bluuebunny/crossref_metadata_2025 huggingface dataset:
Infection of Human Monocyte-Derived Macrophages withChlamydia trachomatisInduces Apoptosis of T Cells: a Potential Mechanism for Persistent Infection
Authors: Jendro Michael C. , Deutsch Tobias , Körber Britta , Köhler Lars , Kuipers Jens G. , Krausse-Opatz Birgit , Westermann Jürgen , Raum Elke , Zeidler Henning
Published: July 2002

Viruses can escape T-cell surveillance by infecting macrophages and thereby induce apoptosis of noninfected T cells. This ability had not been demonstrated for bacteria. We investigated whether infection of macrophages with the important human pathogenChlamydia trachomatiscan induce T-cell apoptosis. BecauseChlamydia-Mycoplasmacoinfection is a frequent event, the ability ofMycoplasma fermentans-infected macrophages to induce T-cell apoptosis was also studied. Infected macrophages were cocultivated with autologous T cells in different activation states. Propidium iodide-based fluorescence-activated cell sorter analysis demonstrated that macrophages infected with viable chlamydiae induced T-cell death. Apoptosis was identified as the mode of death induction by using a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay. Induction of T-cell death was macrophage dependent. Incubation of T cells with infectious chlamydiae in the absence of macrophages did not lead to T-cell apoptosis. UV irradiation of chlamydiae diminished the ability to induce death. T-cell death was induced by a cell-free supernatant of infected macrophages. Not only phytohemagglutinin-preactivated T cells but also non-mitogen-preactivated T cells were susceptible toC. trachomatis-induced apoptosis. In contrast,M. fermentansinfection of macrophages did not induce T-cell death. Coinfection had no additional effect. In summary, intracellular chlamydial infection of macrophages can induce T-cell apoptosis. Apoptosis induction by chlamydiae possibly explains how persistently infected macrophages escape T-cell surveillance and why theChlamydia-specific T-cell response is diminished during persistent chlamydial infection.
Repression of Hyphal Proteinase Expression by the Mitogen-Activated Protein (MAP) Kinase Phosphatase Cpp1p of
Candida albicans
Is Independent of the MAP Kinase Cek1p
Authors: Schröppel Klaus , Sprößer Katrin , Whiteway Malcolm , Thomas David Y. , Röllinghoff Martin , Csank Csilla
Published: July 2002

Cpp1p is a putative mitogen-activated protein (MAP) kinase phosphatase that suppresses
Candida albicans
hyphal formation at 25°C through its probable substrate, the Cek1p filamentation MAP kinase. Here we report that expression of the serum-induced genes
SAP4-6
and
HYR1
increased several fold in hyphal forms of a
cpp1/cpp1
null mutant, while the rate and extent of hyphal development up to 5 h were normal. Therefore, we provide evidence that Cpp1p represses hyphal gene expression by acting through a Cek1p-independent mechanism.
SAP4-6
and
HYR1
transcripts were undetectable in a null mutant of another key regulator of filamentation, Efg1p; thus, Efg1p and Cpp1p oppose each other during the expression of these genes in hyphal forms.
Molecular Characterization of
Mycoplasma arthritidis
Membrane Lipoprotein MAA1
Authors: Washburn Leigh Rice , Miller Elizabeth J. , Weaver Keith E.
Published: July 2002

Genes encoding the
Mycoplasma arthritidis
surface-exposed lipoprotein MAA1 were cloned and sequenced from MAA1-expressing strains 158p10p9 and PG6, from a low-adherence (LA) variant derived from 158p10p9 that expresses a truncated version of MAA1 (MAA1Δ) and from two MAA1-negative strains, 158 and H39. The deduced amino acid sequences of
maa1
from 158p10p9 and PG6 predicted, respectively, 86.5- and 86.4-kDa basic, largely hydrophilic lipoproteins with 29-amino-acid signal peptides and predicted cleavage sites for signal peptidase II (Ala-Ala-Ala↓Cys). The truncation in the LA variant resulted from a G→T substitution at nucleotide 695, which created a premature stop codon. This, in turn, generated a predicted 26.6-kDa prolipoprotein (23.6 kDa after processing), consistent with an
M
r
of ∼24,000 calculated for MAA1Δ. Similarly, absence of MAA1 expression in H39 and 158 resulted from C→A substitutions at nucleotide 208, generating premature stop codons at that site in both strains.
Candida albicans-Derived β-1,2-Linked Mannooligosaccharides Induce Desensitization of Macrophages
Authors: Jouault Thierry , Fradin Chantal , Trinel Pierre-André , Poulain Daniel
Published: July 2002

Candida albicansβ-1,2-oligomannosides stimulate macrophage tumor necrosis factor alpha (TNF-α) but not NO release. This stimulation desensitized macrophages by altering β-1,2-oligomannoside-dependent TNF-α production and lipopolysaccharide-dependent TNF-α and NO secretion. Desensitization was not related to tyrosine phosphorylation signal transduction but was transferred by culture supernatants in which arachidonic acid derivatives were evidenced.
Differential Bacterial Survival, Replication, and Apoptosis-Inducing Ability ofSalmonellaSerovars within Human and Murine Macrophages
Authors: Schwan William R. , Huang Xiao-Zhe , Hu Lan , Kopecko Dennis J.
Published: July 2002

Salmonellaserovars are associated with human diseases that range from mild gastroenteritis to host-disseminated enteric fever. Human infections bySalmonella entericaserovar Typhi can lead to typhoid fever, but this serovar does not typically cause disease in mice or other animals. In contrast,S. entericaserovar Typhimurium andS. entericaserovar Enteritidis, which are usually linked to localized gastroenteritis in humans and some animal species, elicit a systemic infection in mice. To better understand these observations, multiple strains of each of several chosen serovars ofSalmonellawere tested for the ability in the nonopsonized state to enter, survive, and replicate within human macrophage cells (U937 and elutriated primary cells) compared with murine macrophage cells (J774A.1 and primary peritoneal cells); in addition, death of the infected macrophages was monitored. The serovar Typhimurium strains all demonstrated enhanced survival within J774A.1 cells and murine peritoneal macrophages, compared with the significant, almost 100-fold declines in viable counts noted for serovar Typhi strains. Viable counts for serovar Enteritidis either matched the level of serovar Typhi (J774A.1 macrophages) or were comparable to counts for serovar Typhimurium (murine peritoneal macrophages). Apoptosis was significantly higher in J774A.1 cells infected with serovar Typhimurium strain LT2 compared to serovar Typhi strain Ty2. On the other hand, serovar Typhi survived at a level up to 100-fold higher in elutriated human macrophages and 2- to 3-fold higher in U937 cells compared to the serovar Typhimurium and Enteritidis strains tested. Despite the differential multiplication of serovar Typhi during infection of U937 cells, serovar Typhi caused significantly less apoptosis than infections with serovar Typhimurium. These observations indicate variability in intramacrophage survival and host cytotoxicity among the various serovars and are the first to show differences in the apoptotic response of distinctSalmonellaserovars residing in human macrophage cells. These studies suggest that nonopsonized serovar Typhimurium enters, multiplies within, and causes considerable, acute death of macrophages, leading to a highly virulent infection in mice (resulting in death within 14 days). In striking contrast, nonopsonized serovar Typhi survives silently and chronically within human macrophages, causing little cell death, which allows for intrahost dissemination and typhoid fever (low host mortality). The type of disease associated with any particular serovar ofSalmonellais linked to the ability of that serovar both to persist within and to elicit damage in a specific host's macrophage cells.
Peripheral Cytokine Responses toTrichuris murisReflect Those Occurring Locally at the Site of Infection
Authors: Taylor Matthew D. , Betts Catherine J. , Else Kathryn J.
Published: July 2002

The study of human cellular immune responses to parasite infection under field conditions is very complex. Often, the only practical site from which to sample the cellular responses is the peripheral blood. Sampling peripheral blood lymphocytes (PBL) relies on the assumption that these peripheral responses accurately reflect the immune responses acting locally at the site of infection. This is a particularly important point for the human intestinal helminthTrichuris trichiura, which solely inhabits the cecum and large intestine and so will stimulate a localized immune response. Using the well-defined model ofT. trichiura,T. murisin the mouse, we have demonstrated that the dominant cytokine responses of the mesenteric lymph nodes (MLN) can be detected by sampling PBL. Resistant mice which mount a type 2 cytokine response in their MLN had PBL producing interleukin-4 (IL-4), IL-5, and IL-9, with negligible levels of gamma interferon (IFN-γ). Conversely, susceptible mice which mount a type 1 cytokine response in their MLN had PBL producing IFN-γ and negligible levels of type 2 cytokines. We have also shown that the PBL are capable of mounting a functional immune response againstT. muris. PBL from immune mice were capable of transferring immunity toT. muris-infected severe combined immunodeficient (C.B-17scid/scid) mice. Sampling PBL responses is therefore a viable option for monitoring human intestinal immune responses duringT. trichiurainfection in the field.
Drastic Reduction of a Filarial Infection in Eosinophilic Interleukin-5 Transgenic Mice
Authors: Martin Coralie , Le Goff Laëtitia , Ungeheuer Marie-Noelle , Vuong Phat N. , Bain Odile
Published: July 2002

In order to establish the role of eosinophils in destroying parasites, transgenic mice have been used in experimental helminthiases but not in filariasis.Litomosoides sigmodontisoffers a good opportunity for this study because it is the only filarial species that completes its life cycle in mice. Its development was compared in transgenic CBA/Ca mice overexpressing interleukin-5 (IL-5) and in wild-type mice following subcutaneous inoculation of 40 infective larvae. An acceleration of larval growth was observed in the IL-5 transgenic mice. However, the recovery rate of adult worms was considerably reduced in these mice, as evidenced 2 months postinoculation (p.i.). The reduction occurs between days 10 and 30 p.i. in the coelomic cavities. As early as day 10, spherical aggregates of eosinophils and macrophages are seen attached on live developing larvae (always similarly localized on the worm) in both wild-type and transgenic mice. However, on day 60 p.i., granulomas were found in the transgenic mice only, probably because of the higher density of eosinophils. Furthermore, on day 30 p.i., young filariae are seen trapped in granulomas, some of them surrounded by Splendore-Hoeppli deposits, which illustrates the release of the major basic protein by eosinophils. The high protection rate obtained (65%) is similar to that observed previously in BALB/c mice following vaccination with irradiated larvae. Both protocols have a common factor, the high production of IL-5 and eosinophilia. However, protection occurs later in primary infected transgenic mice because specific antibodies are not yet present at the time of challenge.
Peptidoglycan and Lipoteichoic Acid fromStaphylococcus aureusInduce Tumor Necrosis Factor Alpha, Interleukin 6 (IL-6), and IL-10 Production in Both T Cells and Monocytes in a Human Whole Blood Model
Authors: Wang J. E. , Jørgensen P. F. , Almlöf M. , Thiemermann C. , Foster S. J. , Aasen A. O. , Solberg R.
Published: July 2002

We have examined the ability of peptidoglycan (PepG) and lipoteichoic acid (LTA) isolated fromStaphylococcus aureusto induce the release of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-10 in whole human blood and identified the cellular origins of these cytokines. Both PepG and LTA induced transient increases in TNF-α and IL-10 in plasma, with peak values at 6 and 12 h, respectively. IL-6 values increased throughout the experimental period (24 h). The TNF-α, IL-6, and IL-10 release induced by PepG and LTA was dose dependent. Only PepG was a potent inducer of TNF-α secretion. After stimulation of whole blood with PepG or LTA, very pure populations of monocytes (CD14 positive), T cells (CD2 positive), B cells (CD19 positive), and granulocytes (CD15 positive) were isolated by immunomagnetic separation and analyzed by reverse transcription-PCR for mRNA transcripts encoding TNF-α, IL-6, and IL-10. The TNF-α mRNA results were inconclusive. In contrast, PepG induced IL-6 and IL-10 mRNA accumulation in both T cells and monocytes. LTA, as well as lipopolysaccharide, induced IL-6 and IL-10 mRNA production in monocytes and possibly in T cells. Whether granulocytes and B cells produce cytokines in response to bacterial stimuli remains obscure. Blockade of the CD14 receptors with monoclonal antibodies (18D11) had no influence on the PepG-induced release of TNF-α but attenuated the LTA-induced release of the same cytokine. In conclusion, our data indicate that circulating T cells and monocytes contribute to cytokine production in sepsis caused by gram-positive bacteria.
Prophylactic and Therapeutic Efficacy of Antibodies to a Capsular Polysaccharide Shared among Vancomycin-Sensitive and -Resistant Enterococci
Authors: Huebner Johannes , Quaas Alexander , Krueger Wolfgang A. , Goldmann Donald A. , Pier Gerald B.
Published: July 2002

Enterococci are important nosocomial pathogens that are increasingly difficult to treat due to intrinsic and acquired resistance to antibiotics, including vancomycin. A recently described capsular polysaccharide (CP) isolated from
Enterococcus faecalis
12030 was used to evaluate the potential efficacy of active or passive immunotherapy regimens as adjunctive treatments. Evaluation of protective efficacy was carried out in immunocompetent mice challenged intravenously (i.v.) with live enterococci. In nonimmune mice, i.v. inoculations resulted in high levels of bacteria in kidneys, spleens, and livers 5 days after challenge. Mice immunized with four 10-μg doses of CP antigen/mouse were protected against challenge with the homologous
E. faecalis
strain. High-titer opsonic immunoglobulin G was also induced by immunizing rabbits with the purified CP, and passive transfer of this antiserum to mice produced significantly lower bacterial counts in organs than did normal rabbit serum or sterile saline. Antibodies to the polysaccharide isolated from
E. faecalis
12030 were protective against
Enterococcus faecalis
OG1RF and against two serologically related, vancomycin-resistant
Enterococcus faecium
clinical isolates. Antibodies to this CP antigen were also effective as a therapeutic reagent in mice when passive therapy was initiated 48 h after live bacterial challenge. These data indicate that CP antigens from enterococci are potential targets of protective antibodies and that these antibodies may be useful for prophylaxis and treatment of enterococcal infections.
Structural Analysis of Phage-Borne
stx
Genes and Their Flanking Sequences in Shiga Toxin-Producing
Escherichia coli
and
Shigella dysenteriae
Type 1 Strains
Authors: Unkmeir Alexandra , Schmidt Herbert
Published: July 2002

The
stx
-flanking regions of 49 Shiga toxin-producing
Escherichia coli
strains and nine
Shigella dysenteriae
serotype 1 strains containing either
stx
,
stx
1
,
stx
2
, or
stx
2
variant genes, were examined. We analyzed these regions by PCR using a set of primers with one primer specific for the respective
stx
gene and a second primer complementary to sequences of Stx phages H-19B and 933W. We further characterized the amplification products by restriction endonuclease digestion and nucleotide sequencing. PCR products of
stx
1
-containing
E. coli
strains of serogroups O157, O26, and 0103 showed the same lengths and similar restriction patterns. However, we failed to amplify the 3′
stx
-flanking region in
stx
1
-harboring
E. coli
O111:H
−
strains. Stx2-producing
E. coli
strains revealed amplification products of different lengths and restriction patterns, suggesting greater heterogeneity than in
stx
1
-positive strains. We also obtained specific PCR products for two Stx2c-producing and seven Stx2f-producing
E. coli
strains when they were subjected to PCR analysis. In nine
S. dysenteriae
type 1 strains, H-19B- and 933W-specific primers amplified only the 3′
stx
-flanking region. The results of our study demonstrate that the
stx
genes of all strains investigated are continuous with phage sequences. Whereas almost all strains except
E. coli
O111:H
−
strains were associated with a S-like gene, association with Q could not be demonstrated in nine
S. dysenteriae
type 1 strains and three
E. coli
strains. Furthermore, we showed that the organization of the
stx
-flanking regions is similar in all strains investigated, whereas fine-structure analysis showed subtle differences among the sequences examined. Our results support the hypothesis that
stx
genes in
E. coli
and
S. dysenteriae
are generally phage-borne.
Invasion Genes Are Not Required forSalmonella entericaSerovar Typhimurium To Breach the Intestinal Epithelium: Evidence ThatSalmonellaPathogenicity Island 1 Has Alternative Functions during Infection
Authors: Murray Rose Ann , Lee Catherine A.
Published: July 2002

Salmonella entericaserovar Typhimurium invasion genes are necessary for bacterial invasion of intestinal epithelial cells and are thought to allow salmonellae to enter and cross the intestinal epithelium during infection. Many invasion genes are encoded onSalmonellapathogenicity island 1 (SPI1), and their expression is activated by HilA, a transcription factor also encoded on SPI1. We have studied the role ofSalmonellainvasion genes during infection of mice following intragastric inoculation. We have found that strains containing a mutation inhilAorinvGwere recovered from the intestinal contents, intestinal tissues, and systemic tissues at a lower frequency than their parental wild-type strain. In contrast, a strain in which SPI1 is deleted was recovered from infected mice at a frequency similar to that of its parental wild-type strain. The ΔSPI1 phenotype indicates thatS. entericadoes not require invasion genes to cross the intestinal epithelium and infect systemic tissues. This result has forced us to reconsider the long-held belief that invasion genes directly mediate bacterial infection of the intestinal mucosa and traversion of the intestinal barrier during infection. Instead, our results suggest thathilAis required for bacterial colonization of the host intestine. The seemingly contradictory phenotype of the ΔSPI1 mutant suggests that deletion of another gene(s) encoded on SPI1 suppresses thehilAmutant defect. We propose a model forS. entericapathogenesis in whichhilAand invasion genes are required for salmonellae to overcome a host clearance response elicited by another SPI1 gene product(s).
Lipooligosaccharide P
k
(Galα1-4Galβ1-4Glc) Epitope of
Moraxella catarrhalis
Is a Factor in Resistance to Bactericidal Activity Mediated by Normal Human Serum
Authors: Zaleski Anthony , Scheffler N. Karoline , Densen Peter , Lee Frank K. N. , Campagnari Anthony A. , Gibson Bradford W. , Apicella Michael A.
Published: July 2002

Moraxella catarrhalis
is a respiratory pathogen responsible for acute bacterial otitis media in children and exacerbation of chronic bronchitis in adults.
M. catarrhalis
strains are frequently resistant to the bactericidal activity of normal human serum. In order to determine if the lipooligosaccharide (LOS) of
M. catarrhalis
has a role in serum resistance, the UDP-glucose-4-epimerase (
galE
) gene was identified, cloned, and sequenced and a deletion/insertion mutation was introduced into
M. catarrhalis
strain 2951. GalE enzymatic activity, measured in whole-cell lysates, was ablated in
M. catarrhalis
2951
galE
. Mass spectrometric analysis of LOS isolated with hot phenol-water confirmed that strain 2951 produced a type A LOS. These studies showed that the LOS from 2951
galE
had lost two hexose residues due to the
galE
mutation and that the resultant LOS structure lacked the (Galα1-4Galβ1-4Glc) P
k
epitope found on
M. catarrhalis
2951. Wild-type
M. catarrhalis
2951 is resistant to complement-mediated serum bactericidal activity. In contrast, a greater than 2-log
10
-unit reduction in CFU occurred after incubation of 2951
galE
in either 50 or 25% pooled human serum (PNHS), and CFU in 10% PNHS decreased by about 1 log
10
unit. These studies suggest that the P
k
epitope of the LOS may be an important factor in the resistance of
M. catarrhalis
to the complement-mediated bactericidal effect of normal human serum.
Decreased Infectivity in
Borrelia burgdorferi
Strain B31 Is Associated with Loss of Linear Plasmid 25 or 28-1
Authors: Labandeira-Rey Maria , Skare Jonathan T.
Published: July 2002

Previous reports indicated a correlation between loss of plasmids and decreased infectivity of
Borrelia burgdorferi
strain B31, suggesting that plasmids may encode proteins that are required for pathogenesis. In this study, we expand on this correlation. Using the
B. burgdorferi
genomic sequence, we designed primers specific for each plasmid, and by using PCR we catalogued 11 linear and 2 circular plasmids from 49 clonal isolates of a mid-passage
B. burgdorferi
strain B31, initially derived from infected mouse skin, and 20 clones obtained from mouse skin infected with a low-passage isolate of
B. burgdorferi
strain B31. Among the 69 clones analyzed, nine distinct genotypes were identified relative to wild-type
B. burgdorferi
strain B31. Among the nine clonal genotypes obtained, only the 9-kb circular plasmid (cp9), the 25-kb linear plasmid (lp25), and either the 28-kb linear plasmid 1 or 4 (lp28-1 and lp28-4, respectively) were missing, in different combinations. We compared the infectivity of the wild-type strain, containing all known
B. burgdorferi
plasmids, with those of single mutants lacking either lp28-1, lp28-4, or lp25 and a double mutant missing both cp9 and lp28-1. The infectivity data indicated that
B. burgdorferi
strain B31 cells lacking lp28-4 were modestly attenuated in all tissues analyzed, whereas samples missing lp25 were completely attenuated in all tissues, even at the highest inoculum tested. Isolates without lp28-1 infected the joint tissue yet were not able to infect other tissues as effectively. In addition, we have observed a selection in vivo in the skin, bladder, and joint for cells containing lp25 and in the skin and bladder for cells containing lp28-1, indicating that lp25 and lp28-1 encode proteins required for colonization and short-term maintenance in these mammalian tissues. In contrast, there was no selection in the joint for cells containing lp28-1, suggesting that genes on lp28-1 are not required for colonization of
B. burgdorferi
within the joint. These observations imply that the dynamic nature of the
B. burgdorferi
genome may provide the genetic heterogeneity necessary for survival in the diverse milieus that this pathogen occupies in nature and may contribute to tropism in certain mammalian host tissues.
Effect of Influenza A Virus Infection on Nasopharyngeal Colonization and Otitis Media Induced by Transparent or Opaque Phenotype Variants of
Streptococcus pneumoniae
in the Chinchilla Model
Authors: Tong H. H. , Weiser J. N. , James M. A. , DeMaria T. F.
Published: July 2002

Phase variation in the colonial opacity of
Streptococcus pneumoniae
has been implicated as a factor in bacterial adherence, colonization, and invasion in the pathogenesis of pneumococcal disease. Additionally, the synergistic effects of influenza A virus and
S. pneumoniae
in the development of otitis media (OM) have been reported. This study examined the ability of opaque or transparent
S. pneumoniae
from the same strain in combination with an antecedent influenza A virus infection to colonize the nasopharynx and invade the middle ear in the chinchilla model. Our data indicated that there was no significant difference in the level of nasopharyngeal colonization and induction of OM between the opaque and transparent variants unless there was a prior challenge with influenza A virus. Subsequent to influenza A virus infection, there was a significant difference between the variants in the ability to colonize and persist in the nasopharynx and middle ear. The concentrations of the opaque variant in nasopharyngeal-lavage samples and middle-ear fluid remained consistently higher than those of the transparent variant for 10 days postinoculation. Data from this study indicate that the effects of influenza A virus on the pathogenesis of experimental
S. pneumoniae
-induced OM differ depending on the opacity phenotype involved.
Modification of Lipid A Biosynthesis in
Neisseria meningitidis lpxL
Mutants: Influence on Lipopolysaccharide Structure, Toxicity, and Adjuvant Activity
Authors: van der Ley Peter , Steeghs Liana , Hamstra Hendrik Jan , ten Hove Jan , Zomer Bert , van Alphen Loek
Published: July 2002

Two genes homologous to
lpxL
and
lpxM
from
Escherichia coli
and other gram-negative bacteria, which are involved in lipid A acyloxyacylation, were identified in
Neisseria meningitidis
strain H44/76 and insertionally inactivated. Analysis by tandem mass spectrometry showed that one of the resulting mutants, termed
lpxL1
, makes lipopolysaccharide (LPS) with penta- instead of hexa-acylated lipid A, in which the secondary lauroyl chain is specifically missing from the nonreducing end of the GlcN disaccharide. Insertional inactivation of the other (
lpxL2
) gene was not possible in wild-type strain H44/76 expressing full-length immunotype L3 lipopolysaccharide (LPS) but could be readily achieved in a
galE
mutant expressing a truncated oligosaccharide chain. Structural analysis of
lpxL2
mutant lipid A showed a major tetra-acylated species lacking both secondary lauroyl chains and a minor penta-acylated species. The
lpxL1
mutant LPS has retained adjuvant activity similar to wild-type meningococcal LPS when used for immunization of mice in combination with LPS-deficient outer membrane complexes from
N. meningitidis
but has reduced toxicity as measured in a tumor necrosis factor alpha induction assay with whole bacteria. In contrast, both adjuvant activity and toxicity of the
lpxL2
mutant LPS are strongly reduced. As the combination of reduced toxicity and retained adjuvant activity has not been reported before for either
lpxL
or
lpxM
mutants from other bacterial species, our results demonstrate that modification of meningococcal lipid A biosynthesis can lead to novel LPS species more suitable for inclusion in human vaccines.
Salmonella
DNA Adenine Methylase Mutants Confer Cross-Protective Immunity
Authors: Heithoff Douglas M. , Enioutina Elena Y. , Daynes Raymond A. , Sinsheimer Robert L. , Low David A. , Mahan Michael J.
Published: July 2002

Salmonella
isolates that lack or overproduce DNA adenine methylase (Dam) elicited a cross-protective immune response to different
Salmonella
serovars. The protection afforded by the
Salmonella enterica
serovar Typhimurium Dam vaccine was greater than that elicited in mice that survived a virulent infection.
S. enterica
serovar Typhimurium Dam mutant strains exhibited enhanced sensitivity to mediators of innate immunity such as antimicrobial peptides, bile salts, and hydrogen peroxide. Also,
S. enterica
serovar Typhimurium Dam
−
vaccines were not immunosuppressive; unlike wild-type vaccines, they failed to induce increased nitric oxide levels and permitted a subsequent robust humoral response to diptheria toxoid antigen in infected mice. Dam mutant strains exhibited a low-grade persistence which, coupled with the nonimmunosuppression and the ectopic protein expression caused by altered levels of Dam, may provide an expanded source of potential antigens in vaccinated hosts.
Diminished Virulence of an Alpha-Toxin Mutant ofStaphylococcus aureusin Experimental Brain Abscesses
Authors: Kielian Tammy , Cheung Ambrose , Hickey William F.
Published: July 2002

Staphylococcus aureusis one of the major etiologic agents of brain abscesses in humans, occasionally leading to focal neurological deficits and even death. The objective of the present study was to identify key virulence determinants contributing to the pathogenesis ofS. aureusin the brain using a murine brain abscess model. The importance of virulence factor production in disease development was demonstrated by the inability of heat-inactivatedS. aureusto induce proinflammatory cytokine or chemokine expression or brain abscess formation in vivo. To directly address the contribution of virulence determinants in brain abscess development, the abilities ofS. aureusstrains with mutations in the global regulatory locisarAandagrwere examined. AnS.aureus sarAagrdouble mutant exhibited reduced virulence in vivo, as demonstrated by attenuated proinflammatory cytokine and chemokine expression and bacterial replication. Subsequent studies focused on the expression of factors that are altered in thesarA agrdouble mutant. Evaluation of an alpha-toxin mutant revealed a phenotype similar to that of thesarA agrmutant in vivo, as evidenced by lower bacterial burdens and attenuation of cytokine and chemokine expression in the brain. This suggested that alpha-toxin is a central virulence determinant in brain abscess development. Another virulence mechanism utilized by staphylococci is intracellular survival. Cells recovered from brain abscesses were shown to harborS. aureusintracellularly, providing a means by which the organism may establish chronic infections in the brain. Together, these data identify alpha-toxin as a key virulence determinant for the survival ofS. aureusin the brain.
Host Response to Infection: the Role of CpG DNA in Induction of Cyclooxygenase 2 and Nitric Oxide Synthase 2 in Murine Macrophages
Authors: Ghosh Dipak K. , Misukonis Mary A. , Reich Charles , Pisetsky David S. , Weinberg J. Brice
Published: July 2002

Depending on sequence, bacterial and synthetic DNAs can activate the host immune system and influence the host response to infection. The purpose of this study was to determine the abilities of various phosphorothioate oligonucleotides with cytosine-guanosine-containing motifs (CpG DNA) to activate macrophages to produce nitric oxide (NO) and prostaglandin E2(PGE2) and to induce expression of NO synthase 2 (NOS2) and cyclooxygenase 2 (COX2). As little as 0.3 μg of CpG DNA/ml increased NO and PGE2production in a dose- and time-dependent fashion in cells of the mouse macrophage cell line J774. NO and PGE2production was noted by 4 to 8 h after initiation of cultures with the CpG DNA, with the kinetics of NO production induced by CpG DNA being comparable to that induced by a combination of lipopolysaccharide and gamma interferon. CpG DNA-treated J774 cells showed enhanced expression of NOS2 and COX2 proteins as determined by immunoblotting, with the relative potencies of the CpG DNAs generally corresponding to those noted for the induction of NO and PGE2production as well as to those noted for the induction of interleukin-6 (IL-6), IL-12, and tumor necrosis factor. Extracts from CpG DNA-treated cells convertedl-arginine tol-citrulline, but the NOS inhibitorNG-monomethyl-l-arginine (NMMA) inhibited this reaction. The COX2-specific inhibitor NS398 inhibited CpG DNA-induced PGE2production and inhibited NO production to various degrees. The NOS inhibitors NMMA, 1400W, andN-iminoethyl-l-lysine effectively blocked NO production and increased the production of PGE2in a dose-dependent fashion. Thus, analogues of microbial DNA (i.e., CpG DNA) activate mouse macrophage lineage cells for the expression of NOS2 and COX2, with the production of NO and that of PGE2occurring in an interdependent manner.
Serum Resistance in an Invasive, NontypeableHaemophilus influenzaeStrain
Authors: Williams Bryan J. , Morlin Gregory , Valentine Nathan , Smith Arnold L.
Published: July 2002

A common feature of many different organisms causing bacteremia is the ability to avoid the bactericidal effects of normal human serum. InHaemophilus influenzaeencapsulated strains are particularly serum resistant; however, we found that a nonencapsulated strain (R2866) isolated from the blood of an immunocompetent child with meningitis who had been successfully immunized withH. influenzaetype b conjugate vaccine was serum resistant. Since serum resistance usually involves circumventing the action of the complement system, we defined the deposition of various complement components on the surfaces of thisH. influenzaestrain (R2866), a nonencapsulated avirulent laboratory strain (Rd), and a virulent type b encapsulated strain (Eagan). Membrane attack complex (MAC) accumulation correlated with the loss of bacterial viability; correspondingly, the rates of MAC deposition on the serum-sensitive strain Rd and the serum-resistant strains differed. Analysis of cell-associated immunoglobulin G (IgG), C1q, C3b, and C5b indicated that serum-resistantH. influenzaeprevents MAC accumulation by delaying the synthesis of C3b through the classical pathway. Among the initiators of the classical pathway, IgG deposition contributes most of the C3 convertase activity necessary to start the cascade ending with MAC deposition. Despite similar IgG binding, strain R2866 delays C3 convertase activity compared to strain Rd. We conclude that strain R2866 can persist in the bloodstream, in part by inhibiting or delaying C3 deposition on the cell surface, escaping complement mediated killing.
Safety and Immunogenicity of Increasing Doses of a
Clostridium difficile
Toxoid Vaccine Administered to Healthy Adults
Authors: Kotloff Karen L. , Wasserman Steven S. , Losonsky Genevieve A. , Thomas William , Nichols Richard , Edelman Robert , Bridwell Margaret , Monath Thomas P.
Published: July 2002

Clostridium difficile
is a major cause of nosocomial diarrhea in industrialized countries. Although most illnesses respond to available therapy, infection can increase morbidity, prolong hospitalization, and produce life-threatening colitis. Vaccines are being explored as an alternative means for protecting high-risk individuals. We assessed the safety, immunogenicity, and dose response of a parenteral vaccine containing
C. difficile
toxoids A and B. Thirty healthy adults were assigned to receive four spaced inoculations on days 1, 8, 30, and 60 with one of three doses of vaccine (6.25, 25, or 100 μg). At each dose level, subjects were randomized, in a double-blind fashion, to receive either the soluble toxoids (
n
= 5) or toxoids adsorbed to alum (
n
= 5). Subjects were monitored for clinical and immunologic responses to vaccination. Vaccination was generally well tolerated, with occasional, usually mild, systemic reactions (abdominal pain, arthralgia, and diarrhea). The most common local reaction, mild arm pain, was reported by all recipients of the toxoid-alum formulation. Nearly all subjects (≥90%) developed vigorous serum antibody responses to both toxins, as measured by immunoglobulin G (IgG) enzyme-linked immunosorbent assay and neutralization of cytotoxicity, whereas fecal IgA increases occurred in approximately 50%. Statistically significant effects of dose and formulation on immunogenicity were not seen, although antibody levels tended to be higher with the alum-adjuvanted formulations and with increasing doses of soluble toxoid. Serum antibody responses among the toxoid-alum group appeared to plateau at 25 μg. We concluded that the
C. difficile
toxoid vaccine is safe and immunogenic in healthy volunteers. Further development as a prophylactic vaccine or for producing
C. difficile
hyperimmune globulin is justified.
Passive Transfer of Immunoglobulin Y Antibody to
Streptococcus mutans
Glucan Binding Protein B Can Confer Protection against Experimental Dental Caries
Authors: Smith Daniel J. , King William F. , Godiska Ronald
Published: July 2002

Active immunization with
Streptococcus mutans
glucan binding protein B (GBP-B) has been shown to induce protection against experimental dental caries. This protection presumably results from continuous secretion of salivary antibody to GBP-B, which inhibits accumulation of
S. mutans
within the oral biofilm. The purpose of this study was to explore the influence of short-term (9- or 24-day) passive oral administration of antibody to
S. mutans
GBP-B on the longer-term accumulation and cariogenicity of
S. mutans
in a rat model of dental caries. Preimmune chicken egg yolk immunoglobulin Y (IgY) or IgY antibody to
S. mutans
GBP-B was supplied in lower (experiment 1) and higher (experiment 2) concentrations in the diet and drinking water of rats for 9 (experiment 1) or 24 (experiment 2) days. During the first 3 days of IgY feeding, all animals were challenged with 5 × 10
6
streptomycin-resistant
S. mutans
strain SJ-r organisms. Rats remained infected with
S. mutans
for 78 days, during which rat molars were sampled for the accumulation of
S. mutans
SJ-r bacteria and total streptococci. Geometric mean levels of
S. mutans
SJ-r accumulation on molar surfaces were significantly lower in antibody-treated rats on days 16 and 78 of experiment 2 and were lower on all but the initial (day 5) swabbing occasions in both experiments. Relative to controls, the extent of molar dental caries measured on day 78 was also significantly decreased. The decrease in molar caries correlated with the amount and duration of antibody administration. This is the first demonstration that passive antibody to
S. mutans
GBP-B can have a protective effect against cariogenic
S. mutans
infection and disease. Furthermore, this decrease in infection and disease did not require continuous antibody administration for the duration of the infection period. This study also indicates that antibody to components putatively involved only in cellular aggregation can have a significant effect on the incorporation of mutans streptococci in dental biofilm.
Isogenic Serotypes ofBorrelia turicataeShow Different Localization in the Brain and Skin of Mice
Authors: Cadavid Diego , Pachner Andrew R. , Estanislao Lydia , Patalapati Ramaprasad , Barbour Alan G.
Published: July 2002

Mice with severe combined immunodeficiency (scidmice) and infected with the relapsing fever agentBorrelia turicataedevelop manifestations that resemble those of disseminated Lyme disease. We have characterized two isogenic serotypes, A and B, which differ in their variable small proteins (Vsps) and disease manifestations. Serotype A but not serotype B was cultured from the brain during early infection, and serotype B caused more severe arthritis, myocarditis, and vestibular dysfunction than serotype A. Here we compared the localization and number of spirochetes and the severity of inflammation inscidmice, using immunostained and hematoxylin-and-eosin-stained coronal sections of decalcified heads. Spirochetes in the brain localized predominantly to the leptomeninges, and those in peripheral tissues localized mainly to the extracellular matrix. There were significantly more serotype A than B spirochetes in the leptomeninges and more serotype B than A spirochetes in the skin. The first tissue where spirochetes were observed outside the vasculature was the dura mater. Inflammation was more severe in the skin than in the brain. VspA, VspB, and the periplasmic flagellin protein were expressed in all tissues examined. These findings indicate that isogenic but antigenically distinctBorreliaserotypes can have marked differences in their localization in tissues.
Influence of Vector-Encoded Cytokines on Anti-SalmonellaImmunity: Divergent Effects of Interleukin-2 and Tumor Necrosis Factor Alpha
Authors: al-Ramadi Basel K. , Al-Dhaheri Mariam H. , Mustafa Nada , AbouHaidar Mounir , Xu Damu , Liew F. Y. , Lukic Miodrag L. , Fernandez-Cabezudo Maria J.
Published: July 2002

AttenuatedSalmonellastrains are of interest as new vaccine candidates and as vectors of cloned genes of other organisms. Attenuated strains expressing specific cytokines were constructed as a means of manipulating the immune response in various disease settings. In the present study, interleukin-2 (IL-2)-expressing (GIDIL2) or tumor necrosis factor alpha (TNF-α)-expressing (GIDTNF) strains were compared with the parent strain (BRD509) for the effect of cytokines on anti-Salmonellaimmunity. Expression of IL-2 resulted in a rapid clearance of the organism soon after vaccination. The reduction in GIDIL2 CFU was 50- to 300-fold higher than that of BRD509 and correlated with a markedly decreased splenomegaly. Furthermore, no evidence for any significant activation, including upregulation of surface markers and production of nitric oxide (NO), was observed in spleens of GIDIL2-injected mice. In contrast, the host response to GIDTNF was marked by an early, strong, splenic cellular influx, but surprisingly, the degree of induced splenomegaly and NO secretion was only 50% of that observed in BRD509-treated mice. Despite this, bacterial colonization of the spleen in GIDTNF-immunized animals was either slightly decreased from or equivalent to that of the BRD509-treated group, suggesting the induction of additional antimicrobial mechanisms by TNF-α. In vivo protection studies demonstrated that, at limiting doses, GIDIL2 was inferior to GIDTNF and BRD509 in its capacity to protect against virulent challenge. At high doses, however, all three strains exhibited equal protective efficacy. These results demonstrate that the immune response against intracellular bacteria can be manipulated by pathogen-expressed cytokines and open the way for further fine tuning of immune responses not only toSalmonellastrains themselves but also to the heterologous gene(s) carried by them.
Vaccination with FimA from
Streptococcus parasanguis
Protects Rats from Endocarditis Caused by Other Viridans Streptococci
Authors: Kitten Todd , Munro Cindy L. , Wang Aijuan , Macrina Francis L.
Published: July 2002

The FimA protein of
Streptococcus parasanguis
is a virulence factor in the rat model of endocarditis, and immunization with FimA protects rats against homologous bacterial challenge. Because FimA-like proteins are widespread among the oral streptococci, the leading cause of native valve endocarditis, we evaluated the ability of this vaccinogen to protect rats when challenged by other streptococcal species. Here we report that FimA vaccination produced antibodies that cross-reacted with and protected against challenge by the oral streptococci
S. mitis
,
S. mutans
, and
S. salivarius
. FimA thus has promise as a vaccinogen to control infective endocarditis caused by oral streptococci.
Porins fromSalmonella entericaSerovar Typhimurium Activate the Transcription Factors Activating Protein 1 and NF-κB through the Raf-1-Mitogen-Activated Protein Kinase Cascade
Authors: Galdiero Massimiliano , Vitiello Mariateresa , Sanzari Emma , D’Isanto Marina , Tortora Annalisa , Longanella Anna , Galdiero Stefania
Published: July 2002

In this study we examined the ability ofSalmonella entericaserovar Typhimurium porins to activate activating protein 1 (AP-1) and nuclear factor κB (NF-κB) through the mitogen-activated protein kinase (MAPK) cascade, and we identified the AP-1-induced protein subunits. Our results demonstrate that these enzymes may participate in cell signaling pathways leading to AP-1 and NF-κB activation following porin stimulation of cells. Raf-1 was phosphorylated in response to the treatment of U937 cells with porins; moreover, the porin-mediated increase in Raf-1 phosphorylation is accompanied by the phosphorylation of MAPK kinase 1/2 (MEK1/2), p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase. We used three different inhibitors of phosphorylation pathways: 2′-amino-3′-methoxyflavone (PD-098059), a selective inhibitor of MEK1 activator and the MAPK cascade; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of the p38 pathway; and 7β-acetoxy-1α,6β,9α-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), an inhibitor at the level of Raf-1 kinase. PD-098059 pretreatment of cells decreases AP-1 and NF-κB activation by lipopolysaccharide (LPS) but not by porins, and SB203580 pretreatment of cells decreases mainly AP-1 and NF-κB activation by porins; in contrast, forskolin pretreatment of cells does not affect AP-1 and NF-κB activation following either porin or LPS stimulation. Our data suggest that the p38 signaling pathway mainly regulates AP-1 and NF-κB activation in cells treated withS. entericaserovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only after porin stimulation. These data suggest different molecular mechanisms of activation induced by porins or by LPS.
Hydrolysis of Epithelial Junctional Proteins byPorphyromonas gingivalisGingipains
Authors: Katz Jannet , Yang Qiu-Bo , Zhang Ping , Potempa Jan , Travis James , Michalek Suzanne M. , Balkovetz Daniel F.
Published: July 2002

Porphyromonas gingivalishas been implicated as an etiologic agent of adult periodontitis. We have previously shown thatP. gingivaliscan degrade the epithelial cell-cell junction complexes, thus suggesting that this bacterium can invade the underlying connective tissues via a paracellular pathway. However, the precise mechanism(s) involved in this process has not been elucidated. The purpose of this study was to determine if the arginine- and lysine-specific gingipains ofP. gingivalis(i.e., HRgpA and RgpB, and Kgp, respectively) were responsible for the degradation of E-cadherin, the cell-cell adhesion protein in the adherens junctions. In addition, we compared the degradative abilities of the whole gingipains HRgpA and Kgp to those of their catalytic domains alone. In these studies, immunoprecipitated E-cadherin as well as monolayers of polarized Madin-Darby canine kidney (MDCK) epithelial cell cultures were incubated with the gingipains and hydrolysis of E-cadherin was assessed by Western blot analysis. Incubation ofP. gingivaliscells with immunoprecipitated E-cadherin resulted in degradation, whereas prior exposure ofP. gingivaliscells to leupeptin and especially acetyl-Leu-Val-Lys-aldehyde (which are arginine- and lysine-specific inhibitors, respectively) reduced this activity. Furthermore, incubation of E-cadherin immunoprecipitates with the different gingipains resulted in an effective and similar hydrolysis of the protein. However, when monolayers of MDCK cells were exposed to the gingipains, Kgp was most effective in hydrolyzing the E-cadherin molecules in the adherens junction. Kgp was more effective than its catalytic domain in degrading E-cadherin at 500 nM but not at a lower concentration (250 nM). These results suggest that the hemagglutinin domain of Kgp plays a role in degradation and that there is a critical threshold concentration for this activity. Taken together, these results provide evidence that the gingipains, especially Kgp, are involved in the degradation of the adherens junction of epithelial cells, which may be important in the invasion of periodontal connective tissue byP. gingivalis.