bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterium bovisBCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens
Authors: Pedras-Vasconcelos João A. , Chapdelaine Yvan , Dudani Renu , van Faassen Henk , Smith Dean K. , Sad Subash
Published: July 2002

Type 1 T-cell responses against intracellular pathogens play a crucial role in mediating protection. We examined whether the induction of a strong type 1 T-cell response during a chronic bacterial infection influences responses to superantigens capable of inducing acute shock. Intravenous infection of mice withMycobacterium bovisBCG appeared to induce a progressive anergy towards staphylococcal enterotoxin B (SEB) and towards antigen preparation of BCG (BCG-Ag) itself, based on diminished gamma interferon (IFN-γ) production by SEB- and BCG-Ag-stimulated splenocytes from infected mice. In contrast to these in vitro results, injection of SEB into BCG-infected mice led to a dramatic increase in the serum IFN-γ levels and the death of infected but not of control mice. In vitro hyporesponsiveness towards SEB and BCG-Ag occurred only with unfractionated splenocyte cultures, as purified T cells from infected mice produced higher levels of IFN-γ. Hyporesponsiveness towards SEB and BCG-Ag in unfractionated splenocyte cultures was not due to suppressive antigen-presenting cells (APCs), as APCs from infected mice stimulated higher levels of IFN-γ from purified T cells. The diminished IFN-γ levels observed with bulk splenocytes appear to be due to changes in the T-cell-to-APC ratio that result in a decreased proportion of T cells, coupled to reduced proliferative responses and an increased susceptibility of effector T cells to activation-induced cell death in vitro. Our results indicate that the reported phenomena of T-cell anergy during mycobacterial infection may be an in vitro consequence of the development of a strong type 1 response in vivo.
Evidence That the Variable Regions of the Central Domain of VlsE Are Antigenic during Infection with Lyme Disease Spirochetes
Authors: McDowell John V. , Sung Shian-Ying , Hu Linden T. , Marconi Richard T.
Published: July 2002

It has been postulated that thevlssystem of the Lyme disease spirochetes contributes to immune evasion through antigenic variation. While it is clear thatvlsEundergoes sequence change within its variable regions at a high frequency during the early stages of infection, a definitive role in immune evasion has not been demonstrated. In this report we assessed the murine and human humoral immune response to recombinant (r)-VlsE variants that originally arose during infection in mice. Immunoblot analyses of r-VlsE variants were conducted by using serum samples collected from mice infected withBorrelia burgdorfericlones that carried differentvlsEvariants. All of the r-VlsE variants were recognized by infection sera regardless of the identity of the infecting clone or isolate. In addition, all variants were immunoreactive with a panel of human Lyme disease patient serum samples. It is evident from these analyses that the infection-induced VlsE variants share common epitopes that reside within conserved segments of these proteins. However, preabsorption experiments revealed that the variable regions of the central domain of VlsE, which undergo rapid mutation during infection, also influence the antigenic properties of the protein. A subset of the antibodies elicited againstvlsEvariants that differ in the sequences of their variable regions were found to be variant specific. Hence, in spite of a robust antibody response to conserved segments of VlsE, infection-induced sequence changes within the variable regions alter the antigenicity of VlsE. These results provide the first direct evidence of antigenic variation in the VlsE protein.
Streptococcus suisInteractions with the Murine Macrophage Cell Line J774: Adhesion and Cytotoxicity
Authors: Segura Mariela , Gottschalk Marcelo
Published: July 2002

Streptococcus suiscapsular type 2 is an important etiological agent of swine meningitis, and it is also a zoonotic agent. Since one hypothesis of the pathogenesis ofS. suisinfection is that bacteria enter the bloodstream and invade the meninges and other tissues in close association with mononuclear phagocytes, the objective of the present study was to evaluate the capacity ofS. suistype 2 to adhere to macrophages. An enzyme-linked immunosorbent assay technique was standardized to simply and accurately measure the rate of bacterial attachment to phagocytic cells. Results were confirmed by plate counting. Adhesion was dependent on bacterial concentration and incubation time and was not affected by cytochalasin pretreatment of macrophages. Inhibition studies showed that the sialic acid moiety of theS. suiscapsule would be, at least in part, responsible for bacterial recognition by macrophages. Serum preopsonization of bacteria increased adhesion levels. Complement would be partially implicated in the serum-enhanced binding ofS. suisto cells. Adhesion varied among differentS. suistype 2 isolates. However, high bacterial concentrations of several isolates were cytotoxic for cells, and these cytotoxic effects correlated with suilysin production. Indeed, hemolytic strain supernatants, as well as purified suilysin, reproduced cytotoxic effects observed with live bacteria, and these effects were inhibited by cholesterol pretreatment. Bacterial adhesion and cytotoxicity were confirmed by scanning and transmission electron microscopy. We hypothesize that attachment of bacteria to phagocytes could play an important role in the pathogenesis ofS. suisinfection by allowing bacterial dissemination and causing a bacteremia and/or septicemia. This interaction could also be related to the activation of the host inflammatory response observed during meningitis.
Contribution of Phospholipomannan to the Surface Expression of β-1,2-Oligomannosides inCandida albicansand Its Presence in Cell Wall Extracts
Authors: Poulain D. , Slomianny C. , Jouault T. , Gomez J. M. , Trinel P. A.
Published: July 2002

β-1,2-Oligomannosides (β-1,2-Man) derived fromCandida albicansmannan have been shown to act as adhesins and to induce protective antibodies. We used monoclonal antibodies specific for β-1,2-Man in electron, confocal, and fluorescence microscopy to study the surface expression of β-1,2-Man epitopes. These monoclonal antibodies were also used for Western blotting of cell surface extracts to study the nature of the molecules expressing the β-Man epitopes. Evidence was obtained for the contribution of a glycolipid, phospholipomannan (PLM), to the complex expression of β-1,2-Man epitopes at the cell wall surfaces of yeasts grown on solid media. PLM was present in intercellular matrixes of colonies grown on agar and was detected as a contaminant in mannan batches prepared by conventional methods.
Protection of Rhesus Macaques against LethalPlasmodium knowlesiMalaria by a Heterologous DNA Priming and Poxvirus Boosting Immunization Regimen
Authors: Rogers William O. , Weiss Walter R. , Kumar Anita , Aguiar João C. , Tine John A. , Gwadz Robert , Harre Joseph G. , Gowda Kalpana , Rathore Dharmendar , Kumar Sanjai , Hoffman Stephen L.
Published: July 2002

We tested a cytokine-enhanced, multiantigen, DNA priming and poxvirus boosting vaccine regimen for prevention of malaria in thePlasmodium knowlesi-rhesus macaque model system. Animals were primed with a mixture of DNA plasmids encoding two preerythrocytic-stage proteins and two erythrocytic-stage proteins fromP. knowlesiand combinations of the cytokines granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor alpha and were boosted with a mixture of four recombinant, attenuated vaccinia virus strains encoding the fourP. knowlesiantigens. Two weeks after boosting, the geometric mean immunofluorescence titers in the immunized groups against sporozoites and infected erythrocytes ranged from 160 to 8,096 and from 1,810 to 5,120, respectively. The geometric mean anti-P. knowlesicircumsporozoite protein (PkCSP) titers ranged from 1,761 to 24,242. Peripheral blood mononuclear cells (PBMC) from the immunized monkeys produced gamma interferon (IFN-γ) in response to incubation with pooled peptides from the PkCSP at frequencies of 10 to 571 spot-forming cells/106PBMC. Following challenge with 100 infectiousP. knowlesisporozoites, 2 of 11 immunized monkeys were sterilely protected, and 7 of the 9 infected monkeys resolved their parasitemias spontaneously. In contrast, all four controls became infected and required treatment for overwhelming parasitemia. Early protection was strongly associated with IFN-γ responses against a pool of peptides from the preerythrocytic-stage antigen, PkCSP. These findings demonstrate that a multistage, multiantigen, DNA priming and poxvirus boosting vaccine regimen can protect nonhuman primates from an otherwise lethal malaria sporozoite challenge.
Differential Expression of Virulence-Related Genes in
Enterococcus faecalis
in Response to Biological Cues in Serum and Urine
Authors: Shepard Brett D. , Gilmore Michael S.
Published: July 2002

Enterococci rank among leading causes of nosocomial bacteremia and urinary tract infection and are also a leading cause of community acquired subacute endocarditis. Limited evidence suggests that biological cues in serum and urine may play an important role in modulating enterococcal virulence at sites of infection. To determine the extent to which biological cues affect enterococcal virulence-associated gene expression, we used quantitative real-time PCR to compare mRNA levels in
Enterococcus faecalis
cultures grown in serum or urine to that achieved in laboratory medium. Both environment- and growth phase-specific variations were observed, demonstrating the occurrence of as-yet-uncharacterized mechanisms for control of gene expression in
E. faecalis
that may play an important role in vivo.
AhagMutant ofMoraxella catarrhalisStrain O35E Is Deficient in Hemagglutination, Autoagglutination, and Immunoglobulin D-Binding Activities
Authors: Pearson Melanie M. , Lafontaine Eric R. , Wagner Nikki J. , St. Geme Joseph W. , Hansen Eric J.
Published: July 2002

Previous studies correlated the presence of a 200-kDa protein on the surface ofMoraxella catarrhaliswith the ability of this organism to agglutinate human erythrocytes (M. Fitzgerald, R. Mulcahy, S. Murphy, C. Keane, D. Coakley, and T. Scott, FEMS Immunol. Med. Microbiol. 18:209-216, 1997). In the present study, the gene encoding the 200-kDa protein (designated Hag) ofM. catarrhalisstrain O35E was subjected to nucleotide sequence analysis and then was inactivated by insertional mutagenesis. The isogenichagmutant was unable to agglutinate human erythrocytes and lost its ability to autoagglutinate but was still attached at wild-type levels to several human epithelial cell lines. Thehagmutation also eliminated the ability of this mutant strain to bind human immunoglobulin D. The presence of the Hag protein on theM. catarrhaliscell surface, as well as that of the UspA1 and UspA2 proteins (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367-4377, 1997), was investigated by transmission electron and cryoimmunoelectron microscopy. Wild-typeM. catarrhalisstrain O35E possessed a dense layer of surface projections, whereas an isogenicuspA1 uspA2 hagtriple mutant version of this strain did not possess any detectable surface projections. Examination of auspA1 uspA2double mutant that expressed the Hag protein revealed the presence of a relatively sparse layer of surface projections, similar to those seen on auspA2 hagmutant that expressed UspA1. In contrast, auspA1 hagmutant that expressed UspA2 formed a very dense layer of relatively short surface projections. These results indicate that the surface-exposed Hag protein and UspA1 and UspA2 have the potential to interact both with each other and directly with host defense systems.
Opsonization Modulates Rac-1 Activation during Cell Entry byLeishmania amazonensis
Authors: Morehead J. , Coppens I. , Andrews N. W.
Published: July 2002

Lesions caused byLeishmania amazonensisnormally heal, but relapses occur due to parasite persistence in host tissues. It has been proposed that infection of fibroblasts plays an important role in this process by providing the parasites with a safe haven in which to replicate. However, most previous studies have focused on the entry ofLeishmaniainto macrophages, a process mediated by serum opsonins. To gain insight into a possible role of nonopsonic entry in the intracellular persistence of amastigotes, we examined the invasion of Chinese hamster ovary (CHO) cells. Amastigotes entered CHO cells by a cytochalasin D, genistein, wortmannin, and 2,3-butanedione monoxime-sensitive pathway and replicated within phagolysosomes. However, unlike most phagocytic processes described to date, amastigote internalization in CHO cells involved activation of the GTPases Rho and Cdc42 but not Rac-1. When uptake was mediated by fibronectin or when amastigotes were opsonized with immunoglobulin G and internalized by Fc receptor-expressing CHO cells, Rac-1 activation was restored and found to be required for parasite internalization. Given the essential role of Rac in assembly of the respiratory burst oxidase, invasion through this nonopsonic, Rac-1-independent pathway may play a central role in the intracellular survival ofLeishmaniain immune hosts.
The
Enterococcus faecalis fsrB
Gene, a Key Component of the
fsr
Quorum-Sensing System, Is Associated with Virulence in the Rabbit Endophthalmitis Model
Authors: Mylonakis Eleftherios , Engelbert Michael , Qin Xiang , Sifri Costi D. , Murray Barbara E. , Ausubel Frederick M. , Gilmore Michael S. , Calderwood Stephen B.
Published: July 2002

We used a rabbit endophthalmitis model to explore the role of
fsrB
, a gene required for the function of the
fsr
quorum-sensing system of
Enterococcus faecalis
, in pathogenicity. A nonpolar deletion mutant of
fsrB
had significantly reduced virulence compared to wild type. Complementation of mutation restored virulence. These data corroborate the role of
fsrB
in
E. faecalis
pathogenesis and suggest that the rabbit endophthalmitis model can be used to study the in vivo role of quorum sensing.
Network of Hydrogenase Maturation in
Escherichia coli
: Role of Accessory Proteins HypA and HybF
Authors: Hube Michaela , Blokesch Melanie , Böck August
Published: July 2002

We have studied the roles of the auxiliary protein HypA and of its homolog HybF in hydrogenase maturation. A mutation in
hypA
leads to the nearly complete blockade of maturation solely of hydrogenase 3 whereas a lesion in
hybF
drastically but not totally reduces maturation and activity of isoenzymes 1 and 2. The residual level of matured enzymes in the
hybF
mutant was shown to be due to the function of HypA; HybF, conversely, was responsible for a minimal residual activity of hydrogenase 3 in the mutant
hypA
strain. Accordingly, a
hypA
Δ
hybF
double mutant was completely blocked in the maturation process. However, the inclusion of high nickel concentrations in the medium could restore limited activity of all three hydrogenases. The results of this study and of previous work (M. Blokesch, A. Magalon, and A. Böck, J. Bacteriol.
189:
2817-2822, 2001) show that the maturation of the three functional hydrogenases from
Escherichia coli
is intimately connected via the activity of proteins HypA and HypC and of their homologs HybF and HybG, respectively. The results also support the suggestion of Olson et al. (J. W. Olson, N. S. Mehta, and R. J. Maier, Mol. Microbiol.
39:
176-182, 2001) that HypA cooperates with HypB in the insertion of nickel into the precursor of the large hydrogenase subunit. Whereas HypA is predominantly involved in the maturation of hydrogenase 3, HybF takes over its function in the maturation of isoenzymes 1 and 2.
Patterns of Sequence Conservation in the S-Layer Proteins and Related Sequences in
Clostridium difficile
Authors: Calabi Emanuela , Fairweather Neil
Published: July 2002

Clostridium difficile
is the etiological agent of antibiotic-associated diarrhea. Among the factors that may play a role in infection are S-layer proteins (SLPs). Previous work has shown these to consist mainly of two components, resulting from the cleavage of a precursor encoded by the
slpA
gene. The high-molecular-weight (MW) subunit is related both to amidases from
B. subtilis
and to at least another 28 gene products in
C. difficile
strain 630. To gain insight into the functions of the SLPs and related proteins, we have further investigated the pattern of variability both at the
slpA
locus and at six nearby paralogs. Sequencing of the
slpA
gene from an S-layer group II strain and a variant S-layer group strain confirms a high degree of divergence in the low-MW SLP, which may result from diversifying selection. A highly conserved motif, however, is found at the C terminus in all low-MW subunits and may be essential for SlpA precursor cleavage. In strain 167, a variant cleavage product is present, suggesting a secondary processing site. Southern blotting analysis shows
slpA
-like open reading frames (ORFs) 2 to 7 to be conserved in all nine strains tested, with one exception: ORF2, which encodes a 66-kDa polypeptide coextracted at low pH with the main SLPs in strain 630, may be partially deleted in strain 167. Polymorphism within the
slpA
-ORF7 cluster may be more pronounced in the region proximal to the
slpA
gene. Unexpectedly, a high-MW subunit probe cross hybridizes to sequences outside the
slpA
locus, which appear to vary in number in different strains.
Microarray Analysis of the
Mycobacterium tuberculosis
Transcriptional Response to the Acidic Conditions Found in Phagosomes
Authors: Fisher Mark A. , Plikaytis Bonnie B. , Shinnick Thomas M.
Published: July 2002

We used microarrays and real-time reverse transcription-PCR to analyze the global transcriptional response of
Mycobacterium tuberculosis
to low pH in vitro, which may mimic an environmental signal encountered by phagocytosed mycobacteria. Eighty-one genes were differentially expressed >1.5-fold, including many involved in fatty acid metabolism. The most highly induced genes showed homology with nonribosomal peptide synthetases/polyketide synthases.
The BaeSR Two-Component Regulatory System Activates Transcription of the
yegMNOB
(
mdtABCD
) Transporter Gene Cluster in
Escherichia coli
and Increases Its Resistance to Novobiocin and Deoxycholate
Authors: Baranova Natalya , Nikaido Hiroshi
Published: July 2002

Screening of random fragments of
Escherichia coli
genomic DNA for their ability to increase the novobiocin resistance of a hypersusceptible Δ
acrAB
mutant resulted in the isolation of a plasmid containing
baeR
, which codes for the response regulator of the two-component regulatory system BaeSR. When induced for expression,
baeR
cloned in multicopy plasmid pTrc99A significantly increased the resistance of the Δ
acrAB
host strain to novobiocin (16-fold) and to deoxycholate (8-fold). Incubation of cells with novobiocin followed by a chromatographic assay for intracellular drug showed that overproduced BaeR decreased drastically the drug accumulation, presumably via increased active efflux. The genes
baeSR
are part of a putative operon,
yegMNOB baeSR
. Direct binding of BaeR to the
yegM
promoter was demonstrated in vitro by gel retardation assay. The gene
yegB
, which codes for a major facilitator superfamily transporter, was not necessary for increased resistance, but deletion of
yegO
or an in-frame deletion of
yegN
, both of which code for resistance-nodulation-cell division-type multidrug transporters, abolished the BaeR-induced increase in resistance. It is likely that both YegN and YegO produce a complex(es) with the membrane fusion protein family member YegM and pump out novobiocin and deoxycholate. We accordingly propose to rename
yegMNOB
as
mdtABCD
(
mdt
for multidrug transporter). Finally, the expression of two other genes,
yicO
and
ygcL
, was shown to be regulated by BaeR, but it is not known if they play any roles in resistance.
Cooperativity in Signal Transfer through the Uhp System ofEscherichia coli
Authors: Verhamme Daniël T. , Postma Pieter W. , Crielaard Wim , Hellingwerf Klaas J.
Published: July 2002

The UhpABC regulatory system in enterobacteria controls the expression of the hexose phosphate transporter UhpT. Signaling is initiated through sensing of extracellular glucose 6-phosphate by membrane-bound UhpC, which in turn modulates the histidine-protein kinase UhpB. Together with the cytoplasmic response regulator UhpA, they constitute a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Activated (i.e., phosphorylated) UhpA binds to the promoter region ofuhpT, resulting in initiation of transcription. We have investigated the contribution of transmembrane signaling (through UhpBC) and intracellular activation (through UhpA) to the overall Uhp response (UhpT expression) in vivo. UhpA activation could be made independent of transmembrane signaling when ΔuhpBCcells were grown on pyruvate. Inorganic phosphate interfered with glucose 6-phosphate-dependent, UhpBC-mediated, as well as pyruvate-mediated activation of UhpA. The relationship between the concentration of inducer (glucose 6-phosphate) and the Uhp induction rate was nonhyperbolic, indicating positive cooperativity. The degree of cooperativity was affected by the carbon or energy source available to the cells for growth. As pyruvate-mediated activation of UhpA in ΔuhpBCcells could result in considerably stronger UhpT expression than glucose 6-phosphate-dependent activation through UhpBC, the observed positive cooperativity for the overall pathway in wild-type cells may reflect the previously described cooperative binding of UhpA to theuhpTpromoter (J. L. Dahl et al., J. Biol. Chem.272:1910-1919, 1997).
Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase
Authors: Smith Kerry S. , Ingram-Smith Cheryl , Ferry James G.
Published: July 2002

The roles of an aspartate and an arginine, which are completely conserved in the active sites of β-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the β-class enzyme (Cab) from the archaeon
Methanobacterium thermoautotrophicum.
Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the
k
cat
/

K
m

value depends only on the rate constants for the CO
2
hydration step, whereas
k
cat
also depends on rate constants from the proton transfer step (K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, and J. G. Ferry, J. Bacteriol.
182:
6605-6613, 2000). The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P. Strop, K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J. Biol. Chem.
276:
10299-10305, 2001). The
k
cat
/

K
m

values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO
2
hydration step. Parallel decreases in
k
cat
and
k
cat
/

K
m

values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the
k
cat
of the D34A variant was chemically rescued by replacement of 2-(
N
-morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step. The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variants had both
k
cat
and
k
cat
/

K
m

values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue. Imidazole was unable to rescue the
k
cat
of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this residue is important.
Genomic and Functional Analyses of SXT, an Integrating Antibiotic Resistance Gene Transfer Element Derived from
Vibrio cholerae
Authors: Beaber John W. , Hochhut Bianca , Waldor Matthew K.
Published: July 2002

SXT is representative of a family of conjugative-transposon-like mobile genetic elements that encode multiple antibiotic resistance genes. In recent years, SXT-related conjugative, self-transmissible integrating elements have become widespread in Asian
Vibrio cholerae
. We have determined the 100-kb DNA sequence of SXT. This element appears to be a chimera composed of transposon-associated antibiotic resistance genes linked to a variety of plasmid- and phage-related genes, as well as to many genes from unknown sources. We constructed a nearly comprehensive set of deletions through the use of the one-step chromosomal gene inactivation technique to identify SXT genes involved in conjugative transfer and chromosomal excision. SXT, unlike other conjugative transposons, utilizes a conjugation system related to that encoded by the F plasmid. More than half of the SXT genome, including the composite transposon-like structure that contains its antibiotic resistance genes, was not required for its mobility. Two SXT loci, designated
setC
and
setD
, whose predicted amino acid sequences were similar to those of the flagellar regulators FlhC and FlhD, were found to encode regulators that activate the transcription of genes required for SXT excision and transfer. Another locus, designated
setR
, whose gene product bears similarity to lambdoid phage CI repressors, also appears to regulate SXT gene expression.
Glycine Betaine Transmethylase Mutant of
Pseudomonas aeruginosa
Authors: Serra Ana L. , Mariscotti Javier F. , Barra José L. , Lucchesi Gloria I. , Domenech Carlos E. , Lisa Angela T.
Published: July 2002

The gene for glycine betaine transmethylase (
gbt
) was identified in
Pseudomonas aeruginosa
strain Fildes III by biochemical, physiological, and molecular approaches. Based on sequence analysis, the knockout gene corresponded to an open reading frame (ORF) named PA3082 in the genome of
P. aeruginosa
PAO1. The translated product of this ORF displayed similarity to transferases of different microorganisms. Mutation in
gbt
blocked the utilization of choline and glycine betaine as carbon and nitrogen sources.
The MexR Repressor of themexAB-oprMMultidrug Efflux Operon inPseudomonas aeruginosa: Characterization of Mutations Compromising Activity
Authors: Adewoye Lateef , Sutherland Ainsley , Srikumar Ramakrishnan , Poole Keith
Published: July 2002

Mutations inmexRyield a multidrug resistance phenotype innalBmutants ofPseudomonas aeruginosaas a result of derepression of themexAB-oprMmultidrug efflux operon. MexR produced by severalnalBstrains carried single amino acid changes that compromised MexR stability or its ability to dimerize. Changes at residues L95 and R21, however, produced a stable MexR protein capable of dimerization and, thus, likely compromised DNA binding.
Structural Evidence that the P/Q Domain of ZipA Is an Unstructured, Flexible Tether between the Membrane and the C-Terminal FtsZ-Binding Domain
Authors: Ohashi Tomoo , Hale Cynthia A. , de Boer Piet A. J. , Erickson Harold P.
Published: July 2002

The cell division protein ZipA has an N-terminal transmembrane domain and a C-terminal globular domain that binds FtsZ. Between them are a charged domain and a P/Q domain rich in proline and glutamine that has been proposed to be an unfolded polypeptide. Here we provide evidence obtained by electron microscopy that the P/Q domain is a flexible tether ranging in length from 8 to 20 nm and invisible in rotary shadowing electron microscopy. We estimated a persistence length of 0.66 nm, which is similar to the persistence lengths of other unfolded and unstructured polypeptides.
Mutant Analysis Shows that Alanine Racemases from
Pseudomonas aeruginosa
and
Escherichia coli
Are Dimeric
Authors: Strych Ulrich , Benedik Michael J.
Published: July 2002

Alanine racemases are ubiquitous prokaryotic enzymes providing the essential peptidoglycan precursor
d
-alanine. We present evidence that the enzymes from
Pseudomonas aeruginosa
and
Escherichia coli
function exclusively as homodimers. Moreover, we demonstrate that expression of a K35A Y235A double mutation of
dadX
in
E. coli
suppresses bacterial growth in a dominant negative fashion.
Rapid Detection of
Leishmania infantum
Infection in Dogs: Comparative Study Using an Immunochromatographic Dipstick Test, Enzyme-Linked Immunosorbent Assay, and PCR
Authors: Reithinger Richard , Quinnell Rupert J. , Alexander Bruce , Davies Clive R.
Published: July 2002

Current zoonotic visceral leishmaniasis (ZVL) control programs in Brazil include the culling of
Leishmania infantum-
infected reservoir dogs, a strategy that has failed to prevent a rise of canine and human ZVL cases over the past decade. One of the main reasons this strategy has failed is because of a long delay between sample collection, sample analysis, and control implementation. A rapid, sensitive, and specific diagnostic tool would be highly desirable, because it would allow control interventions to be implemented in situ. We compared an immunochromatographic dipstick test to enzyme-linked immunosorbent assay (ELISA) and PCR for detecting
L. infantum
infections in dogs from an area of ZVL endemicity in Brazil. The dipstick test was shown to have 61 to 75% specificity and 72 to 77% sensitivity, compared to 100% specificity for both ELISA and PCR and 71 to 88% and 51 to 64% sensitivity for ELISA and PCR, respectively. Of the field samples tested, 92 of 175 (53%), 65 of 175 (37%), and 47 of 175 (27%) were positive by dipstick, ELISA, and PCR, respectively. The positive and negative predictive values for the tested dipstick were 58 to 77% and 75%, respectively. Efforts should be made to develop a more specific dipstick test for diagnosis of leishmaniasis, because they may ultimately prove more cost-effective than currently used diagnostic tests when used in mass-screening surveys.
Real-Time PCR for Quantification of Human Herpesvirus 6 DNA from Lymph Nodes and Saliva
Authors: Collot S. , Petit B. , Bordessoule D. , Alain S. , Touati M. , Denis F. , Ranger-Rogez S.
Published: July 2002

A real-time quantitative PCR assay has been developed to measure human herpesvirus 6 (HHV-6) DNA in biological specimens. The assay sensitivity was 10 copies of DNA per well, with a linear dynamic range of 10 to 10
7
copies of HHV-6 DNA. Intra- and interassay variations were, respectively, 0.88 and 0.8% for samples containing 10
2
DNA copies, 0.99 and 0.96% for samples containing 10
4
copies, and 0.76 and 0.9% for samples containing 10
6
copies. Among 34 saliva samples from healthy subjects, 26 were found to contain HHV-6 DNA (76.5%; median, 23,870 copies/ml), and following a single freeze-thaw cycle, 25 of the same samples were found to be positive for HHV-6 DNA, although at a statistically significantly lower concentration (median, 3,497 copies/ml). The assay enabled detection of HHV-6 DNA in lymph node biopsies from patients with Hodgkin's disease (HD) (13 of 37 patients [35.1%]), B-cell neoplasms (8 of 36 patients [22.2%]), and T- or NK-cell neoplasms (3 of 13 patients [23.1%]), with concentrations ranging from 100 to 864,640 HHV-6 copies per μg of DNA (HHV-6B being found in every case except two). All HD patients infected with HHV-6 presented clinically with the nodular sclerosis subtype of HD. The real-time quantitative PCR assay developed here was simple to perform and was sensitive over a wide range of HHV-6 concentrations. It therefore appears to be of potential value in clinical investigation or diagnosis of HHV-6 infection.
Detection of
Tropheryma whipplei
DNA in Feces by PCR Using a Target Capture Method
Authors: Maibach Romana C. , Dutly Fabrizio , Altwegg Martin
Published: July 2002

Whipple's disease is a rare multisystemic bacterial infection with variable clinical manifestations. For decades, the laboratory diagnosis was based on the demonstration of periodic acid Schiff-positive inclusions in macrophages of gastrointestinal biopsies. PCR has improved the diagnosis of Whipple's disease due to its increased sensitivity compared to histopathological analysis. To avoid invasive procedures for taking specimens, we have investigated the possibility of detecting
Tropheryma whipplei
DNA in feces rather than in biopsies or gastric aspirate of patients with and without Whipple's disease. Total bacterial DNA was isolated from stool specimens using Qiagen columns followed by a
T. whipplei
-specific hybridization step with a biotinylated capture probe and streptavidin-coated magnetic particles. The captured DNA was then amplified using the same seminested PCR targeting the 16S rRNA gene of the organism that had been applied to other specimens without capturing. For five of eight patients with Whipple's disease, duodenal biopsies and stool samples were PCR positive, whereas for the three other patients, both specimens were PCR negative. Of 84 patients without Whipple's disease, 75 tested negative in the duodenal biopsy and in the stool sample. For four, both specimens were positive. Five patients tested positive in the stool sample but not in the biopsy. However, for three of these five patients, the gastric aspirate had been PCR positive, indicating that the stool PCR result was true rather than false positive. Compared to PCR from duodenal biopsies, stool PCR has a sensitivity of 100% and a specificity of 97.3%. Additionally, 15 PCR-positive and 22 PCR-negative stool samples were extracted using the Invisorb Spin Stool DNA kit. The simplified stool extraction showed 93.3% sensitivity and 95.5% specificity compared to the target capture method. We conclude that PCR with stool specimens with either extraction method is a sensitive and specific diagnostic tool for the detection of
T. whipplei
DNA and one not requiring invasive sampling procedures.
Comparison of an Assay Using Signal Amplification of the Heat-Dissociated p24 Antigen with the Roche Monitor Human Immunodeficiency Virus RNA Assay
Authors: Pascual Alvaro , Cachafeiro Ada , Funk Michele L. , Fiscus Susan A.
Published: July 2002

We compared an assay using signal amplification of a heat-dissociated p24 antigen (HDAg) with the Roche Monitor human immunodeficiency virus (HIV) RNA assay. The two assays gave comparable results when 130 specimens from 130 patients were tested (
r
= 0.60,
P
< 0.0001). The HDAg assay was almost as sensitive (85%) as the Roche HIV RNA kit (95%), just as specific (25 negative results from 25 HIV seronegative volunteers [100%]), less variable (mean log standard deviation of 0.07 compared to 0.11 when eight specimens were tested three or four times), and less expensive (reagent and labor costs, $8 versus $75). The assay appeared to be useful for monitoring established patients (
n
= 17) and identifying seroconverters (
n
= 4). HIV subtypes A to F were all recognized. This assay should be useful for monitoring patients in resource-poor countries and for monitoring vaccine recipients.
Helicobacter marmotae
sp. nov. Isolated from Livers of Woodchucks and Intestines of Cats
Authors: Fox James G. , Shen Zeli , Xu Shilu , Feng Yan , Dangler Charles A. , Dewhirst Floyd E. , Paster Bruce J. , Cullen John M.
Published: July 2002

Woodchucks (
Marmota monax
) have a high incidence of hepatocellular carcinoma (HCC) associated with chronic infection with woodchuck hepatitis virus (WHV) and serve as a model of hepatitis B virus-associated HCC in humans.
Helicobacter hepaticus
, an enterohepatic helicobacter in mice, is known to cause hepatocellular adenomas and carcinomas in susceptible mouse strains. In long-term chemical bioassays conducted with B6C3F
1
mice,
H
.
hepaticus
has been regarded as a confounding factor because of its tumor-promoting activity. In order to determine if woodchucks harbor a
Helicobacter
sp. that might play a role in potentiating hepatic inflammation or neoplasia, a study was undertaken to determine whether woodchucks' livers were infected with a
Helicobacter
sp. Frozen liver samples from 20 (17 WHV-infected and 3 noninfected) woodchucks, 10 with WHV-associated hepatic tumors and 10 without tumors, were cultured by microaerobic techniques and analyzed by using genus- and species-specific helicobacter PCR primers. A 1,200-bp
Helicobacter
sp.-specific sequence was amplified from 14 liver samples. Southern hybridization confirmed the specific identity of the PCR products. Nine of the 10 livers with tumors had positive
Helicobacter
sp. identified by PCR, whereas 5 of the 10 livers without tumors were positive. By use of 16S rRNA species-specific primers for
H
.
marmotae
, two additional liver samples from the nontumor group had positive PCR amplicons confirmed by Southern hybridization. A urease-, catalase-, and oxidase-positive bacterium was isolated from one liver sample from a liver tumor-positive woodchuck. By 16S rRNA analysis and biochemical and phenotypic characteristics, the organism was classified as a novel
Helicobacter
sp. Subsequently, four additional bacterial strains isolated from feces of cats and characterized by biochemical, phenotypic, and 16S rRNA analysis were determined to be identical to the woodchuck isolate. We propose the name
Helicobacter marmotae
sp. nov. for these organisms. Further studies are required to ascertain if this novel
Helicobacter
sp. plays a tumor promotion role in hepadnavirus-associated tumors in woodchucks or causes enterohepatic disease in cats.
Detection of Epstein-Barr Virus Genomes in Peripheral Blood B Cells from Solid-Organ Transplant Recipients by Fluorescence In Situ Hybridization
Authors: Rose Camille , Green Michael , Webber Steven , Kingsley Lawrence , Day Roger , Watkins Simon , Reyes Jorges , Rowe David
Published: July 2002

Resolution of Epstein-Barr Virus (EBV) infection in pediatric solid-organ transplant recipients often leads to an asymptomatic carrier state characterized by a persistently elevated circulating EBV load that is 2 to 4 orders of magnitude greater than the load typical of healthy latently infected individuals. Elevated EBV loads in immunosuppressed individuals are associated with an increased risk for development of posttransplant lymphoproliferative disease. We have performed fluorescence in situ hybridization (FISH) studies with peripheral blood B cells from carriers of persistent EBV loads in order to directly quantitate the number of EBV genomes per infected cell. Patients were assigned to two groups on the basis of the level of the persistent load (low-load carriers, 8 to 200 genomes/105peripheral blood lymphocytes; high-load carriers, >200 genomes/105peripheral blood lymphocytes). FISH analysis revealed that the low-load carriers predominantly had circulating virus-infected cells harboring one or two genome copies/cell. High-load carriers also had cells harboring one or two genome copies/cell; in addition, however, they carried a distinct population of cells with high numbers of viral genome copies. The increased viral loads correlated with an increase in the frequency of cells containing high numbers of viral genomes. We conclude that low-load carriers possess EBV-infected cells that are in a state similar to normal latency, whereas high-load carriers possess two populations of virus-positive B cells, one of which carries an increased number of viral genomes per cell and is not typical of normal latency.
Differentiation of Porcine
Brachyspira
Species by a Novel
nox
PCR-Based Restriction Fragment Length Polymorphism Analysis
Authors: Rohde Judith , Rothkamp Anja , Gerlach Gerald F.
Published: July 2002

A novel PCR-based restriction fragment length polymorphism analysis of the
Brachyspira nox
gene was developed. The restriction patterns for
Brachyspira hyodysenteriae
,
B
.
pilosicoli
,
B
.
intermedia
,
B
.
murdochii
, and
B
.
innocens
were highly distinct with two restriction endonucleases only. The assay proved to be user-friendly and robust.
Virulence Genes in
Helicobacter pylori
Strains from West Bengal Residents with Overt
H. pylori
-Associated Disease and Healthy Volunteers
Authors: Chattopadhyay Santanu , Datta Simanti , Chowdhury Abhijit , Chowdhury Sujit , Mukhopadhyay Asish K. , Rajendran K. , Bhattacharya S. K. , Berg Douglas E. , Nair G. Balakrish
Published: July 2002

We compared putative molecular markers of virulence (
vacA
,
cagA
, and
iceA
) of
Helicobacter pylori
strains isolated from 52 adult duodenal ulcer patients from West Bengal, India, with those of
H
.
pylori
strains isolated from 48 adult healthy volunteers from the same region. On the basis of genotyping by PCR, we conclude that the
H. pylori
strains isolated from the two study groups were indistinguishable and that there are geographic variations in the association of certain putative
H. pylori
virulence genes with clinical status.
Escalating Association of
Vibrio cholerae
O139 with Cholera Outbreaks in India
Authors: Sinha Sutapa , Chakraborty Rupa , De Keya , Khan Asis , Datta Simanti , Ramamurthy T. , Bhattacharya S. K. , Takeda Yoshifumi , Nair G. Balakrish
Published: July 2002

Between December 1999 and December 2000, teams from the National Institute of Cholera and Enteric Diseases, Calcutta, India, examined eight outbreaks of cholera, which occurred in different parts of the country distant from each other. In two of these outbreaks each, only
V. cholerae
O1 biotype ElTor or
V. cholerae
O139 could be isolated, while in the remaining four outbreaks, both O1 and O139 were isolated. The interesting feature is the escalating association of
V. cholerae
O139 with outbreaks of cholera; two of the most recent outbreaks, one in Calcutta and one in Orissa, were caused exclusively by O139. The O139 strains from the six different outbreaks were genotypically closely related. These trends indicate a shift in the outbreak propensity of
V. cholerae
O139.
Laboratory Contamination of Specimens with Quality Control Strains of Vancomycin-Resistant Enterococci in Ontario
Authors: Katz K. C. , McGeer A. , Low D. E. , Willey B. M.
Published: July 2002

Cross-contamination with laboratory control strains of vancomycin-resistant
Enterococcus faecalis
was documented in 15 clinical specimens from nine clinical microbiology laboratories in Ontario, Canada. Laboratories should be alert to the possibility of contamination of specimens with vancomycin-resistant enterococci from the laboratory environment. Molecular typing of strains may assist in elucidating the source of such contamination.
Trichuris vulpis
Recovered from a Patient with Chronic Diarrhea and Five Dogs
Authors: Dunn James J. , Columbus Sharon T. , Aldeen William E. , Davis Mark , Carroll Karen C.
Published: July 2002

We report a case of human infection with the whipworm of dogs,
Trichuris vulpis
, in a woman with duodenal ulcer disease, chronic diarrhea, and close contact with dogs. Morphologically,
T. vulpis
ova resemble those of the human whipworm (
T. trichiura
) but are nearly twice their size.
Species-Specific Receptor Recognition by a Minor-Group Human Rhinovirus (HRV): HRV Serotype 1A Distinguishes between the Murine and the Human Low-Density Lipoprotein Receptor
Authors: Reithmayer Manuela , Reischl Andrea , Snyers Luc , Blaas Dieter
Published: July 2002

Human rhinoviruses (HRV) of the minor receptor group use several members of the low-density lipoprotein receptor superfamily for cell entry. These proteins are evolutionarily highly conserved throughout species and are almost ubiquitously expressed. Their common building blocks, cysteine-rich ligand binding repeats about 40 amino acids in length, exhibit considerable sequence similarity. Various numbers of these repeats are present in the different receptors. We here demonstrate that HRV type 1A (HRV1A) replicates in mouse cells without adaptation. Furthermore, although closely related to HRV2, it fails to bind to the human low-density lipoprotein receptor but recognizes the murine protein, whereas HRV2 binds equally well to both homologues. This difference went unnoticed due to the presence of other receptors, such as the low-density lipoprotein receptor-related protein, which allow species-independent attachment. The species specificity of HRV1A reported here will aid in defining amino acid residues establishing the contact between the viral surface and the receptor.
Cell Surface Major Histocompatibility Complex Class II Proteins Are Regulated by the Products of the γ134.5 and UL41 Genes of Herpes Simplex Virus 1
Authors: Trgovcich Joanne , Johnson David , Roizman Bernard
Published: July 2002

Modulation of host immune responses has emerged as a common strategy employed by herpesviruses both to establish life-long infections and to affect recovery from infection. Herpes simplex virus 1 (HSV-1) blocks the major histocompatibility complex (MHC) class I antigen presentation pathway by inhibiting peptide transport into the endoplasmic reticulum. The interaction of viral gene products with the MHC class II pathway, however, has not been thoroughly investigated, although CD4+T cells play an important role in human recovery from infection. We have investigated the stability, distribution, and state of MHC class II proteins in glioblastoma cells infected with wild-type HSV-1 or mutants lacking specific genes. We report the following findings. (i) Wild-type virus infection caused a decrease in the accumulation of class II protein on the surface of cells and a decrease in the endocytosis of lucifer yellow or dextran conjugated to fluorescein isothiocyanate but no decrease in the total amount of MHC class II proteins relative to the levels seen in mock-infected cells. (ii) Although the total amount of MHC class II protein remained unchanged, the amounts of cell surface MHC class II proteins were higher in cells infected with the UL41-negative mutant, which lacks the virion host shutoff protein, and especially high in cells infected with the γ134.5-negative mutant. We conclude that infected cells attempt to respond to infection by increased acquisition of antigens and transport of MHC class II proteins to the cell surface and that these responses are blocked in part by the virion host shutoff protein encoded by the UL41 gene and in large measure by the direct or indirect action of the infected cell protein 34.5, the product of the γ134.5 gene.
Translational Regulation of Human Papillomavirus Type 16 E7 mRNA by the Peptide SEQIKA, Shared by Rabbit α1-Globin and Human Cytokeratin 7
Authors: Kanduc Darja
Published: July 2002

The possible biochemical factors able to affect the in vitro expression of the high-risk human papillomavirus type 16 (HPV16) E7 oncoprotein have been analyzed. Evidence is provided that E7 mRNA stability is increased and, conversely, transcript translation is inhibited by binding to a 32-kDa protein from rabbit reticulocyte lysate; sequence analysis identified the 32-kDa binding protein as rabbit α1-globin protein; and interaction between rabbit α1-globin and E7 mRNA occurs through the 6-mer peptide SEQIKA present in human cytokeratin 7 protein. The in vitro data were confirmed by the occurrence of HPV16 E7 mRNA-cytokeratin 7 binding in squamous cervical cancer SiHa cells.