bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence Polymorphism, Predicted Secondary Structures, and Surface-Exposed Conformational Epitopes of
Campylobacter
Major Outer Membrane Protein
Authors: Zhang Qijing , Meitzler Jerrel C. , Huang Shouxiong , Morishita Teresa
Published: July 2002

The major outer membrane protein (MOMP), a putative porin and a multifunction surface protein of
Campylobacter jejuni
, may play an important role in the adaptation of the organism to various host environments. To begin to dissect the biological functions and antigenic features of this protein, the gene (designated
cmp
) encoding MOMP was identified and characterized from 22 strains of
C. jejuni
and one strain of
C. coli
. It was shown that the single-copy
cmp
locus encoded a protein with characteristics of bacterial outer membrane proteins. Prediction from deduced amino acid sequences suggested that each MOMP subunit consisted of 18 β-strands connected by short periplasmic turns and long irregular external loops. Alignment of the amino acid sequences of MOMP from different strains indicated that there were seven localized variable regions dispersed among highly conserved sequences. The variable regions were located in the putative external loop structures, while the predicted β-strands were formed by conserved sequences. The sequence homology of
cmp
appeared to reflect the phylogenetic proximity of
C. jejuni
strains, since strains with identical
cmp
sequences had indistinguishable or closely related macrorestriction fragment patterns. Using recombinant MOMP and antibodies recognizing linear or conformational epitopes of the protein, it was demonstrated that the surface-exposed epitopes of MOMP were predominantly conformational in nature. These findings are instrumental in the design of MOMP-based diagnostic tools and vaccines.
Pathogenesis of Infection by Clinical and Environmental Strains of
Vibrio vulnificus
in Iron-Dextran-Treated Mice
Authors: Starks Angela M. , Schoeb Trenton R. , Tamplin Mark L. , Parveen Salina , Doyle Thomas J. , Bomeisl Philip E. , Escudero Gloria M. , Gulig Paul A.
Published: July 2002

Vibrio vulnificus
is an opportunistic pathogen that contaminates oysters harvested from the Gulf of Mexico. In humans with compromising conditions, especially excess levels of iron in plasma and tissues, consumption of contaminated seafood or exposure of wounds to contaminated water can lead to systemic infection and disfiguring skin infection with extremely high mortality.
V. vulnificus
-associated diseases are noted for the rapid replication of the bacteria in host tissues, with extensive tissue damage. In this study we examined the virulence attributes of three virulent clinical strains and three attenuated oyster or seawater isolates in mouse models of systemic disease. All six
V. vulnificus
strains caused identical skin lesions in subcutaneously (s.c.) inoculated iron dextran-treated mice in terms of numbers of recovered CFU and histopathology; however, the inocula required for identical frequency and magnitude of infection were at least 350-fold higher for the environmental strains. At lethal doses, all strains caused s.c. skin lesions with extensive edema, necrosis of proximate host cells, vasodilation, and as many as 10
8
CFU/g, especially in perivascular regions. These data suggest that the differences between these clinical and environmental strains may be related to growth in the host or susceptibility to host defenses. In non-iron dextran-treated mice, strains required 10
5
-fold-higher inocula to cause an identical disease process as with iron dextran treatment. These results demonstrate that s.c. inoculation of iron dextran-treated mice is a useful model for studying systemic disease caused by
V. vulnificus.
Early Induction of Gamma Interferon and Interleukin-10 Production in Draining Lymph Nodes from Mice Infected withBorrelia burgdorferi
Authors: Ganapamo Frederic , Dennis Vida A. , Philipp Mario T.
Published: July 2002

Lymph node (LN) cells from C3H/HeJ mice (Lyme disease susceptible) infected for 1 week withBorrelia burgdorferistrain JD1 produced higher levels of gamma interferon (IFN-γ) when stimulated in vitro withB. burgdorferispirochetes than equivalent cells fromB. burgdorferi-infected C57BL/6J mice (disease resistant). The interleukin-10 (IL-10) levels were comparable in the two strains, whereas the IL-4 levels were below detection limits.B. burgdorferi-stimulated LN cells from C57BL/6J mice produced significantly higher levels of IFN-γ in the presence of neutralizing anti-IL-10 antibody than cells cultured withB. burgdorferialone. No effect of IL-10 neutralization on IFN-γ production by LN cells from C3H/HeJ mice was observed. Neutralizing antibody to IFN-γ had no effect on the production of IL-10 by LN cells from C57BL/6J mice. A slight decrease in IL-10 production was detected in culture supernatants of equivalent cells from C3H/HeJ mice. The differential effect of IL-10 on IFN-γ production in C57BL/6J and C3H/HeJ mice suggests that IL-10 is probably involved in the regulation of IFN-γ production by LN cells during infection and may be at the root of the differential susceptibility to Lyme arthritis in these two strains of mice.
Analysis of a Gene Cluster ofEnterococcus faecalisInvolved in Polysaccharide Biosynthesis
Authors: Xu Yi , Singh Kavindra V. , Qin Xiang , Murray Barbara E. , Weinstock George M.
Published: July 2002

Previously, we described a gene cluster ofEnterococcus faecalisOG1RF that produced an antigenic polysaccharide when cloned inEscherichia coli. The polysaccharide antigen was not detectable inE. faecalisstrains, however. Here, we show by reverse transcriptase-PCR that the 16 genes in this region are transcribed in OG1RF. Gene disruption oforfde4, encoding a putative glycosyl transferase, andorfde6, a putative dTDP-rhamnose biosynthesis gene, generated two OG1RF mutants. The mutants showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. In addition, two mucoidE. faecalisisolates from patients with chronic urinary tract infections were found to produce the polysaccharide antigen.
Identification of Novel Serine/Threonine Protein Phosphatases in
Trypanosoma cruzi
: a Potential Role in Control of Cytokinesis and Morphology
Authors: Orr George A. , Werner Craig , Xu Jun , Bennett Marcia , Weiss Louis M. , Takvorkan Peter , Tanowitz Herbert B. , Wittner Murray
Published: July 2002

We cloned two novel
Trypanosoma cruzi
proteins by using degenerate oligonucleotide primers prepared against conserved domains in mammalian serine/threonine protein phosphatases 1, 2A, and 2B. The isolated genes encoded proteins of 323 and 330 amino acids, respectively, that were more homologous to the catalytic subunit of human protein phosphatase 1 than to those of human protein phosphatase 2A or 2B. The proteins encoded by these genes have been tentatively designated TcPP1α and TcPP1β. Northern blot analysis revealed the presence of a major 2.3-kb mRNA transcript hybridizing to each gene in both the epimastigote and metacyclic trypomastigote developmental stages. Southern blot analysis suggests that each protein phosphatase 1 gene is present as a single copy in the
T. cruzi
genome. The complete coding region for TcPP1β was expressed in
Escherichia coli
by using a vector, pTACTAC, with the
trp-lac
hybrid promoter. The recombinant protein from the TcPP1β construct displayed phosphatase activity toward phosphorylase
a
, and this activity was preferentially inhibited by calyculin A (50% inhibitory concentration [IC
50
], ∼2 nM) over okadaic acid (IC
50
, ∼100 nM). Calyculin A, but not okadaic acid, had profound effects on the in vitro replication and morphology of
T. cruzi
epimastigotes. Low concentrations of calyculin A (1 to 10 nM) caused growth arrest. Electron microscopic studies of the calyculin A-treated epimastigotes revealed that the organisms underwent duplication of organelles, including the flagellum, kinetoplast, and nucleus, but were incapable of completing cell division. At concentrations higher than 10 nM, or upon prolonged incubation at lower concentrations, the epimastigotes lost their characteristic elongated spindle shape and had a more rounded morphology. Okadaic acid at concentrations up to 1 μM did not result in growth arrest or morphological alterations to
T. cruzi
epimastigotes. Calyculin A, but not okadaic acid, was also a potent inhibitor of the dephosphorylation of
32
P-labeled phosphorylase
a
by
T. cruzi
epimastigotes and metacyclic trypomastigote extracts. These inhibitor studies suggest that in
T. cruzi
, type 1 protein phosphatases are important for the completion of cell division and for the maintenance of cell shape.
Assessing the Binding of FourPlasmodium falciparumT Helper Cell Epitopes to HLA-DQ and Induction of T-Cell Responses in HLA-DQ Transgenic Mice
Authors: Pimtanothai Nattiya , Parra Marcela , Johnson Armead H. , David Chella S. , Katovich Hurley Carolyn
Published: July 2002

A subunit vaccine forPlasmodium falciparummalaria will need to contain well-defined T helper cell epitopes that induce protective immune responses to the parasite. One major barrier to the use of subunit vaccines is the requirement for T helper cell epitopes to be presented by the HLA class II molecules that are present in the population being vaccinated. Since the majority of malaria studies have focused on HLA-DR, little information on the role of HLA-DQ in the binding and immune response to malarial epitopes is available. This study used an in vitro peptide-binding assay to predict the extent of HLA-DQ binding of four conserved T helper cell epitopes identified from asexual-stage malaria vaccine candidate antigens. Epstein-Barr virus (EBV)-transformed human B-cell lines expressing 14 different DQ molecules (DQ2.1, -2.2, -4.1, -4.2, -5.1 to -5.3, -6.1, -6.2, -6.4, -7.1, -7.3, -8, and -9) representing all broad serological specificities, including common DQ molecules present in populations in areas where malaria is endemic, were used in the binding assay. Moreover, an HLA-DQ transgenic mouse model was employed to evaluate the correlation between the in vitro DQ binding of the peptides and the generation of in vivo immune responses following peptide immunization. This study identified two broad DQ-binding peptides, ABRA#14 and SERA#9. ABRA#14 also induced T-cell proliferation and Th1-associated cytokine production in DQ8+transgenic mice. The combination of peptide binding to EBV-transformed cell lines and DQ transgenic mice provides a method for identifying additional T-cell epitopes for inclusion in a vaccine.
Characterization of
Porphyromonas gingivalis
-Induced Degradation of Epithelial Cell Junctional Complexes
Authors: Katz Jannet , Sambandam Vijaya , Wu John H. , Michalek Suzanne M. , Balkovetz Daniel F.
Published: July 2002

Porphyromonas gingivalis
is considered among the etiological agents of human adult periodontitis. Although in vitro studies have shown that
P. gingivalis
has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (β1-integrin) transmembrane proteins. These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells. In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier. Using the MDCK cell system, we examined the effect of
P. gingivalis
on epithelial barrier function. Exposure of the basolateral surfaces of MDCK cells to
P. gingivalis
(>10
9
bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and β1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateral
P. gingivalis
. Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure. Cell viability was not affected by either basolateral or apical exposure to
P. gingivalis
. Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and β1-integrin in lysates derived from MDCK cells exposed to
P. gingivalis
. Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by
P. gingivalis
, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins. Collectively, these data suggest that
P. gingivalis
is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium. These results also indicate the importance of a critical threshold concentration of
P. gingivalis
to initiate epithelial barrier destruction.
Long-Term Immunological Memory Induced by Recombinant Oral
Salmonella
Vaccine Vectors
Authors: Kohler James J. , Pathangey Latha , Hasona Adnan , Progulske-Fox Ann , Brown Thomas A.
Published: July 2002

We have previously shown that
Salmonella enterica
serovar Typhimurium expressing the
hagB
hemagglutinin gene from
Porphyromonas gingivalis
can induce primary and recall immune responses in serum and secretions in mice; however, the longevity of memory induced by oral
Salmonella
carriers has not been adequately demonstrated. In this study, we examined the capacity of mice to mount a recall response 52 weeks after primary immunization. Recall responses were seen in serum immunoglobulin G (IgG) and IgA following boosting at week 52, and in most cases, they were equal to or greater than the primary responses. Significant mucosal IgA recall responses in saliva and vaginal wash were also detected following boosting at week 52. In addition, there was a considerable residual response in secretions at week 51, prior to boosting. These results indicate that oral
Salmonella
vectors can induce long-term memory to recombinant HagB and are particularly effective at inducing long-lasting mucosal responses as well as at inducing the capacity for mucosal recall responses.
Yersinia pestis
YbtU and YbtT Are Involved in Synthesis of the Siderophore Yersiniabactin but Have Different Effects on Regulation
Authors: Geoffroy Valerie A. , Fetherston Jacqueline D. , Perry Robert D.
Published: July 2002

One prerequisite for the virulence of
Yersinia pestis
, causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the
pgm
locus of the
Y. pestis
chromosome. Several gene products within the HPI have demonstrated functions in the synthesis or transport of Ybt. Here we examine the roles of
ybtU
and
ybtT
. In-frame mutations in
ybtT
or
ybtU
yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37°C but could be cross-fed by culture supernatants from a Ybt-producing strain of
Y. pestis
. The
ybtU
mutant failed to express four indicator Ybt proteins (HMWP1, HMWP2, YbtE, and Psn), a pattern similar to those for other
ybt
biosynthetic mutants. In contrast, strains carrying mutations in
ybtT
or
ybtS
(a previously identified gene required for Ybt biosynthesis) produced all four proteins at wild-type levels under iron-deprived conditions. To assess the effects of
ybtT
, -
U
, and -
S
mutations on transcription of
ybt
genes, reporter plasmids with
ybtP
or
psn
promoters controlling
lacZ
expression were introduced into these mutants. Normal iron-regulated β-galactosidase activity was observed in the
ybtT
and
ybtS
mutants, whereas a significant loss of expression occurred in the Δ
ybtU
strain. These results show that
ybtT
and
ybtU
genes are involved in the biosynthesis of the Ybt siderophore and that a
ybtU
mutation but not
ybtT
or
ybtS
mutations affects transcription from the
ybtP
and
psn
promoters.
Characterization of
Porphyromonas gingivalis
Insertion Sequence-Like Element IS
Pg5
Authors: Califano Joseph V. , Kitten Todd , Lewis Janina P. , Macrina Francis L. , Fleischmann Robert D. , Fraser Claire M. , Duncan Margaret J. , Dewhirst Floyd E.
Published: July 2002

Porphyromonas gingivalis
, a black-pigmented, gram-negative anaerobe, is found in periodontitis lesions, and its presence in subgingival plaque significantly increases the risk for periodontitis. In contrast to many bacterial pathogens,
P. gingivalis
strains display considerable variability, which is likely due to genetic exchange and intragenomic changes. To explore the latter possibility, we have studied the occurrence of insertion sequence (IS)-like elements in
P. gingivalis
W83 by utilizing a convenient and rapid method of capturing IS-like sequences and through analysis of the genome sequence of
P. gingivalis
strain W83. We adapted the method of Matsutani et al. (S. Matsutani, H. Ohtsubo, Y. Maeda, and E. Ohtsubo, J. Mol. Biol. 196:445–455, 1987) to isolate and clone rapidly annealing DNA sequences characteristic of repetitive regions within a genome. We show that in
P. gingivalis
strain W83, such sequences include (i) nucleotide sequence with homology to tRNA genes, (ii) a previously described IS element, and (iii) a novel IS-like element. Analysis of the
P. gingivalis
genome sequence for the distribution of the least used tetranucleotide, CTAG, identified regions in many of the initial 218 contigs which contained CTAG clusters. Examination of these CTAG clusters led to the discovery of 11 copies of the same novel IS-like element identified by the repeated sequence capture method of Matsutani et al. This new 1,512-bp IS-like element, designated IS
Pg5
, has features of the IS
3
family of IS elements. When a recombinant plasmid containing much of IS
Pg5
was used in Southern analysis of several
P. gingivalis
strains, including clinical isolates, diversity among strains was apparent. This suggests that IS
Pg5
and other IS elements may contribute to strain diversity and can be used for strain fingerprinting.
Soluble Egg Antigen-Stimulated T Helper Lymphocyte Apoptosis and Evidence for Cell Death Mediated by FasL+T and B Cells during MurineSchistosoma mansoniInfection
Authors: Lundy Steven K. , Lerman Stephen P. , Boros Dov L.
Published: July 2002

Granuloma formation around schistosomal eggs is induced by soluble egg antigens (SEA) and mediated by the activity of CD4+Th lymphocytes and their cytokines. Regulation of the inflammatory Th cell response during infection is still insufficiently understood. The hypothesis of this study was that activation-induced cell death (AICD) of CD4+T cells is involved in the immune inflammatory response. This study investigated the dynamics of splenic and granuloma CD4+Th cell apoptosis and Fas ligand (FasL) expression during the acute and chronic stages of murine schistosomal infection. Enhanced apoptosis of freshly isolated CD4+Th lymphocytes commenced after egg deposition and persisted during the peak and modulated phases of granuloma formation. After oviposition, CD4+, CD8+, and CD19+splenocytes and granuloma cells expressed elevated levels of FasL but FasL expression declined during the downmodulated stage of infection. In culture, SEA induced splenic and granuloma CD4+T-cell apoptosis and stimulated expression of FasL on splenic but not granuloma CD4+T cells, CD8+T cells, and CD19+B cells. SEA-stimulated splenocytes and granuloma cells preferentially lysed a Fas-transfected target cell line. Depletion of B cells from SEA-stimulated splenic cultures decreased CD4+T cell apoptosis. Coculture of purified splenic B cells with CD4+T cells and adoptive transfer of purified B cells indicated that antigen-stimulated B cells can kill CD4+Th cells. However, CD4+T cells were the dominant mediators of apoptosis in the granuloma. This study indicates that AICD is involved in the apoptosis of CD4+T cells during schistosomal infection.
Identification of Proteins from
Plasmodium falciparum
That Are Homologous to Reticulocyte Binding Proteins in
Plasmodium vivax
Authors: Triglia Tony , Thompson Jenny , Caruana Sonia R. , Delorenzi Mauro , Speed Terry , Cowman Alan F.
Published: July 2002

Plasmodium falciparum
infections can be fatal, while
P. vivax
infections usually are not. A possible factor involved in the greater virulence of
P. falciparum
is that this parasite grows in red blood cells (RBCs) of all maturities whereas
P. vivax
is restricted to growth in reticulocytes, which represent only approximately 1% of total RBCs in the periphery. Two proteins, expressed at the apical end of the invasive merozoite stage from
P. vivax
, have been implicated in the targeting of reticulocytes for invasion by this parasite. A search of the
P. falciparum
genome databases has identified genes that are homologous to the
P. vivax rbp-1
and -
2
genes. Two of these genes are virtually identical over a large region of the 5′ end but are highly divergent at the 3′ end. They encode high-molecular-mass proteins of >300 kDa that are expressed in late schizonts and localized to the apical end of the merozoite. To test a potential role in merozoite invasion of RBCs, we analyzed the ability of these proteins to bind to mature RBCs and reticulocytes. No binding to mature RBCs or cell preparations enriched for reticulocytes was detected. We identified a parasite clone that lacks the gene for one of these proteins, showing that the gene is not required for normal in vitro growth. Antibodies to these proteins can inhibit merozoite invasion of RBCs.
Lack of Adherence of Clinical Isolates of
Pseudomonas aeruginosa
to Asialo-GM
1
on Epithelial Cells
Authors: Schroeder Torsten H. , Zaidi Tanweer , Pier Gerald B.
Published: July 2002

Numerous studies have reported that asialo-GM
1
, gangliotetraosylceramide, or moieties serve as epithelial cell receptors for
Pseudomonas aeruginosa
. Usually this interaction is confirmed with antibodies to asialo-GM
1
. However, few, if any, of these reports have evaluated the binding of fresh clinical isolates of
P. aeruginosa
to asialo-GM
1
or the specificity of the antibodies for the asialo-GM
1
antigen. We confirmed that asialo-GM
1
dissolved in dimethyl sulfoxide could be added to the apical membrane of Madin-Darby canine kidney cells growing as a polarized epithelium on Transwell membranes (J. C. Comolli, L. L. Waite, K. E. Mostov, and J. N. Engel, Infect. Immun. 67:3207–3214, 1999) and that such treatment enhanced the binding of
P. aeruginosa
strain PA103. However, no other
P. aeruginosa
strain, including eight different clinical isolates, exhibited enhanced binding to asialo-GM
1
-treated cells. Studies with commercially available antibodies to asialo-GM
1
showed that these preparations had high titers of antibody to
P. aeruginosa
antigens, including whole cells, purified lipopolysaccharide (LPS), and pili. Inhibition studies showed that adsorption of an antiserum to asialo-GM
1
with
P. aeruginosa
cells could remove the reactivity of antibodies to asialo-GM
1
, and adsorption of this serum with asialo-GM
1
removed antibody binding to
P. aeruginosa
LPS. Antibodies in sera raised to asialo-GM
1
were observed to bind to
P. aeruginosa
cells by immunoelectron microscopy. Antibodies to asialo-GM
1
inhibited formation of a biofilm by
P. aeruginosa
in the absence of mammalian cells, indicating a direct inhibition of bacterial cell-cell interactions. These findings demonstrate that asialo-GM
1
is not a major cellular receptor for clinical isolates of
P. aeruginosa
and that commercially available antibodies raised to this antigen contain high titers of antibody to multiple
P. aeruginosa
antigens, which do not interfere with the binding of
P. aeruginosa
to mammalian cells but possibly interfere with the binding of
P. aeruginosa
cells to each other.
Implication of Mitogen-Activated Protein Kinases in T84 Cell Responses to EnteropathogenicEscherichia coliInfection
Authors: Czerucka Dorota , Dahan Stéphanie , Mograbi Baharia , Rossi Bernard , Rampal Patrick
Published: July 2002

EnteropathogenicEscherichia coli(EPEC) infection of T84 cells induces a decrease in transepithelial resistance, the formation of attaching and effacing (A/E) lesions, and cytokine production. The purpose of this study was to investigate the ability of EPEC to activate mitogen-activated protein (MAP) kinases in T84 cells and to correlate these signaling pathways with EPEC-induced cell responses. T84 cells were infected with either the wild-type (WT) EPEC strain E2348/69 or two mutants, intimin deletion strain CVD206 (ΔeaeA) and type III secretion apparatus mutant strain CVD452 (ΔescN::aphA). Infection of T84 cells with WT but not mutant EPEC strains induced tyrosine phosphorylation of several proteins in T84 cells, including the p46 and p52 Shc isoforms. Kinetics studies revealed that ERK1/2, p38, and c-Jun N-terminal kinase (JNK) MAP kinases were activated in cells infected with strain E2348/69 but not with the mutant strains. Inhibition of MAP kinases with PD98059 or SB203580 did not affect the EPEC-induced decrease in transepithelial resistance or actin accumulation beneath the WT bacteria, but these two inhibitors significantly decreased interleukin-8 (IL-8) synthesis. We demonstrate that EPEC induces activation of ERK1/2, p38, and JNK cascades, which all depend on bacterial adhesion and expression of the bacterial type III secretion system. ERK1/2 and p38 MAP kinases were equally implicated in IL-8 expression but did not participate in A/E lesion formation or transepithelial resistance modification, indicating that the signaling pathways involved in these events are distinct.
Essential Role for Cellular Phosphoglucomutase in Virulence of Type 3
Streptococcus pneumoniae
Authors: Hardy Gail G. , Magee Ashalla D. , Ventura Christy L. , Caimano Melissa J. , Yother Janet
Published: July 2002

Synthesis of the
Streptococcus pneumoniae
type 3 capsule requires the pathway glucose-6-phosphate (Glc-6-P) → Glc-1-P → UDP-Glc → UDP-glucuronic acid (UDP-GlcUA) → (GlcUA-Glc)

n

. The UDP-Glc dehydrogenase and synthase necessary for the latter two steps, and essential for capsule production, are encoded by genes (
cps3D
and
cps3S
, respectively) located in the type 3 capsule locus. The phosphoglucomutase (PGM) and Glc-1-P uridylyltransferase activities necessary for the first two steps are derived largely through the actions of cellular enzymes. Homologues of these enzymes, encoded by
cps3M
and
cps3U
in the type 3 locus, are not required for capsule production. Here, we show that
cps3M
and
cps3U
also are not required for mouse virulence. In contrast, nonencapsulated isolates containing defined mutations in
cps3D
and
cps3S
were avirulent, as were reduced-capsule isolates containing mutations in
pgm
. Insertion mutants that lacked PGM activity were avirulent in both immunologically normal (BALB/cByJ) and immunodeficient (CBA/N) mice. In contrast, a mutant (JY1060) with reduced PGM activity was avirulent in the former but had only modestly reduced virulence in the latter. The high virulence in CBA/N mice was not due to the lack of antibodies to phosphocholine but reflected a growth environment distinct from that found in BALB/cByJ mice. The reduced PGM activity of JY1060 resulted in enhanced binding of complement and antibodies to surface antigens. However, decomplementation of BALB/cByJ mice did not enhance the virulence of this mutant. Suppressor mutations, only some of which resulted in increased capsule production, increased the virulence of JY1060 in BALB/cByJ mice. The results suggest that PGM plays a critical role in pneumococcal virulence by affecting multiple cellular pathways.
PersistentChlamydia trachomatisInfections Resist Apoptotic Stimuli
Authors: Dean Deborah , Powers Virginia C.
Published: July 2002

Microbial modulation of apoptosis has added a new dimension of understanding to the dynamic interaction between the human host and its microbial invaders. Persistent infection can be a by-product of inhibition of apoptosis and may significantly impact the pathogenesis of diseases caused by organisms such asChlamydia trachomatis. We compared apoptotic responses among HeLa 229 cells acutely and persistently infected and mock infected with serovar A/HAR-13. Persistence was induced by gamma interferon at 0.2 and 2.0 ng/ml. Cells were treated with etoposide or staurosporine at 24-h intervals and assayed for apoptosis by cell count, DNA ladder formation, and cytochromectranslocation. From the 24- to 120-h time points, infected cultures were 87 and 90% viable for etoposide and staurosporine treatment, respectively, and produced no DNA ladder, and cytochromecremained in the mitochondria. In contrast, mock-infected cells were 22 and 37% viable for etoposide (P= 0.0001) and staurosporine (P= 0.01), respectively, and displayed characteristic DNA ladders, and cytochromecwas translocated. We found that resistance to apoptotic stimuli was identical in acute and persistent infections. Since cytochromecwas not translocated from the mitochondrion, caspase-9 activity was likely not involved. The expression of chlamydial hsp60, a known stimulator of inflammation in vivo, was measured in both active and persistent infections by Western blot, with increased production in the latter with or without staurosporine treatment. Chlamydial disregulation of apoptosis and the ensuing persistence of organisms offer an alternative pathogenic mechanism for chlamydial scarring observed in trachoma and infertility populations via sustained inflammation induced by immunoreactive molecules such as hsp60.
Subinhibitory Clindamycin Differentially Inhibits Transcription of Exoprotein Genes in
Staphylococcus aureus
Authors: Herbert Silvia , Barry Peter , Novick Richard P.
Published: July 2002

It has long been known that certain antibiotics, at subinhibitory concentrations, differentially inhibit the synthesis of α-hemolysin and other staphylococcal virulence factors. In this report, we show that subinhibitory clindamycin (SBCL) eliminates production of nearly all exoproteins by
Staphylococcus aureus
but has virtually no effect on cytoplasmic proteins. The effect was abolished by a gene conferring resistance to macrolides-lincosamides-streptogramin B, showing that differential inhibition of protein synthesis is responsible; remarkably, however, subinhibitory clindamycin blocked production of several of the individual exoprotein genes, including
spa
(encoding protein A),
hla
(encoding α-hemolysin), and
spr
(encoding serine protease), at the level of transcription, suggesting that the primary effect must be differential inhibition of the synthesis of one or more regulatory proteins. In contrast to earlier reports, however, we found that subinhibitory clindamycin stimulates synthesis of coagulase and fibronectin binding protein B, also at the level of transcription.
agr
and
sar
expression was minimally affected by subinhibitory clindamycin. These effects varied from strain to strain and do not seem to be responsible for the effects of subinhibitory clindamycin on the overall exoprotein pattern.
Directional Gene Movement from Human-Pathogenic to Commensal-Like Streptococci
Authors: Kalia Awdhesh , Enright Mark C. , Spratt Brian G. , Bessen Debra E.
Published: July 2002

Group A streptococci (GAS) are highly pathogenic for humans, and their closest genetic relatives, group C and G streptococci (GCS and GGS, respectively), are generally regarded as commensals, although they can be found in association with human disease. As part of an effort to better understand the evolution of virulence, the phylogenetic relationships between GAS, GCS, and GGS were examined. The nucleotide sequence was determined for an internal portion of seven housekeeping (neutral) loci among >200 isolates of GAS and 34 isolates of GCS or GGS obtained from human subjects. Genotypic analysis failed to show support for the separation of GCS and GGS into two distinct populations. Unlike GAS, there was poor concordance betweenemmtype and genetic relatedness among GCS and GGS. All housekeeping genes within GAS displayed relatively low levels of sequence diversity. In contrast, individual GCS and GGS strains had mosaic genomes, containing alleles at some loci that were similar or identical to GAS alleles, whereas the alleles at other loci were about 10 to 30% diverged. The data provide evidence for a history of recent interspecies transfer of neutral genes that exhibits a strong net directionality from GAS donors to GCS and GGS recipients. A model for the evolution of GAS and of GCS and GGS is described.
Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene inPseudomonas aeruginosa
Authors: Wilderman Paula J. , Vasil Adriana I. , Johnson Zaiga , Wilson Megan J. , Cunliffe Heather E. , Lamont Iain L. , Vasil Michael L.
Published: July 2002

The expression of many virulence factors inPseudomonas aeruginosais dependent upon environmental conditions, including iron levels, oxygen, temperature, and osmolarity. The virulence ofP. aeruginosaPAO1 is influenced by the iron- and oxygen-regulated gene encoding the alternative sigma factor PvdS, which is regulated through the ferric uptake regulator (Fur). We observed that overexpression of PvdS in strain PAO1 and a ΔpvdS::Gmmutant resulted in increased pyoverdine production and proteolytic activity compared to when PvdS was not overexpressed. To identify additional PvdS-regulated genes, we compared extracellular protein profiles from PAO1 and the ΔpvdS::Gmmutant grown under iron-deficient conditions. A protein present in culture supernatants from PAO1 but not in supernatants from ΔpvdS::Gmwas investigated. Amino acid sequence analysis and examination of the genomic database of PAO1 revealed that the N terminus of this 27-kDa protein is identical to that of protease IV ofP. aeruginosastrain PA103-29 and is homologous to an endoprotease produced byLysobacter enzymogenes.In this study, the gene encoding an endoprotease was cloned from PAO1 and designatedprpL(PvdS-regulated endoprotease, lysyl class). All (n= 41) but one of the strains ofP. aeruginosa, including clinical and environmental isolates, examined carryprpL. Moreover, PrpL production among these strains was highly variable. Analysis of RNase protection assays identified the transcription initiation site ofprpLand confirmed that its transcription is iron dependent. In the ΔpvdS::Gmmutant, the level ofprpLtranscription was iron independent and decreased relative to the level in PAO1. Furthermore, transcription ofprpLwas independent of PtxR, a PvdS-regulated protein. Finally, PrpL cleaves casein, lactoferrin, transferrin, elastin, and decorin and contributes to PAO1's ability to persist in a rat chronic pulmonary infection model.
Development of a Competitive Index Assay To Evaluate the Virulence ofListeria monocytogenes actAMutants during Primary and Secondary Infection of Mice
Authors: Auerbuch Victoria , Lenz Laurel L. , Portnoy Daniel A.
Published: July 2002

We developed a competitive index assay for murine listeriosis that tests the virulence ofListeria monocytogenesstrains in different organs and at various times postinoculation. Studies presented here demonstrate the reproducibility of this assay during primary and secondary infection of inbred and outbred mice. We verified the validity of this assay by performing competitive index analysis of a well-characterized strain ofL. monocytogeneslacking theactAgene. In addition, we found that whileL. monocytogenesstrains unable to recruit vasodilator-stimulated phosphoprotein (VASP) to their surface exhibit a 10-fold virulence attenuation in the livers of naive animals, they display a 50-fold survival defect in the liver during secondary listeriosis.
The Dot/Icm Type IV Secretion System of
Legionella pneumophila
Is Essential for the Induction of Apoptosis in Human Macrophages
Authors: Zink Steven D. , Pedersen Lisa , Cianciotto Nicholas P. , Abu Kwaik Yousef
Published: July 2002

We have previously shown that
Legionella pneumophila
induces caspase 3-dependent apoptosis in mammalian cells during early stages of infection. In this report, we show that nine
L. pneumophila
strains with mutations in the
dotA
,
dotDCB
,
icmT
,
icmGCD,
and
icmJB
loci are completely defective in the induction of apoptosis, in addition to their severe defects in intracellular replication and pore formation-mediated cytotoxicity. Importantly, all nine
dot
/
icm
mutants were complemented for all their defective phenotypes with the respective wild-type loci. We show that the role of the Dot/Icm type IV secretion system in the induction of apoptosis is independent of the RtxA toxin, the
dot
/
icm
-regulated pore-forming toxin, and the type II secretion system. However, the pore-forming toxin, which is triggered upon entry into the postexponential growth phase, enhances the ability of
L. pneumophila
to induce apoptosis. Our data provide the first example of the role of a type IV secretion system of a bacterial pathogen in the induction of apoptosis in the host cell.
The
Neisseria
Lipooligosaccharide-Specific α-2,3-Sialyltransferase Is a Surface-Exposed Outer Membrane Protein
Authors: Shell Dawn M. , Chiles Lisa , Judd Ralph C. , Seal Samar , Rest Richard F.
Published: July 2002

Neisseria gonorrhoeae
and
Neisseria meningitidis
express an ∼43-kDa α-2,3-sialyltransferase (Lst) that sialylates the surface lipooligosaccharide (LOS) by using exogenous (in all
N. gonorrhoeae
strains and some
N. meningitidis
serogroups) or endogenous (in other
N. meningitidis
serogroups) sources of 5′-cytidinemonophospho-
N
-acetylneuraminic acid (CMP-NANA). Sialylation of LOS can protect
N. gonorrhoeae
and
N. meningitidis
from complement-mediated serum killing and from phagocytic killing by neutrophils. The precise subcellular location of Lst has not been determined. We confirm and extend previous studies by demonstrating that Lst is located in the outer membrane and is surface exposed in both
N. gonorrhoeae
and
N. meningitidis
. Western immunoblot analysis of subcellular fractions of
N. gonorrhoeae
strain F62 and
N. meningitidis
strain MC58⊄3 (an acapsulate serogroup B strain) performed with rabbit antiserum raised against recombinant Lst revealed an ∼43-kDa protein exclusively in outer membrane preparations of both pathogens. Inner membrane, periplasmic, cytoplasmic, and culture supernatant fractions were devoid of Lst, as determined by Western blot analysis. Consistent with this finding, outer membrane fractions of
N. gonorrhoeae
were significantly enriched for sialyltransferase enzymatic activity. A trace of enzymatic activity was detected in inner membrane fractions, which may have represented Lst in transit to the outer membrane or may have represented inner membrane contamination of outer membrane preparations. Subcellular preparations of an isogenic
lst
insertion knockout mutant of
N. gonorrhoeae
F62 (strain ST01) expressed neither a 43-kDa immunoreactive protein nor sialyltransferase activity. Anti-Lst rabbit antiserum bound to whole cells of
N. meningitidis
MC58⊄3 and wild-type
N. gonorrhoeae
F62 but not to the Lst mutant ST01, indicating the surface exposure of the enzyme. Although the anti-Lst antiserum avidly bound enzymatically active, recombinant Lst, it inhibited Lst (sialyltransferase) activity by only about 50% at the highest concentration of antibody used. On the contrary, anti-Lst antiserum did not inhibit sialylation of whole
N. gonorrhoeae
cells in the presence of exogenous CMP-NANA, suggesting that the antibody did not bind to or could not access the enzyme active site on the surface of viable
Neisseria
cells. Taken together, these results indicate that Lst is an outer membrane, surface-exposed glycosyltransferase. To our knowledge, this is the first demonstration of the localization of a bacterial glycosyltransferase to the outer membrane of gram-negative bacteria.
The
rel
Gene Is Essential for In Vitro Growth of
Staphylococcus aureus
Authors: Gentry Daniel , Li Tong , Rosenberg Martin , McDevitt Damien
Published: July 2002

The stringent response in
Staphylococcus aureus
is mediated by the nucleotide guanosine pentaphosphate, whose synthesis is catalyzed by the product of the
rel
gene. We report here that the
rel
gene is essential for the in vitro growth of
S. aureus
, distinguishing it from all other bacteria tested for this requirement.
glnE
Is an Essential Gene in
Mycobacterium tuberculosis
Authors: Parish Tanya , Stoker Neil G.
Published: July 2002

Mycobacterium tuberculosis
possesses a homologue of
glnE
, potentially encoding a regulator of glutamine synthetase activity. We attempted to construct
glnE
-disrupted mutants using a two-step strategy, whereby a single-crossover strain was first isolated, followed by
sacB
counterselection to isolate the double-crossover strain. Of 192 sucrose-resistant colonies tested, none were mutants, although the wild-type double crossover could be easily isolated. When a second copy of the wild-type
glnE
was integrated into the chromosome, we could isolate both wild-type and mutant double-crossover strains. Thus, the chromosomal gene could only be replaced with a disrupted copy when another functional copy of the gene was provided, demonstrating that this gene is essential under the conditions tested.
New Virulence-Activated and Virulence-Repressed Genes Identified by Systematic Gene Inactivation and Generation of Transcriptional Fusions in
Bordetella pertussis
Authors: Antoine Rudy , Alonso Sylvie , Raze Dominique , Coutte Loïc , Lesjean Sarah , Willery Eve , Locht Camille , Jacob-Dubuisson Françoise
Published: July 2002

An in silico scan of the partially completed genome sequence of
Bordetella pertussis
and analyses of transcriptional fusions generated with a new integrational vector were used to identify new potential virulence genes. The genes encoding a putative siderophore receptor, adhesins, and an autotransporter protein appeared to be regulated in a manner similar to
Bordetella
virulence genes by the global virulence regulator BvgAS. In contrast, the gene encoding a putative intimin-like protein appeared to be repressed under conditions of virulence.
Development and Dynamics of
Pseudomonas
sp. Biofilms
Authors: Tolker-Nielsen Tim , Brinch Ulla C. , Ragas Paula C. , Andersen Jens Bo , Jacobsen Carsten Suhr , Molin Søren
Published: July 2002

Pseudomonas
sp. strain B13 and
Pseudomonas putida
OUS82 were genetically tagged with the green fluorescent protein and the
Discosoma
sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of
P. putida
OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated
P. putida
OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.
The Multifunctional Bacteriophage P2 Cox Protein Requires Oligomerization for Biological Activity
Authors: Eriksson Jesper M. , Haggård-Ljungquist Elisabeth
Published: July 2002

The Cox protein of bacteriophage P2 is a multifunctional protein of 91 amino acids. It is directly involved in the site-specific recombination event leading to excision of P2 DNA out of the host chromosome. In this context, it functions as an architectural protein in the formation of the excisome. Cox is also a transcriptional repressor of the P2 Pc promoter, thereby ensuring lytic growth. Finally it promotes derepression of prophage P4, a nonrelated defective satellite phage, by activating the P4 P
LL
promoter that controls P4 DNA replication. In this case it binds upstream of the P
LL
promoter, which normally is activated by the P4 Delta protein. In this work we have analyzed the native form of the Cox protein in vivo, using a bacteriophage λ
c
I-based oligomerization assay system, and in vitro, using gel filtration, cross-linking agents, and gel retardation assays. We found that P2 Cox has a strong oligomerization function in vivo as well as in vitro. The in vitro analysis indicates that its native form is a tetramer that can self-associate to octamers. Furthermore we show that oligomerization is necessary for the biological activity by characterizing different
cox
mutants and that oligomerization is mediated by the C-terminal region.
The Metabolic Network of
Lactococcus lactis
: Distribution of
14
C-Labeled Substrates between Catabolic and Anabolic Pathways
Authors: Novák L. , Loubiere P.
Published: July 2002

Lactococcus lactis
NCDO 2118 was grown in a simple synthetic medium containing only six essential amino acids and glucose as carbon substrates to determine qualitatively and quantitatively the carbon fluxes into the metabolic network. The specific rates of substrate consumption, product formation, and biomass synthesis, calculated during the exponential growth phase, represented the carbon fluxes within the catabolic and anabolic pathways. The macromolecular composition of the biomass was measured to distribute the global anabolic flux into the specific anabolic pathways. Finally, the distribution of radiolabeled substrates, both into the excreted fermentation end products and into the different macromolecular fractions of biomass, was monitored. The classical end products of lactic acid metabolism (lactate, formate, and acetate) were labeled with glucose, which did not label other excreted products, and to a lesser extent with serine, which was deaminated to pyruvate and represented approximately 10% of the pyruvate flux. Other minor products, keto and hydroxy acids, were produced from glutamate and branched-chain amino acids via deamination and subsequent decarboxylation and/or reduction. Glucose labeled all biomass fractions and accounted for 66% of the cellular carbon, although this represented only 5% of the consumed glucose.
The Stability Region of the Large Virulence Plasmid of
Shigella flexneri
Encodes an Efficient Postsegregational Killing System
Authors: Sayeed Sameera , Reaves Lucretia , Radnedge Lyndsay , Austin Stuart
Published: July 2002

The large virulence plasmid pMYSH6000 of
Shigella flexneri
contains a determinant that is highly effective in stabilizing otherwise unstable plasmids in
Escherichia coli
. Expression of two small contiguous genes,
mvpA
and
mvpT
(formerly termed STBORF1 and STBORF2), was shown to be sufficient for stability. Mutations in
mvpT
abolished plasmid stability, and plasmids expressing only
mvpT
killed the cells unless
mvpA
was supplied from a separate plasmid or from the host chromosome. When replication of a plasmid carrying the minimal
mvp
region was blocked, growth of the culture stopped after a short lag and virtually all of the surviving cells retained the plasmid. Thus, the
mvp
system stabilizes by a highly efficient postsegregational killing (PSK) mechanism, with
mvpT
encoding a cell toxin and
mvpA
encoding an antidote. The regions that surround the
mvp
genes in their original context have an inhibitory effect that attenuates plasmid stabilization and PSK. The region encompassing the
mvp
genes also appears to contain an additional element that can aid propagation of a pSC101-based plasmid under conditions where replication initiation is marginal. However, this appears to be a relatively nonspecific effect of DNA insertion into the plasmid vector.