bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct Defensin Profiles inNeisseria gonorrhoeaeandChlamydia trachomatisUrethritis Reveal Novel Epithelial Cell-Neutrophil Interactions
Authors: Porter Edith , Yang Huixia , Yavagal Sujata , Preza Gloria C. , Murillo Omar , Lima Heriberto , Greene Sheila , Mahoozi Laily , Klein-Patel Marcia , Diamond Gill , Gulati Sunita , Ganz Tomas , Rice Peter A. , Quayle Alison J.
Published: July 2005

Defensins are key participants in mucosal innate defense. The varied antimicrobial activity and differential distribution of defensins at mucosal sites indicate that peptide repertoires are tailored to site-specific innate defense requirements. Nonetheless, few studies have investigated changes in peptide profiles and function after in vivo pathogen challenge. Here, we determined defensin profiles in urethral secretions of healthy men and men withChlamydia trachomatis-andNeisseria gonorrhoeae-mediated urethritis by immunoblotting for the epithelial defensins HBD1, HBD2, and HD5 and the neutrophil defensins HNP1 to -3 (HNP1-3). HBD1 was not detectable in secretions, and HBD2 was only induced in a small proportion of the urethritis patients; however, HD5 and HNP1-3 were increased inC. trachomatisinfection and significantly elevated inN. gonorrhoeaeinfection. When HNP1-3 levels were low, HD5 appeared mostly as the propeptide; however, when HNP1-3 levels were >10 μg/ml, HD5 was proteolytically processed, suggesting neutrophil proteases might contribute to HD5 processing. HD5 and HNP1-3 were bactericidal againstC. trachomatisandN. gonorrhoeae, but HD5 activity was dependent upon N-terminal processing of the peptide. In vitro proteolysis of proHD5 by neutrophil proteases and analysis of urethral secretions by surface-enhanced laser desorption ionization substantiated that neutrophils contribute the key convertases for proHD5 in the urethra during these infections. This contrasts with the small intestine, where Paneth cells secrete both proHD5 and its processing enzyme, trypsin. In conclusion, we describe a unique defensin expression repertoire in response to inflammatory sexually transmitted infections and a novel host defense mechanism wherein epithelial cells collaborate with neutrophils to establish an antimicrobial barrier during infection.
Global Gene Expression and the Role of Sigma Factors in
Neisseria gonorrhoeae
in Interactions with Epithelial Cells
Authors: Du Ying , Lenz Jonathan , Arvidson Cindy Grove
Published: July 2005

Like many bacterial pathogens,
Neisseria gonorrhoeae
must adapt to environmental changes in order to successfully colonize and proliferate in a new host. Modulation of gene expression in response to environmental signals is an efficient mechanism used by bacteria to achieve this goal. Using DNA microarrays and a tissue culture model for gonococcal infection, we examined global changes in gene expression in
N. gonorrhoeae
in response to adherence to host cells. Among those genes induced upon adherence to human epithelial cells in culture was
rpoH
, which encodes a homolog of the heat shock sigma factor, σ
32
(RpoH), as well as genes of the RpoH regulon,
groEL
and
groES
. Attempts to construct an
rpoH
null mutant in
N. gonorrhoeae
were unsuccessful, suggesting that RpoH is essential for viability of
N. gonorrhoeae
. The extracytoplasmic sigma factor, RpoE (σ
E
), while known to regulate
rpoH
in other bacteria, was found not to be necessary for the up-regulation of
rpoH
in gonococci upon adherence to host cells. To examine the role of RpoH in host cell interactions, an
N. gonorrhoeae
strain conditionally expressing
rpoH
was constructed. The results of our experiments showed that while induction of
rpoH
expression is not necessary for adherence of gonococci to epithelial cells, it is important for the subsequent invasion step, as gonococci depleted for
rpoH
invade cells two- to threefold less efficiently than a wild-type strain. Taken together, these results indicate that σ
32
, but not σ
E
, is important for the response of gonococci in the initial steps of an infection.
Involvement of Ganglioside GM3 in G2/M Cell Cycle Arrest of Human Monocytic Cells Induced byActinobacillus actinomycetemcomitansCytolethal Distending Toxin
Authors: Mise Koji , Akifusa Sumio , Watarai Shinobu , Ansai Toshihiro , Nishihara Tatsuji , Takehara Tadamichi
Published: July 2005

Actinobacillus actinomycetemcomitansproduces a toxin called cytolethal distending toxin (CDT), which causes host cell DNA damage leading to the induction of DNA damage checkpoint pathways. CDT consists of three subunits, CdtA, CdtB, and CdtC. CdtB is the active subunit of CDT and exerts its effect as a nuclease that damages nuclear DNA, triggering cell cycle arrest. In the present study, we confirmed that the only combination of toxin proteins causing cell cycle arrest was that of all three recombinant CDT (rCDT) protein subunits. Furthermore, in order for rCDT to demonstrate toxicity, it was necessary for CdtA and CdtC to access the cell before CdtB. The coexistence of CdtA and CdtC was necessary for these subunits to bind to the cell. Cells treated with the glucosylceramide synthesis inhibitor 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol showed resistance to the cytotoxicity induced by rCDT. Furthermore, LY-B cells, which are deficient in the biosynthesis of sphingolipid, also showed resistance to the cytotoxicity induced by rCDT. To evaluate the binding of each subunit for glucosylceramides, we performed thin-layer chromatography immunostaining. The results indicated that each subunit reacted with the glycosphingolipids GM1, GM2, GM3, Gb3, and Gb4. The rCDT mixture incubated with liposomes containing GM3 displayed partially reduced toxicity. These results indicate that GM3 can act as a CDT receptor.
Surfaceome of
Leptospira
spp
Authors: Cullen Paul A. , Xu Xiaoyi , Matsunaga James , Sanchez Yolanda , Ko Albert I. , Haake David A. , Adler Ben
Published: July 2005

The identification of the subset of outer membrane proteins exposed on the surface of a bacterial cell (the surfaceome) is critical to understanding the interactions of bacteria with their environments and greatly narrows the search for protective antigens of extracellular pathogens. The surfaceome of
Leptospira
was investigated by biotin labeling of viable leptospires, affinity capture of the biotinylated proteins, two-dimensional gel electrophoresis, and mass spectrometry (MS). The leptospiral surfaceome was found to be predominantly made up of a small number of already characterized proteins, being in order of relative abundance on the cell surface: LipL32 > LipL21 > LipL41. Of these proteins, only LipL32 had not been previously identified as surface exposed. LipL32 surface exposure was subsequently verified by three independent approaches: surface immunofluorescence, whole-cell enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Three other proteins, Q8F8Q0 (a putative transmembrane outer membrane protein) and two proteins of 20 kDa and 55 kDa that could not be identified by MS, one of which demonstrated a high degree of labeling potentially representing an additional, as-yet-uncharacterized, surface-exposed protein. Minor labeling of p31
LipL45
, GroEL, and FlaB1 was also observed. Expression of the surfaceome constituents remained unchanged under a range of conditions investigated, including temperature and the presence of serum or urine. Immunization of mice with affinity-captured surface components stimulated the production of antibodies that bound surface proteins from heterologous leptospiral strains. The surfaceomics approach is particularly amenable to protein expression profiling using small amounts of sample (<10
7
cells) offering the potential to analyze bacterial surface expression during infection.
Expression of Arg-Gingipain RgpB Is Required for Correct Glycosylation and Stability of Monomeric Arg-Gingipain RgpA fromPorphyromonas gingivalisW50
Authors: Rangarajan Minnie , Hashim Ahmed , Aduse-Opoku Joseph , Paramonov Nikolay , Hounsell Elizabeth F. , Curtis Michael A.
Published: July 2005

Arg-gingipains are extracellular cysteine proteases produced by the gram-negative periodontal pathogenPorphyromonas gingivalisand are encoded byrgpAandrgpB. Three Arg-gingipains, heterodimeric high-molecular-mass Arg-gingipain HRgpA comprising the α-catalytic chain and the β-adhesin chain, the monomeric soluble Arg-gingipain comprising only the α-catalytic chain (RgpAcat), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the α-catalytic chain (mt-RgPAcat), are derived fromrgpA. The monomeric enzymes contain between 14 and 30% carbohydrate by weight.rgpBencodes two monomeric enzymes, RgpB and mt-RgpB. Earlier work indicated thatrgpBis involved in the glycosylation process, since inactivation ofrgpBresults in the loss of not only RgpB and mt-RgpB but also mt-RgpAcat. This work aims to confirm the role of RgpB in the posttranslational modification of RgpAcatand the effect of aberrant glycosylation on the properties of this enzyme. Two-dimensional gel electrophoresis of cellular proteins from W50 and an inactivatedrgpBstrain (D7) showed few differences, suggesting that loss of RgpB has a specific effect on RgpA maturation. Inactivation of genes immediately upstream and downstream ofrgpBhad no effect onrgpA-derived enzymes, suggesting that the phenotype of thergpBmutant is not due to a polar effect on transcription at this locus. Matrix-assisted laser desorption ionization-time of flight analysis of purified RgpAcatfrom W50 and D7 strains gave identical peptide mass fingerprints, suggesting that they have identical polypeptide chains. However, RgpAcatfrom D7 strain had a higher isoelectric point and a dramatic decrease in thermostability and did not cross-react with a monoclonal antibody which recognizes a glycan epitope on the parent strain enzyme. Although it had the same total sugar content as the parent strain enzyme, there were significant differences in the monosaccharide composition and linking sugars. These data suggest that RgpB is required for the normal posttranslational glycosylation of Arg-gingipains derived fromrgpAand that this process is required for enzyme stabilization.
Clostridium perfringens
Epsilon-Toxin Increases Permeability of Single Perfused Microvessels of Rat Mesentery
Authors: Adamson R. H. , Ly J. C. , Fernandez-Miyakawa M. , Ochi S. , Sakurai J. , Uzal F. , Curry F. E.
Published: July 2005

Epsilon-toxin, the primary virulence factor of
Clostridium perfringens
type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (

L
p

) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 μg ml
−1
the

L
p

increased irreversibly to more than 15 times the control value by 10 min. At 0.3 μg ml
−1
no increase in

L
p

was observed for up to 90 min. The toxin-induced increase in

L
p

was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium.
Comparison ofHelicobacter pyloriVirulence Gene Expression In Vitro and in the Rhesus Macaque
Authors: Boonjakuakul Jenni K. , Canfield Don R. , Solnick Jay V.
Published: July 2005

We used a quantitative real-time reverse transcriptase PCR assay to measure the transcript abundance of 46 known and putativeHelicobacter pylorivirulence genes, including 24 genes on the Cag pathogenicity island. The expression profile ofH. pyloricells grown in vitro was also compared to expression in vivo after experimental infection of rhesus macaques. Transcript abundance in vitro (mid-log phase) ranged from about 0.004 (feoBandhpaA) to 20 (ureAB,napA, andcag25) copies/cell. Expression of most genes was repressed during the transition from logarithmic- to stationary-phase growth, but several well-characterizedH. pylorivirulence genes (katA,napA,vacA, andcagA) were induced. Comparison of results in the rhesus macaque with similar data from humans showed a strong correlation (r= 0.89). The relative in vivo expression in the rhesus monkey was highly correlated with in vitro expression during mid-log (r= 0.89)- and stationary (r= 0.88)-phase growth. Transcript abundance was on average three- to fourfold reduced in vivo compared to in vitro during mid-log phase. However, when compared to stationary phase, increased expression in vivo was observed for 6 of 7 genes on a contiguous portion of the pathogenicity island, several of which are thought to encode theH. pyloritype IV structural pilus and its accessory proteins. These results suggest the possibility that some genes encoding theH. pyloritype IV structural pilus and accessory proteins may form an operon that is induced during growth in vivo.
Divergence of Protection Induced by Bacterial Products and Sepsis-Induced Immune Suppression
Authors: Sterns Theo , Pollak Nils , Echtenacher Bernd , Männel Daniela N.
Published: July 2005

Susceptibility to bacterial infections after a primary immune stimulation differs drastically depending on the presensitization of the innate immune system. To determine the conditions that either induce protection or enhanced susceptibility to infection withSalmonella entericaserovar Typhimurium, we pretreated mice either with tumor necrosis factor (TNF), whole killed bacteria, or sublethal cecal ligation and puncture (CLP) as a mouse model for septic peritonitis. Impaired production of the cytokines TNF, interleukin-6 (IL-6), and IL-10 was induced by these pretreatment schedules, with TNF-signaling not being essential for this effect. Injection of TNF or killed bacteria enhanced survival of mice infected subsequently with serovar Typhimurium. In contrast, sepsis such as that induced by CLP only protected from shock induced byd-galactosamine and lipopolysaccharide or by a high dose of bacteria but sensitized to a secondary bacterial infection. Such sepsis-induced enhanced susceptibility to infection was critically dependent on TNF function.
NK Cells Help To Induce CD8+-T-Cell Immunity againstToxoplasma gondiiin the Absence of CD4+T Cells
Authors: Combe Crescent L. , Curiel Tyler J. , Moretto Magali M. , Khan Imtiaz A.
Published: July 2005

CD8+T-cell immunity plays an important role in protection against intracellular infections. Earlier studies have shown that CD4+T-cell help was needed for launching in vivo CD8+T-cell activity against these pathogens and tumors. However, recently CD4+T-cell-independent CD8 responses during several microbial infections including those withToxoplasma gondiihave been described, although the mechanism is not understood. We now demonstrate that, in the absence of CD4+T cells,T. gondii-infected mice exhibit an extended NK cell response, which is mediated by continued interleukin-12 (IL-12) secretion. This prolonged NK cell response is critical for priming parasite-specific CD8+T-cell immunity. Depletion of NK cells inhibited the generation of CD8+T-cell immunity in CD4−/−mice. Similarly neutralization of IL-12 reduces NK cell numbers in infected animals and leads to the down-regulation of CD8+T-cell immunity againstT. gondii. Adoptive transfer of NK cells into the IL-12-depleted animals restored their CD8+T-cell immune response, and animals exhibited reduced mortality. NK cell gamma interferon was essential for cytotoxic T-lymphocyte priming. Our studies for the first time demonstrate that, in the absence of CD4+T cells, NK cells can play an important role in induction of primary CD8+T-cell immunity against an intracellular infection. These observations have therapeutic implications for immunocompromised individuals, including those with human immunodeficiency virus infection.
Cryptococcus neoformans
α Strains Preferentially Disseminate to the Central Nervous System during Coinfection
Authors: Nielsen Kirsten , Cox Gary M. , Litvintseva Anastasia P. , Mylonakis Eleftherios , Malliaris Stephanie D. , Benjamin Daniel K. , Giles Steven S. , Mitchell Thomas G. , Casadevall Arturo , Perfect John R. , Heitman Joseph
Published: July 2005

Cryptococcus neoformans
is a fungal pathogen that has evolved over the past 40 million years into three distinct varieties or sibling species (
gattii
,
grubii
, and
neoformans
). Each variety manifests differences in epidemiology and disease, and var.
grubii
strains are responsible for the vast majority of human disease. In previous studies, α strains were more virulent than congenic
a
strains in var.
neoformans
, whereas var.
grubii
congenic
a
and α strains exhibited equivalent levels of virulence. Here the role of mating type in the virulence of var.
grubii
was further characterized in a panel of model systems. Congenic var.
grubii
a
and α strains had equivalent survival rates when cultured with amoebae, nematodes, and macrophages. No difference in virulence was observed between
a
and α congenic strains in multiple inbred-mouse genetic backgrounds, and there was no difference in accumulations in the central nervous system (CNS) late in infection. In contrast, during coinfections,
a
and α strains are equivalent in peripheral tissues but α cells have an enhanced predilection to penetrate the CNS. These studies reveal the first virulence difference between congenic
a
and α strains in the most common pathogenic variety and suggest an explanation for the prevalence of α strains in clinical isolates.
Type 1 Immunity Provides Both Optimal Mucosal and Systemic Protection against a Mucosally Invasive, Intracellular Pathogen
Authors: Hoft Daniel F. , Eickhoff Chris S.
Published: July 2005

It has been hypothesized that optimal vaccine immunity against mucosally invasive, intracellular pathogens may require the induction of different types of immune responses in mucosal and systemic lymphoid tissues. Mucosal type 2/3 responses (producing interleukin-4 [IL-4], IL-6 and/or transforming growth factor β) could be necessary for optimal induction of protective secretory immunoglobulin A responses. On the other hand, systemic type 1 responses (including gamma interferon [IFN-γ], tumor necrosis factor alpha, and optimal cytotoxic T-cell responses) are likely to be critical for protection against the disseminated intracellular replication that occurs after mucosal invasion. Despite these predictions, we recently found that vaccines inducing highly polarized type 1 immunity in both mucosal and systemic tissues provided optimal mucosal and systemic protection against the protozoan pathogenTrypanosoma cruzi. To further address this important question in a second model system, we now have studied the capacity of knockout mice to develop protective immune memory.T. cruziinfection followed by nifurtimox treatment rescue was used to immunize CD4, CD8, β2-microglobulin, inducible nitric oxide synthase (iNOS), IL-12, IFN-γ, and IL-4 knockout mice. Despite the previously demonstrated importance of CD4+T cells, CD8+T cells, and nitric oxide forT. cruziimmunity, CD4, CD8, and iNOS knockout mice developed mucosal and systemic protective immunity. However, IL-12, IFN-γ, and β2-microglobulin-deficient mice failed to develop mucosal or systemic protection. In contrast, IL-4 knockout mice developed maximal levels of both mucosal and systemic immune protection. These results strongly confirm our earlier conclusion from studies with polarizing vaccination protocols that type 1 immunity provides optimal mucosal and systemic protection against a mucosally invasive, intracellular pathogen.
Phagocyte-Derived Reactive Oxygen Species Do Not Influence the Progression of Murine Blood-Stage Malaria Infections
Authors: Potter S. M. , Mitchell A. J. , Cowden W. B. , Sanni L. A. , Dinauer M. , de Haan J. B. , Hunt N. H.
Published: July 2005

Phagocyte-derived reactive oxygen species have been implicated in the clearance of malaria infections. We investigated the progression of five different strains of murine malaria in gp91phox−/−mice, which lack a functional NADPH oxidase and thus the ability to produce phagocyte-derived reactive oxygen species. We found that the absence of functional NADPH oxidase in the gene knockout mice had no effect on the parasitemia or total parasite burden in mice infected with either resolving (Plasmodium yoeliiandPlasmodium chabaudiK562) or fatal (Plasmodium bergheiANKA,Plasmodium bergheiK173 andPlasmodium vinckei vinckei) strains of malaria. This lack of effect was apparent in both primary and secondary infections withP. yoeliiandP. chabaudi. There was also no difference in the presentation of clinical or pathological signs between the gp91phox−/−or wild-type strains of mice infected with malaria. Progression ofP. bergheiANKA andP. bergheiK173 infections was unchanged in glutathione peroxidase-1 gene knockout mice compared to their wild-type counterparts. The rates of parasitemia progression in gp91phox−/−mice and wild-type mice were not significantly different when they were treated withl-NG-methylarginine, an inhibitor of nitric oxide synthase. These results suggest that phagocyte-derived reactive oxygen species are not crucial for the clearance of malaria parasites, at least in murine models.
Prevention and Treatment of Cutaneous Leishmaniasis in Primates by Using Synthetic Type D/A Oligodeoxynucleotides Expressing CpG Motifs
Authors: Flynn Barbara , Wang Vivian , Sacks David L. , Seder Robert A , Verthelyi Daniela
Published: July 2005

Oligodeoxynucleotides (ODN) containing CpG motifs mimic microbial DNA and are recognized by toll-like receptor 9 on immune cells. The resulting response limits the early spread of infectious organisms and promotes the development of adaptive immunity. In this regard, CpG ODN show promise as immunoprotective agents and as vaccine adjuvants. Previous studies of nonhuman primates showed that administration of CpG ODN type D (also known as type A) at the site of infection 3 days before and after a challenge withLeishmania majorenhanced host resistance and reduced the lesion's severity. In this study, we show that systemic administration of D/A ODN limits the size of lesions following an intradermal infection withL. major. Importantly, the reduced morbidity was not associated with a reduction in long-term immunity, as such treated macaques were still protected following a secondary challenge. Finally, administration of D/A ODN to macaques that had established cutaneous lesions reduced the severity of the lesions, suggesting a potential role for CpG ODN inL. majortreatment. Together, these findings support the development of clinical studies to assess the use of CpG ODN types D/A as immunoprotective and therapeutic agents.
Impaired Neutrophil Migration Associated with Specific Bovine CXCR2 Genotypes
Authors: Rambeaud M. , Pighetti G. M.
Published: July 2005

Bovine mastitis continues to be the most detrimental factor for profitable dairying. Recent research conducted within our laboratory has identified a genetic marker in the CXCR2 gene associated with mastitis susceptibility. The objective of the present study was to evaluate the migratory ability of neutrophils from cows with different CXCR2 +777 genotypes. Neutrophils isolated from peripheral blood of 30 Holstein cows were tested for in vitro migration and adhesion molecule expression. Cows with the CC or GC genotype at CXCR2 +777 showed significantly lower neutrophil migration to recombinant human interleukin-8 (rhIL-8) than cows with the GG genotype (P< 0.05). Cows with the CC genotype at CXCR2 +777 also showed decreased neutrophil migration to zymosan-activated serum compared to these same cows (P< 0.05). Decreased upregulation of CD18 expression was observed after stimulation with rhIL-8 in cows expressing the CXCR2 +777 CC genotype compared to cows expressing the GG genotype (P< 0.05). A similar trend was observed for CD11b (P< 0.10). However, no difference in CD62 downregulation was observed with respect to genotype. These results provide initial evidence for a phenotypic association between a single nucleotide polymorphism and neutrophil function in dairy cows, as well as potential insight into specific mechanisms affected in cows more susceptible to mastitis.
Characterization of the Filamentous Hemagglutinin-Like Protein FhaS in
Bordetella bronchiseptica
Authors: Julio Steven M. , Cotter Peggy A.
Published: July 2005

Filamentous hemagglutinin (FHA) is a large (>200 kDa), rod-shaped protein expressed by bordetellae that is both surface-associated and secreted. FHA mediates bacterial adherence to epithelial cells and macrophages in vitro and is absolutely required for tracheal colonization in vivo. The recently sequenced
Bordetella bronchiseptica
genome revealed the presence of a gene,
fhaS
, that is nearly identical to
fhaB
, the FHA structural gene. We show that although
fhaS
expression requires the BvgAS virulence control system, it is maximal only under a subset of conditions in which BvgAS is active, suggesting an additional level of regulation. We also show that, like FHA, FhaS undergoes a C-terminal proteolytic processing event and is both surface-associated and secreted and that export across the outer membrane requires the channel-forming protein FhaC. Unlike FHA, however, FhaS was unable to mediate adherence of
B. bronchiseptica
to epithelial cell lines in vitro and was not required for respiratory tract colonization in vivo. In a coinfection experiment, a Δ
fhaS
strain was out-competed by wild-type
B. bronchiseptica
, indicating that
fhaS
is expressed in vivo and that FhaS contributes to bacterial fitness in a manner revealed when the mutant must compete with wild-type bacteria. These data suggest that FHA and FhaS perform distinct functions during the
Bordetella
infectious cycle. A survey of various
Bordetella
strains revealed two distinct
fhaS
alleles that segregate according to pathogen host range and that
B. parapertussis
hu
most likely acquired its
fhaS
allele from
B. pertussis
horizontally, suggesting
fhaS
may contribute to host-species specificity.
Borrelia burgdorferi rel
Is Responsible for Generation of Guanosine-3′-Diphosphate-5′-Triphosphate and Growth Control
Authors: Bugrysheva Julia V. , Bryksin Anton V. , Godfrey Henry P. , Cabello Felipe C.
Published: July 2005

The global transcriptional regulator (p)ppGpp (guanosine-3′-diphosphate-5′-triphosphate and guanosine-3′,5′-bisphosphate, collectively) produced by the
relA
and
spoT
genes in
Escherichia coli
allows bacteria to adapt to different environmental stresses. The genome of
Borrelia burgdorferi
encodes a single chromosomal
rel
gene (BB0198) (
B. burgdorferi rel
[

rel
Bbu

]) homologous to
relA
and
spoT
of
E. coli
. Its role in (p)ppGpp synthesis, bacterial growth, and modulation of gene expression has not been studied in detail. We constructed a

rel
Bbu

deletion mutant in an infectious
B. burgdorferi
297 strain and isolated an extrachromosomally complemented derivative of this mutant. The mutant did not synthesize

rel
Bbu

mRNA, Rel
Bbu
protein, or (p)ppGpp. This synthesis was restored in the complemented derivative, confirming that

rel
Bbu

is necessary and sufficient for (p)ppGpp synthesis and degradation in
B. burgdorferi
. The

rel
Bbu

mutant grew well during log phase in complete BSK-H but reached lower cell concentrations in the stationary phase than the wild-type parent, suggesting that (p)ppGpp may be an important factor in the ability of
B. burgdorferi
to adapt to stationary phase. Deletion of

rel
Bbu

did not eliminate the temperature-elicited OspC shift, nor did it alter
bmp
gene expression or
B. burgdorferi
antibiotic susceptibility. Although deletion of

rel
Bbu

eliminated
B. burgdorferi
virulence for mice, which was not restored by complementation, we suggest that

rel
Bbu

-dependent accumulation of (p)ppGpp may be important for in vivo survival of this pathogen.
Characterization of the
cciIR
Quorum-Sensing System in
Burkholderia cenocepacia
Authors: Malott Rebecca J. , Baldwin Adam , Mahenthiralingam Eshwar , Sokol Pamela A.
Published: July 2005

Several transmissible
Burkholderia cenocepacia
strains that infect multiple cystic fibrosis patients contain a genomic island designated as the
c
eno
c
epacia
i
sland (cci). The cci contains a predicted
N-
acylhomoserine lactone (AHL) synthase gene,
cciI
, and a predicted response regulator gene,
cciR.
AHL production profiles indicated that CciI catalyzes the synthesis of
N
-hexanoyl-
l
-homoserine lactone and minor amounts of
N-
octanoyl-
l
-homoserine lactone. The
cciI
and
cciR
genes were found to be cotranscribed by reverse transcription-PCR analysis, and the expression of a
cciIR
::
luxCDABE
fusion in a
cciR
mutant suggested that the
cciIR
system negatively regulates its own expression.
B. cenocepacia
strains also have a
cepIR
quorum-sensing system. Expression of
cepI
::
luxCDABE
or
cepR
::
luxCDABE
fusions in a
cciR
mutant showed that CciR negatively regulates
cepI
but does not regulate
cepR.
Expression of the
cciIR
::
luxCDABE
fusion in a
cepR
mutant indicated that functional CepR is required for
cciIR
expression. Phylogenetic analysis suggested that the
cciIR
system was acquired by horizontal gene transfer from a distantly related organism and subsequently incorporated into the ancestral
cepIR
regulatory network. Mutations in
cciI
,
cciR
,
cepI cciI
, and
cepR cciR
were constructed in
B. cenocepacia
K56-2. The
cciI
mutant had greater protease activity and less swarming motility than the parent strain. The
cciR
mutant had less protease activity than the parent strain. The phenotypes of the
cepI cciI
and
cepR cciR
mutants were similar to
cepI
or
cepR
mutants, with less protease activity and swarming motility than the parent strain.
Characterization of Enterohemorrhagic
Escherichia coli
Strains Based on Acid Resistance Phenotypes
Authors: Bhagwat Arvind A. , Chan Lynn , Han Rachel , Tan Jasmine , Kothary Mahendra , Jean-Gilles Junia , Tall Ben D.
Published: July 2005

Acid resistance is perceived to be an important property of enterohemorrhagic
Escherichia coli
strains, enabling the organisms to survive passage through the acidic environment of the stomach so that they may colonize the mammalian gastrointestinal tract and cause disease. Accordingly, the organism has developed at least three genetically and physiologically distinct acid resistance systems which provide different levels of protection. The glutamate-dependent acid resistance (GDAR) system utilizes extracellular glutamate to protect cells during extreme acid challenges and is believed to provide the highest protection from stomach acidity. In this study, the GDAR system of 82 pathogenic
E. coli
isolates from 34 countries and 23 states within the United States was examined. Twenty-nine isolates were found to be defective in inducing GDAR under aerobic growth conditions, while five other isolates were defective in GDAR under aerobic, as well as fermentative, growth conditions. We introduced
rpoS
on a low-copy-number plasmid into 26 isolates and were able to restore GDAR in 20 acid-sensitive isolates under aerobic growth conditions. Four isolates were found to be defective in the newly discovered LuxR-like regulator GadE (formerly YhiE). Defects in other isolates could be due to a mutation(s) in a gene(s) with an as yet undefined role in acid resistance since GadE and/or RpoS could not restore acid resistance. These results show that in addition to mutant alleles of
rpoS
, mutations in
gadE
exist in natural populations of pathogenic
E. coli
. Such mutations most likely alter the infectivity of individual isolates and may play a significant role in determining the infective dose of enterohemorrhagic
E. coli
.
Immunogenicity of the
Mycobacterium tuberculosis
PPE55 (Rv3347c) Protein during Incipient and Clinical Tuberculosis
Authors: Singh Krishna K. , Dong Yuxin , Patibandla Sai A. , McMurray David N. , Arora Vijay K. , Laal Suman
Published: July 2005

Clinical tuberculosis (TB), whether noncavitary or cavitary, is the late stage of a chronic disease process, since
Mycobacterium tuberculosis
is a slowly growing organism. Our studies have shown that the profiles of antigenic proteins expressed by the in vivo bacteria that elicit antibodies differ in cavitary and noncavitary TB (K. Samanich, J. T. Belisle, and S. Laal, Infect. Immun.
69:
4600-4609, 2001). To gain insight into antigenic proteins expressed during incipient, subclinical TB, an expression library of
M. tuberculosis
genomic DNA was screened with sera obtained during subclinical TB from guinea pigs infected with aerosols of
M. tuberculosis
H37Rv. One of the proteins recognized by antibodies elicited during subclinical TB infection of guinea pigs is the 309-kDa PPE55 (Rv3347c) protein. Genomic hybridization studies suggest that the PPE55 gene is specific to the
M. tuberculosis
complex and is present in a majority of clinical isolates tested. Antibodies to the C-terminal, ∼100-kDa fragment of PPE55 (PPE-C) were detectable in sera from 29/30 (97%) human immunodeficiency virus-negative/TB-positive (HIV
−
TB
+
) patients and 17/24 (71%) HIV
+
TB
+
patients tested but not in sera from purified-protein derivative-positive healthy controls, suggesting that the in vivo expression of PPE55 protein correlates with active
M. tuberculosis
infection. Anti-PPE-C antibodies were also detected in retrospective sera obtained months prior to manifestation of clinical TB from 17/21 (81%) HIV
+
TB
+
individuals tested, providing evidence that the protein is expressed during incipient, subclinical TB in HIV-infected humans. Thus, PPE55 is a highly immunogenic protein that may be useful for differentiating between latent TB and incipient, subclinical TB.
Endocarditis-Associated Oral Streptococci Promote Rapid Differentiation of Monocytes into Mature Dendritic Cells
Authors: Hahn Chin-Lo , Schenkein Harvey A. , Tew John G.
Published: July 2005

Endocarditis is frequently attributable to oral streptococci, but mechanisms of pathogenesis are not well understood, although monocytes appear to be important. High titers of interleukin-12 (IL-12) are produced by peripheral blood mononuclear cells (PBMC) after engagingStreptococcus mutans, but monocytes in developing endocardial vegetations tend to disappear rather than become macrophages. These data prompted the hypothesis that streptococcus-infected monocytes differentiate into short-lived IL-12-producing dendritic cells (DCs) rather than macrophages. PBMC from healthy subjects were stimulated with six isolates of oral streptococci, three nonstreptococcal oral bacteria, or IL-4 plus granulocyte-macrophage colony-stimulating factor, and the appearance of cells with markers typical of mature DCs (CD83+, CD86+, CD11c+, and CD14−) was monitored. Supernatant fluids from the PBMC cultures were harvested and IL-12 p70 levels were determined.S. mutans-stimulated monocytes were analyzed for their ability to elicit allogeneic mixed-lymphocyte reactions. All streptococci examined, except one strain ofStreptococcus oralis(35037), rapidly induced up-regulation of CD83 and CD86 and a loss of CD14 in the CD11c+monocyte population within 20 h. Induction of IL-12 was CD14 dependent and correlated with streptococcal isolates that promoted the DC phenotype. Major histocompatibility complex (MHC) class II expression was up-regulated byS. mutans, and these cells were short-lived and elicited potent allogeneic mixed-lymphocyte reactions typical of DCs. In summary, monocytes stimulated with endocarditis-associated oral streptococci rapidly exhibited the DC phenotype and functions. These data suggest that the initiation of bacterial endocarditis by oral streptococci may involve monocyte-to-DC differentiation, and this may help explain the low levels of macrophages in the site.
Identification and Characterization of anSKN7Homologue inCryptococcus neoformans
Authors: Wormley F. L. , Heinrich G. , Miller J. L. , Perfect J. R. , Cox G. M.
Published: July 2005

Cryptococcus neoformansis an encapsulated fungal pathogen that primarily infects the central nervous system of immunocompromised individuals, causing life-threatening meningoencephalitis. The capacity ofC. neoformansto subvert host defenses and disseminate by intracellular parasitism of alveolar macrophages in the immune-compromised host has led to studies to evaluate genes associated withC. neoformansresistance to oxidative stress. In the present study, we identify and characterize aC. neoformanshomologue toSKN7, a transcription factor inSaccharomyces cerevisiaethat regulates the oxidative stress response, cell cycle, and cell wall biosynthesis. To examine the contribution ofSKN7in the pathogenesis of fungal infections, we createdskn7mutants via targeted disruption. Theskn7mutants were observed to be more susceptible to reactive oxygen species in vitro and were significantly less virulent than the wild-type strain and a reconstituted strain as measured by cumulative survival in the mouse inhalational model. The Skn7 protein was observed to be important for expression of thioredoxin reductase in response to oxidative challenge. Interestingly,skn7mutants were also observed to flocculate following in vitro culture, a novel phenotype not observed inskn7mutants derived from other fungi. These findings demonstrate thatSKN7contributes to the virulence composite but is not required for pathogenicity inC. neoformans. In addition, flocculation ofC. neoformans skn7mutants suggests a potentially unique function ofSKN7not previously observed in other cryptococcal strains orskn7mutants.
Intranasal Delivery of Group B Meningococcal Native Outer Membrane Vesicle Vaccine Induces Local Mucosal and Serum Bactericidal Antibody Responses in Rabbits
Authors: Shoemaker David R. , Saunders Nancy B. , Brandt Brenda L. , Moran E. Ellen , LaClair Andrew D. , Zollinger Wendell D.
Published: July 2005

We have previously shown that intranasal immunization of mice with meningococcal native outer membrane vesicles (NOMV) induces both a good local mucosal antibody response and a good systemic bactericidal antibody response. However, in the intranasal mouse model, some of the NOMV entered the lung and caused an acute granulocytic response. We therefore developed an alternate animal model using the rabbit. This model reduces the probability of lung involvement and more closely mimics intranasal immunization of humans. Rabbits immunized intranasally with doses of 100 μg of NOMV in 0.5 ml of saline developed serum bactericidal antibody levels comparable to those of rabbits immunized intramuscularly with 25-μg doses, particularly when the primary intranasal immunization was given daily for 3 days. Intranasal immunization also induced a local mucosal response as evidenced by immunoglobulin A antibody in saliva, nasal washes, and lung lavage fluids. NOMV from a capsule-deficient mutant induced higher serum bactericidal antibody responses than NOMV from the encapsulated parent. Meningococcal NOMV could be administered intranasally at 400 μg with no pyrogenic activity, but as little as 0.03 μg/kg of body weight administered intravenously or 25 μg administered intramuscularly induced a pyrogenic response. These data indicate that the rabbit is a useful model for preclinical testing of intranasal meningococcal NOMV vaccines, and this immunization regimen produces a safe and substantial systemic and local mucosal antibody response.
Scrub Typhus Vaccine Candidate Kp r56 Induces Humoral and Cellular Immune Responses in Cynomolgus Monkeys
Authors: Chattopadhyay Suchismita , Jiang Ju , Chan Teik-Chye , Manetz T. Scott , Chao Chien-Chung , Ching Wei-Mei , Richards Allen L.
Published: July 2005

A truncated recombinant 56-kDa outer membrane protein of the Karp strain ofOrientia tsutsugamushi(Kp r56) was evaluated in cynomolgus monkeys (Macaca fascicularis) for immunogenicity and safety as a vaccine candidate for the prevention of scrub typhus. This recombinant antigen induced strong humoral and cellular immune responses in two monkeys and was found to be well tolerated. Antigen-specific immunoglobulin M (IgM) and IgG were produced to almost maximal levels within 1 week of a single immunization. Peripheral blood mononuclear cells from vaccinated animals showed an induction of antigen-specific proliferation and gamma interferon production. The Kp r56 was not as efficient as infection with live organisms in preventing reinfection but was able to reduce the inflammation produced at the site of challenge. This report describes the results of the first systematic study of the immunogenicity of a recombinant scrub typhus vaccine candidate in a nonhuman primate model.
Experimental Infection withTrypanosoma cruziIncreases the Population of CD8+, but not CD4+, Immunoglobulin G Fc Receptor-Positive T Lymphocytes
Authors: Henriques-Pons Andrea , Olivieri Bianca P. , Oliveira Gabriel M. , Daëron Marc , de Araújo-Jorge Tania C.
Published: July 2005

It is well established that activating-type Fc receptors for immunoglobulin G (FcγR), such as FcγRI and FcγRIII, are essential for inducing inflammatory responses. On the other hand, a unique inhibitory FcγR, FcγRIIB, inhibits intracellular signaling upon engagement of immunoglobulin G-immune complexes, suppressing inflammation and autoimmunity. The expression of FcγRIIB on B lymphocytes, natural killer cells, macrophages, mast cells, and a number of other cell types has been demonstrated for many years. However, the expression on T lymphocytes is probably restricted to activated cells in a narrow window of time. The controversy regarding the FcγR expression on T lymphocytes is attributable to considerable heterogeneity of cellular subpopulations and activation stages during immune responses in vivo. We addressed here this question by using mice experimentally infected withTrypanosoma cruzi, and we found an increase in the CD8+FcγR+population but not in the CD4+FcγR+population. Moreover, CD8+FcγR+T cells predominantly composed the cardiac inflammatory infiltration induced by the infection. These results indicate a novel pattern of FcγR expression on T cells in a pathological situation, and possible functional roles of this phenomenon are discussed.
Identification of a Novel Cathelicidin Gene in the Rainbow Trout,Oncorhynchus mykiss
Authors: Chang Chin-I , Pleguezuelos Olga , Zhang Yong-An , Zou Jun , Secombes Christopher J.
Published: July 2005

We report the cloning of a novel antimicrobial peptide gene, termedrtCATH_1, found in the rainbow trout,Oncorhynchus mykiss. The predicted 216-residuertCATH_1 prepropeptide consists of three domains: a 22-residue signal peptide, a 128-residue cathelin-like region containing two identifiable cathelicidin family signatures, and a predicted 66-residue C-terminal cationic antimicrobial peptide. This predicted mature peptide was unique in possessing features of different known (mammalian) cathelicidin subgroups, such as the cysteine-bridged family and the specific amino-acid-rich family. ThertCATH_1 gene comprises four exons, as seen in all known mammalian cathelicidin genes, and several transcription factor binding sites known to be of relevance to host defenses were identified in the 5′ flanking region. By Northern blot analysis, the expression ofrtCATH_1 was detected in gill, head kidney, and spleen of bacterially challenged fish. Primary cultures of head kidney leukocytes from rainbow trout stimulated with lipopolysaccharide or poly(I · C) also expressedrtCATH_1. A 36-residue peptide corresponding to the core part of the fish cathelicidin was chemically synthesized and shown to exhibit potent antimicrobial activity and a low hemolytic effect. Thus,rtCATH_1 represents a novel antimicrobial peptide gene belonging to the cathelicidin family and may play an important role in the innate immunity of rainbow trout.
Conditional Lethality Yields a New Vaccine Strain ofListeria monocytogenesfor the Induction of Cell-Mediated Immunity
Authors: Li Zhongxia , Zhao Xinyan , Higgins Darren E. , Frankel Fred R.
Published: July 2005

Listeria monocytogenesis a gram-positive intracellular pathogen that can enter phagocytic and nonphagocytic cells and colonize their cytosols. Taking advantage of this property to generate an intracellular vaccine delivery vector, we previously described a mutant strain ofL. monocytogenes, ΔdalΔdat, which is unable to synthesize cell wall by virtue of deletions in two genes (dalanddat) required ford-alanine synthesis. This highly attenuated strain induced long-lived protective systemic and mucosal immune responses in mice when administered in the transient presence ofd-alanine. We have now increased the usefulness of this organism as a vaccine vector by use of an inducible complementation system that obviates the need for exogenousd-alanine administration. The strain expresses a copy of theBacillus subtilisracemase gene under the control of a tightly regulated isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter present on a multicopy plasmid. This bacterium demonstrates strict dose-dependent growth in the presence of IPTG. After removal of inducer, bacterial growth ceased within two replication cycles. Following infection of mice in the absence of IPTG ord-alanine, the bacterium survived in vivo for less than 3 days. Nevertheless, a single immunization elicited a state of long-lasting protective immunity against wild-typeL. monocytogenesand induced a subset of effector listeriolysin O-specific CD11a+CD8+T cells in spleen and other tissues that was strongly enhanced after secondary immunization. This improvedL. monocytogenesvector system may have potential use as a live vaccine against human immunodeficiency virus, other infectious diseases, and cancer.
Heme Transfer from Streptococcal Cell Surface Protein Shp to HtsA of Transporter HtsABC
Authors: Liu Mengyao , Lei Benfang
Published: July 2005

Human pathogen group A streptococcus (GAS) can take up heme from host heme-containing proteins as a source of iron. Little is known about the heme acquisition mechanism in GAS. We recently identified a streptococcal cell surface protein (designated Shp) and the lipoprotein component (designated HtsA) of an ATP-binding cassette (ABC) transporter made by GAS as heme-binding proteins. In an effort to delineate the molecular mechanism involved in heme acquisition by GAS, heme-free Shp (apo-Shp) and HtsA (apo-HtsA) were used to investigate heme transfer from heme-containing proteins (holo proteins) to the apo proteins. In addition, the interaction between holo-Shp and holo-HtsA was examined using native polyacrylamide gel electrophoresis. Heme was efficiently transferred from holo-Shp to apo-HtsA but not from holo-HtsA to apo-Shp. Apo-Shp acquired heme from human hemoglobin, and holo-Shp and holo-HtsA were able to form a complex, suggesting that Shp actively relays heme from hemoglobin to apo-HtsA. These findings demonstrate for the first time complex formation and directional heme transfer between a cell surface heme-binding protein and the lipoprotein of a heme-specific ABC transporter in gram-positive bacteria.