PCR in the liver for up to 12 weeks after infection. Granulomatous lesions developed in livers of infected mice, reached maximal density at 12 weeks after infection, and persisted for up to 20 weeks, indicating thatB. henselaeinduced a chronic granulomatous hepatitis in the immunocompetent murine host. T-cell-mediated immune responses were analyzed in vitro by means of spleen cell proliferation and cytokine release assays as well as analysis of immunoglobulin G (IgG) isotypes. Spleen cells from infected mice proliferated specifically upon stimulation with heat-killedBartonellaantigen. Proliferative responses were mainly mediated by CD4+T cells, increased during the course of infection, peaked at 8 weeks postinfection, and decreased thereafter. Gamma interferon, but not interleukin-4, was produced in vitro by spleen cells from infected animals upon stimulation withBartonellaantigens.Bartonella-specific IgG was detectable in serum of infected mice by 2 weeks, and the antibody concentration peaked at 12 weeks postinfection. IgG2b was the prominent isotype among theBartonella-specific serum IgG antibodies. These data indicate thatB. henselaeinduces cell-mediated immune responses with a Th1 phenotype in immunocompetent C57BL/6 mice.
Candida albicansIs Phagocytosed, Killed, and Processed for Antigen Presentation by Human Dendritic Cells
Authors: Newman Simon L. , Holly Angela
Published: July 2002

Candida albicansis a component of the normal flora of the alimentary tract and also is found on the mucocutaneous membranes of the healthy host.Candidais the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients, and of oropharyngeal disease in AIDS patients. As the induction of cell-mediated immunity toCandidais of critical importance in host defense, we sought to determine whether human dendritic cells (DC) could phagocytose and degradeCandidaand subsequently presentCandidaantigens to T cells. Immature DC obtained by culture of human monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 phagocytosed unopsonizedCandidain a time-dependent manner, and phagocytosis was not enhanced by opsonization ofCandidain serum. Like macrophages (Mφ), DC recognizedCandidaby the mannose-fucose receptor. Upon ingestion, DC killedCandidaas efficiently as human Mφ, and fungicidal activity was not enhanced by the presence of fresh serum. Although phagocytosis ofCandidaby DC stimulated the production of superoxide anion, inhibitors of the respiratory burst (or NO production) did not inhibit killing ofCandida, even when phagocytosis was blocked by preincubation of DC with cytochalasin D. Further, although apparently only modest phagolysosomal fusion occurred upon DC phagocytosis ofCandida, killing ofCandidaunder anaerobic conditions was almost equivalent to killing under aerobic conditions. Finally, DC stimulatedCandida-specific lymphocyte proliferation in a concentration-dependent manner after phagocytosis of both viable and heat-killedCandidacells. These data suggest that, in vivo, such interactions between DC andC. albicansmay facilitate the induction of cell-mediated immunity.
cDNA Array Analysis ofcagPathogenicity Island-AssociatedHelicobacter pyloriEpithelial Cell Response Genes
Authors: Cox Joanne M. , Clayton Christopher L. , Tomita Toshihiko , Wallace Don M. , Robinson Philip A. , Crabtree Jean E.
Published: July 2002

Helicobacter pyloristrains containing thecagpathogenicity island (PAI) induce NF-κB activation and interleukin-8 secretion in gastric epithelial cells. The aim of this study was to investigate changes in epithelial gene expression induced bycagPAI-positive and -negative strains ofH. pyloriusing high-density cDNA array hybridization technology. Radio-labeled cDNA prepared fromH. pylori-infected Kato 3 gastric epithelial cells was hybridized to high-density cDNA arrays to identify changes in epithelial gene expression compared to noninfected controls. In vivo expression of selected, differentially expressed genes was examined by reverse transcription-PCR analysis ofH. pylori-positive and -negative gastric mucosa. Screening of ca. 57,800 cDNAs identified 208 known genes and 48 novel genes and/or expressed sequence tags of unknown function to be differentially expressed in Kato 3 cells followingH. pyloriinfection. Marked differences in gene expression profiles were observed followingcagPAI-positive andcagPAI-negative infection with 15 novel cDNAs and 92 known genes being differentially expressed.H. pyloriwas found to change the expression of genes encoding growth factors and cytokine/chemokines and their receptors, apoptosis proteins, transcription factors and metalloprotease-disintegrin proteins (ADAMs), and tissue inhibitors of metalloproteinases. Gastric differential expression of selected known genes (amphiregulin and ADAM 10) and a novel gene (HPYR1) was confirmed in vivo in patients withH. pyloriinfection. Confirmation of the in vivo expression of selected genes demonstrates the usefulness of this approach for investigating pathogen-induced changes in host gene expression.
Role of
Streptococcus gordonii
Amylase-Binding Protein A in Adhesion to Hydroxyapatite, Starch Metabolism, and Biofilm Formation
Authors: Rogers Jeffrey D. , Palmer Robert J. , Kolenbrander Paul E. , Scannapieco Frank A.
Published: July 2002

Interactions between bacteria and salivary components are thought to be important in the establishment and ecology of the oral microflora. α-Amylase, the predominant salivary enzyme in humans, binds to
Streptococcus gordonii
, a primary colonizer of the tooth. Previous studies have implicated this interaction in adhesion of the bacteria to salivary pellicles, catabolism of dietary starches, and biofilm formation. Amylase binding is mediated at least in part by the amylase-binding protein A (AbpA). To study the function of this protein, an erythromycin resistance determinant [
erm
(AM)] was inserted within the
abpA
gene of
S. gordonii
strains Challis and FAS4 by allelic exchange, resulting in
abpA
mutant strains Challis-E1 and FAS4-E1. Comparison of the wild-type and mutant strains did not reveal any significant differences in colony morphology, biochemical metabolic profiles, growth in complex or defined media, surface hydrophobicity, or coaggregation properties. Scatchard analysis of adhesion isotherms demonstrated that the wild-type strains adhered better to human parotid-saliva- and amylase-coated hydroxyapatite than did the AbpA mutants. In contrast, the mutant strains bound to whole-saliva-coated hydroxyapatite to a greater extent than did the wild-type strains. While the wild-type strains preincubated with purified salivary amylase grew well in defined medium with potato starch as the sole carbohydrate source, the AbpA mutants did not grow under the same conditions even after preincubation with amylase. In addition, the wild-type strain produced large microcolonies in a flow cell biofilm model, while the
abpA
mutant strains grew much more poorly and produced relatively small microcolonies. Taken together, these results suggest that AbpA of
S. gordonii
functions as an adhesin to amylase-coated hydroxyapatite, in salivary-amylase-mediated catabolism of dietary starches and in human saliva-supported biofilm formation by
S. gordonii.
Identification of Genes Induced In Vivo during
Klebsiella pneumoniae
CG43 Infection
Authors: Lai Yi-Chyi , Peng Hwei-Ling , Chang Hwan-You
Published: July 2002

A novel in vivo expression technology (IVET) was performed to identify
Klebsiella pneumoniae
CG43 genes that are specifically expressed during infection of BALB/c mice. The IVET employed a UDP glucose pyrophosphorylase (
galU
)-deficient mutant of
K. pneumoniae
which is incapable of utilizing galactose and synthesizing capsular polysaccharide, as demonstrated by its low virulence to BALB/c mice and a white nonmucoid colony morphology on MacConkey-galactose agar. By using a functional
galU
gene as the reporter, an IVE promoter could render the
galU
mutant virulent while maintaining the white nonmucoid colony phenotype. A total of 20 distinct sequences were obtained through the in vivo selection. Five of them have been identified previously as virulence-associated genes in other pathogens, while another five with characterized functions are involved in regulation and transportation of nutrient uptake, biosynthesis of isoprenoids, and protein folding. No known functions have been attributed to the other 10 sequences. We have also demonstrated that 2 of the 20 IVE genes turn on under iron deprivation, whereas the expression of another five genes was found to be activated in the presence of paraquat, a superoxide generator.
Pertussis Toxin and Lipopolysaccharide Influence Phagocytosis ofBordetella pertussisby Human Monocytes
Authors: Schaeffer Lyndsay M. , Weiss Alison A.
Published: July 2002

The potential of human monocytes to mediate the clearance ofBordetella pertussisinfection was examined. Bacteria expressing green fluorescent protein were incubated with adherent peripheral blood monocytes, and phagocytosis was quantified by using fluorescence microscopy. Monocytes internalized only a small percentage of the adherent bacteria. Surface-associated Bvg-regulated virulence factors, including adenylate cyclase toxin and filamentous hemagglutinin, did not affect attachment or phagocytosis. However, 1-h pretreatment with purified pertussis toxin inhibited the ability of monocytes to internalize wild-type bacteria. Mutations affecting the terminal trisaccharide of lipopolysaccharide resulted in reduced internalization without affecting adherence of bacteria to monocytes. Opsonization with human serum played only a modest role in promoting phagocytosis. The viability of internalized bacteria was determined by colony counts following treatment with polymyxin B and gentamicin. Less than 1% of internalized bacteria remained viable. These results suggest that pertussis toxin plays a role in the evasion of monocyte phagocytosis and that these cells represent a potential mediator of the clearance ofB. pertussisinfection.
Salmonella
DNA Adenine Methylase Mutants Elicit Protective Immune Responses to Homologous and Heterologous Serovars in Chickens
Authors: Dueger E. L. , House J. K. , Heithoff D. M. , Mahan M. J.
Published: July 2002

Salmonella
DNA adenine methylase (Dam) mutants that lack or overproduce Dam are highly attenuated for virulence in mice and confer protection against murine typhoid fever. To determine whether vaccines based on Dam are efficacious in poultry, a
Salmonella
Dam
−
vaccine was evaluated in the protection of chicken broilers against oral challenge with homologous and heterologous
Salmonella
serovars. A
Salmonella enterica
serovar Typhimurium Dam
−
vaccine strain was attenuated for virulence in day-of-hatch chicks more than 100,000-fold. Vaccination of chicks elicited cross-protective immune responses, as evidenced by reduced colonization (10- to 10,000-fold) of the gastrointestinal tract (ileum, cecum, and feces) and visceral organs (bursa and spleen) after challenge with homologous (Typhimurium F98) and heterologous (Enteritidis 4973 and
S. enterica
O6,14,24: e,h-monophasic)
Salmonella
serovars that are implicated in
Salmonella
infection of poultry. The protection conferred was observed for the organ or the maximum CFU/tissue/bird as a unit of analysis, suggesting that Dam mutant strains may serve as the basis for the development of efficacious poultry vaccines for the containment of
Salmonella
.
Capsular Reactions ofCryptococcus neoformanswith Polyspecific and Oligospecific Polyclonal Anticapsular Antibodies
Authors: MacGill Tracy C. , MacGill Randall S. , Kozel Thomas R.
Published: July 2002

Monoclonal antibodies to the capsular polysaccharide ofCryptococcus neoformansproduce distinct capsular reactions and have biological activities that are determined by serotype specificity. In the present study, polyclonal rabbit anticapsular antibodies were cross-absorbed to produce serotype specificities similar to those of monoclonal antibodies. The results showed that polyclonal and monoclonal antibodies with similar serotype specificities have similar capsular reactions and biological activities.
C-Terminal Invariable Domain of VlsE May Not Serve as Target for Protective Immune Response againstBorrelia burgdorferi
Authors: Liang Fang Ting , Jacobs Mary B. , Philipp Mario T.
Published: July 2002

VlsE, the variable surface antigen of the Lyme disease spirochete,Borrelia burgdorferi, contains two invariable domains, at the amino and carboxyl termini, respectively, which collectively account for approximately one-half of the entire molecule's length and remain unchanged during antigenic variation. It is not known if these two invariable domains are exposed at the surface of either the antigen or the spirochete. If they are exposed at the spirochete's surface, they may elicit a protective immune response againstB. burgdorferiand serve as vaccine candidates. In this study, a 51-mer synthetic peptide that reproduced the entire sequence of the C-terminal invariable domain of VlsE was conjugated to the carrier keyhole limpet hemocyanin and used to immunize mice. Generated mouse antibody was able to immunoprecipitate native VlsE extracted from culturedB. burgdorferiB31 spirochetes, indicating that the C-terminal invariable domain was exposed at the antigen's surface. However, this domain was inaccessible to antibody binding at the surface of cultured intact spirochetes, as demonstrated by both an immunofluorescence experiment and an in vitro killing assay. Mouse antibody to the C-terminal invariable domain was not able to confer protection againstB. burgdorferiinfection, indicating that this domain was unlikely exposed at the spirochete's surface in vivo. We concluded that the C-terminal invariable domain was exposed at the antigen's surface but not at the surface of either cultured or in vivo spirochetes and thus cannot elicit protection againstB. burgdorferiinfection.