bluuebunny/crossref_metadata_2025 huggingface dataset:
Active Immunization in the United States: Developments over the Past Decade
Authors: Dennehy Penelope H.
Published: July 2002

The Centers for Disease Control and Prevention has identified immunization as the most important public health advance of the 20th century. The purpose of this article is to review the changes that have taken place in active immunization in the United States over the past decade. Since 1990, new vaccines have become available to prevent five infectious diseases: varicella, rotavirus, hepatitis A, Lyme disease, and Japanese encephalitis virus infection. Improved vaccines have been developed to prevent Haemophilus influenzae type b, pneumococcus, pertussis, rabies, and typhoid infections. Immunization strategies for the prevention of hepatitis B, measles, meningococcal infections, and poliomyelitis have changed as a result of the changing epidemiology of these diseases. Combination vaccines are being developed to facilitate the delivery of multiple antigens, and improved vaccines are under development for cholera, influenza, and meningococcal disease. Major advances in molecular biology have enabled scientists to devise new approaches to the development of vaccines against diseases ranging from respiratory viral to enteric bacterial infections that continue to plague the world's population.
Streptococcus suis
Serotype 2 Interactions with Human Brain Microvascular Endothelial Cells
Authors: Charland Nathalie , Nizet Victor , Rubens Craig E. , Kim Kwang Sik , Lacouture Sonia , Gottschalk Marcelo
Published: July 2002

Streptococcus suis
serotype 2 is a worldwide causative agent of many forms of swine infection and is also recognized as a zoonotic agent causing human disease, including meningitis. The pathogenesis of
S. suis
infections is poorly understood. Bacteria circulate in the bloodstream in the nonimmune host until they come in contact with brain microvascular endothelial cells (BMEC) forming the blood-brain barrier. The bacterial polysaccharide capsule confers antiphagocytic properties. It is known that group B streptococci (GBS) invade and damage BMEC, which may be a primary step in the pathogenesis of neonatal meningitis. Interactions between
S. suis
and human endothelial cells were studied to determine if they differ from those between GBS and endothelial cells. Invasion assays performed with BMEC and human umbilical vein endothelial cells demonstrated that unlike GBS,
S. suis
serotype 2 could not invade either type of cell. Adherence assays showed that
S. suis
adhered only to BMEC, whereas GBS adhered to both types of cell. These interactions were not affected by the presence of a capsule, since acapsular mutants from both bacterial species adhered similarly compared to the wild-type strains. Lactate dehydrogenase release measurements indicated that some
S. suis
strains were highly cytotoxic for BMEC, even more than GBS, whereas others were not toxic at all. Cell damage was related to suilysin (
S. suis
hemolysin) production, since only suilysin-producing strains were cytotoxic and cytotoxicity could be inhibited by cholesterol and antisuilysin antibodies. It is possible that hemolysin-positive
S. suis
strains use adherence and suilysin-induced BMEC injury, as opposed to direct cellular invasion, to proceed from the circulation to the central nervous system.
Expression and Immunogenicity of Hemagglutinin A from
Porphyromonas gingivalis
in an Avirulent
Salmonella enterica
Serovar Typhimurium Vaccine Strain
Authors: Kozarov Emil , Miyashita Naohisa , Burks Jacob , Cerveny Karen , Brown Thomas A. , McArthur William P. , Progulske-Fox Ann
Published: July 2002

Porphyromonas gingivalis
is a major etiologic agent of periodontitis, a chronic inflammatory disease that ultimately results in the loss of the supporting tissues of the teeth. Previous work has demonstrated the usefulness of avirulent
Salmonella enterica
serovar Typhimurium strains as antigen delivery systems for protective antigens of pathogens that colonize or cross mucosal surfaces. In this study, we constructed and characterized a recombinant
S. enterica
serovar Typhimurium avirulent vaccine strain which expresses hemagglutinin A and carries no antibiotic resistance markers. HagA, a major virulence-associated surface protein, is a potentially useful immunogen that contains an antigenic epitope which, in humans, elicits an immune response that is protective against subsequent colonization by
P. gingivalis
. The
hagA
gene, including its promoter, was cloned into a balanced-lethal
Salmonella
vector and transferred to the vaccine strain. Heterologous expression of HagA was demonstrated in both
Escherichia coli
JM109 and
S. enterica
serovar Typhimurium vaccine strain χ4072. The HagA epitope was present in its native configuration as determined by immunochemistry and immunoelectron microscopy. Purified recombinant HagA was recognized by sera from mice immunized with the
S. enterica
serovar Typhimurium vaccine strain. The HagA-specific antigen of the vaccine was also found to be recognized by serum from a periodontal patient. This vaccine strain, which expresses the functional hemagglutinin protein, induces a humoral immune response against HagA and may be useful for developing a protective vaccine against periodontal diseases associated with
P. gingivalis
.
Interferon Consensus Sequence Binding Protein Confers Resistance againstYersinia enterocolitica
Authors: Hein Joachim , Kempf Volkhard A. J. , Diebold Joachim , Bücheler Nicole , Preger Sonja , Horak Ivan , Sing Andreas , Kramer Uwe , Autenrieth Ingo B.
Published: July 2002

Interferon consensus sequence binding protein (ICSBP)-deficient mice display enhanced susceptibility to intracellular pathogens. At least two distinct immunoregulatory defects are responsible for this phenotype. First, diminished production of reactive oxygen intermediates in macrophages results in impaired intracellular killing of microorganisms. Second, defective early interleukin-12 (IL-12) production upon microbial challenge leads to a failure in gamma interferon (IFN-γ) induction and subsequently in T helper 1 immune responses. Here, we investigated the role of ICSBP in resistance against the extracellular bacteriumYersinia enterocolitica. ICSBP−/−mice failed to produce IL-12 and IFN-γ, but also IL-4, afterYersiniachallenge. In addition, granuloma formation was highly disturbed in infected ICSBP−/−mice, leading to multiple necrotic abscesses in affected organs. Consequently, ICSBP−/−mice rapidly succumbed to acuteYersiniainfection. In vitro treatment of spleen cells from ICSBP−/−mice with recombinant IL-12 (rIL-12) or rIL-18 in combination with a second stimulus resulted in IFN-γ induction. In experimental therapy of infected ICSBP−/−mice, we observed that administration of rIL-12 induced IFN-γ production which was associated with improved resistance toYersinia. In contrast, treatment with rIL-18 failed to enhance endogenous IFN-γ production but nevertheless reduced bacterial burden in ICSBP−/−mice. Although cytokine therapy with rIL-12 or rIL-18 ameliorated the course ofYersiniainfection in ICSBP−/−mice, both cytokines failed to completely restore impaired immunity. Taken together, the results indicate that the transcription factor ICSBP is essential for efficient host immune defense againstYersinia. These results are important for understanding the complex host immune responses in bacterial infections.
Identification of Secreted Proteins ofMycobacterium tuberculosisby a Bioinformatic Approach
Authors: Gomez Manuel , Johnson Sadie , Gennaro Maria Laura
Published: July 2002

Proteins secreted byMycobacterium tuberculosisare usually targets of immune responses in the infected host. Here we describe a search for secreted proteins that combined the use of bioinformatics andphoA′ fusion technology. The 3,924 proteins deduced from theM. tuberculosisgenome were analyzed with several computer programs. We identified 52 proteins carrying an NH2-terminal secretory signal peptide but lacking additional membrane-anchoring moieties. Of these 52 proteins—the TM1 subgroup—only 7 had been previously reported to be secreted proteins. Our predictions were confirmed in 9 of 10 TM1 genes that were fused toEscherichia coli phoA′, a marker of subcellular localization. These findings demonstrate that the systematic computer search described in this work identified secreted proteins ofM. tuberculosiswith high efficiency and 90% accuracy.
Inactivation of
Pasteurella
(
Mannheimia
)
haemolytica
Leukotoxin Causes Partial Attenuation of Virulence in a Calf Challenge Model
Authors: Highlander Sarah K. , Fedorova Natalie D. , Dusek David M. , Panciera Roger , Alvarez Laura E. , Rinehart Carol
Published: July 2002

The leukotoxin of
Pasteurella
(
Mannheimia
)
haemolytica
is believed to play a significant role in pathogenesis, causing cell lysis and apoptosis that lead to the lung pathology characteristic of bovine shipping fever. Using a system for Cre-
lox
recombination, a nonpolar mutation within the
lktC
transacylase gene of the leukotoxin operon was created. The
lktC
locus was insertionally inactivated using a
loxP-aph3-loxP
cassette, and then the
aph3
marker was excised from the chromosome by Cre recombinase expressed from a
P. haemolytica
plasmid. The resulting
lktC
strain (SH2099) secretes inactive leukotoxin and carries no known antibiotic resistance genes. Strain SH2099 was tested for virulence in a calf challenge model. We inoculated 3 × 10
8
or 3 × 10
9
CFU of wild-type or mutant bacteria into the lungs of healthy, colostrum-deprived calves via transthoracic injection. Animals were observed for clinical signs and for nasal colonization for 4 days, after which they were euthanized and necropsied. The lower inoculum (3 × 10
8
CFU) caused significantly fewer deaths and allowed lung pathology to be scored and compared, while the 3 × 10
9
CFU dose of either the wild-type or mutant was lethal to ≥50% of the calves. The estimated 50% lethal dose of SH2099 was four times higher than that of the wild-type strain. Lung lesion scores were reduced twofold in animals inoculated with the mutant, while clinical scores were nearly equivalent for both strains. The wild-type and mutant strains were equally capable of colonizing the upper respiratory tracts of the calves. In this study, the
P. haemolytica lktC
mutant was shown to be less virulent than the parent strain.
Helicobacter pylori
Urease Suppresses Bactericidal Activity of Peroxynitrite via Carbon Dioxide Production
Authors: Kuwahara Hideo , Miyamoto Yoichi , Akaike Takaaki , Kubota Tatsuo , Sawa Tomohiro , Okamoto Shinichiro , Maeda Hiroshi
Published: July 2002

Helicobacter pylori
can produce a persistent infection in the human stomach, where chronic and active inflammation, including the infiltration of phagocytes such as neutrophils and monocytes, is induced.
H. pylori
may have a defense system against the antimicrobial actions of phagocytes. We studied the defense mechanism of
H. pylori
against host-derived peroxynitrite (ONOO
−
), a bactericidal metabolite of nitric oxide, focusing on the role of
H. pylori
urease, which produces CO
2
and NH
3
from urea and is known to be an essential factor for colonization. The viability of
H. pylori
decreased in a time-dependent manner with continuous exposure to 1 μM ONOO
−
, i.e., 0.2% of the initial bacteria remained after a 5-min treatment without urea. The bactericidal action of ONOO
−
against
H. pylori
was significantly attenuated by the addition of 10 mM urea, the substrate for urease, whereas ONOO
−
-induced killing of a urease-deficient mutant of
H. pylori
or
Campylobacter jejuni
, another microaerophilic bacterium lacking urease, was not affected by the addition of urea. Such a protective effect of urea was potentiated by supplementation with exogenous urease, and it was almost completely nullified by 10 μM flurofamide, a specific inhibitor of urease. The bactericidal action of ONOO
−
was also suppressed by the addition of 20 mM NaHCO
3
but not by the addition of 20 mM NH
3
. In addition, the nitration of
l
-tyrosine of
H. pylori
after treatment with ONOO
−
was significantly reduced by the addition of urea or NaHCO
3
, as assessed by high-performance liquid chromatography with electrochemical detection. These results suggest that
H. pylori
-associated urease functions to produce a potent ONOO
−
scavenger, CO
2
/HCO
3
−
, that defends the bacteria from ONOO
−
cytotoxicity. The protective effect of urease may thus facilitate sustained bacterial colonization in the infected gastric mucosa.
Antiviral Activity of Shiga Toxin 1: Suppression of Bovine Leukemia Virus-Related Spontaneous Lymphocyte Proliferation
Authors: Ferens Witold A. , Hovde Carolyn J.
Published: July 2002

Human infections with Shiga toxin (Stx)-producing
Escherichia coli
(STEC) cause hemorrhagic colitis. The Stxs belong to a large family of ribosome-inactivating proteins (RIPs) that are found in a variety of higher plants and some bacteria. Many RIPs have potent antiviral activity for the plants that synthesize them. STEC strains, both virulent and nonvirulent to humans, are frequently isolated from healthy cattle. Interestingly, despite intensive investigations, it is not known why cattle carry STEC. We tested the hypothesis that Stx has antiviral properties for bovine viruses by assessing the impact of Stx type 1 (Stx1) on bovine peripheral blood mononuclear cells (PBMC) from cows infected with bovine leukemia virus (BLV). PBMC from BLV-positive animals invariably displayed spontaneous lymphocyte proliferation (SLP) in vitro. Stx1 or the toxin A subunit (Stx1A) strongly inhibited SLP. Toxin only weakly reduced the pokeweed mitogen- or interleukin-2-induced proliferation of PBMC from normal (BLV-negative) cows and had no effect on concanavalin A-induced proliferation. The toxin activity in PBMC from BLV-positive cattle was selective for viral SLP and did not abrogate cell response to pokeweed mitogen- or interleukin-2-induced proliferation. Antibody to virus or Stx1A was most effective at inhibiting SLP if administered at the start of cell culture, indicating that both reagents likely interfere with BLV-dependent initiation of SLP. Stx1A inhibited expression of BLV p24 protein by PBMC. A well-defined mutant Stx1A (E167D) that has decreased catalytic activity was not effective at inhibiting SLP, suggesting the inhibition of protein synthesis is likely the mechanism of toxin antiviral activity. Our data suggest that Stx has potent antiviral activity and may serve an important role in BLV-infected cattle by inhibiting BLV replication and thus slowing the progression of infection to its malignant end stage.
Modulation of Cardiocyte Functional Activity by Antibodies againstTrypanosoma cruziRibosomal P2 Protein C Terminus
Authors: Sepulveda P. , Liegeard P. , Wallukat G. , Levin M. J. , Hontebeyrie M.
Published: July 2002

Antibodies against theTrypanosoma cruziribosomal P2β protein (TcP2β) have been associated with the chronic cardiac pathology of Chagas' disease in humans. Using synthetic peptides spanning the entire TcP2β molecule, we investigated their epitope recognition by antibodies from mice chronically infected withT. cruziand from mice immunized with two recombinant TcP2βs. We found clear differences in epitope recognition between antibodies fromT. cruzi-infected mice and mice immunized with two different recombinant TcP2βs associated with different schedules of immunization. Major epitopes recognized by antibodies from mice immunized with recombinant glutathioneS-transferase (GST) or histidine (Hist) fusion TcP2β (GST-TcP2β or Hist-TcP2β) are located in the central and hinge regions of the molecule. Nevertheless, mice immunized with Hist-TcP2β were also able to elicit antibodies against the TcP2β C terminus, a region which is highly conserved in bothT. cruziand mammal ribosomal P proteins. Strikingly, antibodies from infected animals recognized only the TcP2β C terminus. By using these antisera with distinct profiles of epitope recognition, it could be shown that only C terminus-specific antibodies were able to increase the beating frequency of cardiomyocytes from neonatal rats in vitro by selective stimulation of the β1-adrenergic receptor. Thus, antibodies against the TcP2β C terminus elicited in the absence of infection are able to modulate a functional activity of host cells through a molecular mimicry mechanism.
Gonococcal Nitric Oxide Reductase Is Encoded by a Single Gene,
norB
, Which Is Required for Anaerobic Growth and Is Induced by Nitric Oxide
Authors: Householder Tracey C. , Fozo Elizabeth M. , Cardinale Jean A. , Clark Virginia L.
Published: July 2002

The gene encoding a nitric oxide reductase has been identified in
Neisseria gonorrhoeae
. The
norB
gene product shares significant identity with the nitric oxide reductases in
Ralstonia eutropha
and
Synechocystis sp.
and, like those organisms, the gonococcus lacks a
norC
homolog. The gonococcal
norB
gene was found to be required for anaerobic growth, but the absence of
norB
did not dramatically decrease anaerobic survival. In a wild-type background, induction of
norB
expression was seen anaerobically in the presence of nitrite but not anaerobically without nitrite or aerobically.
norB
expression is not regulated by FNR or NarP, but a functional
aniA
gene (which encodes an anaerobically induced outer membrane nitrite reductase) is necessary for expression. When
aniA
is constitutively expressed,
norB
expression can be induced both anaerobically and aerobically, but only in the presence of nitrite, suggesting that nitric oxide, which is likely to be produced by AniA as a product of nitrite reduction, is the inducing agent. This was confirmed with the use of the nitric oxide donor, spermine-nitric oxide complex, in an
aniA
null background both anaerobically and aerobically. NorB is important for gonococcal adaptation to an anaerobic environment, a physiologically relevant state during gonococcal infection. The presence of this enzyme, which is induced by nitric oxide, may also have implications in immune evasion and immunomodulation in the human host.
Inhibition of Caspase 3 Abrogates Lipopolysaccharide-Induced Nitric Oxide Production by Preventing Activation of NF-κB and c-Jun NH2-Terminal Kinase/Stress-Activated Protein Kinase in RAW 264.7 Murine Macrophage Cells
Authors: Chakravortty Dipshikha , Kato Yutaka , Sugiyama Tsuyoshi , Koide Naoki , Mu Mya Mya , Yoshida Tomoaki , Yokochi Takashi
Published: July 2002

The effect of caspase inhibitors on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 267.4 murine macrophage cells was investigated. Pretreatment of RAW cells with a broad caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), resulted in a striking reduction in LPS-induced NO production. Z-VAD-FMK inhibited LPS-induced NF-κB activation. Furthermore, it blocked phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) but not that of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases. Similarly, a caspase 3-specific inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone, inhibited NO production, NF-κB activation, and JNK/SAPK phosphorylation in LPS-stimulated RAW cells. The attenuated NO production was due to inhibition of the expression of an inducible-type NO synthase (iNOS). The overexpression of the dominant negative mutant of JNK/SAPK and the addition of a JNK/SAPK inhibitor blocked iNOS expression but did not block LPS-induced caspase 3 activation. It was therefore suggested that the inhibition of caspase 3 might abrogate LPS-induced NO production by preventing the activation of NF-κB and JNK/SAPK. The caspase family, especially caspase 3, is likely to play an important role in the signal transduction for iNOS-mediated NO production in LPS-stimulated mouse macrophages.
Antigenic Properties and Processing Requirements of 65-Kilodalton Mannoprotein, a Major Antigen Target of Anti-CandidaHuman T-Cell Response, as Disclosed by Specific Human T-Cell Clones
Authors: Nisini Roberto , Romagnoli Giulia , Gomez Maria Jesus , La Valle Roberto , Torosantucci Antonella , Mariotti Sabrina , Teloni Raffaela , Cassone Antonio
Published: July 2002

T-cell-mediated immunity is known to play a central role in the host response toCandida albicans.T-cell clones are useful tools for the exact identification of fungal T-cell epitopes and the processing requirements ofC. albicansantigens. We isolated human T-cell clones from an HLA-DRB1*1101 healthy donor by using an antigenic extract (MP-F2) of the fungus. Specific clones were T-cell receptor α/β and CD4+/CD8−and showed a T-helper type 1 cytokine profile (production of gamma interferon and not interleukin-4). The large majority of these clones recognized both the natural (highly glycosylated) and the recombinant (nonglycosylated) 65-kDa mannoprotein (MP65), an MP-F2 minor constituent that was confirmed to be an immunodominant antigen of the human T-cell response. Surprisingly, most of the clones recognized two synthetic peptides of different MP65 regions. However, the peptides shared the amino acid motif IXSXIXXL, which may be envisaged as a motif sequence representing the minimal epitope recognized by these clones. Three clones recognized natural and pronase-treated MP65 but did not detect nonglycosylated, recombinant MP65 or the peptides, suggesting a possible role for polysaccharides in T-cell recognition ofC. albicans. Finally, lymphoblastoid B-cell lines were efficient antigen-presenting cells (APC) for recombinant MP65 and peptides but failed to present natural, glycosylated antigens, suggesting that nonprofessional APC might be defective in processing highly glycosylated yeast proteins. In conclusion, this study provides the first characterization ofC. albicans-specific human T-cell clones and provides new clues for the definition of the cellular immune response againstC. albicans.
Model of Differential Susceptibility to Mucosal
Burkholderia pseudomallei
Infection
Authors: Liu Boping , Koo Ghee Chong , Yap Eu Hian , Chua Kim Lee , Gan Yunn-Hwen
Published: July 2002

Burkholderia pseudomallei
is the causative agent of melioidosis, an infectious disease with protean clinical manifestations. The major route of infection is thought to be through subcutaneous inoculation of contaminated soil and water, although ingestion and inhalation of contaminated aerosols are also possible. This study examines infection through the intranasal route in a murine model to mimic infection through inhalation. Two strains of mice, C57BL/6 and BALB/c, exhibit differential susceptibilities to the infection, with the C57BL/6 mice being considerably more resistant. To examine host factors that could contribute to this difference, bacterial loads and cytokine profiles in the two strains of mice were compared. We found that infected BALB/c mice exhibited higher bacterial loads in the lung and spleen and that they produced significantly higher levels of gamma interferon (IFN-γ) in the serum than C57BL/6 mice. Although tumor necrosis factor alpha and interleukin-1 could be detected in the nasal washes and sera of both strains of mice, the production in serum was transient and much lower than that of IFN-γ. C57BL/6 mice also exhibited memory responses to bacteria upon reinfection, with the production of serum immunoglobulin G (IgG) and mucosal IgA antibodies. Thus, it is possible that the production of systemic and mucosal antibodies is important for protection against disease in C57BL/6 mice.
Role of Fraction 1 Antigen ofYersinia pestisin Inhibition of Phagocytosis
Authors: Du Yidong , Rosqvist Roland , Forsberg Åke
Published: July 2002

Yersinia pestis, the causative agent of plague, expresses a capsule-like antigen, fraction 1 (F1), at 37°C. F1 is encoded by thecaf1gene located on the large 100-kb pFra plasmid, which is unique toY. pestis. F1 is a surface polymer composed of a protein subunit, Caf1, with a molecular mass of 15.5 kDa. The secretion and assembly of F1 require thecaf1Mandcaf1Agenes, which are homologous to the chaperone and usher protein families required for biogenesis of pili. F1 has been implicated to be involved in the ability ofY. pestisto prevent uptake by macrophages. In this study we addressed the role of F1 antigen in inhibition of phagocytosis by the macrophage-like cell line J774. TheY. pestisstrain EV76 was found to be highly resistant to uptake by J774 cells. An in-frame deletion of thecaf1Mgene of theY. pestisstrain EV76 was constructed and found to be unable to express F1 polymer on the bacterial surface. This strain had a somewhat lowered ability to prevent uptake by J774 cells. Strain EV76C, which is cured for the virulence plasmid common to the pathogenicYersiniaspecies, was, as expected, much reduced in its ability to resist uptake. A strain lacking both the virulence plasmid andcaf1Mwas even further hampered in the ability to prevent uptake and, in this case, essentially all bacteria (95%) were phagocytosed. Thus, F1 and the virulence plasmid-encoded type III system act in concert to makeY. pestishighly resistant to uptake by phagocytes. In contrast to the type III effector proteins YopE and YopH, F1 did not have any influence on the general phagocytic ability of J774 cells. Expression of F1 also reduced the number of bacteria that interacted with the macrophages. This suggests that F1 prevents uptake by interfering at the level of receptor interaction in the phagocytosis process.
Changes in Temporal and Spatial Patterns of Outer Surface Lipoprotein Expression Generate Population Heterogeneity and Antigenic Diversity in the Lyme Disease Spirochete,
Borrelia burgdorferi
Authors: Hefty P. Scott , Jolliff Sarah E. , Caimano Melissa J. , Wikel Stephen K. , Akins Darrin R.
Published: July 2002

Borrelia burgdorferi
differentially expresses many of the OspE/F/Elp paralogs during tick feeding. These findings, combined with the recent report that stable
B. burgdorferi
infection of mammals occurs only after 53 h of tick attachment, prompted us to further analyze the expression of the OspE/F/Elp paralogs during this critical period of transmission. Indirect immunofluorescence analysis revealed that OspE, p21, ElpB1, ElpB2, and OspF/BbK2.11 are expressed in the salivary glands of ticks allowed to feed on mice for 53 to 58 h. Interestingly, many of the spirochetes in the salivary glands that expressed abundant amounts of these antigens were negative for OspA and OspC. Although prior reports have indicated that OspE/F/Elp orthologs are surface exposed, none of the individual lipoproteins or combinations of the lipoproteins protected mice from challenge infections. To examine why these apparently surface-exposed lipoproteins were not protective, we analyzed their genetic stability during infection and their cellular locations after cultivation in vitro and within dialysis membrane chambers, mimicking a mammalian host-adapted state. Combined restriction fragment length polymorphism and nucleotide sequence analyses revealed that the genes encoding these lipoproteins are stable for at least 8 months postinfection. Interestingly, cellular localization experiments revealed that while all of these proteins can be surface localized, there were significant populations of spirochetes that expressed these lipoproteins only in the periplasm. Furthermore, host-specific signals were found to alter the expression patterns and final cellular location of these lipoproteins. The combined data revealed a remarkable heterogeneity in populations of
B. burgdorferi
during tick transmission and mammalian infection. The diversity is generated not only by temporal changes in antigen expression but also by modulation of the surface lipoproteins during infection. The ability to regulate the temporal and spatial expression patterns of lipoproteins throughout infection likely contributes to persistent infection of mammals by
B. burgdorferi
.
Identification of the
mob
Genes of Plasmid pSC101 and Characterization of a Hybrid pSC101-R1162 System for Conjugal Mobilization
Authors: Meyer Richard
Published: July 2002

Similarities in DNA base sequence indicate that pSC101 and R1162 encode related systems for conjugal mobilization, although these plasmids are otherwise very different. The
mob
region of pSC101 was cloned, and two genes that are required for transfer were identified. One gene,
mobA
, encodes a protein similar in amino acid sequence to the DNA processing domain of the R1162 MobA protein. The other gene,
mobX
, is within the same transcriptional unit as the pSC101
mobA
and is located just downstream, at the same position occupied by
mobB
in R1162. Despite this, the MobB and MobX proteins do not appear to be closely related based on a comparison of their amino acid sequences. Complementation analysis indicated that neither of the pSC101 Mob proteins could substitute for, or be replaced by, their R1162 counterparts, nor were they active together at the R1162 origin of transfer (
oriT
). However, the full set of R1162 Mob proteins did recognize the pSC101
oriT
. A hybrid system for mobilization, active at the R1162
oriT
site, was constructed. This system consists of MobX and a chimeric protein made up of the DNA cleaving-ligating domain of the R1162 MobA protein joined to a fragment of pSC101 MobA. Previous results suggested that MobB and a region of MobA distinct from the DNA processing domain together formed a functional unit in transfer. The present results support this model because the chimeric MobA, although active on R1162
oriT
, requires the pSC101 protein MobX for efficient plasmid mobilization.
Regulation of Transcription of themph(A) Gene for Macrolide 2′-Phosphotransferase I inEscherichia coli: Characterization of the Regulatory GenemphR(A)
Authors: Noguchi Norihisa , Takada Katsutoshi , Katayama Jin , Emura Ayako , Sasatsu Masanori
Published: July 2002

The synthesis of macrolide 2′-phosphotransferase I [Mph(A)], which inactivates erythromycin, is inducible by erythromycin. The expression of high-level resistance to erythromycin requires themph(A) andmrxgenes, which encode Mph(A) and an unidentified protein, respectively. We have studied themphR(A) gene, which regulates the inducible expression ofmph(A). An analysis of the synthesis of Mph(A) in minicells and results of a complementation test indicated thatmphR(A) is located downstream frommrxand that its product, MphR(A), represses the production of Mph(A). DNA sequencing indicated that themph(A),mrx, andmphR(A) genes exist as a cluster that begins withmph(A) and that the deduced amino acid sequence of MphR(A) can adopt an α-helix–turn–α-helix structure. To study the regulation of gene expression by MphR(A), we performed Northern blotting and primer extension. A transcript of 2.9 kb that corresponded to the transcript ofmph(A) throughmphR(A) was detected, and its level was elevated upon exposure of cells to erythromycin. Gel mobility shift assays and DNase I footprinting indicated that MphR(A) binds specifically to the promoter region ofmph(A), and the amount of DNA shifted as a results of the binding of MphR(A) decreased as the concentration of erythromycin was increased. These results indicate that transcription of themph(A)-mrx-mphR(A) operon is negatively regulated by the binding of a repressor protein, MphR(A), to the promoter of themph(A) gene and is activated upon inhibition of binding of MphR(A) to the promoter in the presence of erythromycin.
trp
RNA-Binding Attenuation Protein-5′ Stem-Loop RNA Interaction Is Required for Proper Transcription Attenuation Control of the
Bacillus subtilis trpEDCFBA
Operon
Authors: Du Hansen , Yakhnin Alexander V. , Dharmaraj Subramanian , Babitzke Paul
Published: July 2002

The
trp
RNA-binding attenuation protein (TRAP) regulates expression of the
Bacillus subtilis trpEDCFBA
operon by a novel transcription attenuation mechanism. Tryptophan-activated TRAP binds to the nascent
trp
leader transcript by interacting with 11 (G/U)AG repeats, 6 of which are present in an antiterminator structure. TRAP binding to these repeats prevents formation of the antiterminator, thereby promoting formation of an overlapping intrinsic terminator. A third stem-loop structure that forms at the extreme 5′ end of the
trp
leader transcript also plays a role in the transcription attenuation mechanism. The 5′ stem-loop increases the affinity of TRAP for
trp
leader RNA. Results from RNA structure mapping experiments demonstrate that the 5′ stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bp upper stem, and a hexaloop at the apex of the structure. Footprinting results indicate that TRAP interacts with the 5′ stem-loop and that this interaction differs depending on the number of downstream (G/U)AG repeats present in the transcript. Expression studies with
trpE′-′lacZ
translational fusions demonstrate that TRAP-5′ stem-loop interaction is required for proper regulation of the
trp
operon. 3′ RNA boundary experiments indicate that the 5′ structure reduces the number of (G/U)AG repeats required for stable TRAP-
trp
leader RNA association. Thus, TRAP-5′ stem-loop interaction may increase the likelihood that TRAP will bind to the (G/U)AG repeats in time to block antiterminator formation.
Heterologous Inducible Expression of
Enterococcus faecalis
pCF10 Aggregation Substance Asc10 in
Lactococcus lactis
and
Streptococcus gordonii
Contributes to Cell Hydrophobicity and Adhesion to Fibrin
Authors: Hirt Helmut , Erlandsen Stanley L. , Dunny Gary M.
Published: July 2002

Aggregation substance proteins encoded by the sex pheromone plasmid family of
Enterococcus faecalis
have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further,
prgB
, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible
nisA
promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of
E. faecalis
and the heterologous bacteria
Lactococcus lactis
and
Streptococcus gordonii
was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of
E. faecalis
and
L. lactis
but not on that of
S. gordonii
. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of
E. faecalis
and
L. lactis
revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on
L. lactis
also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the
nisA
promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.
Mu and IS
1
Transpositions Exhibit Strong Orientation Bias at the
Escherichia coli bgl
Locus
Authors: Manna Dipankar , Wang Xiuhua , Higgins N. Patrick
Published: July 2002

The region upstream of the
Escherichia coli bgl
operon is an insertion hot spot for several transposons. Elements as distantly related as Tn
1
, Tn
5
, and phage Mu home in on this location. To see what characteristics result in a high-affinity site for transposition, we compared in vivo and in vitro Mu transposition patterns near the
bgl
promoter. In vivo, Mu insertions were focused in two narrow zones of DNA near
bgl
, and both zones exhibited a striking orientation bias. Five hot spots upstream of the
bgl
cyclic AMP binding protein (CAP) binding site had Mu insertions exclusively with the phage oriented left to right relative to the direction of
bgl
transcription. One hot spot within the CAP binding domain had the opposite (right-to-left) orientation of phage insertion. The DNA segment lying between these two Mu hot-spot clusters is extremely A/T rich (80%) and is an efficient target for insertion sequences during stationary phase. IS
1
insertions that activate the
bgl
operon resulted in a decrease in Mu insertions near the CAP binding site. Mu transposition in vitro differed significantly from the in vivo transposition pattern, having a new hot-spot cluster at the border of the A/T-rich segment. Transposon hot-spot behavior and orientation bias may relate to an asymmetry of transposon DNA-protein complexes and to interactions with proteins that produce transcriptionally silenced chromatin.
Identification of Some DNA Damage-Inducible Genes of
Mycobacterium tuberculosis
: Apparent Lack of Correlation with LexA Binding
Authors: Brooks Patricia C. , Movahedzadeh Farahnaz , Davis Elaine O.
Published: July 2002

The repair of DNA damage is expected to be particularly important to intracellular pathogens such as
Mycobacterium tuberculosis
, and so it is of interest to examine the response of
M. tuberculosis
to DNA damage. The expression of
recA
, a key component in DNA repair and recombination, is induced by DNA damage in
M. tuberculosis
. In this study, we have analyzed the expression following DNA damage in
M. tuberculosis
of a number of other genes which are DNA damage inducible in
Escherichia coli
. While many of these genes were also induced by DNA damage in
M. tuberculosis
, some were not. In addition, one gene (
ruvC
) which is not induced by DNA damage in
E. coli
was induced in
M. tuberculosis
, a result likely linked to its different transcriptional arrangement in
M. tuberculosis
. We also searched the sequences upstream of the genes being studied for the mycobacterial SOS box (the binding site for LexA) and assessed LexA binding to potential sites identified. LexA is the repressor protein responsible for regulating expression of these SOS genes in
E. coli
. However, two of the genes which were DNA damage inducible in
M. tuberculosis
did not have identifiable sites to which LexA bound. The absence of binding sites for LexA upstream of these genes was confirmed by analysis of LexA binding to overlapping DNA fragments covering a region from 500 bp upstream of the coding sequence to 100 bp within it. Therefore, it appears most likely that an alternative mechanism of gene regulation in response to DNA damage exists in
M. tuberculosis
.
Identification of the
varR
Gene as a Transcriptional Regulator of Virginiamycin S Resistance in
Streptomyces virginiae
Authors: Namwat Wises , Lee Chang-Kwon , Kinoshita Hiroshi , Yamada Yasuhiro , Nihira Takuya
Published: July 2002

A gene designated
varR
(for
virginiae
antibiotic resistance regulator) was identified in
Streptomyces virginiae
89 bp downstream of a
varS
gene encoding a virginiamycin S (VS)-specific transporter. The deduced
varR
product showed high homology to repressors of the TetR family with a conserved helix-turn-helix DNA binding motif. Purified recombinant VarR protein was present as a dimer in vitro and showed clear DNA binding activity toward the
varS
promoter region. This binding was abolished by the presence of VS, suggesting that VarR regulates transcription of
varS
in a VS-dependent manner. Northern blot analysis revealed that
varR
was cotranscribed with upstream
varS
as a 2.4-kb transcript and that VS acted as an inducer of bicistronic transcription. Deletion analysis of the
varS
promoter region clarified two adjacent VarR binding sites in the
varS
promoter.
Roles of therecJandrecNGenes in Homologous Recombination and DNA Repair Pathways ofNeisseria gonorrhoeae
Authors: Skaar Eric P. , Lazio Matthew P. , Seifert H. Steven
Published: July 2002

The paradigm of homologous recombination comes fromEscherichia coli, where the genes involved have been segregated into pathways. In the human pathogenNeisseria gonorrhoeae(the gonococcus), the pathways of homologous recombination are being delineated. To investigate the roles of the gonococcalrecNandrecJgenes in the recombination-based processes of the gonococcus, these genes were inactivated in theN. gonorrhoeaestrain FA1090. We report that bothrecNandrecJloss-of-function mutants show decreased DNA repair ability. In addition, therecJmutant was decreased in pilus-dependent colony morphology variation frequency but not DNA transformation efficiency, while therecNmutant was decreased in DNA transformation efficiency but not pilus-dependent variation frequency. We were able to complement all of these deficiencies by supplying an ectopic functional copy of eitherrecJorrecNat an irrelevant locus. These results describe the role ofrecJandrecNin the recombination-dependent processes of the gonococcus and further define the pathways of homologous recombination in this organism.
Identification and Characterization of CAMP Cohemolysin as a Potential Virulence Factor of
Riemerella anatipestifer
Authors: Crasta Karen C. , Chua Kim-Lee , Subramaniam Sumathi , Frey Joachim , Loh Hilda , Tan Hai-Meng
Published: July 2002

Riemerella anatipestifer
is responsible for exudative septicemia in ducks. The genetic determinant of the CAMP cohemolysin,
cam
, from a strain of
R. anatipestifer
was cloned and expressed in
Escherichia coli.
Chromosomal DNA from serotype 19 strain 30/90 was used to construct a gene library in pBluescript II SK(−) vector in
E. coli
XL-1-Blue strain. The clones containing recombinant plasmids were screened for the CAMP reaction with
Staphylococcus aureus
. Those that showed cohemolysis were chosen for further analysis by sequencing. One of these clones, JFRA8, was subcloned to identify the smallest possible DNA fragment containing the CAMP cohemolysin determinant, which was located on a 3,566-bp
Bam
HI-
Bst
XI fragment which specified a 1,026-bp open reading frame. Clones containing recombinant plasmids carrying
cam
obtained by PCR cloning into
E. coli
M15 strain secreted an active CAMP cohemolysin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses confirmed that the recombinant strain expressed a protein with a molecular mass of 37 kDa and that strains from serotypes 1, 2, 3, 5, 6, and 19 expressed the cohemolysin. The deduced amino acid sequence showed high homology to those of
O
-sialoglycoprotein endopeptidases. Hydrolysis of radioiodinated glycophorin A confirmed that Cam is a sialoglycoprotease.
Nosocomial Outbreak Due to a Multiresistant Strain of
Pseudomonas aeruginosa
P12: Efficacy of Cefepime-Amikacin Therapy and Analysis of β-Lactam Resistance
Authors: Dubois Véronique , Arpin Corinne , Melon Monique , Melon Bernard , Andre Catherine , Frigo Cécile , Quentin Claudine
Published: July 2002

Over a 3-year period, 67 patients of the Hospital of Pau (Pau, France), including 64 patients hospitalized in the adult intensive care unit (ICU), were colonized and/or infected by strains of
Pseudomonas aeruginosa
P12, resistant to all potentially active antibiotics except colistin. Most patients were mechanically ventilated and presented respiratory tract infections. Since cefepime and amikacin were the least inactive antibiotics by MIC determination, all ICU patients were treated with this combination, and most of them benefited. Cefepime-amikacin was found highly synergistic in vitro. Ribotyping and arbitrary primer-PCR analysis confirmed the presence of a single clonal isolate. Isoelectrofocusing revealed that the epidemic strain produced large amounts of the chromosomal cephalosporinase and an additional enzyme with a pI of 5.7, corresponding to PSE-1, as demonstrated by PCR and sequencing. Outer membrane protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the absence of a ca. 46-kDa protein, likely to be OprD, and increased production of two ca. 49- and 50-kDa proteins, consistent with the outer membrane components of the efflux systems, MexAB-OprM and MexEF-OprN. Thus, we report here a nosocomial outbreak due to multiresistant
P. aeruginosa
P12 exhibiting at least four mechanisms of β-lactam resistance, i.e., production of the penicillinase PSE-1, overproduction of the chromosomal cephalosporinase, loss of OprD, and overexpression of efflux systems, associated with a better activity of cefepime than ceftazidime.
Molecular Epidemiology of
Mycobacterium malmoense
Infections in Scotland
Authors: Doig C. , Muckersie L. , Watt B. , Forbes K. J.
Published: July 2002

Clinical isolates of
Mycobacterium malmoense
collected over 5 years from patients across Scotland with a variety of diseases have been characterized by pulsed-field gel electrophoresis (PFGE), ribotyping, and 16S ribosomal DNA gene sequencing. Results indicate that this species harbors little genetic diversity and that the different strain types that were identified by PFGE showed no correlation with geographical origin or date of isolation.
Detection of
Mycobacterium avium
in Blood Samples of Patients with AIDS by Using PCR
Authors: Rodrigues Vívian de F. Sumnienski , Queiroz Mello Fernanda C. , Ribeiro Marta Osório , Fonseca Leila , Kritski Afrânio Lineu , Rossetti Maria Lucia , Zaha Arnaldo
Published: July 2002

Sixty-nine blood samples from 47 patients infected with human immunodeficiency virus were analyzed by using PCR to detect
Mycobacterium avium
. The sensitivity can be up to 95.7%, depending on the detection method used and the number of blood samples analyzed from each patient. The procedure can be helpful in the diagnosis of mycobacterial disease.
Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein
Authors: Boyko Vitaly , van der Laak Jessica , Ferralli Jacqueline , Suslova Elena , Kwon Myoung-Ok , Heinlein Manfred
Published: July 2002

Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.
Retroviral Vector Targeting to Human Immunodeficiency Virus Type 1-Infected Cells by Receptor Pseudotyping
Authors: Somia Nikunj V. , Miyoshi Hiroyuki , Schmitt Mark J. , Verma Inder M.
Published: July 2002

We report the generation of retroviral vectors based on Moloney murine leukemia virus that specifically transduce cells infected with T-cell-tropic human immunodeficiency virus type 1 (HIV-1). This vector was pseudotyped with T-cell-tropic HIV-1 receptors CD4 and CXCR4. We demonstrate that transduction is contingent upon HIV-1 gp120 and gp41 expression.