Establishment of an Animal Model Using Recombinant NOD.B10.D2 Mice To Study Initial Adhesion of Oral Streptococci

An oral biofilm is a community of surface-attached microorganisms that coats the oral cavity, including the teeth, and provides a protective reservoir for oral microbial pathogens, which are the primary cause of persistent and chronic infectious diseases in patients with dry mouth or Sjögren''s syndrome (SS). The purpose of this study was to establish an animal model for studying the initial adhesion of oral streptococci that cause biofilm formation in patients with dry mouth and SS in an attempt to decrease the influence of cariogenic organisms and their substrates. In nonobese diabetogenic (NOD) mice that spontaneously develop insulin-dependent diabetes mellitus (IDDM) and SS, we replaced major histocompatibility complex (MHC) class II (A^g7 E^g7) and class I D^b with MHC class II (A^d E^d) and class I D^d from nondiabetic B10.D2 mice to produce an animal model that inhibited IDDM without affecting SS. The adhesion of oral streptococci, including Streptococcus mutans, onto tooth surfaces was then investigated and quantified in homologous recombinant N5 (NOD.B10.D2) and N9 (NOD.B10.D2) mice. We found that a higher number of oral streptococci adhered to the tooth surfaces of N5 (NOD.B10.D2) and N9 (NOD.B10.D2) mice than to those of the control C57BL/6 and B10.D2 mice. On the basis of our observation, we concluded that these mouse models might be useful as animal models of dry mouth and SS for in vivo biological studies of oral biofilm formation on the tooth surfaces.Oral streptococci are present in large numbers in dental plaque, and several types interact with the enamel salivary pellicle to form a biofilm on tooth surfaces (9, 16, 17, 21, 29). Streptococci account for approximately 20% of the total number of salivary bacteria (24), with Streptococcus salivarius being the primary organism. Further, the densities of Streptococcus mutans and Streptococcus sanguis in saliva are more than 1 × 10⁵ cells per ml. S. mutans is a pioneering organism that plays an important role in biofilm formation on tooth surfaces and is a primary causative agent of dental caries (9, 16, 21). The mechanical forces of salivary flow and tongue movement tend to dislodge and expel bacteria from tooth surfaces and the oral cavity (3, 5, 6), and their importance in controlling microbial colonization in the oral cavity has been well demonstrated in individuals with diabetes mellitus, Sjögren''s syndrome (SS), and dry mouth, who suffer from a rapid overgrowth of biofilm and rampant caries, making them highly susceptible to oral infections (1-2, 6). Thus, attempts to investigate the initial adhesion by oral streptococci, including S. mutans, in mouse models are likely to aid in the understanding and prevention of oral infectious diseases caused by the components of oral biofilm.Previous studies of S. mutans infections in the oral cavities of mice have been performed by feeding the animals diets containing sucrose in the presence of glucans (13, 15, 30, 43). Since the adherence of S. mutans to the tooth surface may depend on the balance between physical adherence and synthesis of insoluble glucans in a natural environment, that infection method may be inappropriate for investigation of natural biofilm formation associated with streptococci, including S. mutans (18, 39).The nonobese diabetogenic (NOD) mouse strain is currently the best available model for the study of insulin-dependent type 1 diabetes mellitus (IDDM) and SS (11, 31), both of which develop spontaneously and are characterized by lymphatic infiltration of the pancreas and salivary glands. Oral changes are prominent features of these diseases, which are manifested by dry mouth and hyposalivation (6, 7, 37). NOD mice are also used as an animal model for the study of oral infectious diseases associated with systemic diseases such as diabetes and SS or dry mouth.The unique major histocompatibility complex (MHC) class II genes (I-A^g7, no expression of I-E) represent dominant susceptibility factors and mediate activated T cells during the development of diabetes in NOD mice (11, 22, 25, 36, 41, 42). In the NOD model of SS, histopathological analyses of the salivary glands in MHC-congenic strains of NOD mice have indicated that the I-A^g7 region is not required for lymphocytic infiltration (26, 31). Further, replacement of the NOD MHC class I K^d region with another haplotype, MHC class I K^wm7, as well as replacement of the MHC class II A^g7 E^g7 and class I D^d regions with the corresponding region from the other MHC haplotype, has been shown to prevent diabetes (12). However, replacement with MHC class I K does not completely prevent development of insulitis. In another report, NOD mice pretreated nasally by using peptides restricted with MHC class I K^d showed a delayed onset of spontaneous IDDM, though insulitis could not be prevented by the induction of tolerance (23).In the present study, we attempted to establish an animal model for oral infectious diseases such as dental caries by focusing on replacement of the MHC class II and class I D region but not the class I K region in nondiabetic NOD mice by outcrossing B10.D2 mice (K^d, I-A^d, and D^d) with NOD mice (K^d, I-A^g7, and D^b) because the MHC class I K region in B10.D2 mice is identical with that in NOD mice (12). The present backcrossed and intercrossed NOD mice with the MHC class II and MHC class I D region replaced with that from B10.D2 mice developed SS, however, not diabetes. We then attempted to determine whether these mice would be useful as animal models for a sucrose-free study of the initial adhesion of oral streptococci on tooth surfaces in humans.     

Association of Single Nucleotide Polymorphisms in Cytotoxic T-Lymphocyte Antigen 4 and Susceptibility to Autoimmune Type 1 Diabetes in Tunisians

In addition to HLA and insulin genes, the costimulatory molecule CTLA-4 gene is a confirmed type 1 diabetes (T1D) susceptibility gene. Previous studies investigated the association of CTLA-4 genetic variants with the risk of T1D, but with inconclusive findings. Here, we tested the contributions of common CTLA-4 gene variants to T1D susceptibility in Tunisian patients and control subjects. The study subjects comprised 228 T1D patients (47.8% females) and 193 unrelated healthy controls (45.6% females). Genotyping for CTLA-4 CT60A/G (rs3087243), +49A/G (rs231775), and −318C/T (rs5742909) was performed by PCR-restriction fragment length polymorphism (RFLP) analysis. The minor-allele frequencies (MAF) for the three CTLA-4 variants were significantly higher in T1D patients, and significantly higher frequencies of homozygous +49G/G and homozygous CT60G/G genotypes were seen in patients, which was confirmed by univariate regression analysis (taking the homozygous wild type as a reference). Of the eight possible three-locus CTLA-4 haplotypes (+49A/G, −318C/T, and CT60A/G) identified, multivariate regression analysis confirmed the positive association of ACG (odds ratio [OR], 1.93; 95% confidence interval [CI], 1.26 to 2.94), GCG (OR, 2.40; 95% CI, 1.11 to 5.21), and GTA (OR, 4.67; 95% CI, 1.52 to 14.39) haplotypes with T1D, after confounding variables were adjusted for. Our results indicate that CTLA-4 gene variants are associated with increased T1D susceptibility in Tunisian patients, further supporting a central role for altered T-cell costimulation in T1D pathogenesis.Type 1 (insulin-dependent) diabetes (T1D) is the most prevalent form of diabetes in children and young adults and results from autoimmune CD4⁺ and CD8⁺ T-cell-directed destruction of insulin-producing pancreatic β islet cells in genetically susceptible individuals (3, 12), leading to irreversible hyperglycemia and related complications (13). There is a strong genetic component to T1D pathogenesis, evidenced by its clustering in families and by the contributions of a number of susceptibility gene variants to its pathogenesis (10, 12, 29). They include the human leukocyte antigen (HLA) locus, in particular the class II region (DR and DQ), which accounts for 40 to 50% of T1D familial clustering (1, 12, 18), and non-HLA susceptibility loci, several of which were mapped by genome-scanning (11, 29) and/or candidate gene (7, 18, 31) approaches. They include insulin promoter gene variants, which reportedly may modulate immunological tolerance by controlling the expansion of the autoreactive cell pool (26), and the T-cell costimulator cytotoxic T-lymphocyte antigen 4 (CTLA-4) transmembrane glycoprotein, which plays a key role in the fine tuning of T-cell immunity (9, 32, 33).CTLA-4 is a 40-kDa transmembrane glycoprotein expressed on resting and activated T cells and nonlymphoid cells (33), and along with the related CD28 costimulatory molecule, it regulates T-cell activation (and is itself primarily mediated by engagement of the T-cell receptor [TCR]) but does recognize major histocompatibility complex (MHC)-bound antigenic peptides (9, 33). CTLA-4 negatively regulates T-cell activation and effector function, in part by inhibiting Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-γ]) cytokine production and IL-2 receptor α-chain (p55; Tac) expression by engaging antigen-presenting cell (APC)-bound B7.1 (CD80) and B7.2 (CD86) ligands (9, 33). Functionally, CTLA-4 attenuates T-cell signaling by interference with intracellular signal transduction events, including TCR signaling, and reduced CTLA-4 expression and/or activity results in uncontrolled T-cell-associated autoimmunity and lymphoproliferative disease (9, 21). In this regard, it was shown that CTLA-4 polymorphisms significantly influence the risk of autoimmune diseases, including Graves'' disease, systemic lupus erythematosus, autoimmune hypothyroidism, celiac disease, and type 1 diabetes (15, 21, 32).First observed in Italian subjects (25), and confirmed subsequently by case control and family studies, CTLA-4 polymorphic variants were linked with T1D pathogenesis (14, 20, 31, 32). While this association was detected in different ethnic groups (14, 23, 30), it appears more likely to be Caucasian selective (10, 29, 33) and absent from non-Caucasians (5, 6, 8, 19, 22). A recent report from the Type I Diabetes Genetics Consortium bearing on 2,300 affected sib pair families demonstrated that among the 24 single nucleotide polymorphisms (SNPs) genotyped in the CTLA-4 region, only the +49A/G and CT60 SNPs were replicated in the nine combined collections (27). In the present study, we investigated the association of three common CTLA-4 SNPs (−318C/T; +49A/G, and CT60A/G) and the corresponding haplotypes with T1D in Tunisian Arab patients.     

Involvement of SOCS3 in Regulation of CD11c+ Dendritic Cell-Derived Osteoclastogenesis and Severe Alveolar Bone Loss

To investigate the role of suppressor of cytokine signaling (SOCS) molecules in periodontal immunity and RANKL-mediated dendritic cell (DC)-associated osteoclastogenesis, we analyzed SOCS expression profiles in CD4⁺ T cells and the effect of SOCS3 expression in CD11c⁺ DCs during periodontal inflammation-induced osteoclastogenesis and bone loss in nonobese diabetic (NOD) versus humanized NOD/SCID mice. Our results of ex vivo and in vitro analyses showed that (i) there is significantly higher SOCS3 expression associated with RANKL⁺ T-cell-mediated bone loss in correlation with increased CD11c⁺ DC-mediated osteoclastogenesis; (ii) the transfection of CD11c⁺ DC using an adenoviral vector carrying a dominant negative SOCS3 gene significantly abrogates TRAP and bone-resorptive activity; and (iii) inflammation-induced TRAP expression, bone resorption, and SOCS3 activity are not associated with any detectable change in the expression levels of TRAF6 and mitogen-activated protein kinase signaling adaptors (i.e., Erk, Jnk, p38, and Akt) in RANKL⁺ T cells. We conclude that SOCS3 plays a critical role in modulating cytokine signaling involved in RANKL-mediated DC-derived osteoclastogenesis during immune interactions with T cells and diabetes-associated severe inflammation-induced alveolar bone loss. Therefore, the development of SOCS3 inhibitors may have therapeutic potential as the target to halt inflammation-induced bone loss under pathological conditions in vivo.Inflammatory bone diseases affect a large portion of the skeletal system, particularly those portions underlying mucosal surfaces, where inflammation-induced bone loss is closely associated with elevated osteoclast (OC) activity (15, 48). Inflammation-induced bone loss is readily manifested in periodontal disease (i.e., periodontitis), resulting in attachments loss, including periodontal connective tissues and supporting alveolar bone, as a consequence of the interplays between the specific subgingival biofilm and the host''s immune/inflammatory responses (36, 37). The tumor necrosis factor (TNF) family member receptor activator of NF-κB ligand (RANKL), its receptor, RANK, and the natural antagonist, osteoprotegrin (OPG), have been shown to be the key regulators of the differentiation, activation, and survival of OCs and OC. precursors (5, 16-18, 23-25, 44, 55, 58). In addition, it is now clear that the host responses, especially T-cell immunity, play a pivotal role in regulating osteoclastogenesis and homeostasis of the bone tissues (termed osteoimmunology [5]). Activated CD4⁺ T cells express RANKL, which can directly trigger osteoclastogenesis and bone loss, and they can also negatively regulate RANKL activity via OPG production. For instance, it has been shown that OPG treatment results in significantly reduced bone loss in arthritis, osteoporosis, and cancer-related bone metastasis (16-18, 24, 45, 48, 55, 58). In murine models of periodontitis, OPG injections yield a robust inhibition of alveolar bone loss of up to 80 to 100%, suggesting that the RANKL-RANK/OPG axis is the key pathway controlling periodontal osteoclastogenesis (6, 27, 46, 49, 53). In addition to its importance in regulating bone remodelling, RANKL-RANK signaling is also critical for lymph node formation and organogenesis and is involved with dendritic cell (DC) survival and DC-T-cell interactions (23, 55, 59) in which modulation of a complex cytokine network in osteoclastogenesis in vivo has been suggested and shown (45, 52, 53).DCs are efficient antigen-presenting cells (APCs) necessary for regulating T-cell immunity (9). They are detected in diseased tissues of periodontitis and arthritic joints, where they form aggregates with T-cell infiltrates in the inflammatory foci (9, 40, 53) and are believed to contribute indirectly to inflammation-induced bone loss. Recent studies showed that Flt3⁺ monocyte/macrophage (Mo/MQ) precursors may differentiate to OCs, microglia, or DCs, thereby suggesting that these cell types may share common progenitors (2, 9, 26, 32). We recently reported the development of functional OCs from a CD11c⁺ DC subset(s) capable of inducing bone resorption in vitro and post-adoptive transfer in vivo (called DC-derived OCs [DDOC]) (1-4). Moreover, under arthritic conditions, human Mo-derived DCs have been shown to trans-differentiate into OCs in vitro, collectively suggesting that DCs may contribute directly to pathological bone loss (1, 2, 38). Thus, further understanding of the molecular mechanisms during DC-T-cell interactions driving disease pathogenesis and the resulting bone loss is imperative to facilitate the development of novel therapeutics for future treatments.Suppressors of cytokine signaling (SOCS) family and cytokine-inducible SH2 domain-containing protein (CIS) are cytoplasmic adaptor proteins that regulate various cytokine responses in leukocytes via negative feedback loops to inhibit inflammatory stimuli (62). There are eight members, including SOCS 1 to 7 and CIS, and each contains an N terminus, a central SH2 domain, and a C-terminal SOCS box. SOCS inhibits cytokine signaling by binding to phosphorylated JAKs and cytokine receptors and interacting with E3 ubiquitin ligases to polyubiquitylate JAKs for degradation (62). Several studies have investigated the roles of SOCS family members in regulating the development and function of immune cells. For instance, SOCS1 and -3 contain a kinase inhibitory region (KIR) in the N terminus, which inhibits JAK tyrosine kinase activity (35, 60, 61). As cytokine signaling activates JAK/STAT pathways, yielding activated SOCS molecules, SOCS1 is known to bind to JAKs with inhibitory catalytic activity and SOCS3 binds to its proximal sites on cytokine receptors, thereby directly inhibiting JAK signaling (62). These studies established the involvement of SOCS1 and SOCS3 molecules in regulating APCs (i.e., DCs) and T-cell functions during both innate and adaptive immunity (61, 62). Meanwhile, researchers have just begun to elucidate the effects of SOCS expression on the development of OCs and OC precursors, whereby specific SOCS molecules regulating osteoclastogenesis via cytokine signaling have been shown (35, 62). To date, however, the role(s) of SOCS family molecules and their impact on modulating RANKL expression and alveolar bone destruction in periodontal lesions remain unclear. Of particular interest to us is SOCS3, as it has been previously shown to be involved during inflammation-induced osteoclastogenesis and its activity is closely associated with key osteotropic cytokines, such as interleukin-1 (IL-1), TNF-α, transforming growth factor β, IL-6, IL-17, etc. (8, 10, 11, 35, 43, 60).We and others have established the NOD and humanized NOD/SCID mouse models to study periodontal inflammation and immunity and discovered that diabetic NOD mice manifest enhanced alveolar bone loss associated with increases in (i) T-cell proliferation and RANKL expression postinfection by anaerobic Aggregatibactor actinomycetemcomitans compared to prediabetic and nondiabetic NOD mice, and (ii) the Th1-inducing capability of the CD11c⁺ DC subset (14, 27-29). The enhanced alveolar bone loss in diabetic NOD and the physiological relevance of humanized NOD/SCID to periodontitis (14, 27, 49, 63) provide robust systems to study disease pathogenesis during the host-microbe interactions and alveolar bone loss. Herein, we employed these established animal models to assess the role of key SOCS molecules during periodontal immunity and RANKL-mediated alveolar bone loss by studying SOCS expression profiles in pathogen-reactive CD4⁺ T cells and the impact of halting functional SOCS3 on the development and activity of CD11c⁺ DDOC.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Transitions in Oral and Intestinal Microflora Composition and Innate Immune Receptor-Dependent Stimulation during Mouse Development

Commensal bacteria possess immunostimulatory activities that can modulate host responses to affect development and homeostasis in the intestine. However, how different populations of resident bacteria stimulate the immune system remains largely unknown. We characterized here the ability of intestinal and oral microflora to stimulate individual pattern recognition receptors (PRRs) in bone marrow-derived macrophages and mesothelial cells. The intestinal but not oral microflora elicited age- and cell type-specific immunostimulation. The immunostimulatory activity of the intestinal microflora varied among individual mice but was largely mediated via Toll-like receptor 4 (TLR4) during breast-feeding, whereas it became TLR4 independent after weaning. This transition was associated with a change from a microflora rich in TLR4-stimulatory proteobacteria to one dominated by Bacteroidales and/or Clostridiales that poorly stimulate TLR4. The major stimulatory activity of the intestinal microflora was still intact in NOD1-, NOD2-, TLR2-, TLR4-, TLR5-, TLR9-, TLR11-, ASC-, or RICK-deficient cells but still relied on the adaptor MyD88. These studies demonstrate a transition in the intestinal microflora accompanied by a dynamic change of its ability to stimulate different PRRs which control intestinal homeostasis.Accumulating evidence indicates that environmental bacteria can regulate the development and homeostasis of the host immune system, particularly within the gut, and affect susceptibility to a variety of diseases (3, 5, 6, 9, 10, 40, 44). Both humans and animals harbor a large number of nonpathogenic residential bacteria, especially in the intestine and oral cavity (41). Uncontrolled translocation of bacteria or bacterial components into systemic tissues of the host often results in bacteremia and sepsis (8), which causes significant mortality worldwide each year. On the other hand, intestinal bacteria contain immunostimulatory molecules that can regulate local immunity, epithelial development, immunotolerance, and susceptibility to inflammatory bowel disease (2, 41). The bacterium-derived molecules are recognized by innate immune receptors, including Toll-like receptors (TLRs) and Nod-like receptors (NLRs) (8, 20). TLRs and NLRs, often referred as pattern recognition receptors (PRRs), are involved in the recognition of commensal and pathogenic bacteria, as well as in the clearance of pathogens through interaction with their cognate microbial molecules (8, 20). Interactions between PRRs and commensal bacteria have been demonstrated to be important for gut homeostasis. MyD88, an essential adaptor for TLR signaling, has been shown to be important for epithelial homeostasis (40) and IgA secretion in the intestine (3, 44), and TLR9 has been shown to be important for the balance of regulatory T/Th17/Th1 cells (10). In addition, NOD1, an NLR family member, was shown to play a role in the development of intestinal lymphoid tissue via the recognition of commensal bacteria (5). Finally, genetic variation in NOD1 affects the susceptibility to allergic disease (17, 49) and Crohn''s disease (31) and that in NOD2 regulates susceptibility to Crohn''s disease (11, 16, 36) and graft-versus-host disease (14). In the oral cavity, the immunostimulatory properties of periodontal bacteria are believed to be important for the development of dental diseases (47).Although beneficial interactions between commensal bacteria and innate immune receptors have been demonstrated, the dominant bacterial species that are responsible for PRR stimulation in the normal intestine and oral cavity are unknown. Moreover, the commensal-dependent immunostimulatory activity for the various innate immune receptors has not been characterized. In humans, both genetic factors and the environment, including the diet, modulate the composition of the microflora (7, 15, 23, 33, 41, 48). Consequently, the intestinal microflora of individual humans is highly diverse (7, 15, 23, 33, 41). By taking advantage of the mouse model which allows a more uniform environmental and genetic platform, we analyzed the immunostimulatory activity induced by the oral and intestinal microflora and linked the activity to specific populations of commensal bacteria that emerge during postnatal development. We study provide here a comprehensive resource on microflora analysis in the mouse that is expected to facilitate future studies of the interaction between commensal bacteria and host immunity.     

Molecular Identification and Susceptibility of Trichosporon Species Isolated from Clinical Specimens in Qatar: Isolation of Trichosporon dohaense Taj-Aldeen,Meis & Boekhout sp. nov.

Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid leukemia. Sequence analysis revealed the isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp. nov., with CBS 10761^T as the holotype strain, belonging to the Ovoides clade. In the D1-D2 large-subunit rRNA gene analysis, T. dohaense is a sister species to T. coremiiforme, and in the internal transcribed spacer analysis, the species is basal to the other species of this clade. Molecular identification of the strains yielded 17 T. asahii, 3 T. inkin, 2 T. japonicum, 2 T. faecale, and 3 T. dohaense isolates. The former four species exhibited low MICs for five antifungal azoles but showed high MICs for amphotericin B. T. dohaense demonstrated the lowest amphotericin B MIC (1 mg/liter). For the majority of T. asahii isolates, amphotericin B MICs were high (MIC at which 90% of isolates were inhibited [MIC₉₀], ≥16 mg/liter), and except for fluconazole (MIC₉₀, 8 mg/liter), the azole MICs were low: MIC₉₀s were 0.5 mg/liter for itraconazole, 0.25 mg/liter for voriconazole, 0.25 mg/liter for posaconazole, and 0.125 mg/liter for isavuconazole. The echinocandins, caspofungin and anidulafungin, demonstrated no activity against Trichosporon species.Trichosporon species are yeast-like fungi, widely distributed in nature and commonly isolated from soil and other environmental sources, which have been involved in a variety of opportunistic infections and have been recognized as emerging fungal pathogens in immunocompromised hosts (19, 79, 80). Disseminated Trichosporon infections are potentially life-threatening and are often fatal in neutropenic patients (7, 22). Although uncommon, pathogenic species of this genus have been reported increasingly, mostly in patients with malignant diseases (3, 6, 9, 10, 11, 20, 32, 44, 47, 48, 63, 77), neonates (18, 56, 84), a bone marrow transplant recipient (22), a solid organ transplant recipient (50), and patients with human immunodeficiency virus (34, 35, 46). Trichosporon has also been reported to cause fungemia (5, 9, 25, 29, 30, 33, 53, 62). Members of the genus Trichosporon have occasionally been implicated as nail pathogens (16, 28, 74) and in subcutaneous infections (66). Trichosporon is considered an opportunistic agent, and therefore, recovery of Trichosporon species capable of growing at 37°C, especially from immunocompromised patients, should be regarded as potentially significant. Several reports have addressed the difficulty of identifying Trichosporon to the species level by physiological and biochemical characteristics (2, 64); therefore, molecular methods based on the sequencing of the internal transcribed spacer (ITS) have been developed (15, 69, 71, 72).In the present paper, we report the isolation of Trichosporon species from clinical specimens over a 4-year period in Qatar, the poor performance of biochemical identification methods, the significance of molecular identification, and the antifungal susceptibility data for the isolates. While investigating the molecular identification of Trichosporon species, we found three strains that do not match any of the published strains in the literature. We describe this organism as Trichosporon dohaense Taj-Aldeen, Meis & Boekhout, sp. nov., the name proposed for this species.     

Glyceraldehyde-3-Phosphate Dehydrogenase Is a Surface-Associated,Fibronectin-Binding Protein of Trichomonas vaginalis

Trichomonas vaginalis colonizes the urogenital tract of humans and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. We have shown an association of T. vaginalis with basement membrane extracellular matrix components, a property which we hypothesize is important for colonization and persistence. In this study, we identify a fibronectin (FN)-binding protein of T. vaginalis. A monoclonal antibody (MAb) from a library of hybridomas that inhibited the binding of T. vaginalis organisms to immobilized FN was identified. The MAb (called ws1) recognized a 39-kDa protein and was used to screen a cDNA expression library of T. vaginalis. A 1,086-bp reactive cDNA clone that encoded a protein of 362 amino acids with identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained. The gapdh gene was cloned, and recombinant GAPDH (rGAPDH) was expressed in Escherichia coli cells. Natural GAPDH and rGAPDH bound to immobilized FN and to plasminogen and collagen but not to laminin. MAb ws1 inhibited binding to FN. GAPDH was detected on the surface of trichomonads and was upregulated in synthesis and surface expression by iron. Higher levels of binding to FN were seen for organisms grown in iron-replete medium than for organisms grown in iron-depleted medium. In addition, decreased synthesis of GAPDH by antisense transfection of T. vaginalis gave lower levels of organisms bound to FN and had no adverse effect on growth kinetics. Finally, GAPDH did not associate with immortalized vaginal epithelial cells (VECs), and neither GAPDH nor MAb ws1 inhibited the adherence of trichomonads to VECs. These results indicate that GAPDH is a surface-associated protein of T. vaginalis with alternative functions.Trichomonas vaginalis, an extracellular protozoan parasite, is the cause of trichomonosis, the most prevalent nonviral sexually transmitted disease (47). In women, vaginitis due to T. vaginalis clinically manifests with symptoms of vaginal itching, odor, and discharge. Adverse health outcomes for women with this sexually transmitted disease include cervical cancer (46) and preterm delivery and low-birth-weight infants (25). There is a relationship between seropositivity to T. vaginalis and prostate cancer (43). This disease is significant due to its association with human immunodeficiency virus (33, 45). More recently, persistent, undetected T. vaginalis infections associated with asymptomatic carriage were found among women (40).T. vaginalis penetration of the mucous layer (28), followed by adherence to vaginal epithelial cells (VECs), is preparatory for colonization (9, 10). VEC adherence by parasites is mediated by numerous distinct trichomonad surface adhesins (5, 10, 18). Brief contact of T. vaginalis with VECs and fibronectin (FN) elicited dramatic changes in parasite morphology, suggesting a host-specific signaling of parasites (8, 9). Importantly, iron and cell contact by parasites each upregulated the expression of adhesins in a coordinated fashion via distinct mechanisms (2, 4, 6, 21, 29). Genetic approaches using antisense (AS) inhibition of synthesis (36, 37) and heterologous expression in Tritrichomonas foetus (26, 36) have reaffirmed the role of these T. vaginalis proteins as adhesins. T. vaginalis organisms secrete or release numerous metabolic enzymes, including adhesin AP65 (decarboxylating malic enzyme), α-enolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during growth and multiplication (27). AP65 and α-enolase were found to reassociate with the parasite surface for the expression of adhesin function (19) and binding to plasminogen (35), respectively.There is an increased awareness of the existence of metabolic enzymes on the surfaces of bacterial pathogens, yeast, and parasites (12, 24, 35). These surface-associated enzymes appear to be novel virulence factors (17, 22, 38, 39). The anchorless glycolytic enzymes GAPDH (13, 31, 38) and α-enolase (39) are present on the surface of group A streptococcus. The surface-associated GAPDH of Candida albicans binds with strong affinity to FN and laminin (22). In enterohemorrhagic Escherichia coli and enteropathogenic E. coli, GAPDH is an extracellular protein that binds human plasminogen and fibrinogen and also interacts with intestinal epithelial cells (17).We demonstrate that GAPDH is another enzyme on the surface of T. vaginalis. A monoclonal antibody (MAb) that inhibited parasite associations with FN was immunoreactive with GAPDH. Importantly, iron was found to regulate gene expression and synthesis and surface placement of GAPDH. Both low-iron-grown trichomonads and AS-transfected parasites with decreased amounts of GAPDH had smaller amounts of surface GAPDH and corresponding lower levels of binding to FN. GAPDH was not involved in adherence of trichomonads to immortalized VECs. Interestingly, as with other microbial pathogens, T. vaginalis GAPDH also bound plasminogen and collagen but not laminin (17, 22).     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Contribution of Vibrio parahaemolyticus Virulence Factors to Cytotoxicity,Enterotoxicity, and Lethality in Mice

Vibrio parahaemolyticus, one of the human-pathogenic vibrios, causes three major types of clinical illness: gastroenteritis, wound infections, and septicemia. Thermostable direct hemolysin (TDH) secreted by this bacterium has been considered a major virulence factor of gastroenteritis because it has biological activities, including cytotoxic and enterotoxic activities. Previous reports revealed that V. parahaemolyticus strain RIMD2210633, which contains tdh, has two sets of type III secretion system (T3SS) genes on chromosomes 1 and 2 (T3SS1 and T3SS2, respectively) and that T3SS1 is responsible for cytotoxicity and T3SS2 is involved in enterotoxicity, as well as in cytotoxic activity. However, the relative importance and contributions of TDH and the two T3SSs to V. parahaemolyticus pathogenicity are not well understood. In this study, we constructed mutant strains with nonfunctional T3SSs from the V. parahaemolyticus strain containing tdh, and then the pathogenicities of the wild-type and mutant strains were evaluated by assessing their cytotoxic activities against HeLa, Caco-2, and RAW 264 cells, their enterotoxic activities in rabbit ileal loops, and their lethality in a murine infection model. We demonstrated that T3SS1 was involved in cytotoxic activities against all cell lines used in this study, while T3SS2 and TDH had cytotoxic effects on a limited number of cell lines. T3SS2 was the major contributor to V. parahaemolyticus-induced enterotoxicity. Interestingly, we found that both T3SS1 and TDH played a significant role in lethal activity in a murine infection model. Our findings provide new indications that these virulence factors contribute to and orchestrate each distinct aspect of the pathogenicity of V. parahaemolyticus.Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that inhabits estuarine and coastal waters and can be isolated from seafood (6, 7, 9). It causes acute gastroenteritis in humans after they consume contaminated raw or undercooked seafood. Although this microorganism is better known for causing gastroenteritis, it also can cause wound infections and septicemia (5, 23, 36).Most clinical isolates of V. parahaemolyticus from patients with diarrhea show β-hemolysis on Wagatsuma agar (37). This phenomenon is known as the Kanagawa phenomenon (KP) and is considered a good marker to distinguish between pathogenic and nonpathogenic V. parahaemolyticus strains (25, 37). Thermostable direct hemolysin (TDH), which is responsible for the KP (28, 43), has multiple biological activities, including hemolysis, enterotoxicity, cytotoxicity, and cardiotoxicity (11-13, 26, 29, 32, 34, 38). For this reason, TDH has been considered a major virulence factor of V. parahaemolyticus.Whole-genome sequencing of a KP-positive V. parahaemolyticus strain revealed that this strain contains two sets of gene clusters for the type III secretion system (T3SS), T3SS1 and T3SS2, one on each of its two chromosomes (21). T3SS gene clusters have been detected in numerous Gram-negative animal and plant pathogens, where they enable delivery of virulence factor proteins (effectors) into the cytosol of eukaryotic cells (4, 8, 15). V. parahaemolyticus T3SS1 is reportedly involved in cytotoxic activity against a variety of cell lines (1, 2, 19, 31), while T3SS2 has been demonstrated to be involved in enterotoxicity in the rabbit ileal loop test, as well as in cytotoxic activity against Caco-2 and HCT-8 cells (18, 19, 33). However, these studies used a tdhAS deletion mutant strain of V. parahaemolyticus (POR-1) as the parent for construction of the T3SS deletion mutations to exclude any biological activities of TDH. It is therefore still not clear what the contribution of T3SSs to these activities is under conditions in which TDH is produced.V. parahaemolyticus sometimes also causes wound infections and septicemia (5, 23, 36). Unlike the information for gastrointestinal illness, there is no epidemiological information that indicates a correlation between any known virulence factor of V. parahaemolyticus and septicemia. The results of an intraperitoneal challenge experiment indicated that the lethality in mice due to V. parahaemolyticus infection occurred irrespective of the production of TDH, suggesting that another virulence factor(s) besides TDH may contribute to V. parahaemolyticus-induced septicemia (10).In this study, we constructed mutant strains with nonfunctional T3SSs from a V. parahaemolyticus strain possessing tdh to investigate the roles and contributions of the two T3SSs and TDH to the pathogenicity of V. parahaemolyticus (i.e., cytotoxic activity against several cell lines, enterotoxicity in the rabbit ileal loop test, and lethality in mice).     

Virulence and Cellular Interactions of Burkholderia multivorans in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens, but not with Escherichia coli. In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1β. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.Chronic granulomatous disease (CGD) is a rare primary immunodeficiency resulting from genetic defects in the phagocyte NAPDH oxidase. It is characterized by life-threatening infections caused by specific bacteria and fungi, leading to pneumonias, tissue abscesses, and exuberant granuloma formation (38). The Burkholderia cepacia complex (Bcc) includes at least 10 distinct species and is a leading cause of bacterial infections in CGD (44). Patients with cystic fibrosis (CF) also develop Bcc infections with various outcomes, ranging from no change in clinical course to a more rapid deterioration of lung function to the dreadful cepacia syndrome, which is characterized by necrotizing pneumonia and sepsis (25, 45). Interestingly, Bcc rarely causes infection in healthy individuals, but it can infect patients undergoing bronchoscopies and other procedures (4).Within the Bcc, Burkholderia cenocepacia and Burkholderia multivorans are commonly isolated from CF and non-CF patients (4, 32); the rate of B. multivorans infection now exceeds that of B. cenocepacia at several CF centers (15). In contrast to the high transmissibility of some CF B. cenocepacia strains (i.e., the epidemic lineage ET12) (24, 25), CF B. multivorans infections likely reflect independent acquisitions from unrelated sources (24). Curiously, unlike B. cenocepacia, B. multivorans has been recovered from environmental samples only rarely (1, 24), and it is the most frequently found species among CGD patients (16, 17).The mechanisms by which the Bcc causes disease specifically in CF are not known. Bcc isolates can survive within macrophages (28, 33) and respiratory epithelial cells (5, 21) and can invade epithelial cells in vivo (8, 10) and persist in the lung (9, 10). Cell infection assays using monocytes, macrophages, and epithelial cells (10, 11, 29, 46) show great variability among individual Bcc strains, with no clear correlation between those isolated from CF patients and those isolated from the environment (22). For the most part, these studies have been carried out using tissue culture models (28, 29, 43) and, in some cases, CF human or CF mouse cell systems (34, 35).Much less is known about the interaction between the Bcc and CGD despite the availability of animal models for the disease (20, 31). B. cenocepacia induced the necrosis of human CGD neutrophils but not normal controls (6). Similarly to healthy people, normal mice are resistant to the Bcc and usually show only transient infections upon inoculation (8, 37). On the other hand, CGD mice are highly susceptible to Bcc infection and show clinical signs that are similar to those of the human disease (20, 31, 37).To address why B. multivorans is a pathogen in CGD, we initiated studies with strains isolated from CGD patients and CGD cells. Strains of B. multivorans differed in cell association and cell entry. We found a preferential association of bacteria with CGD instead of normal leukocytes as shown by microscopy and culture techniques. This preferential association is shared by another CGD pathogen, Serratia marcescens, but not by Escherichia coli. Finally, we demonstrate dramatic differences in virulence in B. multivorans strains in a mouse model of CGD.     

Immune Response and Alveolar Bone Resorption in a Mouse Model of Treponema denticola Infection

Treponema denticola is considered to be an agent strongly associated with periodontal disease. The lack of an animal infection model has hampered the understanding of T. denticola pathogenesis and the host''s immune response to infection. In this study, we have established an oral infection model in mice, demonstrating that infection by oral inoculation is feasible. The presence of T. denticola in the oral cavities of the animals was confirmed by PCR. Mice given T. denticola developed a specific immune response to the bacterium. The antibodies generated from the infection were mainly of the immunoglobulin G1 subclass, indicating a Th2-tilted response. The antibodies recognized 11 T. denticola proteins, of which a 62-kDa and a 53-kDa protein were deemed immunodominant. The two proteins were identified, respectively, as dentilisin and the major outer sheath protein by mass spectrometry. Splenocytes cultured from the infected mice no longer produced interleukin-10 and produced markedly reduced levels of gamma interferon relative to those produced by naïve splenocytes upon stimulation with T. denticola. Mandibles of infected mice showed significantly greater bone resorption (P < 0.01) than those of mock-infected controls.Treponema denticola is highly implicated as one of the causative agents in periodontal disease in humans (7, 31). The organism is the predominant spirochete identified within the gingival crevice and developing periodontal pocket of various forms of periodontitis (30), infected root canals, and acute alveolar abscesses (28, 29). The organism has been reported to possess several putative virulence factors, such as attachment factors (6, 12, 15), proteolytic activities (13, 20, 34), and an immunosuppressive factor (14, 27). However, the actual role of these factors in the pathogenesis of T. denticola has yet to be proven, because of the lack of an oral infection model in animals. A T. denticola subcutaneous abscess model was described previously, but the model has many fundamental differences from periodontal diseases (15). As well, the host response to T. denticola oral infections is largely unknown. For other periodontal pathogens, such as Porphyromonas gingivalis, animal infection models have been established for a number of years (2). By use of these models, insights into the pathogenesis of P. gingivalis have emerged. A Th1-biased immune response to P. gingivalis infection appears to be responsible for periodontal bone loss (1, 10, 32). In addition, immunization of mice and rats with components of P. gingivalis protected against periodontal bone loss (8, 9, 22). Recently, a T. denticola oral infection model using rats was described; however, the immune response was not adequately investigated, and bone loss was only marginal (16).The purpose of this study is to establish an oral infection model in mice with T. denticola as the infectious agent. The model will serve as a good starting point to promote understanding of the pathogenesis of periodontal disease caused by T. denticola and the host immune responses to T. denticola. The model will allow investigations of the prevention and treatment of T. denticola infections to be pursued.     

Salmonella-Containing Vacuoles Display Centrifugal Movement Associated with Cell-to-Cell Transfer in Epithelial Cells

Intracellular Salmonella enterica serovar Typhimurium (serovar Typhimurium) occupies a Salmonella-containing vacuole (SCV) where bacterial effector proteins are secreted into the host cell using type III secretion systems (T3SS). Cytoskeletal motor proteins and T3SS-delivered effector proteins facilitate SCV positioning to juxtanuclear positions where bacterial replication occurs. Here, we show that this characteristic SCV positioning is not maintained by all SCVs during infection of HeLa cells. Notably, juxtanuclear SCV localization that occurs by 8 to 14 h postinfection is followed by significant centrifugal displacement of a subset of SCVs toward the host cell periphery by 24 h postinfection. This novel phenotype requires bacterial protein synthesis, a functional Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS, intact microtubules, and kinesin-1 motor protein. Bacteria lacking PipB2, a kinesin-recruiting T3SS effector, did not exhibit centrifugal displacement and remained at juxtanuclear positions throughout 24 h of infection. While levels of the SPI-2 effectors PipB2 and SifA increased during 24 h postinfection, a corresponding decrease in levels of the SPI-1 T3SS effectors SipA and SopB, both known to mediate juxtanuclear SCV positioning, was observed. A fluorescence-based assay indicated that wild-type serovar Typhimurium transferred from infected to uninfected epithelial cells while strains deficient in SPI-2 T3SS secretion or PipB2 did not. Our results reveal a novel SCV phenotype implicated in the cell-to-cell spread of serovar Typhimurium during infection.Salmonella enterica serovar Typhimurium (serovar Typhimurium) is a cause of gastroenteritis in humans and typhoid-like disease in certain strains of mice (55). Serovar Typhimurium is a facultative intracellular pathogen that can actively invade nonphagocytic cells through the delivery of bacterial proteins, termed effectors, into the host cell cytosol using the type III secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) (19). Following entry, serovar Typhimurium typically resides in a membrane-bound compartment termed the Salmonella-containing vacuole (SCV) (16, 50, 54). From here, additional effector proteins required for intracellular replication and virulence are delivered into the host cytosol using a second T3SS encoded by another pathogenicity island, SPI-2 (10, 28, 40, 48, 58). These effectors modulate various host cell activities, including endosomal trafficking, actin assembly dynamics, and microtubule-based transport (5, 8, 24, 33, 50, 51, 58).One characteristic trait of SCVs is their localization to a juxtanuclear, Golgi apparatus-associated region of the host cell several hours postinfection (1, 5, 41, 45). In general, with some variation likely due to differing experimental methods, this distinct localization is observed as early as 4 h postinfection (hpi) and is maintained at 8 to 16 hpi (2, 5, 13, 24, 26, 41, 42, 45). A recent study has shown that serovar Typhimurium can redirect host secretory traffic, resulting in an accumulation of post-Golgi vesicles around the SCV (36). It has been proposed that serovar Typhimurium targets the Golgi apparatus to acquire nutrients and/or membrane materials for maintenance of a replicative niche within the SCV (23, 45).Several different effectors secreted by the SPI-2 T3SS appear to mediate this centralized positioning of SCVs, including SseF, SseG, and SifA (2, 5, 13, 41, 42, 45). Deletion of sseF or sseG results in SCVs that are displaced from their usual juxtanuclear, Golgi compartment-associated position (1, 13, 45). SseF and SseG have been shown to interact with each other (13) and appear to promote the recruitment of the minus-end-directed microtubule motor dynein to SCVs to permit their juxtanuclear localization (2). SseG has also been proposed to act by tethering SCVs to the Golgi region (42).Deletion of sifA also results in SCVs that are displaced from the nucleus and located toward the host cell periphery (5). SifA was shown to interact with a host SifA- and kinesin-interacting protein that negatively regulates the recruitment of plus-end-directed kinesin-1 motors to the SCV, thus favoring the inward migration and maintenance of the SCV around the nucleus (5). In apparent opposition to SifA, the SPI-2 effector PipB2 has been shown to recruit kinesin-1 to the SCV (26). However, the characteristic positioning of SCVs to juxtanuclear regions suggests that the kinesin-inhibitory action of SifA may be dominant over the effects of PipB2 (26), at least at 8 to 14 hpi.Interestingly, some effectors secreted by the SPI-1 T3SS that is traditionally associated with Salmonella invasion appear to persist in host cells (6, 14) and are also implicated in modulating intracellular SCV positioning (6, 57). We recently demonstrated a role for the SPI-1 T3SS effector SopB in maintaining the juxtanuclear positioning of SCVs through the action of nonmuscle myosin II actin motors (57). Another SPI-1 effector, SipA, has also been shown to persist in host cells after bacterial entry and appears to act with SifA to ensure perinuclear positioning of SCVs (6). Hence, it appears that stringent control of microtubule and actin motor activity on the SCV by both SPI-1 and SPI-2 T3SS effectors is an important facet of SCV intracellular positioning (26).Overall, much remains to be resolved regarding the mediators and implications of intracellular SCV positioning. By remaining at the juxtanuclear region, the bacteria likely modify their compartments into a replicative niche where nutrient acquisition and SCV maintenance can occur (23, 36, 45). As a result of replication, high numbers of intracellular bacteria would presumably lead to host cell lysis, resulting in bacterial release; however, little is known about any mechanism(s) of Salmonella escape from host cells.The present study was conducted to examine the intracellular positioning of SCVs over the course of a 24-h infection. We show that at later stages of epithelial cell infection, the positioning of a significant proportion of SCVs is not maintained at a juxtanuclear region but is situated closer to the host cell periphery. This outward displacement of SCVs was dependent upon the SPI-2 T3SS, host microtubules and kinesin, and the SPI-2 effector PipB2. Furthermore, the dynamic positioning of SCVs is associated with a decrease in protein levels of SPI-1 effectors previously shown to mediate juxtanuclear positioning. Results from a cell-to-cell infection assay indicate that serovar Typhimurium strains that did not exhibit peripheral displacement at later stages of infection were also impaired in their ability to infect newly introduced host cells. Our results provide new insight into the nature of SCV positioning and demonstrate that intracellular SCV positioning is a dynamic process, with implications for bacterial cell-to-cell transfer.     

Transmission of Toxoplasma gondii from Infected Dendritic Cells to Natural Killer Cells

The obligate intracellular parasite Toxoplasma gondii can actively infect any nucleated cell type, including cells from the immune system. In the present study, we observed that a large number of natural killer (NK) cells were infected by T. gondii early after intraperitoneal inoculation of parasites into C57BL/6 mice. Interestingly, one mechanism of NK cell infection involved NK cell-mediated targeting of infected dendritic cells (DC). Perforin-dependent killing of infected DC led to active egress of infectious parasites that rapidly infected adjacent effector NK cells. Infected NK cells were not efficiently targeted by other NK cells. These results suggest that rapid transfer of T. gondii from infected DC to effector NK cells may contribute to the parasite''s sequestration and shielding from immune recognition shortly after infection.Toxoplasma gondii causes chronic infections in up to one-third of the human population and in animals (22, 31). In healthy individuals, primary T. gondii infection causes relatively mild symptoms, whereas in the immunocompromised patient or in the developing fetus, life-threatening manifestations lead to severe neurological and ocular damage (11, 28, 37). Following oral infection, T. gondii parasites typically pass across restrictive biological barriers and rapidly disseminate (13). In this process, T. gondii actively infects a great variety of cell types, including epithelial cells and blood leukocytes (12, 21). In infected cells, the parasites establish nonfusigenic parasitophorous vacuoles, where they can replicate (27, 32, 38).Natural killer (NK) cells and dendritic cells (DC) are two important cell types of the innate immune system. DC-NK cell interactions are important not only in host defense but also for the development of adaptive immune responses (5, 9). The activation of DC by pathogens leads to cytokine secretion, which activates NK cells, which in turn, via cytokines or by direct cell-cell contact, may determine the adaptive immune responses that follow (9, 29). DC are sensitive to NK cell-mediated lysis in vitro and can be eliminated by NK cells in vivo (4, 6, 17, 19, 33, 43). Viral or bacterial infection of DC can reduce their sensitivity to NK cell-mediated lysis by increasing the expression of classical and nonclassical major histocompatibility complex class I molecules on the cell surface (14, 35, 43).DC and NK cells play critical roles in innate immunity during acute Toxoplasma infection, being early sources of interleukin-12 (IL-12) and gamma interferon (IFN-γ), respectively (16, 20, 24, 34, 40). It has recently been suggested that infected DC, and possibly other leukocytes, can act as Trojan horses, potentiating the dissemination of the parasite from the point of infection to distal parts (8, 26). In the early phase of infection with T. gondii, NK cell recruitment to the site of infection is mediated by CCR5-binding chemokines (24). IFN-γ production by NK cells, induced by IL-12 from infected DC or macrophages, has been suggested to be the primary contribution of NK cells to the host defense against T. gondii (18, 25, 39). It can also drive cytotoxic CD8⁺ T-cell immunity to T. gondii even in the absence of CD4⁺ T cells (7). NK cells can also kill T. gondii-infected target cells (42), and perforin has been demonstrated to be important in protecting mice in the chronic stage of infection (10). In the present study, we investigated NK cell interactions with T. gondii-infected DC and, surprisingly, demonstrated how this interaction leads to T. gondii infection of NK cells.     

Interleukin-17-Mediated Control of Parasitemia in Experimental Trypanosoma congolense Infection in Mice

BALB/c mice are highly susceptible to experimental Trypanosoma congolense infections, whereas C57BL/6 mice are relatively resistant. Infected highly susceptible BALB/c mice die of systemic inflammatory response syndrome. Because interleukin-17 (IL-17) and Th17 cells regulate inflammatory responses, we investigated their role in the pathogenesis of experimental African trypanosomiasis in mice. We show that the production of IL-17 by spleen and liver cells and the serum IL-17 level increased after T. congolense infection in mice. Interestingly, infected highly susceptible BALB/c mice produced more IL-17 and had more Th17 cells than infected relatively resistant C57BL/6 mice. Paradoxically, neutralization of IL-17 with anti-IL-17 monoclonal antibody in vivo induced higher parasitemia in both the susceptible and the relatively resistant mice. Interestingly, anti-IL-17 antibody-treated mice had higher serum levels of alanine aminotransferase and aspartate aminotransferase, and the production of IL-10 and nitric oxide by liver cells was markedly decreased. Moreover, recombinant IL-17-treated mice exhibited significantly faster parasite control and lower peak parasitemia compared to control mice. Collectively, these results suggest that the IL-17/Th17 axis plays a protective role in murine experimental African trypanosomiasis.African trypanosomes are extracellular protozoan parasites that cause fatal disease in humans and domestic livestock in sub-Saharan Africa. The disease is endemic in 36 countries, and millions of people are at risk of suffering from human African trypanosomiasis. Trypanosomiasis in animals is caused by Trypanosoma congolense, Trypanosoma brucei brucei, and Trypanosoma vivax, but T. congolense is the most important cause of disease for livestock (29). It is estimated that the disease costs $1.3 billion to livestock producers and consumers every year (17).African trypanosomes have developed very sophisticated mechanisms to evade the host''s immune defenses (39, 40). The indigenous African and exotic European breeds of cattle are relatively resistant and susceptible, respectively, to African trypanosomiasis (28). In the laboratory, BALB/c mice are highly susceptible to experimental T. congolense infections, whereas C57BL/6 mice are relatively resistant, as measured by levels of parasitemia and survival time. When infected intraperitoneally (i.p.) with 10³ T. congolense, BALB/c mice have a mean survival time of 8.5 ± 0.5 days, whereas C57BL/6 mice survive for more than 100 days (40). Infected BALB/c mice die of systemic inflammatory response syndrome (SIRS), which is associated with high production of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-12, and gamma interferon (IFN-γ) (12, 37, 39, 40, 43, 45). Interestingly, blockade of IL-10 signaling either by anti-IL-10R monoclonal antibody (MAb) treatment (35, 36, 38) or knocking out the IL-10 gene (7) induces early death in the infected relatively resistant C57BL/6 mice. Thus, IL-10 is crucial for controlling proinflammatory cytokine storm that results lethal SIRS.Recently, T helper 17 (Th17) cells have been identified as a new T-helper subset (3, 10, 16, 33). A hallmark of Th17 cells is the production of IL-17A (also called IL-17), a proinflammatory cytokine. IL-17 and Th17 cells play important pathological role in several immune-mediated diseases, including inflammatory bowel disease, experimental autoimmune encephalopathy, and collagen-induced arthritis (5, 13, 30). IL-17A promotes inflammation by inducing the production of various proinflammatory cytokines (IL-6, TNF-α, and IL-1β) (3, 10, 16, 33). However, the protective function of Th17 and their effector cytokines in various infectious diseases has also been reported. For example, Mycobacterium tuberculosis (14), Pneumocystis carinii (34), Candida albicans (9), and Klebsiella pneumoniae (2) can trigger a strong Th17 response that mediates protective effects. These observations indicate that Th17 cells and their effector cytokines have both pathological and protective roles during inflammation and infections, respectively.There is as yet no report on the role of IL-17 and Th17 cells in resistance to African trypanosomes. Because T. congolense-infected BALB/c mice have high serum levels of IL-6, and their macrophages elaborate high amounts of IL-6 after in vitro infection (12), a cytokine that favors Th17 differentiation and IL-17 production (3, 16), we hypothesized that IL-17 and/or Th17 cells play important roles in resistance to T. congolense infection in mice by contributing to excessive inflammatory response. However, the data presented here suggest that IL-17 may be playing some protective role, particularly in controlling early parasitemia in mice infected with T. congolense.     

Generation of Antibody Responses to Pneumococcal Capsular Polysaccharides Is Independent of CD1 Expression in Mice

Streptococcus pneumoniae is a bacterial microorganism that frequently causes serious infection, particularly in children and the elderly. Protection against infection with S. pneumoniae is based mainly on the generation of antibodies to the pneumococcal capsular polysaccharides (caps-PS), but the mechanisms responsible for the generation of anticapsular antibodies remain incompletely understood. The aim of the present study was to evaluate the role of CD1-restricted T cells in the antibody response to caps-PS. When immunized with Pneumo23, wild-type mice and CD1 knockout mice on BALB/c and C57BL/6 backgrounds generated immunoglobulin M (IgM) and IgG antibody responses to soluble caps-PS that were comparable. Similar results were obtained after immunization with heat-inactivated S. pneumoniae. The IgM and IgG antibody response of wild-type mice to Pneumo23 was not affected by an antagonizing monoclonal anti-CD1 antibody treatment. In summary, our data provide evidence that the antibody response to caps-PS is generated independently of CD1 expression.Streptococcus pneumoniae is a major human pathogen. Infections with S. pneumoniae result in substantial morbidity and mortality, particularly in young children, the elderly, and immunocompromised patients (26). In various animal species and in humans, protection against S. pneumoniae infection is mediated by antibodies against pneumococcal capsular polysaccharides (caps-PS) and surface proteins (2, 4, 21).caps-PS are classified as T lymphocyte-independent type 2 (TI-2) antigens (24). While T lymphocytes are not required for the generation of antibody responses against TI-2 antigens, they can influence the antibody response to these antigens (24). In the case of caps-PS, the role of T lymphocytes in the generation of antibody responses might be more important than was initially thought. There is now evidence that T lymphocytes may support the antibody response to TI-2 antigens via several pathways (14). The magnitude of the antibody response to caps-PS is regulated both positively and negatively by distinct subsets of thymus-derived T lymphocytes. It has been reported that CD4⁺ T cells have positive effects on the antibody response to caps-PS, whereas CD8⁺ T cells have a suppressive effect. The presence of these two distinct types of T cells with opposing regulatory functions with respect to the immune response to soluble caps-PS has been demonstrated in vivo in mice and in vitro with human lymphocytes (1, 8, 10). SCID/SCID mice reconstituted with B lymphocytes and CD4⁺ T lymphocytes mounted a higher specific immunoglobulin M (IgM) antibody response to soluble pneumococcal caps-PS than SCID/SCID mice reconstituted with only B lymphocytes (12, 15). Murine spleen cells depleted of CD8⁺ T lymphocytes mounted a higher immune response to soluble caps-PS than total murine spleen cells, whereas spleen cells depleted of CD4⁺ T cells elicited only a weak antibody response (15). Similarly, the human IgM and IgG antibody response to soluble pneumococcal caps-PS was strongly dependent on CD4⁺ T cells (13). Several reports have provided evidence that CD4⁺ T cells enhance the IgG antibody response to pneumococcal polysaccharides after immunization of mice with intact S. pneumoniae (19, 37). The antipolysaccharide antibody response after immunization with conjugated polysaccharide serotype 3 was higher in CD8-deficient mice than in control mice, a finding attributed to CD8 T lymphocyte-mediated suppression of the antipolysaccharide immune response (34).In a provocative study, Kobrynski et al. (20) reported that CD1-restricted T cells and major histocompatibility complex (MHC) class I-dependent CD8⁺ cells are essential for the anti-caps-PS immune response. These findings set forth a new paradigm for humoral responses to caps-PS in which CD1 expression as well as a subset of CD8⁺ cells is required to provide helper function for antibody production against TI-2 caps-PS, akin to the role of MHC class II-restricted CD4⁺ cells for the generation of antibody responses to protein antigens (20). The MHC class I-like protein CD1 is expressed on antigen-presenting cells and is required for the presentation of lipids and glycolipids to T lymphocytes (25, 28, 29).The findings of Kobrynski et al. (20), suggesting that CD8⁺ T cells are essential for the IgG antibody response to caps-PS, are at odds with many other experimental data (1, 8, 10, 12, 13, 15, 19, 34, 37) that support the concept that CD4⁺ T cells have a positive effect on the antipolysaccharide immune response. Because of this controversy and because Kobrynski et al. (20) did not investigate the role of CD1 expression in the generation of IgM anti-caps-PS antibody responses, we reevaluated the role of CD1 expression in the IgM and IgG antibody response to pneumococcal polysaccharides. Our results revealed that CD1 expression was not required for the generation of IgM and IgG antibody responses to caps-PS.     

Yersinia enterocolitica Promotes Robust Mucosal Inflammatory T-Cell Immunity in Murine Neonates

Mucosal immunity to gastrointestinal pathogens in early life has been studied only slightly. Recently, we developed an infection model in murine neonates using the gastroenteric pathogen Yersinia enterocolitica. Here, we report that oral infection of neonatal mice with low doses of virulent Y. enterocolitica leads to vigorous intestinal and systemic adaptive immunity. Y. enterocolitica infection promoted the development of anti-LcrV memory serum IgG1 and IgG2a responses of comparable affinity and magnitude to adult responses. Strikingly, neonatal mesenteric lymph node CD4⁺ T cells produced Yersinia-specific gamma interferon (IFN-γ) and interleukin-17A (IL-17A), exceeding adult levels. The robust T- and B-cell responses elicited in neonates exposed to Y. enterocolitica were associated with long-term protection against mucosal challenge with this pathogen. Using genetically deficient mice, we found that IFN-γ and CD4⁺ cells, but not B cells, are critical for protection of neonates during primary Y. enterocolitica infection. In contrast, adults infected with low bacterial doses did not require either cell population for protection. CD4-deficient neonatal mice adoptively transferred with CD4⁺ cells from wild-type, IFN-γ-deficient, or IL-17AF-deficient mice were equally protected from infection. These data demonstrate that inflammatory CD4⁺ T cells are required for protection of neonatal mice and that this protection may not require CD4-derived IFN-γ, IL-17A, or IL-17F. Overall, these studies support the idea that Y. enterocolitica promotes the development of highly inflammatory mucosal responses in neonates and that intestinal T-cell function may be a key immune component in protection from gastrointestinal pathogens in early life.Host protection against microbial agents ultimately relies on the cooperative action of the innate and adaptive immune systems. In both human and murine neonates, adaptive immune responses are compromised compared to responses in developmentally mature hosts (5, 66). Factors that may contribute to the immunological immaturity reported during neonatal life include the following: the lack of antigen-specific immunological memory (5, 65), reduced levels of antigen presenting cells (APC) (46) and adaptive immune cells (21), delays in the development of lymph node germinal centers (57), and cell-intrinsic differences in immune responsiveness (4, 48, 67). Thus, neonatal immune responses following infection or vaccination often appear to be diminished compared to responses in adults. In particular, B-cell and CD4⁺ T helper (T_h) responses to a variety of antigens may be reduced in magnitude, quality, and duration (5, 65). Neonatal immunization with prototypic protein vaccine antigens often leads to mixed T_h1 and T_h2 primary responses (2), but the development of T_h1-associated memory (3) and production of T_h1-associated IgG2a antibodies are often reduced compared to these responses in adults (9). However, adult-like T_h1 immunity has been achieved in neonatal hosts after Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination (53, 72), DNA vaccines (55, 62), or attenuated vaccinia-derived vectors (44). These observations led to the recognition that immune responsiveness during early life could be greatly enhanced by optimizing the conditions of antigen exposure using highly inflammatory treatments.Activation of the neonatal immune system through microbe-associated molecular pattern receptors has demonstrated remarkable improvements in promoting effective immunity to vaccine antigens. For example, bacterially derived products such as mutated Escherichia coli enterotoxins LT-R192G (70) and LT-K63 (11, 14, 27, 34), CpG oligonucleotides (CpG) (8, 29, 31), and lyophilized bacterial extracts (12) have been described to markedly enhance neonatal vaccine responses. Another approach used to improve immune responses has been the delivery of specific antigens using live attenuated bacterial vectors such as Listeria monocytogenes (42, 50) and Salmonella species (16, 59). Both of these approaches have shown dramatic improvements in CD4⁺ and CD8⁺ IFN-γ production, mucosal IgA production, and systemic IgG1 and IgG2a antibodies to the delivered vaccine antigens. Recently, CD4⁺ T_h17-mediated immunity has been studied in response to vaccination with rotavirus antigen in adjuvant (70) and to Mycobacterium tuberculosis antigens in the presence of non-CpG oligonucleotides (36) or cationic liposomes (37). These vaccines promoted interleukin-17A (IL-17A) levels of the same magnitude in neonatal and adult CD4⁺ cells (36, 37, 70). Altogether, it has become apparent that under the proper stimulation conditions, all arms of the neonatal adaptive immune system can be induced to generate adult-like responses. Importantly, some of these immunization regimens promoted protective immunity against infection with fully pathogenic bacteria (16, 29, 31, 34, 42, 59).Despite the profound maturation of the neonatal immune system through vaccination with live attenuated L. monocytogenes and Salmonella vectors (16, 17, 42, 50, 59), neonatal immune responses to fully virulent pathogens are inefficient in controlling infection (15, 25, 31, 61). This exquisite susceptibility to infection during neonatal life includes both peripheral and mucosal routes of infection. In particular, neonatal animals succumb rapidly to pulmonary infection with Streptococcus pneumoniae (24) and gastrointestinal infection with enteropathogens including Vibrio cholerae (10), Aeromonas hydrophila (76), Shigella flexneri (23), enterotoxigenic E. coli (19), and Salmonella species (15, 61). Thus, mucosal immune responses to most pathogens studied to date are severely compromised in early life.In contrast to the vast majority of experimental systems, we recently demonstrated (20) that murine neonates are highly resistant to oral infection with the Gram-negative enteropathogen Yersinia enterocolitica. The resistance of neonatal mice infected with Y. enterocolitica was associated with robust innate inflammation, characterized by the recruitment of high levels of neutrophils and macrophages into the intestinal tissue (20). We hypothesized that the vigorous innate responses in neonates may promote similarly robust adaptive immunity. Here, we have compared the development of Yersinia-specific B- and CD4⁺ T-cell immunity in neonatal and adult mice. We demonstrate that highly protective intestinal and systemic adaptive immunity can be induced in neonatal mice. Remarkably, neonatal mice developed greater Yersinia-specific T_h1 and T_h17 responses in the mesenteric lymph nodes (MLN) than did adults. Experiments using genetically deficient mice with or without adoptive transfer of donor cells showed that CD4⁺ T cells, but not B cells, appeared to be necessary for resistance of infected neonates. Thus, we extend our earlier studies to further demonstrate the unprecedented inflammatory potential of the neonatal gastrointestinal immune system in response to a fully virulent enteric pathogen.