bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Type 1 Fimbriae by Unlinked FimB- and FimE-Like Recombinases in Uropathogenic
Escherichia coli
Strain CFT073
Authors: Bryan Andrew , Roesch Paula , Davis Lindsay , Moritz Rebecca , Pellett Shahaireen , Welch Rodney A.
Published: January 2006

Genomic DNA sequence analysis of the uropathogenic
Escherichia coli
strain CFT073 revealed that besides the
fimB
and
fimE
recombinase genes that control the type 1 pilus
fim
phase switch, there are three additional
fimB
- and
fimE
-like genes:
ipuA
,
ipuB
, and
ipbA
. Alignment of the predicted amino acid sequences showed that the five recombinases range in sequence similarity from 63 to 70%. An epidemiological survey indicates that
ipuA
and
ipuB
are present and linked next to the
dsdCXA
locus in 24 of 67 uropathogenic
E. coli
strains but are found in only 1 of 15 normal human fecal isolates. The
ipbA
sequence located next to the
betABIT
locus was found in 42 of 67 uropathogenic isolates and 8 of 15 of the commensal strains. We show that two of these recombinases, those encoded by
ipuA
and
ipbA
, can function at the type 1 pilus
fim
switch. In a CFT073 deletion mutant lacking all five recombinase genes, recombinant
ipuA
or
ipbA
provided in
trans
inverted the
fim
element from the off state to the on state. When a
fim
OFF CFT073 Δ
fimBE
mutant was used to infect the urinary tracts of mice, a switch to the
fim
on state was detected within 24 h in bacteria recovered from urine, the bladder, and the kidneys. A
fim
OFF CFT073 Δ
fimBE ipuB ipbA
mutant also demonstrated the ability to switch from the
fim
off state to the on state during mouse infection. CFT073 recombinase mutants derived from isolates in either the
fim
on or off state showed a reciprocal relationship for motility. Switches from a nonmotile to a motile phenotype and from a
fim
on to off genotype were observed in
fim
ON CFT073 Δ
fimBE ipuAB ipbA
mutants when
ipuA
or
fimB
was provided in
trans
. Together these results indicate that
ipuA
has
fimB
-like on-to-off and off-to-on
fim
switching activity and that
ipbA
has the ability to switch
fim
from the off to the on orientation.
Identification of Novel γδ T-Cell Subsets following Bacterial Infection in the Absence of Vγ1+T Cells: Homeostatic Control of γδ T-Cell Responses to Pathogen Infection by Vγ1+T Cells
Authors: Newton Darren J. , Andrew Elizabeth M. , Dalton Jane E. , Mears Rainy , Carding Simon R.
Published: January 2006

Although γδ T cells are a common feature of many pathogen-induced immune responses, the factors that influence, promote, or regulate the response of individual γδ T-cell subsets to infection is unknown. Here we show that in the absence of Vγ1+T cells, novel subsets of γδ T cells, expressing T-cell receptor (TCR)-Vγ chains that normally define TCRγδ+dendritic epidermal T cells (DETCs) (Vγ5+), intestinal intraepithelial lymphocytes (iIELs) (Vγ7+), and lymphocytes associated with the vaginal epithelia (Vγ6+), are recruited to the spleen in response to bacterial infection in TCR-Vγ1−/−mice. By comparison of phenotype and structure of TCR-Vγ chains and/or -Vδ chains expressed by these novel subsets with those of their epithelium-associated counterparts, the Vγ6+T cells elicited in infected Vγ1−/−mice were shown to be identical to those found in the reproductive tract, from where they are presumably recruited in the absence of Vγ1+T cells. By contrast, Vγ5+and Vγ7+T cells found in infected Vγ1−/−mice were distinct from Vγ5+DETCs and Vγ7+iIELs. Functional analyses of the novel γδ T-cell subsets identified for infected Vγ1−/−mice showed that whereas the Vγ5+and Vγ7+subsets may compensate for the absence of Vγ1+T cells by producing similar cytokines, they do not possess cytocidal activity and they cannot replace the macrophage homeostasis function of Vγ1+T cells. Collectively, these findings identify novel subsets of γδ T cells, the recruitment and activity of which is under the control of Vγ1+T cells.
Dendritic Cell Immaturity during Infancy Restricts the Capacity To Express Vaccine-Specific T-Cell Memory
Authors: Upham John W. , Rate Angela , Rowe Julie , Kusel Merci , Sly Peter D. , Holt Patrick G.
Published: January 2006

The capacity of the immune system in infants to develop stable T-cell memory in response to vaccination is attenuated, and the mechanism(s) underlying this developmental deficiency in humans is poorly understood. The present study focuses on the capacity for expression of in vitro recall responses to tetanus and diphtheria antigens in lymphocytes from 12-month-old infants vaccinated during the first 6 months of life. We demonstrate that supplementation of infant lymphocytes with “matured” dendritic cells (DC) cultured from autologous CD14+precursors unmasks previously covert cellular immunity in the form of Th2-skewed cytokine production. Supplementation of adult lymphocytes with comparable prematured autologous DC also boosted vaccine-specific T-cell memory expression, but in contrast to the case for the infants, these cytokine responses were heavily Th1 skewed. Compared to adults, infants had significantly fewer circulating myeloid DC (P< 0.0001) and plasmacytoid DC (P< 0.0001) as a proportion of peripheral blood mononuclear cells. These findings suggest that deficiencies in the numbers of antigen-presenting cells and their functional competence at 12 months of age limit the capacity to express effector memory responses and are potentially a key factor in reduced vaccine responsiveness in infants.
Flagellin Is an Effective Adjuvant for Immunization against Lethal Respiratory Challenge withYersinia pestis
Authors: Honko Anna N. , Sriranganathan Nammalwar , Lees Cynthia J. , Mizel Steven B.
Published: January 2006

Gram-negative flagellin, a Toll-like receptor 5 (TLR5) agonist, is a potent inducer of innate immune effectors such as cytokines and nitric oxide. In the lung, flagellin induces a localized and transient innate immune response characterized by neutrophil infiltration and the production of cytokines and chemokines. In view of the extraordinary potency of flagellin as an inducer of innate immunity and the contribution of innate responses to the development of adaptive immunity, we evaluated the efficacy of recombinantSalmonellaflagellin as an adjuvant in an acellular plague vaccine. Mice immunized intranasally or intratracheally with the F1 antigen ofYersinia pestisand flagellin exhibited dramatic increases in anti-F1 plasma immunoglobulin G (IgG) titers that remained stable over time. In contrast, control mice had low or undetectable antibody responses. The IgG1/IgG2a ratio of antibody titers against F1 in immunized mice is consistent with a Th2 bias. However, no significant antigen-specific IgE production was detected. Interferons, tumor necrosis factor alpha, and interleukin-6 were not essential for the adjuvant effects of flagellin. Preexisting antiflagellin antibodies had no significant effect on the adjuvant activity of flagellin. Importantly, intranasal immunization with flagellin and the F1 antigen was protective against intranasal challenge with virulentY. pestisCO92, with 93 to 100% survival of immunized mice. Lastly, vaccination of cynomolgus monkeys with flagellin and a fusion of the F1 and V antigens ofY. pestisinduced a robust antigen-specific IgG antibody response.
PsrA Is a Positive Transcriptional Regulator of the Type III Secretion System in
Pseudomonas aeruginosa
Authors: Shen D. K. , Filopon D. , Kuhn L. , Polack B. , Toussaint B.
Published: January 2006

The type III secretion system (TTSS) of
Pseudomonas aeruginosa
is induced in vivo upon contact with eukaryotic cells and in vitro by calcium depletion in culture medium. We have observed a previously identified protein, PsrA, necessary for full activation of TTSS gene expression in
P. aeruginosa
. Electrophoretic mobility shift assays showed that recombinant PsrA could bind to the
exsCEBA
promoter region. A mutant with a deletion in the
psrA
gene was constructed. Using transcriptional fusions, we demonstrated that PsrA is required for the full activation of transcription of the TTSS regulatory operon
exsCEBA
and effector
exoS
, although the deletion mutant still responded to calcium depletion, to serum, and to host cell contact. The
psrA
mutant showed a marked decrease in the secretion of the type III effectors and weak resistance to phagocyte-like PLB-985 cells. The defect in TTSS transcription and secretion in the
psrA
mutant could be complemented by expression in
trans
of
psrA
. PsrA was previously identified as a transcriptional activator of RpoS, a central regulator during stationary phase. We confirmed with our strain that RpoS has a negative effect on TTSS gene expression. Taken altogether, these results suggest that PsrA is a newly identified activator that is involved in the expression of the TTSS by enhancing the
exsCEBA
transcriptional level.
Role of Gluconeogenesis and the Tricarboxylic Acid Cycle in the Virulence of
Salmonella enterica
Serovar Typhimurium in BALB/c Mice
Authors: Tchawa Yimga Merlin , Leatham Mary P. , Allen James H. , Laux David C. , Conway Tyrrell , Cohen Paul S.
Published: January 2006

In
Salmonella enterica
serovar Typhimurium, the Cra protein (
c
atabolite
r
epressor/
a
ctivator) regulates utilization of gluconeogenic carbon sources by activating transcription of genes in the gluconeogenic pathway, the glyoxylate bypass, the tricarboxylic acid (TCA) cycle, and electron transport and repressing genes encoding glycolytic enzymes. A serovar Typhimurium SR-11 Δ
cra
mutant was recently reported to be avirulent in BALB/c mice via the peroral route, suggesting that gluconeogenesis may be required for virulence. In the present study, specific SR-11 genes in the gluconeogenic pathway were deleted (
fbp
,
glpX
,
ppsA
, and
pckA
), and the mutants were tested for virulence in BALB/c mice. The data show that SR-11 does not require gluconeogenesis to retain full virulence and suggest that as yet unidentified sugars are utilized by SR-11 for growth during infection of BALB/c mice. The data also suggest that the TCA cycle operates as a full cycle, i.e., a
sucCD
mutant, which prevents the conversion of succinyl coenzyme A to succinate, and an Δ
sdhCDA
mutant, which blocks the conversion of succinate to fumarate, were both attenuated, whereas both an SR-11 Δ
aspA
mutant and an SR-11 Δ
frdABC
mutant, deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent. Moreover, although it appears that SR-11 replenishes TCA cycle intermediates from substrates present in mouse tissues, fatty acid degradation and the glyoxylate bypass are not required, since an SR-11 Δ
fadD
mutant and an SR-11 Δ
aceA
mutant were both fully virulent.
Genetic and Phenotypic Diversity of Quorum-Sensing Systems in Clinical and Environmental Isolates of
Vibrio cholerae
Authors: Joelsson Adam , Liu Zhi , Zhu Jun
Published: January 2006

Vibrio cholerae
is the causative agent of cholera, a severe and devastating diarrheal disease.
V. cholerae
lives naturally in various aquatic habitats during interepidemic periods. Recent studies reveal that quorum-sensing systems, which exist in many bacteria and help them monitor their population densities and regulate various cellular functions, control
V. cholerae
pathogenesis, biofilm formation, and protease production. In this study we surveyed quorum-sensing systems in 16 geographically diverse
V. cholerae
strains from epidemic-causing O1 and O139 strains as well as non-O1/non-O139 and environmental isolates and discovered an unexpectedly high rate of dysfunctional components. We also found that a functional quorum-sensing system conferred a survival advantage on bacteria in biofilms when the bacteria were exposed to seawater, though quorum sensing was less important to survival in a planktonic state under the same conditions. These findings suggest that variations in quorum-sensing systems are due to environmental selective pressures and might be beneficial to
V. cholerae
's fitness under certain conditions found in its natural reservoirs.
Fas Ligand Deficiency Impairs Host Inflammatory Response against Infection with the SpirocheteBorrelia burgdorferi
Authors: Shi Cuixia , Wolfe Julie , Russell Jennifer Q. , Fortner Karen , Hardin Nicholas , Anguita Juan , Budd Ralph C.
Published: January 2006

Lyme disease represents a complex response toBorrelia burgdorferithat involves both bacterial factors as well as host responses. This results in an inflammatory reaction at several sites, including the synovial lining of joints. Synovial tissues of inflamed joints contain cells expressing high levels of Fas and Fas ligand (FasL). Although Fas stimulation is typically associated with cell death, it can also transmit stimulatory signals to certain cell types. Among these are dendritic cells and macrophages, which are abundant in inflamed synovium. To better assess the role of FasL in the pathogenesis of Lyme arthritis, we evaluated the response toB. burgdorferiinfection in C3H/HeJgldmice that bear a nonfunctional mutation in FasL. Compared to wild-type C3H+/+mice, C3Hgldmice had a similar bacterial burden and antibody response 2 weeks and 4 weeks following infection, but they manifested a significantly reducedBorrelia-specific cytokine response. In addition, C3Hgldmice developed a greatly reduced incidence and severity of arthritis. The findings document a contribution of FasL to the host inflammatory response toB. burgdorferi.
Conservation and Diversity of HMW1 and HMW2 Adhesin Binding Domains among Invasive Nontypeable
Haemophilus influenzae
Isolates
Authors: Giufrè Maria , Muscillo Michele , Spigaglia Patrizia , Cardines Rita , Mastrantonio Paola , Cerquetti Marina
Published: January 2006

The pathogenesis of nontypeable
Haemophilus influenzae
(NTHi) begins with adhesion to the rhinopharyngeal mucosa. In almost 80% of NTHi clinical isolates, the HMW proteins are the major adhesins. The prototype HMW1 and HMW2 proteins, identified in NTHi strain 12, exhibit different binding specificities. The two binding domains have been localized in regions of maximal sequence dissimilarity (40% identity, 58% similarity). Two areas within these binding domains have been found essential for full level adhesive activity (designated the core-binding domains). To investigate the conservation and diversity of the HMW1 and HMW2 core-binding domains among isolates, PCR and DNA sequencing were used. First, we separately amplified the
hmw1A
-like and
hmw2A
-like structural genes in nine invasive NTHi isolates, discovering two new
hmwA
alleles, whose sequences are herein reported. Then, the
hmw1A-
like and
hmw2A
-like PCR products were used as the template in nested PCR to produce amplicons encompassing the encoding sequences of the two core-binding domains. In-depth sequence analysis was then performed among sequences of each group, with the support of specific computer programs. Overall, extensive sequence diversity among isolates was highlighted. However, similarity plots showed patterns consisting of peaks of relatively high similarity alternating with strongly divergent regions. The phylogenetic tree clearly indicated the HMW1-like and HMW2-like core-binding domain sequences as two clusters. Distinct sets of conserved amino acid motifs were identified within each group of sequences using the MEME/MOTIFSEARCH tool. Since HMW adhesins could represent candidates for future vaccines, identification of specific patterns of conserved motifs in otherwise highly variable regions is of great interest.
Mn
2+
-Dependent Regulation of Multiple Genes in
Streptococcus pneumoniae
through PsaR and the Resultant Impact on Virulence
Authors: Johnston Jason W. , Briles David E. , Myers Lisa E. , Hollingshead Susan K.
Published: January 2006

The concentration of Mn
2+
is 1,000-fold higher in secretions than it is at internal sites of the body, making it a potential signal by which bacteria can sense a shift from a mucosal environment to a more invasive site. PsaR, a metal-dependent regulator in
Streptococcus pneumoniae
, was found to negatively affect the transcription of
psaBCA
,
pcpA
,
rrgA
,
rrgB
,
rrgC
,
srtBCD
, and
rlrA
in the presence of Mn
2+
.
psaBCA
encode an ABC-type transporter for Mn
2+
. 
pcpA
,
rrgA
,
rrgB
, and
rrgC
encode several outer surface proteins.
srtBCD
encode a cluster of sortase enzymes, and
rlrA
encodes a transcriptional regulator. Steady-state RNA levels are high under low Mn
2+
concentrations in the wild-type strain and are elevated under both high and low Mn
2+
concentrations in a
psaR
mutant strain. RlrA is an activator of
rrgA
,
rrgB
,
rrgC
, and
srtBCD
(D. Hava and A. Camilli, Mol. Microbiol.
45:
1389-1406, 2002), suggesting that PsaR may indirectly control these genes through
rlrA
, while PsaR-dependent repression of
psaBCA
,
pcpA
, and
rlrA
transcription is direct. The impact of Mn
2+
-dependent regulation on virulence was further examined in mouse models of pneumonia and nasopharyngeal carriage. The abilities of Δ
psaR
,
pcpA
, and Δ
psaR
Δ
pcpA
mutant strains to colonize the lung were reduced compared to those of the wild type, confirming that both PcpA-mediated gene regulation and PsaR-mediated gene regulation are required for full virulence in the establishment of pneumonia. Neither PcpA nor PsaR was found to be required for colonization of the nasopharynx in a carriage model. This is the first demonstration of Mn
2+
acting as a signal for the expression of virulence factors within different host sites.
Tumor Necrosis Factor Alpha and Gamma Interferon, but Not Hemorrhage or Pathogen Burden, Dictate Levels of Protective Fibrin Deposition during Infection
Authors: Mullarky Isis K. , Szaba Frank M. , Berggren Kiera N. , Kummer Lawrence W. , Wilhelm Lindsey B. , Parent Michelle A. , Johnson Lawrence L. , Smiley Stephen T.
Published: January 2006

While coagulation often causes pathology during infectious disease, we recently demonstrated that fibrin, a product of the coagulation pathway, performs a critical protective function during acute toxoplasmosis (L. L. Johnson, K. N. Berggren, F. M. Szaba, W. Chen, and S. T. Smiley, J. Exp. Med.197:801-806, 2003). Here, we investigate the mechanisms regulating the formation of this protective fibrin. Through comparisons ofToxoplasma-infected wild-type and cytokine-deficient mice we dissociate, for the first time, the relative fibrin-regulating capacities of pathogen products, host cytokines, and infection-stimulated hemorrhage. Remarkably, neither the pathogen burden nor hemorrhage is a primary regulator of fibrin levels. Rather, two type 1 cytokines exert dominant and counterregulatory roles: tumor necrosis factor alpha (TNF-α), acting via the type 1 TNF-α receptor, promotes fibrin deposition, while gamma interferon (IFN-γ), acting via STAT1 and IFN-γ receptors expressed on radioresistant cells, suppresses fibrin deposition. These findings have important clinical implications, as they establish that cytokines known to regulate pathological coagulation also dictate levels of protective fibrin deposition. We present a novel model depicting mechanisms by which the immune system can destroy infected tissue while independently restraining hemorrhage and promoting tissue repair through the deliberate deposition of protective fibrin.
Identification of aNaegleria fowleriMembrane Protein Reactive with Anti-Human CD59 Antibody
Authors: Fritzinger Angela E. , Toney Denise M. , MacLean Rebecca C. , Marciano-Cabral Francine
Published: January 2006

Naegleria fowleri, the causative agent of primary amebic meningoencephalitis, is resistant to complement lysis. The presence of a complement regulatory protein on the surface ofN. fowleriwas investigated. Southern blot and Northern blot analyses demonstrated hybridization of a radiolabeled cDNA probe for CD59 to genomic DNA and RNA, respectively, from pathogenicN. fowleri. An 18-kDa immunoreactive protein was detected on the membrane ofN. fowleriby Western immunoblot and immunofluorescence analyses with monoclonal antibodies for human CD59. Complement component C9 immunoprecipitated with theN. fowleri“CD59-like” protein from amebae incubated with normal human serum. In contrast, a gene or protein similar to CD59 was not detected in nonpathogenic, complement-sensitiveN. gruberiamebae. Collectively, our studies suggest that a protein reactive with antibodies to human CD59 is present on the surface ofN. fowleriamebae and may play a role in resistance to lysis by cytolytic proteins.
Pathogenesis of B-Cell Superantigen-Induced Immune Complex-Mediated Inflammation
Authors: Anderson Amy L. , Sporici Romeo , Lambris John , LaRosa David , Levinson Arnold I.
Published: January 2006

Staphylococcal protein A (SpA) is representative of a new class of antigens, the B-cell superantigens (SAgs). These antigens bind to the Fab regions of immunoglobulin molecules outside their complementarity-determining regions. SpA, the best-studied B-cell SAg, reacts with the Fabs of most VH3+immunoglobulins, which are expressed on 30 to 60% of human peripheral B cells. Therefore, B-cell SAgs like SpA have great potential to elicit inflammatory responses in vivo. We previously reported that the interaction of SpA with VH3+immunoglobulin molecules leads to activation of the complement cascade and produces a histologic pattern of inflammation in the skin of a rabbit indicative of immune complex injury. To elucidate the cellular and molecular events contributing to this type of unconventional immune complex-mediated inflammation, we established a mouse peritoneal Arthus reaction model. Mice treated intravenously with human polyclonal immunoglobulin G (IgG), followed by intraperitoneal injection of SpA, showed neutrophil influx into the peritoneal cavity with peak numbers appearing at 8 h. This inflammatory reaction was dependent on the interaction of SpA with VH3+IgG. Mast cells, FcγRIII, complement components, and tumor necrosis factor alpha play obligatory roles, and the reaction is associated with the local release of the CXC chemokines macrophage inflammatory protein 2 and KC. The data provide further compelling evidence for the induction of immune complex-mediated injury by a B-cell SAg and highlight important factors contributing to the pathogenesis of this novel type of inflammatory reaction.
Identification of Proteins Differentially Expressed in Human Monocytes Exposed toPorphyromonas gingivalisand Its Purified Components by High-Throughput Immunoblotting
Authors: Zhou Qingde , Amar Salomon
Published: January 2006

To characterize the roles ofPorphyromonas gingivalisand its components in disease processes, we investigated the cytokine profiles induced by liveP. gingivalis, its lipopolysaccharide (LPS), and its major fimbrial protein, fimbrillin (FimA). A cytokine antibody array revealed that human monocyte-derived macrophages were induced to produce chemokines (e.g., monocyte chemoattractant protein 1, macrophage inflammatory protein 1β [MIP-1β], and MIP-3α) as early as 1 h after exposure toP. gingivalis, with production declining after 24 h of exposure. As expected, an extensive repertoire of inflammatory mediators increased subsequent to infection, most predominantly tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor. The induction of cytokines byP. gingivaliswas not triggered simply by bacterial cell surface components, since purifiedP. gingivalisLPS and FimA induced similar patterns of cytokines, while the pattern of cytokines induced by liveP. gingivaliswas significantly different, indicating that the host defense system senses live bacteria differently than it does the cell surface components LPS and FimA. To further understand the mechanisms by which liveP. gingivalisand its components exert their effects, we used a high-throughput immunoblot screening approach (Becton-Dickinson PowerBlot) to analyze intracellular proteins involved inP. gingivalisinfection in human macrophages. Exposure of human macrophages to either liveP. gingivalis, its LPS, or its FimA protein led to the up-regulation of 12, 8, and 10 proteins and the down-regulation of 15, 8, and 17 proteins, respectively. The expression of proteins involved in gene transcription (e.g., monocyte enhancer factor 2D [MEF2D], signal transducer and activator of transcription 1 [STAT1], STAT3, STAT6, and IL enhancer binding factors [ILF3]), of protein kinases (e.g., mitogen-activated protein kinase 3 [MAPK3], MAP3K8, double-stranded RNA-activated protein kinase [PRKR], and MAP2K4), and of proteins involved in immune responses (e.g., TNF super family member 6 [TNFSF6] and interferon-induced protein with tetratricopeptide repeat 4 [IFIT4]), apoptosis (e.g., genes associated with retinoid interferon-induced mortality 19 [GRIM19]), and other fundamental cellular processes (e.g., clathrin heavy-chain polypeptide, culreticulin, and Ras-associated protein RAB27A) was found to be modulated differentially byP. gingivalis, LPS, and FimA. These differential changes are interpreted as preferential signal pathway activation in host immune/inflammatory responses toP. gingivalisinfection.
Cytokine Production Associated with Periportal Fibrosis during Chronic Schistosomiasis Mansoni in Humans
Authors: Alves Oliveira L. F. , Moreno E. C. , Gazzinelli G. , Martins-Filho O. A. , Silveira A. M. S. , Gazzinelli A. , Malaquias L. C. C. , LoVerde P. , Leite P. Martins , Correa-Oliveira R.
Published: January 2006

Volunteers living in an area where schistosomiasis mansoni is endemic were subjected to ultrasound examination and classified into groups according to the levels of fibrosis diagnosed, namely, absence of indications of fibrosis (group 0), incipient fibrosis (group 1), and moderate/severe fibrosis (group 2). Peripheral blood mononuclear cells (PBMC) collected from the volunteers were stimulated with soluble antigens from adult schistosomes or from schistosome eggs, and the production of the cytokines gamma interferon, tumor necrosis factor alpha, transforming growth factor β (TGF-β), interleukin-4 (IL-4), IL-10, and IL-13 was determined. Potential associations of the level of fibrosis with age, sex, intensity of infection, and cytokine production were investigated between the three groups. Univariate analysis identified associations of age (>50), gender (male), and absence of eggs/g of feces with moderate/severe fibrosis and an association of intensity of infection (>100 eggs) with incipient fibrosis. When cytokine production in PBMC cultures stimulated by soluble egg antigens was categorized as low or high, significant differences in the distribution of IL-13 levels were established between groups 0 and 2. No significant differences were detected between the groups in the cytokines produced by PBMC cultures stimulated with soluble antigens from adult schistosomes. When all variables were tested in multivariate analyses, only IL-13 was strongly associated with fibrosis (odds ratio = 5.8; 95% confidence interval [CI] = 1.1 to 30.5). While high levels of TGF-β appeared to be associated with protection against fibrosis, the strength of the association was low.
Identification of Amino Acid Residues Involved in Heme Binding and Hemoprotein Utilization in the
Porphyromonas gingivalis
Heme Receptor HmuR
Authors: Liu Xinyan , Olczak Teresa , Guo Hwai-Chen , Dixon Dabney W. , Genco Caroline Attardo
Published: January 2006

We have previously identified and characterized a heme/hemoglobin receptor, HmuR, in
Porphyromonas gingivalis.
To analyze the conserved amino acid residues of HmuR that may be involved in hemin/hemoprotein binding and utilization, we constructed a series of
P. gingivalis
A7436
hmuR
mutants with amino acid replacements and characterized the ability of these mutants to utilize hemin and hemoproteins. Site-directed mutagenesis was employed to introduce mutations H95A, H434A, H95A-H434A, YRAP420-423YAAA, and NPDL442-445NAAA into HmuR in both
P. gingivalis
and
Escherichia coli
. Point mutations at H95 and H434 and in the NPDL motif of HmuR resulted in decreased binding to hemin, hemoglobin, and human serum albumin-hemin complex. Notably, mutations of these conserved sites and motifs led to reduced growth of
P. gingivalis
when human serum was used as the heme source. Analysis using a three-dimensional homology model of HmuR indicated that H95, H434, and the NPDL motif are present on apical or extracellular loops of HmuR, while the YRAP motif is present on the barrel wall. Taken together, these results support a role for H95, H434, and the NPDL motif of the
P. gingivalis
HmuR protein in heme binding and utilization of serum hemoproteins and the HmuR YRAP motif in serum hemoprotein utilization.
Polynucleotide Phosphorylase Negatively ControlsspvVirulence Gene Expression inSalmonella enterica
Authors: Ygberg Sofia Eriksson , Clements Mark O. , Rytkönen Anne , Thompson Arthur , Holden David W. , Hinton Jay C. D. , Rhen Mikael
Published: January 2006

Mutational inactivation of the cold-shock-associated exoribonuclease polynucleotide phosphorylase (PNPase; encoded by thepnpgene) inSalmonella entericaserovar Typhimurium was previously shown to enable the bacteria to cause chronic infection and to affect the bacterial replication in BALB/c mice (M. O. Clements et al., Proc. Natl. Acad. Sci. USA99:8784-8789, 2002). Here, we report that PNPase deficiency results in increased expression ofSalmonellaplasmid virulence (spv) genes under in vitro growth conditions that allow induction ofspvexpression. Furthermore, whole-genome microarray-based transcriptome analyses of bacteria growing inside murine macrophage-like J774.A.1 cells revealed six genes as being significantly up-regulated in the PNPase-deficient background, which includedspvABC,rtcB,entC, and STM2236. Mutational inactivation of thespvRregulator diminished the increased expression ofspvobserved in thepnpmutant background, implying that PNPase acts upstream of or at the level of SpvR. Finally, competition experiments revealed that the growth advantage of thepnpmutant in BALB/c mice was dependent onspvRas well. Combined, our results support the idea that inS. entericaPNPase, apart from being a regulator of the cold shock response, also functions in tuning the expression of virulence genes and bacterial fitness during infection.
Anthrax Lethal Toxin Induces Ketotifen-Sensitive Intradermal Vascular Leakage in Certain Inbred Mice
Authors: Gozes Yehoshua , Moayeri Mahtab , Wiggins Jason F. , Leppla Stephen H.
Published: January 2006

Bacillus anthracis
lethal toxin (LT) is a bipartite toxin composed of protective antigen (PA) and lethal factor (LF). Injection of LT produces clinical signs characteristic of anthrax infection, including pleural edema and vascular collapse in various animal models. We utilized the classic Miles leakage assay to quantify vascular leakage in mice. LT injected intradermally induced leakage as early as 15 to 25 min in some inbred mouse strains, but not in others, whereas PA or LF individually did not induce leakage. A third component of anthrax toxin, edema factor, did not induce leakage alone or with PA. Leakage was quantified in eight mouse strains, and no correlation was found between sensitivity to intradermal leakage and sensitivity to the lethality of systemically administered LT. The leakage could be inhibited by ketotifen, an inhibitor of mast cell degranulation, but not by azelastine, a histamine receptor 1 antagonist, or by ketanserin, a serotonin 5-HT2A receptor antagonist. LT was cytotoxic to MC/9 mast cells (in vitro) by 7 h after toxin treatment but did not induce histamine release from these cells. Mast cell-deficient mice exhibited the leakage event and had no increased resistance to systemic LT. Human umbilical vein endothelial cells were resistant to LT over 12 h, with only 20% of cells succumbing by 24 h, suggesting that endothelial cell killing is not the cause of the rapid LT-mediated leakage event. We describe here a ketotifen-sensitive vascular leakage event induced by LT which is the most rapid in vivo or in vitro LT-mediated effect reported to date.
Intranasal Exposure to Bacterial Superantigens Induces Airway Inflammation in HLA Class II Transgenic Mice
Authors: Rajagopalan Govindarajan , Iijima Koji , Singh Manisha , Kita Hirohito , Patel Robin , David Chella S.
Published: January 2006

Staphylococcus aureusis widely prevalent in the nasopharynges of healthy individuals (carriers) but can also cause serious infections.S. aureuscan elaborate a variety of superantigen exotoxins in “carrier” or “pathogenic” states.Streptococcus pyogenescan also colonize the nasopharynx and elaborate superantigens. Unlike the acute effects of superantigen exotoxins absorbed through the gut or vaginal mucosa, little is known regarding the pathogenesis of superantigens entering through the intranasal route. In the current study, we evaluated the local and systemic effects of staphylococcal enterotoxin B (SEB) and streptococcal pyrogenic exotoxin A (SPEA) delivered through the intranasal route. Superantigens were administered intranasally on multiple occasions, and experimental animals were sacrificed on day 8 for experimental analyses. SEB-induced airway inflammation was more pronounced for HLA-DR3 transgenic mice than for BALB/c mice, consistent with bacterial superantigens binding more efficiently to human than murine major histocompatibility complex class II. The nature of the airway inflammation in HLA-DR3 mice was determined by the concentration of SEB applied intranasally. Low concentrations (20 ng) induced eosinophilic airway inflammation as well as eosinophil degranulation, whereas intranasal exposure to higher concentrations (2,000 ng) resulted in neutrophilic airway inflammation, permanent airway destruction, toxic shock, and mortality. SEB-induced eosinophilic inflammatory response was enhanced in signal transducer and activator of transcription (STAT)-4-deficient HLA-DQ8 transgenic mice with defective interleukin-12 signaling. Intranasal administration of SPEA induced airway inflammation and systemic immune activation in HLA-DQ8 transgenic mice. In conclusion, repeated chronic intranasal exposure to bacterial superantigens causes airway inflammation and systemic immune activation.
Cathepsin B Homologue at the Interface between a Parasitic Nematode and Its Intermediate Host
Authors: Duffy Michael S. , Cevasco Deanne K. , Zarlenga Dante S. , Sukhumavasi Woraporn , Appleton Judith A.
Published: January 2006

Parelaphostrongylus tenuisis a parasitic nematode that causes a debilitating neurologic disease in many North American cervids and domestic livestock species. We produced a PCR-based cDNA library from infective larvae (L3) in order to identify molecules that mediate parasitism. A dominant 1,250-bp amplicon encoded a homologue of cathepsin B cysteine proteases. The sequence incorporated a C29G substitution in the putative active site. Antibodies generated against a recombinant form detected the native protein (PtCPR-1) in Western blot assays of L3, but not adult worm, extracts. Immunohistochemical methods revealed that PtCPR-1 synthesis was restricted to larval stages within the snail intermediate host (Triodopsissp.), beginning as early as 2 days postinfection (dpi) of snails. The protein was present in the intestine and luminal contents and was lost from larvae over time. Concurrent studies showed that larvae induced an immune response in snails beginning at 1 dpi. Layers of hemocytes encapsulated larvae immediately after infection, and granuloma-like structures formed around parasites in chronic infections. Loss of PtCPR-1 from L3 and its accumulation in host tissues coincided with degeneration of granuloma architecture 90 to 105 dpi. Fully developed L3 emerged from the snail at this time. Our data implicate PtCPR-1 in larval development and possibly in the emergence ofP. tenuisfrom the intermediate host. Emerged L3 survived desiccation and cold stress, suggesting that they could remain infectious in the environment. Molecules promoting emergence would facilitate dispersal of L3 and increase the likelihood of transmission to definitive hosts.
Biogenesis of
Leishmania major
-Harboring Vacuoles in Murine Dendritic Cells
Authors: Körner Ulrich , Fuss Veronika , Steigerwald Jutta , Moll Heidrun
Published: January 2006

In mammalian hosts,
Leishmania
sp. parasites are obligatory intracellular organisms that invade macrophages and dendritic cells (DC), where they reside in endocytic organelles termed parasitophorous vacuoles (PV). Most of the present knowledge of the characteristics of PV harboring
Leishmania
sp. is derived from studies with infected macrophages. Since DC play a key role in host resistance to leishmaniasis, there is a need to understand the properties and biogenesis of PV in
Leishmania
sp.-infected DC. Therefore, we determined the acquisition of endosomal and lysosomal molecules by
Leishmania major
-containing compartments in DC at different maturation stages, using fluorescence labeling and confocal microscopy. The results show that newly formed phagosomes in DC rapidly develop into late endosomal compartments. However, the small GTPase Rab7, which regulates late fusion processes, was found only in PV of mature bone marrow-derived DC (BMDC); it was absent in immature BMDC, suggesting an arrest of their PV biogenesis at the stage of late endosomes. Indeed, fusion assays with endocytic tracers demonstrated that the fusion activity of
L. major
-harboring PV toward lysosomes is higher in mature BMDC than in immature BMDC. The inhibition of PV-lysosome fusion in DC is dependent upon the viability and life cycle stage of the parasite, because live promastigotes blocked the fusion almost completely, whereas killed organisms and amastigotes induced a considerable level of fusion activity. The differences in the fusion competences of immature and mature DC may be relevant for their distinct functional activities in the uptake, transport, and presentation of parasite antigens.
Analysis of Antibodies Directed against Merozoite Surface Protein 1 of the Human Malaria ParasitePlasmodium falciparum
Authors: Woehlbier Ute , Epp Christian , Kauth Christian W. , Lutz Rolf , Long Carole A. , Coulibaly Boubacar , Kouyaté Bocar , Arevalo-Herrera Myriam , Herrera Sócrates , Bujard Hermann
Published: January 2006

The 190-kDa merozoite surface protein 1 (MSP-1) ofPlasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera ofAotusmonkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria.
Intracellular Gene Expression Profile of
Listeria monocytogenes
Authors: Chatterjee Som Subhra , Hossain Hamid , Otten Sonja , Kuenne Carsten , Kuchmina Katja , Machata Silke , Domann Eugen , Chakraborty Trinad , Hain Torsten
Published: January 2006

Listeria monocytogenes
is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality.
L. monocytogenes
is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with
L. monocytogenes
strain EGD-e and examined the gene expression profile of
L. monocytogenes
inside the vacuolar and cytosolic environments of the host cell by using whole-genome microarray and mutant analyses. We found that ∼17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic
Listeria innocua
CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced
L. monocytogenes
F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of
L. monocytogenes
in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by
L. monocytogenes
to escape the host cell responses.
Expression Microarray and Mouse Virulence Analysis of Four Conserved Two-Component Gene Regulatory Systems in Group A
Streptococcus
Authors: Sitkiewicz Izabela , Musser James M.
Published: January 2006

Group A streptococcus (GAS) is a gram-positive human bacterial pathogen that causes diseases ranging from relatively mild epithelial cell surface infections to life-threatening invasive episodes. Much is known about the extracellular molecules that contribute to host-pathogen interactions, but in contrast, far less information is available about regulatory genes that control the expression of individual or multiple GAS virulence factors. The eight GAS genomes that have been sequenced have 12 conserved two-component gene regulatory systems (TCSs), but only 3 of these 12 have been studied in detail. Using an allelic replacement strategy with a nonpolar cassette, we inactivated the response regulator of four TCSs that have only weak homology with TCS genes of known or inferred function in other bacteria. The mutant strains were analyzed by expression microarray analysis at four time points and tested in two mouse infection models. Each TCS influenced expression (directly or indirectly) of 12 to 41% of all chromosomal genes, as assessed by growth in Todd-Hewitt broth and a custom Affymetrix GeneChip. None of the isogenic mutant strains was significantly altered for mouse virulence based on intraperitoneal inoculation. Similarly, compared to the wild-type strain, there was no significant difference in skin lesion size for three of the four mutants. In contrast, the ΔM5005_Spy_0680 mutant strain produced significantly larger abscesses after subcutaneous inoculation into mice, consistent with a hypervirulence phenotype. The mutant strain had significantly higher in vitro expression of several proven and putative virulence genes, including
scpA
, encoding a peptidase that inactivates complement protein C5a. Together, the data provide new information about previously uncharacterized GAS TCSs.
Role of
AFR1
, an ABC Transporter-Encoding Gene, in the In Vivo Response to Fluconazole and Virulence of
Cryptococcus neoformans
Authors: Sanguinetti Maurizio , Posteraro Brunella , La Sorda Marilena , Torelli Riccardo , Fiori Barbara , Santangelo Rosaria , Delogu Giovanni , Fadda Giovanni
Published: January 2006

We have recently demonstrated that upregulation of the ATP binding cassette (ABC) transporter-encoding gene
AFR1
in
Cryptococcus neoformans
is involved in the in vitro resistance to fluconazole of this yeast. In the present study, we investigated the role of
AFR1
in the in vivo response to fluconazole in a mouse model of systemic cryptococcosis. Mice were infected with a wild-type fluconazole-susceptible strain of
C. neoformans
, strain BPY22; an
afr1
mutant, BPY444, which displayed hypersusceptibility to fluconazole in vitro; or an
AFR1-
overexpressing strain, BPY445, which exhibited in vitro resistance to the drug. In each of the three groups, infected animals were randomly assigned to fluconazole treatment or untreated-control subgroups. As expected, fluconazole prolonged survival and reduced fungal tissue burdens (compared with no treatment) in BPY22- and BPY444-infected mice, whereas it had no significant effects in mice infected with BPY445. When the pathogenicities of these strains in mice were investigated, strain BPY445 was significantly more virulent than BPY22 following inhalational or intravenous inoculation, but mice infected with BPY444 survived significantly longer than BPY22-infected animals only when infection was acquired via the respiratory tract. In in vitro macrophage infection studies, strain BPY445 also displayed enhanced intracellular survival compared with strains BPY22 and BPY444, suggesting that its increased virulence may be due to its reduced vulnerability to the antimicrobial factors produced by phagocytic cells. These findings indicate that the upregulation of the
AFR1
gene is an important factor in either determining the in vivo resistance to fluconazole or influencing the virulence of
C. neoformans
.
Lipooligosaccharide Structure Contributes to Multiple Steps in the Virulence ofNeisseria meningitidis
Authors: Plant Laura , Sundqvist Johanna , Zughaier Susu , Lövkvist Lena , Stephens David S. , Jonsson Ann-Beth
Published: January 2006

Lipooligosaccharide (LOS) ofNeisseria meningitidishas been implicated in meningococcal interaction with host epithelial cells and is a major factor contributing to the human proinflammatory response to meningococci. LOS mutants of the encapsulatedN. meningitidisserogroup B strain NMB were used to further determine the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The wild-type strain [NeuNAc-Galβ-GlcNAc-Galβ-Glcβ-Hep2(GlcNAc, Glcα) 3-deoxy-d-manno-2-octulosonic acid (KDO2)-lipid A; 1,4′ bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the mouse model. In contrast, the Hep2(GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB) mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induction of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of this study further define the importance of the LOS structure as a virulence component involved in multiple steps in the pathogenesis ofN. meningitidis.
Exchange of Lipooligosaccharide Synthesis Genes Creates Potential Guillain-Barré Syndrome-Inducible Strains ofCampylobacter jejuni
Authors: Phongsisay Vongsavanh , Perera Viraj N. , Fry Benjamin N.
Published: January 2006

Human ganglioside-like structures, such as GM1, found on someCampylobacter jejunistrains have been linked to inducing the Guillain-Barré Syndrome (GBS). This study shows that aC. jejunistrain without GM1-like molecules acquired large DNA fragments, including lipooligosaccharide synthesis genes, from a strain expressing GM1-like molecules and consequently transformed into a number of potential GBS-inducible transformants, which exhibited a high degree of genetic and phenotypic diversity.