bluuebunny/crossref_metadata_2025 huggingface dataset:
BrucellaLumazine Synthase Elicits a Mixed Th1-Th2 Immune Response and Reduces Infection in Mice Challenged withBrucella abortus544 Independently of the Adjuvant Formulation Used
Authors: Velikovsky Carlos A. , Goldbaum Fernando A. , Cassataro Juliana , Estein Silvia , Bowden Raúl A. , Bruno Laura , Fossati Carlos A. , Giambartolomei Guillermo H.
Published: September 2003

The immunogenicity and protective efficacy of recombinant lumazine synthase fromBrucellaspp. (rBLS) administered with different adjuvants was evaluated in mice. Mice were immunized with rBLS in the absence or the presence of aluminum hydroxide gel (BLS-Al), monophosphoryl lipid A (BLS-MPA), or incomplete Freund's adjuvant (BLS-IFA). rBLS per se induced a vigorous immunoglobulin G (IgG) response, with high titers of IgG1 as well as IgG2. All the adjuvants increased this response; the BLS-IFA formulation was the most effective at inducing BLS-specific IgG antibodies. In addition, after in vitro stimulation with rBLS, spleen cells from BLS-IFA-, BLS-Al-, or BLS-MPA-immunized mice proliferated and produced interleukin-2 (IL-2), gamma interferon (IFN-γ), IL-10, and IL-4, suggesting the induction of a mixed Th1-Th2 response. Immunization with rBLS protected mice against challenge withB. abortus544. The levels of protection in the spleen were similar for all adjuvants, but only BLS-Al and BLS-IFA were effective in the liver. Our results indicate that BLS might be a useful candidate for the development of subunit vaccines against brucellosis, since it elicits antigen-specific cellular responses, with production of IFN-γ and protection, independently of the adjuvant formulation used.
Parenchymal Organ, and Not Splenic, Immunity Correlates with Host Survival during Disseminated Candidiasis
Authors: Spellberg Brad , Johnston Douglas , Phan Quynh Trang , Edwards John E. , French Samuel W. , Ibrahim Ashraf S. , Filler Scott G.
Published: September 2003

We examined the relationship between host survival and renal and splenic immune responses in a murine model of hematogenously disseminated candidiasis. Male BALB/c mice were infected via tail vein injection with wild-typeC. albicansor with an isogenic, Δefg1/Δefg1hypha-deficient mutant. Host survival, organ fungal burden, intracellular cytokine content of splenic and kidney lymphocytes, and whole-organ cytokine profiles were determined. Wild-typeC. albicansinduced type 2 splenocyte responses with both nonfatal and fatal inocula. In the kidney, conversely, wild-type inocula causing no or low mortality induced type 1 responses and 100% fatal inocula induced type 2 or interleukin-10 (IL-10)-dominant responses. Hypha-deficient mutantC. albicanscaused no or low mortality while inducing type 1 responses in both the spleen and kidney. To our knowledge, this is the first demonstration that host survival during systemic infection correlates with the type of immune response engendered in a nonlymphoid, parenchymal organ and not with the response in the spleen. Furthermore, the results provide in vivo confirmation that hyphal formation byC. albicansinduces type 2 or IL-10-dominant host responses in tissues.
Rapid Displacement of
Cryptosporidium parvum
Type 1 by Type 2 in Mixed Infections in Piglets
Authors: Akiyoshi Donna E. , Mor Siobhan , Tzipori Saul
Published: September 2003

Genotypes 1 and 2 of
Cryptosporidium parvum
are the primary types associated with infections in humans, with type 1 being by far the predominant genotype. The frequency of mixed infection with both genotypes in humans is relatively rare, while type 1, which experimentally infects other mammals, has been found to naturally infect almost exclusively humans. One possible explanation for the absence of type 1 in other mammals and the low frequency of mixed infections in humans is the inability of type 1 to compete with type 2 in nature when both occur simultaneously. To investigate this, we challenged gnotobiotic piglets with equal number of oocysts of type 1 and type 2, given either simultaneously or with type 2 given 24 or 48 h after type 1. The genotype of the oocysts excreted in feces and the relative distribution of each of the genotypes throughout the gut at necropsy were determined. Regardless of the time interval between challenges with the two genotypes, type 2 invariably displaced type 1. The rate of displacement was rapid when both genotypes were given simultaneously, after which no traces of type 1 were detected in the feces or in gut sections by PCR. Infection with type 1 24 or 48 h before challenge with type 2, while permitting type 1 to become established, was still rapidly eliminated within 3 days after challenge with type 2. These observations have major implications regarding the relative perpetuation and survival of these two genotypes in mammals.
Identification of Two Eukaryote-Like Serine/Threonine Kinases Encoded by
Chlamydia trachomatis
Serovar L2 and Characterization of Interacting Partners of Pkn1
Authors: Verma Anita , Maurelli Anthony T.
Published: September 2003

Genome sequencing of
C. trachomatis
serovar D revealed the presence of three putative open reading frames (ORFs), CT145 (Pkn1), CT673 (Pkn5), and CT301 (PknD), encoding eukaryote-like serine/threonine kinases (Ser/Thr kinases). Two of these putative kinase genes, CT145 and CT301, were PCR amplified from serovar L2, cloned, and sequenced. Predicted translation products of the ORFs showed the presence of conserved kinase motifs at the N terminus of the proteins. CT145 and CT301 (encoding Pkn1 and PknD, respectively) were expressed in
Escherichia coli
as GST fusion proteins. In vitro kinase assays with
Escherichia coli
-derived glutathione
S
-transferase fusion proteins showed autophosphorylation of Pkn1 and PknD, indicating that they are functional kinases. Gene expression analysis of these kinase genes in
Chlamydia
by reverse transcriptase PCR indicated expression of these kinases at the early mid phase of the developmental cycle. Immunoprecipitated native chlamydial Pkn1 and PknD proteins also showed autophosphorylation in an in vitro kinase assay. Phosphoamino acid analysis by thin-layer chromatography confirmed that Pkn1 and PknD are phosphorylated on both serine and threonine residues. Interaction of Pkn1 and PknD with each other as well as interaction of Pkn1 with inclusion membrane protein G (IncG) was demonstrated by using a bacterial two-hybrid system. These interactions were further suggested by phosphorylation of the proteins in in vitro kinase assays. This report is the first description of the existence of functional Ser/Thr kinases in
Chlamydia
. The results of these findings should lead to a better understanding of how
Chlamydia
interact and interfere with host signaling pathways, since kinases represent potential mediators of the intimate host-pathogen interactions that are essential to the intracellular life cycle of
Chlamydia.
ThePseudomonas aeruginosaAutoinducerN-3-Oxododecanoyl Homoserine Lactone Accelerates Apoptosis in Macrophages and Neutrophils
Authors: Tateda Kazuhiro , Ishii Yoshikazu , Horikawa Manabu , Matsumoto Tetsuya , Miyairi Shinichi , Pechere Jean Claude , Standiford Theodore J. , Ishiguro Masaji , Yamaguchi Keizo
Published: September 2003

Quorum-sensing systems are critical regulators of the expression of virulence factors of various organisms, includingPseudomonas aeruginosa. Las and Rhl are two major quorum-sensing components, and they are regulated by their corresponding autoinducers,N-3-oxododecanoyl homoserine lactone (3-oxo-C12-HSL) andN-butyryl-l-homoserine lactone (C4-HSL). Recent progress has demonstrated the potential of quorum-sensing molecules, especially 3-oxo-C12-HSL, for modulation of the host immune system. Here we show the specific ability of 3-oxo-C12-HSL to induce apoptosis in certain types of cells. When bone marrow-derived macrophages were incubated with synthetic 3-oxo-C12-HSL, but when they were incubated not C4-HSL, significant loss of viability was observed in a concentration (12 to 50 μM)- and incubation time (1 to 24 h)-dependent manner. The cytotoxic activity of 3-oxo-C12-HSL was also observed in neutrophils and monocytic cell lines U-937 and P388D1 but not in epithelial cell lines CCL-185 and HEp-2. Cells treated with 3-oxo-C12-HSL revealed morphological alterations indicative of apoptosis. Acceleration of apoptosis in 3-oxo-C12-HSL-treated cells was confirmed by multiple criteria (caspases 3 and 8, histone-associated DNA fragments, phosphatidylserine expression). Structure-activity correlation experiments demonstrated that the fine structure of 3-oxo-C12-HSL, the HSL backbone, and side chain length are required for maximal activity. These data suggest thatPseudomonas3-oxo-C12-HSL specifically promotes induction of apoptosis, which may be associated with 3-oxo-C12-HSL-induced cytotoxicity in macrophages and neutrophils. Our data suggest that the quorum-sensing molecule 3-oxo-C12-HSL has critical roles in the pathogenesis ofP. aeruginosainfection, not only in the induction of bacterial virulence factors but also in the modulation of host responses.
Role of Alternative Oxidase Gene in Pathogenesis of
Cryptococcus neoformans
Authors: Akhter Shamima , McDade Henry C. , Gorlach Jenifer M. , Heinrich Garrett , Cox Gary M. , Perfect John R.
Published: September 2003

We identified a homologue of the alternative oxidase gene in a screen to identify genes that are preferentially transcribed in response to a shift to 37°C in the human-pathogenic yeast
Cryptococcus neoformans
. Alternative oxidases are nucleus-encoded mitochondrial proteins that have two putative roles: they can function in parallel with the classic cytochrome oxidative pathway to produce ATP, and they may counter oxidative stress within the mitochondria. The
C. neoformans
alternative oxidase gene (
AOX1
) was found to exist as a single copy in the genome, and it encodes a putative protein of 401 amino acids. An
aox1
mutant strain was created using targeted gene disruption, and the mutant strain was reconstituted to wild type using a full-length
AOX1
. Compared to both the wild-type and reconstituted strains, the
aox1
mutant strain was not temperature sensitive but did have significant impairment of both respiration and growth when treated with inhibitors of the classic cytochrome oxidative pathway. The
aox1
mutant strain was also found to be more sensitive to the oxidative stressor tert-butyl hydroperoxide. The
aox1
mutant strain was significantly less virulent than both the wild type and the reconstituted strain in the murine inhalational model, and it also had significantly impaired growth within a macrophage-like cell line. These data demonstrate that the alternative oxidase of
C. neoformans
can make a significant contribution to metabolism, has a role in the yeast's defense against exogenous oxidative stress, and contributes to the virulence composite of this organism, possibly by improving survival within phagocytic cells.
Molecular Characterization of the Acute Inflammatory Response to Infections with Gram-Negative versus Gram-Positive Bacteria
Authors: Feezor Robert J. , Oberholzer Caroline , Baker Henry V. , Novick Daniela , Rubinstein Menachem , Moldawer Lyle L. , Pribble John , Souza Sonia , Dinarello Charles A. , Ertel Wolfgang , Oberholzer Andreas
Published: September 2003

Sepsis caused by gram-negative bacteria and that caused by gram-positive bacteria often manifest similar clinical features. We investigated plasma proinflammatory cytokine profiles in patients with sepsis due to gram-positive and gram-negative bacteria and studied the cytokine production and differential gene regulation of leukocytes stimulated ex vivo withEscherichia colilipopolysaccharide or heat-killedStaphylococcus aureus. Concentrations of tumor necrosis factor alpha, interleukin 1 receptor antagonist (IL-1Ra), IL-8, IL-10, IL-18 binding protein, procalcitonin, and protein C in plasma did not differ between patients with sepsis due to gram-negative and gram-positive bacteria. However, plasma IL-1β, IL-6, and IL-18 concentrations were significantly higher in patients with sepsis due to gram-positive bacteria. Ex vivo stimulation of whole blood with heat-killedS. aureusmarkedly increased IL-1β and IL-18 levels more thanE. colilipopolysaccharide stimulation. Microarray analysis revealed at least 359 cross-validated probe sets (genes) significant at theP< 0.001 level whose expression discriminated among gram-negative-organism-stimulated, gram-positive-organism-stimulated, and unstimulated whole-blood leukocytes. The host inflammatory responses to gram-negative and gram-positive stimuli share some common response elements but also exhibit distinct patterns of cytokine appearance and leukocyte gene expression.
Chlamydia pneumoniaeActivates Epithelial Cell Proliferation via NF-κB and the Glucocorticoid Receptor
Authors: Gencay Mikael M. Cornelsen , Tamm Michael , Glanville Allan , Perruchoud André P. , Roth Michael
Published: September 2003

Chlamydia pneumoniaeis an obligate intracellular eubacterium and a common cause of acute and chronic respiratory tract infections. This study was designed to show the effect ofC. pneumoniaeon transcription factor activation in epithelial cells. The activation of transcription factors byC. pneumoniaewas determined in human epithelial cell lines (HL and Calu3) by electrophoretic DNA mobility shift assay, Western blotting, and luciferase reporter gene assay. The activation of transcription factors was further confirmed by immunostaining ofC. pneumoniae-infected HL cells and mock-infected controls. The effect of transcription factors onC. pneumoniae-induced host cell proliferation was assessed by [3H]thymidine incorporation and direct cell counting in the presence and absence of antisense oligonucleotides targeting transcription factors or the glucocorticoid receptor (GR) antagonist RU486. The activation of the GR, CCAAT-enhancer binding protein (C/EBP), and NF-κB was induced within 1 to 6 h byC. pneumoniae. While the interleukin-6 promoter was not activated byC. pneumoniae, the GR-driven p21(Waf1/Cip1)promoter was increased 2.5- to 3-fold over controls 24 h after infection.C. pneumoniaedose-dependently increased the DNA synthesis of the host cells 2.5- to 2.9-fold, which was partly inhibited either by RU486 or by NF-κB antisense oligonucleotides. Furthermore, we provide evidence that heat-inactivatedC. pneumoniaedoes not cause a significant increase in cell proliferation. Our results demonstrate thatC. pneumoniaeactivates C/EBP-β, NF-κB, and the GR in infected cells. However, only NF-κB and the GR were involved inC. pneumoniae-induced proliferation of epithelial cells.
M1 Protein Triggers a Phosphoinositide Cascade for Group A
Streptococcus
Invasion of Epithelial Cells
Authors: Purushothaman Sai Sudha , Wang Beinan , Cleary P. Patrick
Published: September 2003

Invasion of nonphagocytic cells by bacteria provides a favorable niche for persistence and evasion of host defenses and antibiotics. M protein is a major virulence factor because it promotes high-frequency invasion of epithelial cells by group A
Streptococcus
(GAS) and also renders the bacterium resistant to phagocytosis. In this study, we investigated the role of M1 protein from serotype M1 strain 90-226 in regulating mammalian signal transduction and cytoskeletal rearrangement for bacterial entry. LY294002 and wortmannin, which are inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked invasion of epithelial cells by GAS by 75 and 80%, respectively, but failed to inhibit invasion by
Salmonella enterica
serovar Typhimurium. Also, epithelial cells transiently transfected with dominant negative p85 and p110 genes, the regulatory and catalytic subunits of PI 3-K, respectively, were less able to be invaded by GAS. To separate the influence of other streptococcal virulence factors from M protein,
Lactococcus lactis
was engineered to express M1 protein on its surface.
L. lactis
(pLM1) invaded epithelial cells efficiently in vitro, and PI 3-K inhibitors blocked 90% of this invasion. Purified soluble M1 protein stimulated the formation of stress fibers and actin tuffs on epithelial cells. LY294002 and wortmannin inhibited these cellular changes. A phosphoinositide analogue also inhibited the invasion of epithelial cells by GAS. Therefore, M1 protein, either directly or via bound fibronectin, initiates signals that depend on the lipid kinase PI 3-K pathway, which paves the way for cytoskeletal rearrangement that internalize the bacterium.
ExperimentalMycobacterium tuberculosisInfection of Cynomolgus Macaques Closely Resembles the Various Manifestations of HumanM. tuberculosisInfection
Authors: Capuano Saverio V. , Croix Denise A. , Pawar Santosh , Zinovik Angelica , Myers Amy , Lin Philana L. , Bissel Stephanie , Fuhrman Carl , Klein Edwin , Flynn JoAnne L.
Published: September 2003

Nonhuman primates were used to develop an animal model that closely mimics humanMycobacterium tuberculosisinfection. Cynomolgus macaques were infected with low doses of virulentM. tuberculosisvia bronchoscopic instillation into the lung. All monkeys were successfully infected, based on tuberculin skin test conversion and peripheral immune responses toM. tuberculosisantigens. Progression of infection in the 17 monkeys studied was variable. Active-chronic infection, observed in 50 to 60% of monkeys, was characterized by clear signs of infection or disease on serial thoracic radiographs and in other tests and was typified by eventual progression to advanced disease. Approximately 40% of monkeys did not progress to disease in the 15 to 20 months of study, although they were clearly infected initially. These monkeys had clinical characteristics of latent tuberculosis in humans. Low-dose infection of cynomolgus macaques appears to represent the full spectrum of humanM. tuberculosisinfection and will be an excellent model for the study of pathogenesis and immunology of this infection. In addition, this model will provide an opportunity to study the latentM. tuberculosisinfection observed in ∼90% of all infected humans.
Widespread Bronchogenic Dissemination Makes DBA/2 Mice More Susceptible than C57BL/6 Mice to Experimental Aerosol Infection withMycobacterium tuberculosis
Authors: Cardona Pere-Joan , Gordillo Sergi , Díaz Jorge , Tapia Gustavo , Amat Isabel , Pallarés Ángeles , Vilaplana Cristina , Ariza Aurelio , Ausina Vicenç
Published: September 2003

We have used the murine model of aerosol-induced experimental tuberculosis to assess the effects of four clinical isolates and a reference strain ofMycobacterium tuberculosison resistant C57BL/6 mice and susceptible DBA/2 mice. Histological studies and detection of 25 cytokines potentially involved in the infection were carried out. DBA/2 mice showed higher concentrations of bacilli in bronchoalveolar lavage fluid and lung tissue. Furthermore, these mice evidenced a larger granulomatous infiltration in the parenchyma due to an increased rate of emigration of infected foamy macrophages from the granulomas to the neighboring pulmonary alveolar spaces. The better control of bacillary concentrations and pulmonary infiltration observed in C57BL/6 mice from week 3 postinfection could result from their higher RANTES, ICAM-1, and gamma interferon (IFN-γ) mRNA levels. On the other hand, the higher MIP-2 and MCP-3 mRNA levels seen in DBA/2 mice would result in stronger lung recruitment of macrophages and neutrophils. Additionally, DBA/2 mice showed increased inducible nitric oxide synthase expression, induced by the larger number of foamy macrophages, at weeks 18 and 22. This increment was a consequence of phagocytosed bacillary debris, was independent of IFN-γ expression, and could exert only a bacteriostatic effect. The results of the study suggest that DBA/2 mice are more susceptible than C57BL/6 mice toM. tuberculosisinfection due to a higher bronchial dissemination of bacilli inside poorly activated foamy macrophages.
Rab GTPases Are Recruited to Chlamydial Inclusions in Both a Species-Dependent and Species-Independent Manner
Authors: Rzomp Kimberly A. , Scholtes Luella D. , Briggs Benjamin J. , Whittaker Gary R. , Scidmore Marci A.
Published: September 2003

Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the peri-Golgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFP-Rab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to
Chlamydia trachomatis
,
Chlamydia muridarum
, and
Chlamydia pneumoniae
inclusions, whereas GFP-Rab5, GFP-Rab7, and GFP-Rab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with
C. trachomatis
inclusions but not with
C. pneumoniae
or
C. muridarum
inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11's association with the inclusion is not mediated solely through Rab11's association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with
C. trachomatis
IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion.
Citrobacter koseriBrain Abscess in the Neonatal Rat: Survival and Replication within Human and Rat Macrophages
Authors: Townsend Stacy M. , Pollack Harvey A. , Gonzalez-Gomez Ignacio , Shimada Hiroyuki , Badger Julie L.
Published: September 2003

A unique feature ofCitrobacter koseriis the extremely high propensity to initiate brain abscesses during neonatal meningitis. Previous clinical reports and studies on infant rats have documented manyCitrobacter-filled macrophages within the ventricles and brain abscesses. It has been hypothesized that intracellular survival and replication within macrophages may be a mechanism by whichC. koserisubverts the host response and elicits chronic infection, resulting in brain abscess formation. In this study, we showed thatC. kosericauses meningitis and brain abscesses in the neonatal rat model, and we utilized histology and magnetic resonance imaging technology to visualize brain abscess formation. Histology and electron microscopy (EM) revealed that macrophages (and not fibroblasts, astrocytes, oligodendrocytes, or neurons) were the primary target for long-termC. koseriinfection. To better understandC. koseripathogenesis, we have characterized the interactions ofC. koseriwith human macrophages. We found thatC. koserisurvives and replicates within macrophages in vitro and that uptake ofC. koseriincreases in the presence of human pooled serum in a dose-dependent manner. EM studies lend support to the hypothesis thatC. koseriuses morphologically different methods of uptake to enter macrophages. FcγRI blocking experiments show that this receptor primarily facilitates the entry of opsonizedC. koseriinto macrophages. Further, confocal fluorescence microscopy demonstrates thatC. koserisurvives phagolysosomal fusion and that more than 90% of intracellularC. koseriorganisms are colocalized within phagolysosomes. The ability ofC. koserito survive phagolysosome fusion and replicate within macrophages may contribute to the establishment of chronic central nervous system infection including brain abscesses.
Intracellular Adhesion Molecule 1 Plays a Key Role in Acquired Immunity to Salmonellosis
Authors: Clare Simon , Goldin Robert , Hale Christine , Aspinall Richard , Simmons Cameron , Mastroeni Pietro , Dougan Gordon
Published: September 2003

This study investigated the role of intracellular adhesion molecule 1 (ICAM-1) duringSalmonella entericaserovar Typhimurium infection of mice. We show that ICAM-1 is expressed in and around granulomas on day 4 of infection in wild-type mice. However, when naive ICAM-1−/−mice were challenged with a sublethal dose of serovar Typhimurium, there were no detectable differences in systemic bacterial burden over the first 9 days of infection compared to wild-type control mice. When mice were immunized with theS. entericaserovar Typhimurium vaccine strain SL2361 and then challenged with the virulentS. entericaserovar Typhimurium strain C5, 100% of the ICAM-1−/−mice succumbed to infection, compared to 30% of wild-type mice. T-cell responses, as measured by activation via interleukin-2 production, as well as antibody responses were comparable in the ICAM-1−/−and wild-type mice. Following challenge, counts in organs were significantly higher in the ICAM-1−/−mice, and histological examination of organs showed pathological differences. Strain SL3261-immunized wild-type mice had cellular infiltrate and normal granuloma formation in the liver and spleen on days 5 and 10 after challenge with strain C5. ICAM-1−/−mice had a similar infiltrate on day 5, whereas on day 10 the infiltrate was more widespread and there were fewer macrophages associated with the granulomas. High circulating levels of tumor necrosis factor alpha and gamma interferon, as well as a high burden of strain C5 in the blood, accompanied the differences in histopathology. In this study we show that ICAM-1 plays a critical role during rechallenge of immunized mice with virulentS. entericaserovar Typhimurium.
The Ability To Replicate in Macrophages Is Conserved between
Yersinia pestis
and
Yersinia pseudotuberculosis
Authors: Pujol Céline , Bliska James B.
Published: September 2003

Yersinia pestis
, the agent of plague, has arisen from a less virulent pathogen,
Yersinia pseudotuberculosis
, by a rapid evolutionary process. Although
Y. pestis
displays a large number of virulence phenotypes, it is not yet clear which of these phenotypes descended from
Y. pseudotuberculosis
and which were acquired independently.
Y. pestis
is known to replicate in macrophages, but there is no consensus in the literature on whether
Y. pseudotuberculosis
shares this property. We investigated whether the ability to replicate in macrophages is common to
Y. pestis
and
Y. pseudotuberculosis
or is a unique phenotype of
Y. pestis
. We also examined whether a chromosomal type III secretion system (TTSS) found in
Y. pestis
is present in
Y. pseudotuberculosis
and whether this system is important for replication of
Yersinia
in macrophages. A number of
Y. pestis
and
Y. pseudotuberculosis
strains of different biovars and serogroups, respectively, were tested for the ability to replicate in primary murine macrophages. Two
Y. pestis
strains (EV766 and KIM10
+
) and three
Y. pseudotuberculosis
strains (IP2790c, IP2515c, and IP2666c) were able to replicate in macrophages with similar efficiencies. Only one of six strains tested, the
Y. pseudotuberculosis
YPIII(p
−
) strain, was defective for intracellular replication. Thus, the ability to replicate in macrophages is conserved in
Y. pestis
and
Y. pseudotuberculosis
. Our results also indicate that a homologous TTSS is present on the chromosomes of
Y. pestis
and
Y. pseudotuberculosis
and that this secretion system is not required for replication of these bacteria in macrophages.
Heterogeneous Surface Expression of EspA Translocon Filaments by
Escherichia coli
O157:H7 Is Controlled at the Posttranscriptional Level
Authors: Roe Andrew J. , Yull Helen , Naylor Stuart W. , Woodward Martin J. , Smith David G. E. , Gally David L.
Published: September 2003

Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic
Escherichia coli
O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as “effectors” such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of
LEE4
translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when
E. coli
O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine
E. coli
O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of
LEE1
-
4
operon transcription, as demonstrated by using both β-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when
LEE1-4
translational fusions were used. Despite this, the
LEE4
-
espADB
mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce
LEE1-4
expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in
E. coli
O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on
LEE4
-
espADB
mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.
Iron Superoxide Dismutases Targeted to the Glycosomes ofLeishmania chagasiAre Important for Survival
Authors: Plewes Katherine A. , Barr Stephen D. , Gedamu Lashitew
Published: September 2003

Kinetoplastid glycosomes contain a variety of metabolic activities, such as glycolysis, β-oxidation of fatty acids, lipid biosynthesis, and purine salvage. One advantage of sequestering metabolic activities is the avoidance of cellular oxidative damage by reactive oxygen species produced as a by-product of metabolism. Little is known about how glycosomes themselves withstand these toxic metabolites. We previously isolated an iron superoxide dismutase fromLeishmania chagasithat is expressed at low levels in the early logarithmic promastigote stage and increases toward the stationary promastigote and amastigote stages. We have since identified a second highly homologousLcfesodbgene that is expressed at high levels in the early logarithmic promastigote stage and decreases toward the stationary promastigote and amastigote stages. Localization studies using green fluorescent protein fusions have revealed that LcFeSODB1 and LcFeSODB2 are localized within the glycosomes by the last three amino acids of their carboxyl termini. To better understand the specific role that FeSODB plays in parasite growth and survival, a single-allele knockout of theLcfesodb1gene was generated. The parasites with these genes exhibited a significant reduction in growth when endogenous superoxide levels were increased with paraquat in culture. Furthermore, the FeSODB1-deficient parasites exhibited a significant reduction in survival within human macrophages. Our results suggest that LcFeSODB plays an important role in parasite growth and survival by protecting glycosomes from superoxide toxicity.
Acid-Adaptive Genes of
Helicobacter pylori
Authors: Wen Yi , Marcus Elizabeth A. , Matrubutham Uday , Gleeson Martin A. , Scott David R. , Sachs George
Published: September 2003

Helicobacter pylori
is the only neutralophile that has been able to colonize the human stomach by using a variety of acid-adaptive mechanisms. One of the adaptive mechanisms is increased buffering due to expression of an acid-activated inner membrane urea channel, UreI, and a neutral pH-optimum intrabacterial urease. To delineate other possible adaptive mechanisms, changes in gene expression in response to acid exposure were examined using genomic microarrays of
H. pylori
exposed to different levels of external pH (7.4, 6.2, 5.5, and 4.5) for 30 min in the absence and presence of 5 mM urea. Gene expression was correlated with intrabacterial pH measured using 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein and compared to that observed with exposure to 42°C for 30 min. Microarrays containing the 1,534 open reading frames of
H. pylori
strain 26695 were hybridized with cDNAs from control (pH 7.4; labeled with Cy3) and acidic (labeled with Cy5) conditions. The intrabacterial pH was 8.1 at pH 7.4, fell to 5.3 at pH 4.5, and rose to 6.2 with urea. About 200 genes were up-regulated and ∼100 genes were down-regulated at pH 4.5 in the absence of urea, and about half that number changed in the presence of urea. These genes included pH-homeostatic, transcriptional regulatory, motility, cell envelope, and pathogenicity genes. The up-regulation of some pH-homeostatic genes was confirmed by real-time PCR. There was little overlap with the genes induced by temperature stress. These results suggest that
H. pylori
has evolved multifaceted acid-adaptive mechanisms enabling it to colonize the stomach that may be novel targets for eliminating infection.
An Attenuated Strain of the Facultative Intracellular BacteriumFrancisella tularensisCan Escape the Phagosome of Monocytic Cells
Authors: Golovliov Igor , Baranov Vladimir , Krocova Zuzana , Kovarova Hana , Sjöstedt Anders
Published: September 2003

The facultative intracellular bacteriumFrancisella tularensisis a highly virulent and contagious organism, and little is known about its intracellular survival mechanisms. We studied the intracellular localization of the attenuated human vaccine strain,F. tularensisLVS, in adherent mouse peritoneal cells, in mouse macrophage-like cell line J774A.1, and in human macrophage cell line THP-1. Confocal microscopy of infected J774A.1 cells indicated that during the first hour of infection the bacteria colocalized with the late endosomal-lysosomal glycoprotein LAMP-1, but within 3 h this colocalization decreased significantly from approximately 60% to 30%. Transmission electron microscopy revealed that >90% of bacteria were not enclosed by a phagosomal membrane after 2 h of infection, and some bacteria were in vacuoles that were only partially surrounded by a limiting membrane. Similar findings were obtained with all three host cell types. Immunoelectron microscopy performed with anF. tularensisLVS-specific polyclonal rabbit antiserum showed that the antiserum stained a thick, evenly distributed capsule-like material in bacteria grown in broth. In contrast, intracellularF. tularensisLVS cells were only marginally stained with this antiserum. Instead, most of the immunoreactive material was diffusely localized in the phagosomes or was associated with the phagosomal membrane. Our findings indicate thatF. tularensisLVS is able to escape from the phagosomes of macrophages via a mechanism that may involve degradation of the phagosomal membrane.
Entry and Intracellular Replication ofEscherichia coliK1 in Macrophages Require Expression of Outer Membrane Protein A
Authors: Sukumaran Sunil K. , Shimada Hiroyuki , Prasadarao Nemani V.
Published: September 2003

Interactions betweenEscherichia coliK1, which causes meningitis in neonates, and macrophages have not been explored well. In this study we found thatE. coliK1 was able to enter, survive, and replicate intracellularly in both murine and human macrophage cell lines, as well as in monocytes and macrophages of newborn rats. In addition, we demonstrated that OmpA+E. colialso enters and replicates in human peripheral blood monocytes in vitro. Outer membrane protein A (OmpA) expression onE. colicontributes to binding to macrophages, phagocytosis, and survival within macrophages. Opsonization with either complement proteins or antibody is not required for uptake and survival of the bacteria within the macrophages. Transmission electron microscopy and immunocytochemistry studies with the infected macrophages indicated that OmpA+E. colimultiplies enormously in a single phagosome and bursts the cell. Internalization of OmpA+E. coliby RAW 264.7 cells occurred by both actin- and microtubule-dependent processes, which are independent of RGD-mediated integrin receptors. Internalization and intracellular survival within phagocytic cells thus may play an important role in the development of bacteremia, which is crucial forE. colicrossing of the blood-brain barrier.
Identification and Characterization of HtsA, a Second Heme-Binding Protein Made byStreptococcus pyogenes
Authors: Lei Benfang , Liu Mengyao , Voyich Jovanka M. , Prater Christopher I. , Kala Subbarao V. , DeLeo Frank R. , Musser James M.
Published: September 2003

Group A streptococci (GAS) can use heme and hemoproteins as sources of iron. However, the machinery for heme acquisition in GAS has not been firmly revealed. Recently, we identified a novel heme-associated cell surface protein (Shp) made by GAS. Theshpgene is cotranscribed with eight downstream genes, includingspy1795,spy1794, andspy1793encoding a putative ABC transporter (designated HtsABC). In this study,spy1795(designatedhtsA) was cloned from a serotype M1 strain, and recombinant HtsA was overexpressed inEscherichia coliand purified to homogeneity. HtsA binds 1 heme molecule per molecule of protein. HtsA was produced in vitro and localized to the bacterial cell surface. GAS up-regulated transcription ofhtsAin human blood compared with that in Todd-Hewitt broth supplemented with 0.2% yeast extract. The level of thehtsAtranscript dramatically increased under metal cation-restricted conditions compared with that under metal cation-replete conditions. The cation content, cell surface location, and gene transcription of HtsA were also compared with those of MtsA and Spy0385, the lipoprotein components of two other putative iron acquisition ABC transporters of GAS. Our results suggest that HtsABC is an ABC transporter that may participate in heme acquisition in GAS.
PneumocystisPneumonia Increases the Susceptibility of Mice to Sublethal Hyperoxia
Authors: Beck James M. , Preston Angela M. , Wilcoxen Steven E. , Morris Susan B. , White Eric S. , Paine Robert
Published: September 2003

Patients withPneumocystispneumonia often develop respiratory failure after entry into medical care, and one mechanism for this deterioration may be increased alveolar epithelial cell injury. In vitro, we previously demonstrated thatPneumocystisis not cytotoxic for alveolar epithelial cells. In vivo, however, infection withPneumocystiscould increase susceptibility to injury by stressors that, alone, would be sublethal. We examined transient exposure to hyperoxia as a prototypical stress that does cause mortality in normal mice. Mice were depleted of CD4+T cells and inoculated intratracheally withPneumocystis. Control mice were depleted of CD4+T cells but did not receivePneumocystis. After 4 weeks, mice were maintained in normoxia, were exposed to hyperoxia for 4 days, or were exposed to hyperoxia for 4 days followed by return to normoxia. CD4-depleted mice withPneumocystispneumonia demonstrated significant mortality after transient exposure to hyperoxia, while all uninfected control mice survived this stress. We determined that organism burdens were not different. However, infected mice exposed to hyperoxia and then returned to normoxia demonstrated significant increases in inflammatory cell accumulation and lung cell apoptosis. We conclude thatPneumocystispneumonia leads to increased mortality following a normally sublethal hyperoxic insult, accompanied by alveolar epithelial cell injury and increased pulmonary inflammation.
Parachlamydia acanthamoebaEnters and Multiplies within Human Macrophages and Induces Their Apoptosis
Authors: Greub Gilbert , Mege Jean-Louis , Raoult Didier
Published: September 2003

Parachlamydia acanthamoebais an obligately intracellular bacterium that naturally infects free-living amoebae. It is a potential human pathogen and may survive in human macrophages. We studiedP. acanthamoebaentry into, and multiplication within, human monocyte-derived macrophages. After 8 h of incubation, 80% of macrophages were infected with a mean of 3.8P. acanthamoebaorganisms per cell. Electron microscopy demonstrated that parachlamydiae were in an intracellular vacuole. After infection with living organisms, the number of parachlamydiae per macrophage increased 4 times from day 0 to day 4, whereas heat-inactivated parachlamydiae were eliminated during the same period. Quantitative PCR confirmed thatP. acanthamoebareplicates within macrophages. Transcriptional activity ofP. acanthamoebawas detected by reverse transcription-PCR targeting the gene encoding ADP-ATP translocase (tlc).P. acanthamoebaexerted a cytopathic effect on macrophages. When macrophages were infected with living bacteria, their number decreased significantly from day 0 to day 4 due to apoptosis, as shown by annexin-V binding and electron microscopy. This study shows thatP. acanthamoebaenters and multiplies within human macrophages before inducing their apoptosis.
Host Resistance of CD18 Knockout Mice against Systemic Infection withListeria monocytogenes
Authors: Wu Huaizhu , Prince Joseph E. , Brayton Cory F. , Shah Chirayu , Zeve Daniel , Gregory Stephen H. , Smith C. Wayne , Ballantyne Christie M.
Published: September 2003

Mice with targeted mutations of CD18, the common β2 subunit of CD11/CD18 integrins, have leukocytosis, impaired transendothelial neutrophil emigration, and reduced host defense toStreptococcus pneumoniae, a gram-positive extracellular bacterium. Previous studies using blocking monoclonal antibodies suggested roles for CD18 and CD11b in hepatic neutrophil recruitment and host innate response toListeria monocytogenes, a gram-positive intracellular bacterium. We induced systemic listeriosis in CD18 knockout (CD18-ko) and wild-type (WT) mice by tail vein injection withListeria. By 14 days postinjection (dpi), 8 of 10 WT mice died, compared with 2 of 10 CD18-ko mice (P< 0.01). Quantitative organ culture showed that numbers ofListeriaorganisms in livers and spleens were similar in both groups at 20 min postinfection. By 3, 5, and 7 dpi, however, numbers ofListeriaorganisms were significantly lower in livers and spleens of CD18-ko mice than in WT mice. Histopathology showed that followingListeriainfection, CD18-ko mice had milder inflammatory and necrotizing lesions in both spleens and livers than did WT mice. Cytokine assays indicated that baseline interleukin-1β and granulocyte colony-stimulating factor (G-CSF) levels were higher in CD18-ko mice than in WT mice and that CD18-ko splenocytes produced higher levels of interleukin-1β and G-CSF than WT splenocytes under the same amount ofListeriastimulation. These findings show that CD18 is not an absolute requirement for antilisterial innate immunity or hepatic neutrophil recruitment. We propose that the absence of CD18 in the mice results in the priming of innate immunity, as evidenced by elevated cytokine expression, and neutrophilic leukocytosis, which augments antilisterial defense.
Inhibition of Phagocytosis byHaemophilus ducreyiRequires Expression of the LspA1 and LspA2 Proteins
Authors: Vakevainen Merja , Greenberg Steven , Hansen Eric J.
Published: September 2003

Haemophilus ducreyipreviously has been shown to inhibit the phagocytosis of both secondary targets and itself by certain cells in vitro. Wild-typeH. ducreyistrain 35000HP contains two genes,lspA1andlspA2, whose encoded protein products are predicted to be 456 and 543 kDa, respectively. An isogenic mutant ofH. ducreyi35000HP with inactivatedlspA1andlspA2genes has been shown to exhibit substantially decreased virulence in the temperature-dependent rabbit model for chancroid. ThislspA1 lspA2mutant was tested for its ability to inhibit phagocytosis of immunoglobulin G-opsonized particles by differentiated HL-60 and U-937 cells and by J774A.1 cells. The wild-type strainH. ducreyi35000HP readily inhibited phagocytosis, whereas thelspA1 lspA2mutant was unable to inhibit phagocytosis. Similarly, the wild-type strain was resistant to phagocytosis, whereas thelspA1 lspA2mutant was readily engulfed by phagocytes. This inhibitory effect of wild-typeH. ducreyion phagocytic activity was primarily associated with live bacterial cells but could also be found, under certain conditions, in concentratedH. ducreyiculture supernatant fluids that lacked detectable outer membrane fragments. Both the wild-type strain and thelspA1 lspA2mutant attached to phagocytes at similar levels. These results indicate that the LspA1 and LspA2 proteins ofH. ducreyiare involved, directly or indirectly, in the antiphagocytic activity of this pathogen, and they provide a possible explanation for the greatly reduced virulence of thelspA1 lspA2mutant.
Different Strains ofMycobacterium tuberculosisCause Various Spectrums of Disease in the Rabbit Model of Tuberculosis
Authors: Manabe Yukari C. , Dannenberg Arthur M. , Tyagi Sandeep K. , Hatem Christine L. , Yoder Mark , Woolwine Samuel C. , Zook Bernard C. , Pitt M. Louise M. , Bishai William R.
Published: September 2003

The rabbit model of tuberculosis has been used historically to differentiate betweenMycobacterium tuberculosisandMycobacterium bovisbased on their relative virulence in this animal host.M. tuberculosisinfection in market rabbits is cleared over time, whereas infection withM. bovisresults in chronic, progressive, cavitary disease leading to death. Because of the innate resistance of commercial rabbits toM. tuberculosis, 320 to 1,890 log-phase, actively growing inhaled bacilli were required to form one grossly visible pulmonary tubercle at 5 weeks. The range of inhaled doses required to make one tubercle allows us to determine the relative pathogenicities of different strains. Fewer inhaled organisms of theM. tuberculosisErdman strain were required than ofM. tuberculosisH37Rv to produce a visible lesion at 5 weeks. Furthermore, with the Erdman strain, only 7 of 15 rabbits had healed lesions at 16 to 18 weeks; among the other animals, two had chronic, progressive cavitary disease, a phenotype usually seen only withM. bovisinfection. Genotypic investigation of the Erdman strain with an H37Rv-based microarray identified gene differences in the RD6 region. Southern blot and PCR structural genetic analysis showed significant differences betweenM. tuberculosisstrains in this region. Correlation of the relative pathogenicity, including disease severity, in the rabbit model with the strain genotype may help identify stage-specificM. tuberculosisgenes important in human disease.