Role of Porphyromonas gingivalis Phosphoserine Phosphatase Enzyme SerB in Inflammation,Immune Response,and Induction of Alveolar Bone Resorption in Rats

Porphyromonas gingivalis secretes a serine phosphatase enzyme, SerB, upon contact with gingival epithelial cells in vitro. The SerB protein plays a critical role in internalization and survival of the organism in epithelial cells. SerB is also responsible for the inhibition of interleukin-8 (IL-8) secretion from gingival epithelial cells infected with P. gingivalis. This study examined the ability of a P. gingivalis SerB mutant to colonize the oral cavity and induce gingival inflammation, immune responses, and alveolar bone resorption in a rat model of periodontal disease. Both P. gingivalis ATCC 33277 and an isogenic ΔSerB mutant colonized the oral cavities of rats during the 12-week experimental period. Both of the strains induced significant (P < 0.05) systemic levels of immunoglobulin G (IgG) and isotypes IgG1, IgG2a, and IgG2b, indicating the involvement of both T helper type 1 (Th1) and Th2 responses to infection. Both strains induced significantly (P < 0.05) higher levels of alveolar bone resorption in infected rats than in sham-infected control rats. However, horizontal and interproximal alveolar bone resorption induced by the SerB mutant was significantly (P < 0.05) lower than that induced by the parental strain. Rats infected with the ΔSerB mutant exhibited significantly higher levels of apical migration of the junctional epithelium (P < 0.01) and polymorphonuclear neutrophil (PMN) recruitment (P < 0.001) into the gingival tissues than rats infected with the wild type. In conclusion, in a rat model of periodontal disease, the SerB phosphatase of P. gingivalis is required for maximal alveolar bone resorption, and in the absence of SerB, more PMNs are recruited into the gingival tissues.One of the predominant polymicrobial infections of humans is expressed clinically as periodontal disease. A bacterial etiology for periodontal diseases is well established, and a group of Gram-negative, mostly anaerobic bacteria is associated with the initiation and progression of disease. Porphyromonas gingivalis is considered one of the more pathogenic members of this group, and elevated levels of this organism are associated with an increased risk of periodontal breakdown (4, 54). P. gingivalis produces a variety of virulence factors that enable colonization of the periodontal pocket and destruction of the structural components of the periodontium. These virulence factors include proteolytic enzymes that can damage host cells, tissues, and immune response mediators; toxic metabolites; surface components with immune-modulating activity; and adherence factors that promote colonization and persistence (7, 8, 15, 18, 21, 22, 35, 38). Moreover, the role of some of these virulence factors, such as fimbriae and proteases, has been verified in animal models of periodontal disease (36, 44, 46). P. gingivalis is also an intracellular organism that can invade gingival epithelial cells in culture (37, 39, 58). In addition, P. gingivalis has been observed within epithelial cells in ex vivo samples such as gingival biopsy samples (45, 49), and high numbers of P. gingivalis have been observed within gingival and buccal epithelial cells obtained from healthy and disease subjects (5, 50, 51). Mechanistically, P. gingivalis invasion is mediated through fimbria-mediated attachment to gingival epithelial cell integrin receptors and cytoskeletal rearrangements that allow the bacteria to enter the cell (64, 65). A key effector of the invasive process is SerB, a haloacid dehydrogenase family phosphoserine phosphatase enzyme. SerB is present in the outer membrane of P. gingivalis, and contact with gingival epithelial cells induces secretion into the extracellular milieu (67). Cell-associated and extracellular SerB impacts host cell signal transduction pathways that control the cytoskeletal architecture, and treatment of epithelial cells with SerB induces actin microfilament and tubulin microtubule rearrangements (19, 59). Furthermore, SerB is required for intracellular persistence, as a mutant lacking SerB is compromised in intracellular survival (59). An additional role of SerB concerns involvement in the stealth-like properties of P. gingivalis (18). P. gingivalis is capable of both inhibiting secretion of interleukin-8 (IL-8) from gingival epithelial cells and antagonizing IL-8 production stimulated by other organisms (9, 23, 26, 60). SerB activity is required for this innate immune suppression, known as localized chemokine paralysis (9, 19).While SerB is important for invasion, intracellular survival, and immune suppression in vitro, its contribution to in vivo pathogenicity has not been addressed. Study of the in vivo role of P. gingivalis virulence factors has employed a variety of animal models (16). Among these, the oral infection model in rodents has been used to study colonization, periodontal inflammation, immune responses, and induction of alveolar bone resorption by P. gingivalis (3, 29, 32). The pathological processes induced by oral infection with periodontal pathogens in rodent models, including gingival and periodontal ligament destruction and resorption of the alveolar bone, resemble those that occur in humans (34). In this study, we examine the in vivo role of SerB in colonization, inflammation, immune response, alveolar bone resorption, and induction of periodontal disease in rats.     

The Absence of Hck,Fgr, and Lyn Tyrosine Kinases Augments Lung Innate Immune Responses to Pneumocystis murina

Src family tyrosine kinases (SFKs) phosphorylate immunotyrosine activation motifs in the cytoplasmic tail of multiple immunoreceptors, leading to the initiation of cellular effector functions, such as phagocytosis, reactive oxygen species production, and cytokine production. SFKs also play important roles in regulating these responses through the activation of immunotyrosine inhibitory motif-containing inhibitory receptors. As myeloid cells preferentially express the SFKs Hck, Fgr, and Lyn, we questioned the role of these kinases in innate immune responses to Pneumocystis murina. Increased phosphorylation of Hck was readily detectable in alveolar macrophages after stimulation with P. murina. We further observed decreased phosphorylation of Lyn on its C-terminal inhibitory tyrosine in P. murina-stimulated alveolar macrophages, indicating that SFKs were activated in alveolar macrophages in response to P. murina. Mice deficient in Hck, Fgr, and Lyn exhibited augmented clearance 3 and 7 days after intratracheal administration of P. murina, which correlated with elevated levels of interleukin 1β (IL-1β), IL-6, CXCL1/KC, CCL2/monocyte chemoattractant protein 1, and granulocyte colony-stimulating factor in lung homogenates and a dramatic increase in macrophage and neutrophil recruitment. Augmented P. murina clearance was also observed in Lyn^−/− mice 3 days postchallenge, although the level was less than that observed in Hck^−/− Fgr^−/− Lyn^−/− mice. A correlate to augmented clearance of P. murina in Hck^−/− Fgr^−/− Lyn^−/− mice was a greater ability of alveolar macrophages from these mice to kill P. murina in vitro, suggesting that SFKs regulate the alveolar macrophage effector function against P. murina. Mice deficient in paired immunoglobulin receptor B (PIR-B), an inhibitory receptor activated by SFKs, did not exhibit enhanced inflammatory responsiveness to or clearance of P. murina. Our results suggest that SFKs regulate innate lung responses to P. murina in a PIR-B-independent manner.Although the advent of prophylactic therapy against Pneumocystis jirovecii alone or in combination with highly active antiretroviral therapy has reduced the incidence of pneumonia caused by this fungal pathogen, this infection remains the most prevalent opportunistic infection in individuals with AIDS (25). Furthermore, individuals receiving immunosuppressive therapies, such as individuals undergoing solid organ or hematopoietic cell transplantation, are a growing population susceptible to Pneumocystis pneumonia (34) (33). These observations warrant a more thorough examination of interactions between Pneumocystis and the host in order to define and develop new vaccine and immunotherapeutic approaches to treat Pneumocystis pneumonia.An intense area of research over the last decade is investigating how lung immune cells recognize and respond to inhaled pathogens, such as P. murina. After inhalation of P. murina into the lungs, one of the first interactions with the host is recognition by the alveolar macrophage. Our laboratory has previously reported that alveolar macrophages recognize P. murina via the beta-glucan receptor dectin-1 (37). Recognition by dectin-1 leads to internalization of P. murina and subsequent killing of the organism, as well as elaboration of the neutrophil-attracting chemokine CXCL2/MIP-2 (37). Toll-like receptor 2 (TLR2) is an additional receptor expressed by alveolar macrophages that mediates CXCL2/MIP-2 production (54) and, in humans, interleukin-8 (IL-8) production in cooperation with the macrophage mannose receptor (39). P. murina infection in TLR2^−/− mice is prolonged and associated with a lack of inflammatory responsiveness (51). Other studies have implicated TLR4 in alveolar macrophage recognition of P. murina (6).Immunoreceptors expressed by cells of the innate immune system are abundant and fall into many different categories, such as scavenger receptors, integrins, immunoglobulin (Ig) superfamily receptors, C-type lectin receptors, and TLRs (41). As expected based on this diversity, immunoreceptor signaling in innate cells is a complex process that differs greatly for different types of receptors. For example, receptors in the C-type lectin family often utilize an immunotyrosine activation motif (ITAM) in the cytoplasmic tail for signaling (2). Receptors in the Ig superfamily, such as SIRPα and Siglec3, contain immunotyrosine inhibitory motifs (ITIMs) that initiate regulatory signals, whereas members of the TLR family signal through multiple intermediate proteins, such as TIRAP, TRIF, and MyD88 (28). One commonality in innate immunoreceptor signaling is the role of Src family tyrosine kinases (SFKs). Our current understanding of how SFKs function is due in large part to studies that have characterized ITAM-associated Fc receptor (FcγR) signaling (32). In FcγR signaling, SFKs phosphorylate two tyrosine residues in the ITAM domain, which leads to the recruitment of Syk and subsequent activation of cellular responses, such as phagocytosis and cytokine and chemokine production (32).An equally important function of SFKs is to phosphorylate ITIMs, which leads to the recruitment of SHP-1 or SHIP-1 phosphatases and subsequent regulation or inhibition of responses (21). The phosphorylation of ITIMs by SFKs is often responsible for the regulation of responses initiated by many types of immunoreceptors. Mice deficient in the SFKs Hck and Fgr have enhanced chemokine receptor signaling as a result of lower phosphorylation of the ITIM in the inhibitory receptor paired Ig receptor B (PIR-B) (55). Mice deficient in PIR-B were also found to be hyperresponsive to chemokine signaling (55). PIR-B ITIM activation has also been shown to regulate TLR-mediated macrophage responses to some gram-positive and gram-negative bacteria (26). Other studies have shown that the SFK Lyn phosphorylates an ITIM of platelet endothelial cell adhesion molecule 1 in mast cells, leading to regulated FcɛRI responses (45).Macrophage-mediated recognition of P. murina involves a variety of receptors that may be dependent on SFKs for induction as well as the regulation of responses; therefore, we sought to investigate the role of SFKs in innate immune responses to P. murina. In this study, we made the surprising observation that deficiency of Hck, Fgr, and Lyn resulted in paradoxically augmented lung clearance of P. murina and enhanced cytokine and chemokine production. This response was not due to impaired PIR-B inhibitory responses as PIR-B^−/− mice were not capable of enhanced P. murina lung clearance. We propose that the SFKs Hck, Fgr, and Lyn regulate innate immune responses to P. murina and that, therefore, novel therapeutics to control the activity of SFKs may be beneficial in treating pulmonary infections.     

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.     

Porphyromonas gingivalis RgpA-Kgp Proteinase-Adhesin Complexes Penetrate Gingival Tissue and Induce Proinflammatory Cytokines or Apoptosis in a Concentration-Dependent Manner

The RgpA-Kgp proteinase-adhesin complexes of Porphyromonas gingivalis were observed, using immunostaining, in human gingival tissue associated with periodontitis but not in healthy tissue. The staining pattern suggested a concentration gradient from the subgingival plaque into the subjacent gingival connective tissue. Intense immunostaining was observed in areas displaying gross disturbance of tissue architecture. P. gingivalis cells and the RgpA-Kgp complexes at low concentrations were shown to stimulate secretory intercellular adhesion molecule 1, interleukin-8 (IL-8), IL-6, and macrophage chemoattractant protein secretion from cultured human epithelial (KB) and fibroblast (MRC-5) cells. However, at high concentrations a reduction in the level of these mediators was observed. In contrast, macrophage inflammatory protein 1α and IL-1α were stimulated only at high P. gingivalis cell concentrations. P. gingivalis cells and the RgpA-Kgp complexes were shown to induce apoptosis in KB and MRC-5 cells in a time- and dose-dependent manner. These data suggest that the RgpA-Kgp complexes penetrate the gingival connective tissue; at low concentrations distal from the plaque the complexes stimulate the secretion of proinflammatory mediators, while at high concentrations proximal to the plaque they induce apoptosis and attenuate the secretion of proinflammatory mediators.Chronic periodontitis is an inflammatory disease associated with specific bacteria in subgingival plaque that results in the destruction of the tooth''s supporting tissues. The presence of the three bacterial species Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as a consortium in subgingival plaque has been associated with chronic periodontitis, and of these bacteria, P. gingivalis is reported to be most closely associated with the severity of disease (51, 79). P. gingivalis produces extracellular complexes of proteinases and adhesins, designated the RgpA-Kgp complexes (or high-molecular-weight gingipains). Isogenic mutants of P. gingivalis lacking the RgpA-Kgp complexes are avirulent in an animal periodontitis model, and therefore the complexes have been proposed to be a major virulence factor for this bacterium (51, 56). The chronic interaction of the host immune system with subgingival plaque containing P. gingivalis and the RgpA-Kgp complexes in the subjacent tissue is believed to be a major factor in tissue destruction in chronic periodontitis (6, 21, 51, 83). Compared with healthy subjects, gingival tissues and gingival crevicular fluid of patients with chronic periodontitis are reported to have significantly increased amounts of proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (TNF-α); chemokines such as IL-8, macrophage chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α); and adhesion molecules such as intracellular adhesion molecule 1 (ICAM-1) (12, 25, 27, 28, 32, 33, 37, 44, 46, 62, 84, 87). These cytokines and chemokines play a significant role in mediating the recruitment of a dense mononuclear infiltrate, consisting mainly of T cells and macrophages (21, 22, 39, 55, 81). Furthermore, a concentration gradient of secretory ICAM-1 (sICAM-1) across the junctional epithelium is reported to be an important mechanism leading to leukocyte recruitment into the gingival sulcus (85). Assuma et al. (2) have reported that blocking IL-1 and TNF-α activity in a nonhuman primate model significantly reduced tissue destruction and alveolar bone loss. These studies suggest that the chronic presence of specific pathogens such as P. gingivalis in subgingival plaque results in the secretion of inflammatory mediators which, in turn, may cause inappropriate accumulation and activation of circulating and resident leukocytes at the site of infection, producing chronic inflammation and tissue destruction. Another contributing factor for periodontal tissue destruction is reported to be the induction of host cell apoptosis by specific subgingival plaque pathogens (5). In gingival biopsies from patients with chronic periodontitis, apoptotic cells have been reported to constitute about 10% of the total cell population and included epithelial and fibroblast cells (31, 36). Tonetti et al. (86) have reported that exposure of clinically healthy gingival tissues to plaque bacteria induces apoptosis-associated DNA damage and the expression of proapoptotic p53 protein. Furthermore, in gingival biopsies from patients with periodontitis, epithelial cell apoptosis was reported to be more prevalent in the most apical part of the sulcus closest to the subgingival plaque (31, 86). These observations suggest that interactions with certain bacterial products may play an important role in inducing apoptosis in gingival tissue cells. Furthermore, a high prevalence of apoptotic cells expressing the p53 protein was detected in the subgingival inflammatory infiltrate, suggesting that apoptotic cell death may be important in the regulation of the inflammatory response to chronic bacterial challenge (86).A number of prior studies have investigated the ability of P. gingivalis to induce secretion of proinflammatory mediators from oral epithelial and fibroblast cells. It has been reported that P. gingivalis induces expression of proinflammatory mediators such as IL-1β, IL-8, IL-6, and sICAM-1 from gingival epithelial or fibroblast cells (1, 70, 80, 93). However, in contrast, P. gingivalis cells have also been reported to degrade existing inflammatory cytokines and antagonize IL-1β, IL-8, IL-6, and sICAM-1 production by gingival epithelial or fibroblast cells (13, 16-18, 43). These apparent contradictions may be partially explained by the assay conditions related to factors such as bacterial strain variability and inclusion of human serum (38, 74, 80). However, we have postulated that this paradoxical situation where P. gingivalis both suppresses and induces the host immune response is a concentration-dependent phenomenon that is related to the level of P. gingivalis cells and the P. gingivalis major virulence factor, the RgpA-Kgp proteinase-adhesin complexes (51).In the present study, we demonstrate the presence of RgpA-Kgp in gingival tissue associated with periodontitis, and we investigate the hypothesis that P. gingivalis W50 whole cells and RgpA-Kgp complexes differentially regulate the expression of proinflammatory mediators and the induction of apoptosis in human epithelial and fibroblast cells in a concentration-dependent fashion.     

Local and Systemic Responses in Matrix Metalloproteinase 8-Deficient Mice during Porphyromonas gingivalis-Induced Periodontitis

Periodontitis is a bacterium-induced chronic inflammation that destroys tissues that attach teeth to jaw bone. Pathologically excessive matrix metalloproteinase 8 (MMP-8) is among the key players in periodontal destruction by initiating type I collagen degradation. We studied MMP-8 in Porphyromonas gingivalis-induced periodontitis by using MMP-8-deficient (MMP8^−/−) and wild-type (WT) mice. Alveolar bone loss, inflammatory mediator expression, serum immunoglobulin, and lipoprotein responses were investigated to clarify the role of MMP-8 in periodontitis and systemic inflammatory responses. P. gingivalis infection induced accelerated site-specific alveolar bone loss in both MMP8^−/− and WT mice relative to uninfected mice. The most extensive bone degradation took place in the P. gingivalis-infected MMP8^−/− group. Surprisingly, MMP-8 significantly attenuated (P < 0.05) P. gingivalis-induced site-specific alveolar bone loss. Increased alveolar bone loss in P. gingivalis-infected MMP8^−/− and WT mice was associated with increase in gingival neutrophil elastase production. Serum lipoprotein analysis demonstrated changes in the distribution of high-density lipoprotein (HDL) and very-low-density lipoprotein (VLDL) particles; unlike the WT mice, the MMP8^−/− mice underwent a shift toward a smaller HDL/VLDL particle sizes. P. gingivalis infection increased the HDL/VLDL particle size in the MMP8^−/− mice, which is an indicator of lipoprotein responses during systemic inflammation. Serum total lipopolysaccharide activity and the immunoglobulin G-class antibody level in response to P. gingivalis were significantly elevated in both infected mice groups. Thus, MMP-8 appears to act in a protective manner inhibiting the development of bacterium-induced periodontal tissue destruction, possibly through the processing anti-inflammatory cytokines and chemokines. Bacterium-induced periodontitis, especially in MMP8^−/− mice, is associated with systemic inflammatory and lipoprotein changes that are likely involved in early atherosclerosis.Periodontitis is a chronic infection-induced inflammatory disease that causes tooth loss and is considered a modifying factor in systemic health (1, 6). Several pathogens are associated with periodontitis. Porphyromonas gingivalis is one of the major pathogens in chronic periodontitis (59). P. gingivalis has a number of virulence factors such as capsule, fimbriae, lipopolysaccharide (LPS), and potent proteolytic enzymes, gingipains (23). These factors can induce an inflammatory cascade involving proinflammatory cytokines, reactive oxygen species, and matrix metalloproteinases (MMP), thus leading to the destruction of supportive soft and hard tissues around the teeth.Pathologically excessive MMP plays a significant role in periodontal destruction (48, 50). MMP-8 (collagenase 2) is a collagenolytic enzyme that can initiate the digestion of type I collagen, the most dominant interstitial collagen type in the periodontal tissues. Collagen degradation is regarded as one of the key factors in the uncontrolled tissue destruction in periodontitis (48). In addition to periodontitis (52), elevated MMP-8 levels are attributable to many diseases such as bronchiectasis, asthma (40, 41), atherosclerosis (28, 55), inflammatory bowel disease (39), oral cysts (61), and oral cancer (33). MMP-8 is predominantly synthesized in the bone marrow and stored within the secondary granules of neutrophils (polymorphonuclear leukocytes) (58). Even though MMP-8 in tissues is primarily derived from degranulating neutrophils, de novo expression of MMP-8 has been identified in non-neutrophil-lineage cells such as gingival fibroblasts, odontoblasts, epithelial cells, plasma cells, and monocytes/macrophages (25, 50). Recent studies suggest that in addition to surrogate tissue destructive properties (48, 50), MMP-8 can exert anti-inflammatory effects in the host defense by processing anti-inflammatory cytokines and chemokines (37). MMP-8 can also regulate apoptotic and immune responses and play a protective role in lung inflammation (18), cancer progression (2, 20, 27), and wound healing (19).Although chronic periodontitis is localized to the tissues surrounding the teeth, it is linked to serious systemic conditions such as cardiovascular disease (4, 13), stroke (62), diabetes (10), and complications during pregnancy (12). Increased bacterial burden in inflamed periodontal pockets leads to the presence of oral bacteria and their components, such as LPS, in the systemic circulation (15, 22). Periodontitis is also accompanied by the systemic antibody response against periodontal pathogens and proatherogenic changes in lipoprotein metabolism (42-45).Knockout mouse models are useful in studies of the roles of specific MMPs in physiological and pathological situations. We evaluated the role of MMP-8 in P. gingivalis-induced periodontitis by comparing alveolar bone destruction between MMP-8-deficient (MMP8^−/−) and wild-type (WT) mice. Furthermore, serum antibody level and lipoprotein determinations were performed to clarify the systemic effects of MMP-8 during the inflammatory process of periodontitis.     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Tracking the Emerging Human Pathogen Pseudallescheria boydii by Using Highly Specific Monoclonal Antibodies

Pseudallescheria boydii has long been known to cause white grain mycetoma in immunocompetent humans, but it has recently emerged as an opportunistic pathogen of humans, causing potentially fatal invasive infections in immunocompromised individuals and evacuees of natural disasters, such as tsunamis and hurricanes. The diagnosis of P. boydii is problematic since it exhibits morphological characteristics similar to those of other hyaline fungi that cause infectious diseases, such as Aspergillus fumigatus and Scedosporium prolificans. This paper describes the development of immunoglobulin M (IgM) and IgG1 κ-light chain monoclonal antibodies (MAbs) specific to P. boydii and certain closely related fungi. The MAbs bind to an immunodominant carbohydrate epitope on an extracellular 120-kDa antigen present in the spore and hyphal cell walls of P. boydii and Scedosporium apiospermum. The MAbs do not react with S. prolificans, Scedosporium dehoogii, or a large number of clinically relevant fungi, including A. fumigatus, Candida albicans, Cryptococcus neoformans, Fusarium solani, and Rhizopus oryzae. The MAbs were used in immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to accurately differentiate P. boydii from other infectious fungi and to track the pathogen in environmental samples. Specificity of the DAS-ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of environmental isolates.Pseudallescheria boydii is an infectious fungal pathogen of humans (7, 16, 40, 58, 59). It is the etiologic agent of white grain mycetoma in immunocompetent humans (7) and has emerged over recent years as the cause of fatal disseminated infections in individuals with neutropenia, AIDS, diabetes, renal failure, bone marrow or solid organ transplants, systemic lupus erythematous, and Crohn''s disease; in those undergoing corticosteroid treatment; and in leukemia and lymphoma patients (1, 2, 3, 18, 27, 31, 32, 34, 36, 37, 38, 47, 49, 52). The fungus is the most prevalent species after Aspergillus fumigatus in the lungs of cystic fibrosis patients (8), where it causes allergic bronchopulmonary disease (5) and chronic lung lesions simulating aspergillosis (24). Near-drowning incidents and recent natural disasters, such as the Indonesian tsunami in 2004, have shown P. boydii and the related species Scedosporium apiospermum and Scedosporium aurantiacum to be the causes of fatal central nervous system infections and pneumonia in immunocompetent victims who have aspirated polluted water (4, 11, 12, 21, 22, 25, 30, 33, 57). Its significance as a potential pathogen of disaster evacuees has led to its recent inclusion in the Centers for Disease Control and Prevention list of infectious etiologies in persons with altered mental statuses, central nervous system syndromes, or respiratory illness.P. boydii is thought to be an underdiagnosed fungus (60), and misidentification is one of the reasons that the mortality rate due to invasive pseudallescheriasis is high. Detection of invasive P. boydii infections, based on cytopathology and histopathology, is problematic since it can occur in tissue and bronchoalveolar and bronchial washing specimens with other hyaline septated fungi, such as Aspergillus and Fusarium spp. (7, 23, 53, 60), which exhibit similar morphological characteristics upon microscopic examination (2, 23, 24, 28, 37, 44, 53, 60). Early diagnosis of infection by P. boydii and differentiation from other agents of hyalohyphomycosis is imperative, since it is refractory to antifungal compounds, such as amphotericin B, that are commonly administered for the control of fungal infections (10, 39, 58).The immunological diagnosis of Pseudallescheria infections has focused on the detection of antigens by counterimmunoelectrophoresis, and by immunohistological techniques using polyclonal fluorescent antibodies, but cross-reactions with antigens from other fungi, such as Aspergillus species, occurs (7, 19, 23). Pinto and coworkers (41, 42) isolated a peptidorhamnomannan from hyphae of P. boydii and proposed the antigen as a diagnostic marker for the pathogen. Cross-reactivity with Sporothrix schenckii and with Aspergillus have, however, been noted (23, 41). Furthermore, it is uncertain whether a similar antigen is present in the related pathogenic species S. prolificans, an important consideration in patient groups susceptible to mixed Scedosporium infections (6, 18).Hybridoma technology allows the production of highly specific MAbs that are able to differentiate between closely related species of fungi (54, 55, 56). The purpose of this paper is to report the development of MAbs specific to P. boydii and certain closely related species and their use to accurately discriminate among P. boydii, A. fumigatus, and other human pathogenic fungi by using immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs).Currently, the natural environmental habitat of P. boydii is unknown, but nutrient-rich, brackish waters, such as estuaries, have been suggested (9, 17). In combination with a semiselective isolation procedure, I show how the DAS-ELISA can be used to rapidly and accurately track the pathogen in naturally infested estuarine muds, and in doing so illustrate the potential of the DAS-ELISA as a diagnostic platform for detection of P. boydii and related species within the Pseudallescheria complex.     

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.     

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).     

Transcellular Passage of Neisseria meningitidis across a Polarized Respiratory Epithelium

Neisseria meningitidis is a major cause of sepsis and meningitis but is also a common commensal, present in the nasopharynx of between 8 and 20% of healthy individuals. During carriage, the bacterium is found on the surface of the nasopharyngeal epithelium and in deeper tissues, while to develop disease the meningococcus must spread across the respiratory epithelium and enter the systemic circulation. Therefore, investigating the pathways by which N. meningitidis crosses the epithelial barrier is relevant for understanding carriage and disease but has been hindered by the lack of appropriate models. Here, we have established a physiologically relevant model of the upper respiratory epithelial cell barrier to investigate the mechanisms responsible for traversal of N. meningitidis. Calu-3 human respiratory epithelial cells were grown on permeable cell culture membranes to form polarized monolayers of cells joined by tight junctions. We show that the meningococcus crosses the epithelial cell barrier by a transcellular route; traversal of the layer did not disrupt its integrity, and bacteria were detected within the cells of the monolayer. We demonstrate that successful traversal of the epithelial cell barrier by N. meningitidis requires expression of its type 4 pili (Tfp) and capsule and is dependent on the host cell microtubule network. The Calu-3 model should be suitable for dissecting the pathogenesis of infections caused by other respiratory pathogens, as well as the meningococcus.Neisseria meningitidis is a leading cause of fatal sepsis and meningitis worldwide (61). Despite being an important human pathogen, the bacterium is also a common commensal, found in the nasopharynx of between 8 and 20% of healthy individuals (25, 62). The human nasopharynx is lined by a columnar epithelium that forms the first cellular barrier encountered by the meningococcus following its acquisition. This cell layer consists of differentiated, polarized, respiratory epithelial cells joined by tight junctions that form a barrier which excludes mucosal pathogens. The majority of cells in the layer are ciliated, resulting in a brush border, although areas of nonciliated cells are also present, along with mucus-secreting goblet cells (54). To cause disease the meningococcus must spread from the nasopharynx, its only natural reservoir, penetrate the upper respiratory epithelial cell barrier, and enter the systemic circulation. Furthermore, during asymptomatic colonization bacteria are not confined to the epithelial surface of the nasopharynx but are also found in clusters beneath the epithelial cell layer in tonsillar tissue (57). Therefore, defining the route and mechanisms of passage of the respiratory epithelial cell layer by N. meningitidis are relevant for understanding both meningococcal carriage and disease.Traversal of an epithelial barrier by a pathogen can be considered to consist of a number of steps, which include (i) adhesion to the apical surface, (ii) invasion of epithelial cells, (iii) survival within cells, (iv) movement to the basal side of cells, and (iv) escape from the basolateral aspect of the epithelium. The initial step in traversal by N. meningitidis involves attachment of bacteria to epithelial cells. Work with nasopharyngeal tissue in an organ culture model indicates that meningococcal attachment is largely limited to nonciliated cells of the respiratory epithelium (63). For encapsulated meningococci, adhesion is mediated by bacterial surface-associated filamentous structures called type 4 pili (Tfp) (42, 46, 50, 64), which are also required for colonization of human nasopharyngeal tissue (16). Tfp perform several important functions, including DNA uptake (77), twitching motility (43), and bacterial aggregation (28), and have been proposed to mediate meningococcal attachment to host cells by binding to CD46 (33). After initial adhesion, N. meningitidis forms microcolonies on the apical surface of epithelial cells and the microvilli of infected cells become elongated and interweave through the microcolonies (51, 63). Bacteria replicate within the microcolonies then disperse across the cells in a Tfp-dependent manner (51). After dispersal, the pili retract and bacteria become tightly associated with the host plasma membrane; at this stage bacteria may be internalized (50, 60, 63).Although the mechanisms that mediate attachment of N. meningitidis to epithelial cells are well understood, there is a paucity of information about the subsequent steps involved in traversal of the epithelial cell barrier. Indeed, there are even conflicting data regarding the route of traversal with both paracellular and transcellular routes identified in experiments with endometrial and gastrointestinal cells (3, 42, 50). Although the related pathogen, Neisseria gonorrhoeae, migrates through cells (76), it is not possible to extrapolate findings from this unencapsulated bacterium to the meningococcus, given the proposed role of capsule for survival within cells (60). Therefore, the aim of the present study was to determine the route of traversal of N. meningitidis across the respiratory epithelial cell barrier and define host and pathogen factors involved in this key step in pathogenesis. We used Calu-3 cells to establish a model for meningococcal traversal of the respiratory epithelial barrier. Calu-3 human bronchial epithelial cells are one of the few respiratory cell lines that differentiate into polarized monolayers when grown on porous membranes in vitro (14, 20, 21, 56). Growing the cells in this way allows bacteria to escape from the basolateral surface and provides them with the epithelial cell barrier encountered in the nasopharynx without the need for organ culture models, which are not well suited for defining mechanisms of cell entry and passage. Calu-3 monolayers display features of differentiated human airway epithelium with junctional complexes (24, 56) and apical-basal differentiation and possess transport and metabolism functions (20). These characteristics have led to Calu-3 cells becoming widely used for in vitro studies of drug delivery and toxicology of the human respiratory epithelium (5, 7, 14, 17, 38, 49). We show that N. meningitidis crosses the respiratory epithelial monolayer by a transcellular route without disrupting the barrier function of the layer. We also demonstrate that the bacterial capsule and Tfp are important for this process. These structures are also required for passage when cells are grown with an air interface. On the host side, an intact microtubule network is necessary for efficient traversal, while we observed no effect of the proposed Tfp receptor, CD46.     

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.     

Role of the Corneal Epithelial Basement Membrane in Ocular Defense against Pseudomonas aeruginosa

Pseudomonas aeruginosa can invade corneal epithelial cells and translocates multilayered corneal epithelia in vitro, but it does not penetrate the intact corneal epithelium in vivo. In healthy corneas, the epithelium is separated from the underlying stroma by a basement membrane containing extracellular matrix proteins and pores smaller than bacteria. Here we used in vivo and in vitro models to investigate the potential of the basement membrane to defend against P. aeruginosa. Transmission electron microscopy of infected mouse corneas in vivo showed penetration of the stroma by P. aeruginosa only where the basement membrane was visibly disrupted by scratch injury, suggesting that the intact basement membrane prevented penetration. This hypothesis was explored using an in vitro Matrigel Transwell model to mimic the corneal basement membrane. P. aeruginosa translocation of multilayered corneal epithelia grown on Matrigel was ∼100-fold lower than that of cells grown without Matrigel (P < 0.005, t test). Matrigel did not increase transepithelial resistance. Matrigel-grown cells blocked translocation by a P. aeruginosa protease mutant. Without cells, Matrigel also reduced traversal of P. aeruginosa and the protease mutant. Fluorescence microscopy revealed a relative accumulation of bacteria at the superficial epithelium of cells grown on Matrigel at 3 h compared to cells grown on uncoated filters. By 5 h, bacteria accumulated beneath the cells, suggesting direct trapping by the Matrigel. These findings suggest that the basement membrane helps defend the cornea against infection via physical barrier effects and influences on the epithelium and that these roles could be compromised by P. aeruginosa proteases.Pseudomonas aeruginosa is an important opportunistic pathogen, commonly affecting burn victims, individuals with cystic fibrosis, patients in hospital intensive care units, and contact lens wearers (9-11). In the absence of contact lens wear, the cornea is remarkably resistant to infection, with P. aeruginosa effectively colonizing this tissue only if it is injured or otherwise compromised (41). To initiate clinically significant corneal pathology, P. aeruginosa (and almost all other microbes) must first access the corneal stroma, which is normally protected by a multilayered epithelium and associated basement membrane (1).The corneal epithelial basement membrane is secreted by the overlying epithelia and is comprised of sheets of extracellular matrix constituents, including type IV collagen, heparan sulfate proteoglycan, and various glycoproteins (laminin, entactin, nidogen, and fibronectin) and growth factors that mediate cellular function (1). Quantitative imaging of the corneal epithelial basal membrane in the rhesus macaque has shown a complex cross-linking of fibers and proteins intermingled with pores ranging from 30 to 400 nm in size (2). As shown for other basement membranes in the body (17, 19, 33), the corneal basement membrane anchors epithelial cells and provides positional information for healing, tissue regeneration, and repair (44). In vitro, Matrigel forms an artificial basement membrane with pores ranging from 26 to 359 nm in size, is composed of laminin, collagen IV, heparan sulfate proteoglycans, entactin, nidogen, and naturally occurring growth factors, and closely resembles the natural corneal basement membrane (2, 26).It has been shown that P. aeruginosa isolates can translocate MDCK cell monolayers (6, 22) and that translocation and virulence were reduced by mutation of genes encoding multidrug resistance efflux systems (23). Purified elastase and exotoxin A from P. aeruginosa have each been shown to increase the permeability of MDCK cell monolayers (4), and purified elastase increases alveolar permeability in vivo (5). However, the role of the basement membrane in P. aeruginosa translocation has not been studied.We have previously shown that P. aeruginosa can translocate multilayered corneal epithelia in vitro and that human tear fluid reduced both translocation in vitro and virulence in vivo in murine models of corneal infection (28). While this and other studies have focused on the role of the tear film and corneal epithelium in defense against P. aeruginosa keratitis (14), little attention has been given to the basement membrane. Previous studies have reported that the epithelial basement membranes of other tissues can form a physical barrier to potential pathogens, including human papillomavirus, herpes simplex virus, and Rift Valley fever virus (25, 42, 43, 47). In this study, it was hypothesized that the corneal basement membrane forms a physical barrier to defend against the penetration of P. aeruginosa, since its pores are smaller than the size of bacteria, and that P. aeruginosa proteases can functionally overcome that defense. This hypothesis was examined correlatively using transmission electron microscopy of in vivo-infected mouse corneas and tested directly using a quantitative in vitro Matrigel-based model system to mimic the corneal epithelium and its associated basement membrane.     

The K5 Capsule of Escherichia coli Strain Nissle 1917 Is Important in Mediating Interactions with Intestinal Epithelial Cells and Chemokine Induction

Escherichia coli strain Nissle 1917 has been widely used as a probiotic for the treatment of inflammatory bowel disorders and shown to have immunomodulatory effects. Nissle 1917 expresses a K5 capsule, the expression of which often is associated with extraintestinal and urinary tract isolates of E. coli. In this paper, we investigate the role of the K5 capsule in mediating interactions between Nissle 1917 and intestinal epithelial cells. We show that the loss of capsule significantly reduced the level of monocyte chemoattractant protein 1 (MCP-1), RANTES, macrophage inflammatory protein 2α (MIP-2α), MIP-2β, interleukin-8, and gamma interferon-inducible protein 10 induction by Nissle 1917 in both Caco-2 cells and MCP-1 induction in ex vivo mouse small intestine. The complementation of the capsule-minus mutation confirmed that the effects on chemokine induction were capsule specific. The addition of purified K5, but not K1, capsular polysaccharide to the capsule-minus Nissle 1917 at least in part restored chemokine induction to wild-type levels. The purified K5 capsular polysaccharide alone was unable to stimulate chemokine production, indicating that the K5 polysaccharide was acting to mediate interactions between Nissle 1917 and intestinal epithelial cells. The induction of chemokine by Nissle 1917 was generated predominantly by interaction with the basolateral surface of Caco-2 cells, suggesting that Nissle 1917 will be most effective in inducing chemokine expression where the epithelial barrier is disrupted.A probiotic has been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (20). These benefits include the balancing and restoration of the intestinal microflora, repair of intestinal barrier functions (54), expression of bacteriocins (36), immunomodulatory effects (18, 43, 47, 53), and antagonizing epithelial colonization and invasion by pathogens (2). Escherichia coli strain Nissle 1917 was isolated from the feces of a soldier who did not develop diarrhea during a severe outbreak of shigellosis (38). Despite exhibiting a serotype (O6:K5:H1) that is characteristic of E. coli strains associated with urinary tract infections, Nissle 1917 apparently is nonpathogenic (25, 53) and has been used widely in preventing infectious diarrheal diseases (7, 14, 27, 37, 52, 53), the treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn''s disease (7, 23, 32, 33), and to prevent the colonization of the digestive tract of neonates by pathogens (35). Recently, there has been a growing interest in investigating the immunomodulatory effect of Nissle 1917. Previous studies showed that colonization by Nissle 1917 may lead to an alteration of the hosts'' cytokine repertoire (13, 49), increased immunoglobulin A secretion (14), lymphocyte or macrophage activation (13), the modulation of CD4⁺ clonal expansion (47), the stimulation of antimicrobial peptide production by intestinal epithelial cells (39, 52, 54), and alterations of the pro- and anti-inflammatory balance of local cytokines (49). Recently it has been shown that Nissle 1917 activates γδT cells, stimulating CXCL8 and interleukin-6 (IL-6) release but inhibiting tumor necrosis factor alpha (TNF-α) secretion (26). Following activation, Nissle 1917 induced apoptosis in activated γδT cells, indicating a key role for Nissle 1917 in interacting with the subset of T cells that operate at the interface between the adaptive and innate immune responses (26). Nissle 1917 also has been shown to express a direct anti-inflammatory activity on epithelial cells by blocking TNF-α-induced IL-8 secretion through a NF-κB-independent mechanism (28). Although the immunomodulatory effects of Nissle 1917 are well documented, the contribution of individual microbial components in mediating such effects is less well understood. So far, only a role for flagellin in mediating the induction of human β-defensin expression by Nissle 1917 has been established (44). Nissle 1917 expresses a K5 capsule on its cell surface, and a number of roles for polysaccharide capsules in the virulence of E. coli have been proposed, including resistance to phagocytosis and complement-mediated killing and the increased colonization of the host (42). In contrast, in the case of other encapsulated pathogens, it has been shown that the expression of a polysaccharide capsule can affect the induction of chemokines following attachment to host cells (6, 17, 22, 24, 40, 41, 45, 50). The aim of the present study was to investigate the role of the K5 capsule in mediating the immunomodulatory activity of Nissle 1917.     

Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Induces Apoptosis in Nonproliferating Macrophages by a Phosphatase-Independent Mechanism

Aggregatibacter actinomycetemcomitans strains that express cytolethal distending toxin (Cdt) are associated with localized aggressive periodontitis. However, the in vivo targets of Cdt in the human oral cavity have not been firmly established. Here, we demonstrate that A. actinomycetemcomitans Cdt kills proliferating and nonproliferating U937 monocytic cells at a comparable specific activity, approximately 1.5-fold lower than that against the Cdt-hypersensitive Jurkat T-cell line. Cdt functioned both as a DNase and a phosphatidylinositol 3-phosphate (PIP₃) phosphatase, and these activities were distinguished by site-specific mutagenesis of the active site residues of CdtB. Using these mutants, we determined that the DNase activity of CdtB is required for cell cycle arrest and caspase-dependent induction of apoptosis in proliferating U937 cells. In contrast, Cdt holotoxin induced apoptosis by a mechanism independent of caspase- and apoptosis-inducing factor in nonproliferating U937 cells. Furthermore, apoptosis of nonproliferating U937 cells was unaffected by the Cdt mutant possessing reduced phosphatase activity or by the addition of a specific PIP₃ phosphatase inhibitor, suggesting that the induction of apoptosis is independent of phosphatase activity. These results indicate that Cdt intoxication of proliferating and nonproliferating U937 cells occurs by distinct mechanisms and suggest that macrophages may also be potential in vivo targets of Cdt.Aggregatibacter (Actinobacillus) actinomycetemcomitans is associated with localized aggressive periodontitis (LAP) (40, 47, 48), a severe form of periodontal disease that results in the rapid destruction of the periodontal ligament and resorption of alveolar bone. Furthermore, growing evidence suggests that the oral cavity is a microbial reservoir for various systemic infections. In this regard, A. actinomycetemcomitans has also been associated with a variety of nonoral infections, including endocarditis, bacteremia, pericarditis, septicemia, pneumonia, infectious arthritis, osteomyelitis, synovitis, skin infections, urinary tract infections, and abscesses (45).Although A. actinomycetemcomitans expresses a variety of potential virulence factors, including epithelial cell adhesins (12, 13), an RTX leukotoxin (21, 23), and a cytolethal distending toxin (Cdt) (39, 41), their contributions to disease are not well understood. However, several studies suggest that Cdt may be important in the pathogenesis of LAP. For example, A. actinomycetemcomitans strains that possess the cdt operon are strongly associated with patients diagnosed with LAP (43). In addition, 97% of clinical A. actinomycetemcomitans isolates obtained from periodontitis patients in Sweden, Japan, Kenya, and Brazil possessed the cdt operon and were cytotoxic to CHO cells (10). In a separate study, Ahmed et al. (1) found that 86% of clinical A. actinomycetemcomitans isolates expressed Cdt and were toxic to HEp-2 cells.Cdt holotoxin is a tripartite complex comprised of subunits CdtA, CdtB, and CdtC (25, 33, 34). The CdtB protein is the active subunit and functions as a type I DNase (9, 24). However, a recent study shows that CdtB also functions as a phosphatidylinositol 3-phosphate (PIP₃) phosphatase and that many of the catalytic residues required for DNase activity are also necessary for phosphatase activity (36). CdtB is internalized by target cells, and internalization is inhibited by monensin, suggesting that entry occurs via the endocytic pathway (2). CdtA and CdtC are thought to interact with the target cell surface and may facilitate internalization of CdtB (2, 25, 27, 37). However, Mao and DiRienzo (27) suggest that both CdtB and CdtC are internalized by CHO cells and that CdtC may also possess toxic activity. CdtA is a putative lipoprotein that localizes to the bacterial outer membrane and is processed during secretion of the holotoxin (44).The in vivo cellular targets of the Cdt toxins are not well defined. Cdt holotoxin induces arrest in the G₂ phase of the cell cycle in a variety of proliferating cells, including epithelial cells, fibroblasts, human periodontal ligament cells, and lymphocytes (2, 3, 5, 8, 19, 20, 24, 25, 27, 28, 38, 41). Interestingly, Shenker et al. reported that the specific activity of Cdt against stimulated primary T lymphocytes was five- to 10-fold greater than that against HeLa cells (39) and subsequently showed that the Jurkat T-cell line is hypersensitive to Cdt intoxication (36). These results suggest that lymphocytes may be a primary physiologic target of the A. actinomycetemcomitans Cdt. Additional evidence that cells of the host immune response may be targeted by Cdt came from studies showing that purified Haemophilus ducreyi or Campylobacter jejuni Cdt induced apoptosis in nonproliferating dendritic cells (DCs) and macrophages (16, 26, 42, 46), although the specific activities against these cell types were not determined. Together, these studies suggest that many different cell types are potential targets of Cdt and that active proliferation may not be strictly required for Cdt intoxication.In this report, we show that A. actinomycetemcomitans Cdt induces apoptosis in both proliferating and nonproliferating U937 monocytic cells at a similar specific activity. Reconstituted Cdt holotoxin was shown to possess both DNase and PIP₃ phosphatase activities. Site-specific mutagenesis of CdtB active site residues generated one mutant with reduced DNase but significant phosphatase activity and a second mutant that was reduced in both activities. Cell cycle arrest and caspase 3-dependent induction of apoptosis in proliferating, nondifferentiated U937 cells were dependent on the DNase activity of CdtB. In contrast, Cdt-induced apoptosis in nonproliferating, differentiated U937 cells occurred by a mechanism independent of caspase- and apoptosis-inducing factor (AIF) and did not require a functional PIP₃ phosphatase activity. These results suggest that Cdt intoxication of proliferating and nonproliferating U937 cells occurs by distinct mechanisms and that macrophages may be potential in vivo targets of A. actinomycetemcomitans Cdt.     

Staphylococcal Alpha-Toxin Is Not Sufficient To Mediate Escape from Phagolysosomes in Upper-Airway Epithelial Cells

Intracellular Staphylococcus aureus has been implicated in the establishment of chronic infections. It is therefore imperative to understand by what means S. aureus is able to survive within cells. Here we use two expression systems with a fluorescent readout to assay alpha-toxin expression and function within phagolysosomes of infected upper-airway epithelial cells: avirulent Staphylococcus carnosus TM300 and phenotypically alpha-toxin-negative S. aureus laboratory strains. Data from CFU recovery assays suggest that the presence of alpha-toxin is not beneficial for the intracellular survival of recombinant Staphylococcus strains. This finding was corroborated by immunofluorescence studies: whereas S. carnosus and S. aureus are able to deliver S. aureus alpha-toxin to lumina of host cell phagolysosomes, the membrane integrity of these organelles was not affected. Alpha-toxin-expressing strains were detected exclusively within lysosome-associated membrane protein 1 (LAMP1)-yellow fluorescent protein (YFP)-positive vesicles. Measurements of intraphagosomal pH illustrated that all infected phagolysosomes acidified regardless of alpha-toxin expression. In contrast, S. aureus expressing Listeria monocytogenes listeriolysin O leads to the breakdown of the phagolysosomal membrane, as indicated by staphylococci that are not associated with LAMP1-YFP-decorated vesicles and that do not reside within an acidic cellular environment. Thus, our results suggest that staphylococcal alpha-toxin is not sufficient to mediate phagolysosomal escape in upper-airway epithelial cells.Staphylococcus aureus is readily internalized by nonprofessional phagocytes including keratinocytes and endothelial or epithelial cells (2, 29, 39, 40, 50). Internalization is mediated by the cell wall-linked adhesins fibronectin-binding protein A (FnBPA) and FnBPB (50), via an α₅β₁ integrin-dependent pathway (51). Upon invasion, S. aureus-containing endosomes eventually fuse with phagolysosomes, where a large portion of the internalized bacteria is inactivated by host cells (34, 47). A fraction of bacteria is able to persist within the host cells as so-called small-colony variants, and the ability of S. aureus to persist within human host cells has been implicated in the establishment of chronic infections (e.g., reviewed in references 17, 42, 43, and 48). Cytotoxic strains of S. aureus seem to follow a different strategy: it was previously shown that these strains escape the phagolysosomal vesicles and ultimately lead to host cell death (6, 29, 30, 49). In order to understand the contributions of single virulence factors to intracellular survival strategies of S. aureus such as phagolysosomal escape, detailed analyses on a gene-by-gene basis are necessary. Previous studies suggested that staphylococcal alpha-toxin is involved in the induction of host cell death (12, 24, 25, 56) and phagolysosomal escape (30). Furthermore, alpha-toxin has been described to be a key effector for intracellular pathogen survival within professional phagocytes (35). As a pore former that increases the permeability of cells or vesicles (14, 56) and exhibits enhanced hemolytic activity at low pH (8, 28), staphylococcal alpha-toxin is a prime candidate for mediating phagolysosomal escape (30, 35, 49). The toxin is secreted by S. aureus in a monomeric 27-kDa form. On target membranes, heptamers form and ultimately perforate the membrane for ions and macromolecules (reviewed in references 9, 21, and 53).In order to investigate if alpha-toxin causes phagolysosomal escape, we used apathogenic Staphylococcus carnosus TM300 (20) as well as noncytotoxic S. aureus laboratory strains to deliver alpha-toxin to phagolysosomes of upper-airway epithelial cells. Data from CFU recovery assays suggest that the presence of alpha-toxin is not beneficial for the intracellular survival of recombinant staphylococci. Immunofluorescence assays as well as pH measurements of infected phagolysosomes with confocal fluorescence microscopy demonstrate that the organellar membrane is not disrupted by alpha-toxin. Our results therefore suggest that staphylococcal alpha-toxin is not sufficient to mediate phagolysosomal escape in upper-airway epithelial cells.