bluuebunny/crossref_metadata_2025 huggingface dataset:
Porphyromonas gingivalis
Lipopolysaccharide Contains Multiple Lipid A Species That Functionally Interact with Both Toll-Like Receptors 2 and 4
Authors: Darveau Richard P. , Pham Thu-Thao T. , Lemley Kayde , Reife Robert A. , Bainbridge Brian W. , Coats Stephen R. , Howald William N. , Way Sing Sing , Hajjar Adeline M.
Published: August 2004

The innate host response to lipopolysaccharide (LPS) obtained from
Porphyromonas gingivalis
is unusual in that different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) as well as an antagonist or agonist for TLR4. In this report it is shown that
P. gingivalis
LPS is highly heterogeneous, containing more lipid A species than previously described. In addition, purification of LPS can preferentially fractionate these lipid A species. It is shown that an LPS preparation enriched for lipid A species at
m/z
1,435 and 1,450 activates human and mouse TLR2, TLR2 plus TLR1, and TLR4 in transiently transfected HEK 293 cells coexpressing membrane-associated CD14. The HEK cell experiments further demonstrated that cofactor MD-2 was required for functional engagement of TLR4 but not of TLR2 nor TLR2 plus TLR1. In addition, serum-soluble CD14 effectively transferred
P. gingivalis
LPS to TLR2 plus TLR1, but poorly to TLR4. Importantly, bone marrow cells obtained from TLR2
−/−
and TLR4
−/−
mice also responded to
P. gingivalis
LPS in a manor consistent with the HEK results, demonstrating that
P. gingivalis
LPS can utilize both TLR2 and TLR4. No response was observed from bone marrow cells obtained from TLR2 and TLR4 double-knockout mice, demonstrating that
P. gingivalis
LPS activation occurred exclusively through either TLR2 or TLR4. Although the biological significance of the different lipid A species found in
P. gingivalis
LPS preparations is not currently understood, it is proposed that the presence of multiple lipid A species contributes to cell activation through both TLR2 and TLR4.
Cooperative Interactions between Flagellin and SopE2 in the Epithelial Interleukin-8 Response toSalmonella entericaSerovar Typhimurium Infection
Authors: Huang Fu-Chen , Werne Adam , Li Qian , Galyov Edouard E. , Walker W. Allan , Cherayil Bobby J.
Published: August 2004

Flagellin is an important stimulus for epithelial interleukin-8 (IL-8) secretion because of its ability to activate Toll-like receptor 5 (TLR5). SopE2, aSalmonellaguanine nucleotide exchange factor (GEF), is also involved in intestinal inflammation. To clarify the proinflammatory mechanisms of these proteins, we examined their effects on IL-8 secretion and intracellular signaling in T84 epithelial cells. ASalmonellastrain lacking SopE2 (and its homolog SopE) induced lower levels of IL-8 than the wild type and exhibited reduced activation of mitogen-activated protein kinases (MAPKs). Overexpression of wild-type SopE2 in this strain restored MAPK activation and augmented IL-8 production, whereas a mutant lacking GEF activity failed to increase IL-8 expression. Additional effects on signaling were demonstrated in transient transfection experiments, in which SopE2 enhanced the ability of TRAF6, a signal transducer downstream of TLR5, to activate the NF-κB transcription factor in 293 cells. Flagellin was also found to be required for IL-8 induction in T84 cells. In its absence, the ability of SopE2 overexpression to increase IL-8 secretion was impaired. Part of this impairment was related to the decreased motility of the flagellin-deficient strain, but lack of flagellin also affected translocation of SopE2 into the infected cells. Our results indicate that flagellin and SopE2 interact functionally at multiple levels to increase IL-8 secretion by epithelial cells—flagellin facilitating the translocation of SopE2, and SopE2 enhancing signaling pathways activated by flagellin. These observations offer a mechanistic explanation for the involvement of these proteins in the pathogenesis ofSalmonella-induced gastroenteritis.
Temporal Analysis of the Antigenic Composition of
Borrelia burgdorferi
during Infection in Rabbit Skin
Authors: Crother Timothy R. , Champion Cheryl I. , Whitelegge Julian P. , Aguilera Rodrigo , Wu Xiao-Yang , Blanco David R. , Miller James N. , Lovett Michael A.
Published: August 2004

The numbers of host-adapted
Borrelia burgdorferi
(HAB) organisms in rabbit skin were assessed by real-time PCR over the first 3 weeks of infection. Maximal numbers were found at day 11, while spirochete numbers decreased by more than 30-fold by day 21. The antigenic composition of HAB in skin biopsy samples was determined by use of a procedure termed hydrophobic antigen tissue Triton extraction. Immune sera from rabbits, sera from chronically infected mice, and monospecific antiserum to the antigenic variation protein, VlsE, were used to probe parallel two-dimensional immunoblots representing each time point. Individual proteins were identified using either specific antisera or by matching protein spots to mass spectrometry-identified protein spots from in vitro-cultivated
Borrelia
. There were significant changes in the relative expression of a variety of known and previously unrecognized HAB antigens during the 21-day period. OspC and the outer membrane proteins OspA and OspB were prominent at the earliest time point, day 5, when the antigenic variation protein VlsE was barely detected. OspA and OspB were not detected after day 5. OspC was not detected after day 9. VlsE was the most prominent antigen from day 7 through day 21. BmpA, ErpN, ErpP, LA7, OppA-2, DbpA, and an unidentified 15-kDa protein were also detected from day 7 through day 21. Immunoblot analysis using monospecific anti-VlsE revealed the presence of prominent distinct VlsE lower forms in HAB at days 9, 11, and 14; however, these lower forms were no longer detected at day 21. This marked diminution in VlsE lower forms paralleled the clearance of the spirochete from skin.
Impaired Host Defense against
Sporothrix schenckii
in Mice with Chronic Granulomatous Disease
Authors: Kajiwara Hideko , Saito Mitsumasa , Ohga Shouichi , Uenotsuchi Takeshi , Yoshida Shin-ichi
Published: August 2004

We compared the immune defense of mice with chronic granulomatous disease (CGD mice) with that of wild-type C57BL/6 mice for their response to
Sporothrix schenckii
. A subcutaneous injection of 5 × 10
4
CFU
S. schenckii
strain IFM41598 into CGD mice resulted in systemic infection and death within 84 days. In contrast, injected C57BL/6 mice did not develop systemic infection and were able to survive through 100 days of observation. Differences in host resistance were analyzed in vitro. Neutrophils and macrophages obtained from CGD mice were found to allow greater growth of this organism than did those obtained from C57BL/6 mice. Moreover, macrophages obtained from immunized CGD mice were able to simply inhibit the growth of this fungus whereas macrophages obtained from immunized C57BL/6 mice killed the fungus within 48 h after phagocytosis. These results suggest that (i) the lack of NADPH oxidase function is a risk factor for lethal
S. schenckii
infection and (ii) superoxide anion and its reactive oxidative metabolites produced by neutrophils and macrophages are involved in fungistatic and fungicidal activities.
Molecular Evolutionary Relationships of Enteroinvasive
Escherichia coli
and
Shigella
spp
Authors: Lan Ruiting , Alles M. Chehani , Donohoe Kathy , Martinez Marina B. , Reeves Peter R.
Published: August 2004

Enteroinvasive
Escherichia coli
(EIEC), a distinctive pathogenic form of
E. coli
causing dysentery, is similar in many properties to bacteria placed in the four species of
Shigella. Shigella
has been separated as a genus but in fact comprises several clones of
E. coli
. The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of
Shigella
strains. The EIEC strains were grouped in four clusters with one outlier strain, indicating independent derivation of EIEC several times. Three of the four clusters contain more than one O antigen type. One EIEC strain (an O112ac:H− strain) was found in
Shigella
cluster 3 but is not identical to the
Shigella
cluster 3 D2 and B15 strains with the same O antigen. Two forms of the virulence plasmid pINV have been identified in
Shigella
strains by using the sequences of
ipgD
and
mxiA
genes, and all but two of our EIEC strains have pINV A. The EIEC strains were grouped in two subclusters with a very low level of variation, generally not intermingled with
Shigella
pINV A strains. The EIEC clusters based on housekeeping genes were reflected in the plasmid gene sequences, with some exceptions. Two strains were found in the pINV B form by using the
ipgD
sequence, with one strain having an
mxiA
sequence similar to the divergent sequence of D1. Clearly, EIEC and
Shigella
spp. form a pathovar of
E. coli
.
Dendritic Cells Stimulated withActinobacillus actinomycetemcomitansElicit Rapid Gamma Interferon Responses by Natural Killer Cells
Authors: Kikuchi T. , Hahn C. L. , Tanaka S. , Barbour S. E. , Schenkein H. A. , Tew J. G.
Published: August 2004

Human immunoglobulin G2 (IgG2) responses are gamma interferon (IFN-γ) dependent, and monocyte-derived dendritic cells (mDCs) promote IgG2 production. DCs spontaneously emerge from monocytes in cultures prepared from localized aggressive periodontitis (LagP) patients, and these patients have high levels of IgG2 that is reactive withActinobacillus actinomycetemcomitans. These results prompted the hypothesis that an interaction between mDCs andA. actinomycetemcomitanspromotes IFN-γ production, and IFN-γ is known to promote both immunopathology and protective IgG2.A. actinomycetemcomitansinduced mDCs to produce interleukin-12 (IL-12), and the addition ofA. actinomycetemcomitansand DCs to cultured peripheral blood lymphocytes elicited high levels of IFN-γ within just 24 h. In contrast, IL-4 was not detectable although DC-derived IL-10 production was apparent.A. actinomycetemcomitans-stimulated macrophages prepared from the same monocytes lacked the ability to induce IL-12 or IFN-γ responses. NK cells of the innate immune system were the primary source of this early IFN-γ, although CD8 T cells also contributed some. The NK cell-derived IFN-γ was IL-12 dependent, andA. actinomycetemcomitans-DC interactions were Toll-like receptor 4 dependent.A. actinomycetemcomitansandA. actinomycetemcomitanslipopolysaccharide (LPS) were more potent thanEscherichia coliandE. coliLPS in the ability to induce DC IL-12 and IFN-γ. The ability ofA. actinomycetemcomitans-stimulated DCs to induce NK cells to rapidly produce IFN-γ in the absence of detectable IL-4 suggests their potential for skewing responses toward Th1. This may help explain the presence of Th1-associated cytokines in gingival crevicular fluid (GCF) from LagP patients and the high levels of IgG2 in their serum and GCF that is reactive withA. actinomycetemcomitans.
Major Histocompatibility Complex Class I Peptide Presentation afterSalmonella entericaSerovar Typhimurium Infection Assessed via Stable Isotope Tagging of the B27-Presented Peptide Repertoire
Authors: Ringrose Jeffrey H. , Meiring Hugo D. , Speijer Dave , Feltkamp Theodorus E. W. , van Els Cecile A. C. M. , de Jong Ad P. J. M. , Dankert Jacob
Published: August 2004

Reactive arthritis (ReA) induced by infection with several gram-negative bacteria is strongly associated with expression of the major histocompatibility complex class I molecule HLA-B27. It is thought that due to the intracellular lifestyle of ReA-inducing bacteria, bacterial fragments can be presented by HLA-B27. Cytotoxic T cells recognizing such bacterial peptides or other induced host peptides could cross-react with self peptides presented in the joints, giving rise to disease. Studies to analyze the B27 peptide repertoire in relation to infection were severely hampered, as complex peptide profiles obtained from separate infected and noninfected cell preparations had to be compared. For this study, we applied a new approach to examine the effect ofSalmonella entericaserovar Typhimurium infection on the B27 peptide repertoire presented by the HLA-B*2704 subtype associated with disease. Firstly, we showed that both host cell andS. entericaserovar Typhimurium proteins can be tagged metabolically with stable-isotope-labeled arginine. We then designed experiments so that either the tagged endogenous or tagged bacterial B*2704-presented peptide repertoires from infected cells could be analyzed by mass spectrometry from single peptide preparations that included uninfected controls. Using this new approach, we found no evidence for significant changes in endogenous B*2704 peptide presentation after infection or for anyS. entericaserovar Typhimurium-derived B27-bound peptide. In conclusion, the hypothesis thatS. entericaserovar Typhimurium induces changes in B27 peptide presentation could not be supported.
Senescent BALB/c Mice Are Able To Develop Resistance toLeishmania majorInfection
Authors: Ehrchen Jan , Sindrilaru Anca , Grabbe Stephan , Schönlau Frank , Schlesiger Christian , Sorg Clemens , Scharffetter-Kochanek Karin , Sunderkötter Cord
Published: August 2004

Aging has been associated with a decline in immunocompetence and resistance to infections, partially due to dysregulated NO production by macrophages and deficits in mounting Th2 cell responses. We wondered if these alterations would reverse the immune response in experimental leishmaniasis. Bone-marrow-derived macrophages from 2- and 18-month-old (senescent) C57BL/6 or BALB/c mice showed no marked difference in leishmanicidal functions. In vivo infections of resistant C57BL/6 mice withLeishmania majorrevealed no difference between senescent and young mice. However, among susceptible BALB/c mice, senescent animals showed less foot-pad swelling than young mice, and 40 to 60% of them even showed healing of ulcers, reduced parasite dissemination, and a Th1 cell response. These changes were associated with a spontaneous release of interleukin-12 (IL-12) by macrophages from aged but not from young mice. Since exogenous microbial stimulation can influence immune responses during aging, we also infected senescent mice who were raised under specific-pathogen-free (SPF) conditions. They showed neither resistance nor a Th1 response, but their macrophages still spontaneously released IL-12. A microbiological analysis showed that conventionally kept mice, but not SPF mice, had experienced infection with murine hepatitis virus (MHV), an infection associated with a Th1-like response. We conclude that for the reversal of the immune response, senescence is the premier requirement but needs to be completed by another mandatory event such as microbial stimulation. One of the age-related, but not environment-related, factors is the spontaneous release of IL-12 by macrophages, while confrontation with MHV presents an environment-related difference, with both having the potential to support a Th1 response.
SseK1 and SseK2 Are Novel Translocated Proteins ofSalmonella entericaSerovar Typhimurium
Authors: Kujat Choy Sonya L. , Boyle Erin C. , Gal-Mor Ohad , Goode David L. , Valdez Yanet , Vallance Bruce A. , Finlay B. Brett
Published: August 2004

Salmonella entericais a gram-negative, facultative intracellular pathogen that causes disease symptoms ranging from gastroenteritis to typhoid fever. A key virulence strategy is the translocation of bacterial effector proteins into the host cell, mediated by the type III secretion systems (TTSSs) encoded inSalmonellapathogenicity island 1 (SPI-1) and SPI-2. InS. entericaserovar Typhimurium LT2, we identified the protein products ofSTM4157andSTM2137as novel candidate secreted proteins by comparison to known secreted proteins from enterohemorrhagicEscherichia coliandCitrobacter rodentium. The STM4157 and STM2137 proteins, which we have designated SseK1 and SseK2, respectively, are 61% identical at the amino acid level and differ mainly in their N termini. Western analysis showed that in vitro accumulation and secretion of these proteins in serovar Typhimurium were affected by mutations in the two-component systems SsrA/B and PhoP/Q, which are key mediators of intracellular growth and survival. SPI-2 TTSS-dependent translocation of recombinant SseK1::Cya was evident at 9 h postinfection of epithelial cells, while translocation of SseK2::Cya was not detected until 21 h. Remarkably, the translocation signal for SseK1 was contained within the N-terminal 32 amino acids. Fractionation of infected epithelial cells revealed that following translocation SseK1 localizes to the host cytosol, which is unusual among the currently knownSalmonellaeffectors. Phenotypic analysis of ΔsseK1, ΔsseK2, and ΔsseK1/ΔsseK2mutants provided evidence for a role that was not critical during systemic infection. In summary, this work demonstrates that SseK1 and SseK2 are novel translocated proteins of serovar Typhimurium.
Contribution of
Burkholderia cenocepacia
Flagella to Infectivity and Inflammation
Authors: Urban Teresa A. , Griffith Adam , Torok Anastasia M. , Smolkin Mark E. , Burns Jane L. , Goldberg Joanna B.
Published: August 2004

Burkholderia cenocepacia
is an opportunistic pathogen that can cause severe lung infections in cystic fibrosis patients. To understand the contribution of
B. cenocepacia
flagella to infection, a strain mutated in the major flagellin subunit,
fliCII
, was constructed in
B. cenocepacia
K56-2 and tested in a murine agar bead model of lung infection. C57/BL6 mice infected with ∼10
8
wild-type K56-2 bacteria exhibited 40% mortality after 3 days, whereas no mortality was noted in mice infected with the
fliCII
mutant. Among the mice surviving the infection with either strain, there was no significant difference in the bacterial loads in the lungs and spleen, bacteremia, weight loss, or infiltration of immune effector cells at 3 days postinfection. Similar results were observed at 24 h, prior to expression of the lethality phenotype. KC, a murine interleukin-8 (IL-8) homolog, was elevated in both the bronchoalveolar lavage fluid and serum of mice infected with the wild type compared to the
fliCII
mutant at 24 h, suggesting that flagella stimulated host cells. To demonstrate that flagella contributed to these responses, the interaction between
B. cenocepacia
and Toll-like receptor 5 (TLR5) was investigated. Infection of HEK293 cells with heat-killed wild-type K56-2, but not infection with the
fliCII
mutant, resulted in both NF-κB activation and IL-8 secretion that was dependent upon expression of TLR5. Together, these results demonstrate that
B. cenocepacia
flagella contribute to virulence in an in vivo infection model, and that induction of host immune responses through interaction with TLR5 may contribute to its overall pathogenic potential.
Gamma Interferon Responses toPlasmodium falciparumLiver-Stage Antigen 1 and Thrombospondin-Related Adhesive Protein and Their Relationship to Age, Transmission Intensity, and Protection against Malaria
Authors: John Chandy C. , Moormann Ann M. , Sumba Peter O. , Ofulla Ayub V. , Pregibon Daniel C. , Kazura James W.
Published: August 2004

Gamma interferon (IFN-γ) responses to thePlasmodium falciparumantigens liver-stage antigen 1 (LSA-1) and thrombospondin-related adhesive protein (TRAP) are thought to be important in protection against malaria. Optimal methods of testing and the effects of age and transmission intensity on these responses are unknown. IFN-γ responses to LSA-1 and TRAP peptides were assessed by the enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) in children and adults from areas of stable and unstable malaria transmission in Kenya. Adults in the areas of stable and unstable transmission had similar frequencies and levels of IFN-γ responses to LSA-1 and TRAP as determined by ELISPOT and ELISA. In contrast, IFN-γ responses to the LSA-1 T3 peptide (assessed by ELISPOT) and to any LSA-1 peptide (assessed by ELISA) were less frequent in children in the area of unstable transmission than in children in the area of stable transmission. IFN-γ responses to LSA-1 were more frequently detected by ELISA than by ELISPOT in the stable-transmission area. IFN-γ responses detected by ELISA and ELISPOT did not correlate with each other. In children in the stable-transmission area, IFN-γ responses to LSA-1 peptides assessed by ELISA, but not by ELISPOT, were associated with protection against clinical malaria and anemia. IFN-γ responses to LSA-1 appear to require repeatedP. falciparumexposure and/or increased age and, as measured by ELISA, are associated with protection against clinical malaria and anemia.
Differential Requirements for VirB1 and VirB2 during
Brucella abortus
Infection
Authors: den Hartigh Andreas B. , Sun Yao-Hui , Sondervan David , Heuvelmans Niki , Reinders Marjolein O. , Ficht Thomas A. , Tsolis Renée M.
Published: August 2004

The
Brucella abortus virB
operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the
virB
operon,
virB1
and
virB2
, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of
virB1
and
virB2
. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene
virB5
during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar
virB2
mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar
virB1
mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.
Mycobacterium tuberculosisTriggers Apoptosis in Peripheral Neutrophils Involving Toll-Like Receptor 2 and p38 Mitogen Protein Kinase in Tuberculosis Patients
Authors: Alemán Mercedes , Schierloh Pablo , de la Barrera Silvia S. , Musella Rosa M. , Saab María A. , Baldini Matías , Abbate Eduardo , Sasiain María C.
Published: August 2004

Polymorphonuclear neutrophils (PMN) exposed toMycobacterium tuberculosisdisplay bactericidal responses and produce inflammatory proteins. This PMN-mediated inflammatory response is regulated by an activation of the apoptotic program, which collaborates to avoid tissue injury. In vitro, circulating PMN from patients with tuberculosis (TB) show an increased spontaneous apoptosis, andM. tuberculosis-induced activation accelerates the PMN apoptosis. In this study, we evaluated the mechanisms involved in spontaneous andM. tuberculosis-induced apoptosis. We demonstrate that apoptosis of PMN is not induced by lipoarabinomannan or by a whole-cell lysate ofM. tuberculosisand that neither tumor necrosis factor alpha nor CD11b, CD14, and Fcγ receptors are involved. Apoptosis of PMN from patients with active TB (TB-PMN) is induced by the interaction with the wholeM. tuberculosisvia Toll-like receptor 2 (TLR2), and, in contrast to spontaneous apoptosis, it involves the p38 mitogen-activated protein kinase (MAPK) pathway. These results correlate with a high expression of phosphorylated p38 (p-p38) in circulating TB-PMN and with the ability ofM. tuberculosisto induce in vitro the expression of p-p38 in PMN. Therefore, when the bacterial burden is low, TB-PMN could be detecting nonopsonizedM. tuberculosisvia TLR2, leading to the activation of the p38 MAPK pathway, which in turn would induce PMN activation and apoptosis. This mechanism needs further confirmation at the site of infection.
Proteus mirabilis
ZapA Metalloprotease Degrades a Broad Spectrum of Substrates, Including Antimicrobial Peptides
Authors: Belas Robert , Manos Jim , Suvanasuthi Rooge
Published: August 2004

The 54-kDa extracellular metalloprotease ZapA is an important virulence factor of uropathogenic
Proteus mirabilis
. While ZapA has the ability to degrade host immunoglobulins (Igs), the dramatic attenuation of virulence in ZapA mutants suggests that this enzyme may have a broader spectrum of activity. This hypothesis was tested by in vitro assays with purified ZapA and an array of purified protein or peptide substrates. The data reveal that many proteins found in the urinary tract are substrates of ZapA proteolysis, including complement (C1q and C3), cell matrix (collagen, fibronectin, and laminin), and cytoskeletal proteins (actin and tubulin). Proteolysis of IgA and IgG was significantly enhanced by conditions that denatured the Igs. It was discovered that the antimicrobial peptides human β-defensin 1 (hBD1) and LL-37 are readily cleaved by the enzyme. To the best of our knowledge, this is the first report of a bacterial protease capable of cleaving hBD1, a component of the human renal tubule innate immune response. Proteolysis of hBD1 resulted in ca. six peptides, while proteolysis of LL-37 resulted in at least nine products. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of the molecular masses of the reaction products indicated that ZapA preferred no distinct peptide bond. The antimicrobial activity of hBD1 and LL-37 was significantly reduced following ZapA treatment, suggesting that proteolysis results in inactivation of these peptides. The data suggest that a function of ZapA during urinary tract infections is the proteolysis of antimicrobial peptides associated with the innate immune response.
Chemokine Gene Expression in Toll-Like Receptor-Competent and -Deficient Mice Infected with
Leishmania major
Authors: Antoniazi Simone , Price Helen P. , Kropf Pascale , Freudenberg Marina A. , Galanos Chris , Smith Deborah F. , Müller Ingrid
Published: August 2004

We studied the expression of a subset of chemokines, including RANTES/CCL5, MIP-1α/CCL3, IP-10/CXCL10, and MCP-1/CCL2, in Toll-like receptor (TLR)-competent and -deficient mice after infection with
Leishmania major
. Chemokine expression at the site of infection (the footpad), in the draining lymph nodes and in the spleens of infected animals was determined by using two different methods of analysis. The results indicate that
L. major
infection causes overall upregulation of RANTES/CCL5, MIP-1α/CCL3, IP-10/CXCL10, and MCP-1/CCL2 in the footpads and lymph nodes, while expression of these chemokines is constitutive in the spleens of TLR4-competent mice (C57BL/10ScSn) and TLR4-deficient mice (C57BL10/ScN). Different patterns of expression were detected depending on the time postinfection, but there was little variation in the expression of these four chemokines in the presence or absence of TLR4.
Age-Related Resistance of C57BL/6 Mice toCryptococcus neoformansIs Dependent on Maturation of NKT Cells
Authors: Blackstock Rebecca , Murphy Juneann W.
Published: August 2004

Conflicting results have been reported regarding the ability of C57BL/6 mice to clear infections due toCryptococcus neoformans. Examination of the various experimental protocols used suggested that C57BL/6 mice might develop the ability to resist infection as they mature. We analyzed the ability of C57BL/6 mice of different ages to respond to immunization with cryptococcal antigen or to clear a cryptococcal infection. Mice were immunized with a soluble cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund's adjuvant (CneF-CFA). Delayed-type hypersensitivity (DTH) reactions elicited by the immunization were significantly stronger in 15-week-old C57BL/6 mice than in 7-week-old mice. Analysis of cryptococcal CFU 8 weeks following intratracheal infection of 7-week-old mice or 15-week-old mice revealed a relative inability of the younger animals to control the infection. Six-week-old immunized and infected mice cleared cryptococci from brain, spleen, and liver in a manner similar to that of immunized and infected 15-week-old mice. However, the older mice cleared cryptococci much more efficiently from the lungs. The possible role for NKT cells was determined by passive transfer of thymocytes from 10-week-old mice (containing mature NKT cells) or 2-week-old mice (containing immature NKT cells) to 6-week-old mice. The 10-week-old thymocytes significantly enhanced the ability of the mice to develop a DTH response after immunization with CneF-CFA, while animals treated with 2-week-old thymocytes did not improve their DTH response after immunization. The cells in the 10-week-old thymocyte population responsible for improvement of DTH responses were identified as being NK1.1 positive.
Helicobacter pylori
Infection Targets Adherens Junction Regulatory Proteins and Results in Increased Rates of Migration in Human Gastric Epithelial Cells
Authors: Conlin Victoria S. , Curtis Susan B. , Zhao Ying , Moore Edwin D. W. , Smith Valerie C. , Meloche R. Mark , Finlay B. Brett , Buchan Alison M. J.
Published: August 2004

The human gastric pathogen
Helicobacter pylori
attaches to antral epithelial cells in vivo. Cultured human antral epithelial cells, AGS and NCI-N87 cell lines, were grown in the absence or presence of
H. pylori
and compared with respect to gene transcript levels, protein expression, organization of the actin cytoskeleton, and the regulation of cell migration. The Clontech Neurobiology array detected differentially expressed transcripts, while Western blots were used to investigate related changes in protein levels. Infection with
H. pylori
consistently upregulated annexin II, S100 A7, Rho-GTP, and IQGAP-1, whereas SSTR-1 was downregulated upon
H. pylori
infection. In the adherens junction, E-cadherin and IQGAP-1 were translocated from the plasma membrane to intracellular vesicles. The primary and NCI-N87 cells were similar with respect to cell-cell and cell-matrix adhesion and cell migratory behavior; in contrast the AGS cells were significantly different from the primary gastric epithelial cell preparations, and thus caution must be used when using this cell line for studies of gastric disease. These studies demonstrate a correlation between
H. pylori
infection and alterations to epithelial cell adhesion molecules, including increased levels of Rho-GTP and cell migration. These data indicate that destabilizing epithelial cell adherence is one of the factors increasing the risk of
H. pylori
-infected individuals developing gastric cancer.
The Cyclic AMP-Dependent Protein Kinase A Network Regulates Development and Virulence in
Aspergillus fumigatus
Authors: Liebmann Burghard , Müller Meike , Braun Armin , Brakhage Axel A.
Published: August 2004

Aspergillus fumigatus
is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. To determine the importance of the cyclic AMP (cAMP) signaling pathway for virulence, the
pkaC1
gene encoding a protein kinase A (PKA) catalytic subunit was cloned and characterized. Deletion of
pkaC1
led to reduced conidiation and growth. PKA activity was not detectable in Δ
pkaC1
, Δ
gpaB
, and Δ
acyA
mutant strains.
gpaB
and
acyA
encode a G protein α subunit involved in cAMP signal transduction and adenylate cyclase, respectively. Addition of cAMP led to PKA activity in crude extracts of both the Δ
gpaB
and Δ
acyA
strains but not in crude extracts of the Δ
pkaC1
strain. These findings provide evidence that PKAC1 represents the predominant form of PKA under the conditions tested, and GPAB and ACYA are members of the cAMP signaling cascade. Analysis of a
pksP
p-
lacZ
gene fusion indicated that the expression of the pathogenicity determinant-encoding
pksP
gene was reduced in Δ
pkaC1
mutant strains compared with the expression of the gene fusion in the parental strain. In a low-dose murine inhalation model, conidia of both the Δ
pkaC1
and Δ
gpaB
mutant strains were almost avirulent. Taken together, these findings indicate that the cAMP-PKA signal transduction pathway is required for
A. fumigatus
pathogenicity.
Effects of
Clostridium perfringens
Alpha-Toxin (PLC) and Perfringolysin O (PFO) on Cytotoxicity to Macrophages, on Escape from the Phagosomes of Macrophages, and on Persistence of
C. perfringens
in Host Tissues
Authors: O'Brien David K. , Melville Stephen B.
Published: August 2004

Clostridium perfringens
is the most common cause of clostridial myonecrosis (gas gangrene). Polymorphonuclear cells (PMNs) appear to play only a minor role in preventing the onset of myonecrosis in a mouse animal model of the disease (unpublished results). However, the importance of macrophages in the host defense against
C. perfringens
infections is still unknown. Two membrane-active toxins produced by the anaerobic
C. perfringens
, alpha-toxin (PLC) and perfringolysin O (PFO), are thought to be important in the pathogenesis of gas gangrene and the lack of phagocytic cells at the site of infection. Therefore,
C. perfringens
mutants lacking PFO and PLC were examined for their relative cytotoxic effects on macrophages, their ability to escape the phagosome of macrophages, and their persistence in mouse tissues.
C. perfringens
survival in the presence of mouse peritoneal macrophages was dependent on both PFO and PLC. PFO was shown to be the primary mediator of
C. perfringens
-dependent cytotoxicity to macrophages. Escape of
C. perfringens
cells from phagosomes of macrophage-like J774-33 cells and mouse peritoneal macrophages was mediated by either PFO or PLC, although PFO seemed to play a more important role in escape from the phagosome in peritoneal macrophages. At lethal doses (10
9
) of bacteria only PLC was necessary for the onset of myonecrosis, while at sublethal doses (10
6
) both PFO and PLC were necessary for survival of
C. perfringens
in mouse muscle tissue. These results suggest PFO-mediated cytotoxicity toward macrophages and the ability to escape macrophage phagosomes may be important factors in the ability of
C. perfringens
to survive in host tissues when bacterial numbers are low relative to those of phagocytic cells, e.g., early in an infection.
Gastric Transcription Profile of
Helicobacter pylori
Infection in the Rhesus Macaque
Authors: Huff Jennifer L. , Hansen Lori M. , Solnick Jay V.
Published: August 2004

Infection with
Helicobacter pylori
is usually asymptomatic but sometimes progresses to peptic ulcer disease or gastric adenocarcinoma. The development of disease involves both host and bacterial factors. In order to better understand host factors in pathogenesis, we studied the gastric transcription profile of
H. pylori
infection in the rhesus macaque by using DNA microarrays. Significant changes were found in the expression of genes important for innate immunity, chemokines and cytokines, cell growth and differentiation, apoptosis, structural proteins, and signal transduction and transcription factors. This broad transcription profile demonstrated expected up-regulation of cell structural elements and the host inflammatory and immune response, as well as the novel finding of down-regulation of heat shock proteins. These results provide a unique view of acute
H. pylori
infection in a relevant animal model system and will direct future studies regarding the host response to
H. pylori
infection.
Contribution of the Major Secreted Yops of
Yersinia enterocolitica
O:8 to Pathogenicity in the Mouse Infection Model
Authors: Trülzsch Konrad , Sporleder Thorsten , Igwe Emeka I. , Rüssmann Holger , Heesemann Jürgen
Published: August 2004

Pathogenic yersiniae (
Yersinia pestis
,
Y. pseudotuberculosis
, and
Y. enterocolitica
) harbor a 70-kb virulence plasmid (pYV) that encodes a type III secretion system and a set of at least six effector proteins (YopH, YopO, YopP, YopE, YopM, and YopT) that are injected into the host cell cytoplasm. Yops (
Yersinia
outer proteins) disturb the dynamics of the cytoskeleton, inhibit phagocytosis by macrophages, and downregulate the production of proinflammatory cytokines, which makes it possible for yersiniae to multiply extracellularly in lymphoid tissue.
Y. enterocolitica
serotype O:8 belongs to the highly mouse-pathogenic group of yersiniae in contrast to
Y. enterocolitica
serotype O:9. However, there has been no systematic study of the contribution of Yops to the pathogenicity of
Y. enterocolitica
O:8 in mice. We generated a set of
yop
gene deletion mutants of
Y. enterocolitica
O:8 by using the novel Red cloning procedure. We subsequently analyzed the contribution of
yopH
,
-O
,
-P
,
-E
,
-M
,
-T
, and -
Q
deletions to pathogenicity after oral and intravenous infection of mice. Here we showed for the first time that a Δ
yopT
deletion mutant colonizes mouse tissues to a greater extent than the parental strain. The Δ
yopO
, Δ
yopP
, and Δ
yopE
mutants were only slightly attenuated after oral infection since they were still able to colonize the spleen and liver and cause systemic infection. The Δ
yopO
mutant was lethal for mice, whereas
ΔyopP
and Δ
yopE
mutants were successfully eliminated from the spleen and liver 2 weeks after infection. In contrast the Δ
yopH
, Δ
yopM
, and Δ
yopQ
mutants were highly attenuated and not able to colonize the spleen and liver on any of the days tested. The Δ
yopH
, Δ
yopO
, Δ
yopP
, Δ
yopE
, Δ
yopM
, and Δ
yopQ
mutants had only modest defects in the colonization of the small intestine and Peyer's patches. The Δ
yopE
mutant was eliminated from the small intestine 3 weeks after infection, whereas the Δ
yopH
,
ΔyopP
,
ΔyopM
, and
ΔyopQ
mutants continued to colonize the small intestine at this time.
Activation of Dendritic Cells by Liposomes Prepared from Phosphatidylinositol Mannosides fromMycobacterium bovisBacillus Calmette-Guérin and Adjuvant Activity In Vivo
Authors: Sprott G. Dennis , Dicaire Chantal J. , Gurnani Komal , Sad Subash , Krishnan Lakshmi
Published: August 2004

Liposome vesicles could be formed at 65°C from the chloroform-soluble, total polar lipids (TPL) extracted fromMycobacterium bovisbacillus Calmette-Guérin (BCG). Mice immunized with ovalbumin (OVA) entrapped in TPL liposomes produced both anti-OVA antibody and cytotoxic T lymphocyte responses. Murine bone marrow-derived dendritic cells were activated to secrete interleukin-6 (IL-6), IL-12, and tumor necrosis factor upon exposure to antigen-free TPL liposomes. Three phosphoglycolipids and three phospholipids comprising 96% of TPL were identified as phosphatidylinositol dimannoside, palmitoyl-phosphatidylinositol dimannoside, dipalmitoyl-phosphatidylinositol dimannoside, phosphatidylinositol, phosphatidylethanolamine, and cardiolipin. The activation of dendritic cells by liposomes prepared from each purified lipid component of TPL was evaluated in vitro. A basal activity of phosphatidylinositol liposomes to activate proinflammatory cytokine production appeared to be attributable to the tuberculosteric fatty acyl 19:0 chain characteristic of mycobacterial glycerolipids, as similar lipids lacking tuberculosteric chains showed little activity. Phosphatidylinositol dimannoside was identified as the primary lipid that activated dendritic cells to produce amounts of proinflammatory cytokines several times higher than the basal level, indicating the importance of mannose residues. Although the activity of phosphatidylinositol dimannoside was little influenced by palmitoylation of mannose at C-6, a further palmitoylation at inositol C-3 diminished the induction levels of IL-6 and IL-12. Further, OVA entrapped in palmitoyl-phosphatidylinositol dimannoside liposomes was delivered to dendritic cells for major histocompatibility complex class I presentation more effectively than TPL OVA-liposomes. BCG liposomes containing mannose lipids caused up-regulation of costimulatory molecules and CD40. Thus, the inclusion of pure phosphatidylinositol mannosides of BCG in lipid vesicle vaccines represents a simple and efficient option for targeting antigen delivery and providing immune stimulation.
Involvement of the Lectin Pathway of Complement Activation in Antimicrobial Immune Defense during Experimental Septic Peritonitis
Authors: Windbichler Michaela , Echtenacher Bernd , Hehlgans Thomas , Jensenius Jens C. , Schwaeble Wilhelm , Männel Daniela N.
Published: August 2004

A critical first line of defense against infection is constituted by the binding of natural antibodies to microbial surfaces, activating the complement system via the classical complement activation pathway. In this function, the classical activation pathway is supported and amplified by two antibody-independent complement activation routes, i.e., the lectin pathway and the alternative pathway. We studied the contribution of the different complement activation pathways in the host defense against experimental polymicrobial peritonitis induced by cecal ligation and puncture by using mice deficient in either C1q or factors B and C2. The C1q-deficient mice lack the classical complement activation pathway. While infection-induced mortality of wild-type mice was 27%, mortality of C1q-deficient mice was increased to 60%. Mice with a deficiency of both factors B and C2 lack complement activation via the classical, the alternative, and the lectin pathways and exhibit a mortality of 92%, indicating a significant contribution of the lectin and alternative pathways of complement activation to survival. For 14 days after infection, mannan-binding lectin (MBL)-dependent activation of C4 was compromised. Serum MBL-A and MBL-C levels were significantly reduced for 1 week, possibly due to consumption. mRNA expression profiles did not lend support for either of the two MBL genes to respond as typical acute-phase genes. Our results demonstrate a long-lasting depletion of MBL-A and MBL-C from serum during microbial infection and underline the importance of both the lectin and the alternative pathways for antimicrobial immune defense.
Population Structure of theBacillus cereusGroup as Determined by Sequence Analysis of Six Housekeeping Genes and theplcRGene
Authors: Ko Kwan Soo , Kim Jong-Wan , Kim Jong-Man , Kim Wonyong , Chung Sang-in , Kim Ik Jung , Kook Yoon-Hoh
Published: August 2004

The population structure of theBacillus cereusgroup (52 strains ofB. anthracis,B. cereus, andB. thuringiensis) was investigated by sequencing seven gene fragments (rpoB,gyrB,pycA,mdh,mbl,mutS, andplcR). Most of the strains were classifiable into two large subgroups in six housekeeping gene trees but not in theplcRtree. In addition, several consistent clusters were identified, which were unrelated to species distinction. Moreover, interrelationships among these clusters were incongruent in each gene tree. The incongruence length difference test and split decomposition analyses also showed incongruences between genes, suggesting horizontal gene transfer. TheplcRgene was observed to have characteristics that differed from those of the other genes in terms of phylogenetic topology and pattern of sequence diversity. Thus, we suggest that the evolutionary history of the PlcR regulon differs from those of the other chromosomal genes and that recombination of theplcRgene may be frequent. The homogeneity ofB. anthracis, which is depicted as an independent lineage in phylogenetic trees, is suggested to be of recent origin or to be due to the narrow taxonomic definition of species.
Comparison of Disseminated and Nondisseminated Strains ofBorrelia burgdorferiSensu Stricto in Mice Naturally Infected by Tick Bite
Authors: Dolan Marc C. , Piesman Joseph , Schneider Bradley S. , Schriefer Martin , Brandt Kevin , Zeidner Nordin S.
Published: August 2004

Clinical isolates ofBorrelia burgdorferisensu stricto have been categorized into disseminated and nondisseminated groups based on distinct ribosomal spacer restriction fragment length polymorphism genotypes (RSTs). In order to determine whether transmission by tick bite would alter the dissemination dynamics and disease produced by distinct genotypes, disseminated isolates (RST1), nondisseminated isolates (RST3), and a standard laboratory strain (B-31) were established in a murine cycle utilizing infections transmitted by ticks. B-31 spirochetes circulated in the blood of inbred C3H/HeJ mice longer than in the blood of outbred mice. The majority of C3H mice exposed to RST1-infected ticks contained cultivable spirochetes in their blood for up to 17 days; in contrast, mice exposed to RST3 isolates demonstrated a precipitous decline in infection after day 7 postexposure. A quantitative PCR (q-PCR) assay demonstrated that the densities of spirochetes in blood were similar for the RST1 and RST3 isolates, except during the 2nd week postexposure, when the RST1 isolates displayed a markedly higher density in blood. Spirochete load in the heart and bladder of infected mice was measured by q-PCR at 8 weeks postexposure; the numbers of spirochetes in these tissues were similar for mice infected with either disseminated or nondisseminated strains. Similarly, histopathology samples of heart, bladder, and joint tissue obtained at 8 weeks postexposure did not reveal greater pathology in mice infected with the disseminated isolates. We conclude that although the spirochetemia induced by tick-transmitted disseminated isolates was more intense and of longer duration than that induced by nondisseminated isolates, the resultant pathologies produced by these strains were ultimately similar.
Lack of an Association between Antibodies toPlasmodium falciparumGlycosylphosphatidylinositols and Malaria-Associated Placental Changes in Cameroonian Women with Preterm and Full-Term Deliveries
Authors: Suguitan Amorsolo L. , Gowda D. Channe , Fouda Genevieve , Thuita Lucy , Zhou Ainong , Djokam Rosine , Metenou Simon , Leke Rose G. F. , Taylor Diane Wallace
Published: August 2004

Sequestration ofPlasmodium falciparumparasites within the placenta often leads to an accumulation of macrophages within the intervillous space and increased production of tumor necrosis factor alpha (TNF-α), a cytokine associated with placental pathology and poor pregnancy outcomes.P. falciparumglycosylphosphatidylinositol (GPI) anchors have been shown to be the major parasite component that induces TNF-α production by monocytes and macrophages. Antibodies againstP. falciparumGPI (anti-PfGPI), however, can inhibit the induction of TNF-α and inflammation. Thus, the study was undertaken to determine whether anti-PfGPI antibodies down-regulate inflammatory-type changes in the placentas of women with malaria. Anti-PfGPI immunoglobulin M (IgM) and IgG levels were measured in 380 pregnant women with or without placental malaria, including those who delivered prematurely and at term. Results showed that anti-PfGPI antibody levels increased with gravidity and age and that malaria infection boosted anti-PfGPI antibodies in pregnant women. However, no association was found between anti-PfGPI antibodies and placental TNF-α levels or the presence of acute or chronic placental malaria. Furthermore, anti-PfGPI antibody levels were similar in women with preterm and full-term deliveries and were not associated with an increase in infant birth weight. Thus, these results fail to support a strong role for anti-PfGPI antibodies in the prevention of chronic placental malaria infections and malaria-associated poor birth outcomes.